2020 Book RhizosphereMicrobes

Microorganisms for Sustainability 23
Series Editor: Naveen Kumar Arora
Sushil Kumar Sharma
Udai B. Singh · Pramod Kumar Sahu
Harsh Vardhan Singh
Pawan Kumar Sharma Editors
Rhizosphere
Microbes
Soil and Plant Functions
Microorganisms for Sustainability
Volume 23
Series Editor
Naveen Kumar Arora, Environmental Microbiology, School for Environmental
Science, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India
More information about this series at http://www.springer.com/series/14379
Sushil Kumar Sharma • Udai B. Singh •
Pramod Kumar Sahu •
Harsh Vardhan Singh •
Pawan Kumar Sharma
Editors
Rhizosphere Microbes
Soil and Plant Functions
Editors
Sushil Kumar Sharma Udai B. Singh
NAIMCC Plant-Microbe Interaction and Rhizosphere
ICAR-National Bureau of Agriculturally Biology Lab
Important Microorganisms ICAR-National Bureau of Agriculturally
Maunath Bhanjan, Uttar Pradesh, India Important Microorganisms
Maunath Bhanjan, Uttar Pradesh, India
ICAR-National Institute of Biotic
Stress Management
Raipur, Chhattisgarh, India
Pramod Kumar Sahu Harsh Vardhan Singh

Plant-Microbe Interaction and Rhizosphere Plant-Microbe Interaction and Rhizosphere
Biology Lab Biology Lab
ICAR-National Bureau of Agriculturally ICAR-National Bureau of Agriculturally
Important Microorganisms Important Microorganisms
Maunath Bhanjan, Uttar Pradesh, India Maunath Bhanjan, Uttar Pradesh, India
Pawan Kumar Sharma

Plant Pathology
ICAR-National Bureau of Agriculturally
Important Microorganisms
Maunath Bhanjan, Uttar Pradesh, India
ISSN 2512-1901 ISSN 2512-1898 (electronic)

Microorganisms for Sustainability
ISBN 978-981-15-9153-2 ISBN 978-981-15-9154-9 (eBook)
https://doi.org/10.1007/978-981-15-9154-9
# Springer Nature Singapore Pte Ltd. 2020

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Preface
Plants are an incredible gift of nature not only to mankind but also to a myriad of
other organisms for their survival on the planet Earth. The impact of plants on the
environment including the microbiome and viceversa is enormous and is of great
significance to all the component partners. The system consisting of plants, their
environment, and all the associated organisms including microorganisms in this
environment is cumulatively known as phytobiome, while plants with their
associated extra- and endo-cellular microbiome constitute the holobiont. The
genome of such associated microbiome is referred to as the ‘second genome’ of
the plant. The plant genome and microbial genome collectively constitute
‘hologenome’. Similar to phytobiome, there is a concept of ‘rhizobiome’ which
consists of roots, their surrounding environment and all associated organisms living
in that particular environment. The rhizosphere is the zone around roots of the plants
in the soil that may extend from a millimetre to 3 centimetre and beyond around the
root surface. This zone contains sloughed off root, mucilage, root exudates and
gases. The gases released from roots in the soil dissipate to comparatively longer
distances leading to the extended size of rhizospheric zone up to many centimetres.
The rhizosphere is of paramount importance for ecosystem services, namely carbon
and water cycling, nutrient trapping/cycling or mobilization, carbon uptake and
storage etc. The rhizosphere, one of the most dynamic interfaces on Earth, contains
up to 1011 microbial cells per gram of soil representing over 30,000 bacterial
species. Such rhizomicrobiome plays an important role in the regulation of biogeo-
chemical cycles, global climate and sustaining plant growth. The health and produc-
tivity of plants are governed by various microbial mechanisms such as nutrient
solubilization and mineralization, biological nitrogen fixation, induced systemic
resistance (ISR), systemic acquired resistance (SAR), production of plant growth
regulators, siderophores, proton extrusion, organic acids, secondary metabolites and
volatile organic compounds (VOCs) as well as protection by enzymes like
1-aminocyclopropane-1-carboxylate (ACC)-deaminase, chitinase and glucanase
functioning in the rhizosphere. Ward off biotic stress, the plants protect themselves
by recruiting a group of disease-resistance inducing and plant growth-promoting
microorganisms (PGPM) in the rhizosphere which is under perpetual pressure from
soil-borne pathogens and helps to maintain PGPM descendents in the rhizosphere
soil. This phenomenon is called ‘soil-borne legacy’. Likewise, plants also have a
v
vi Preface
unique mechanism of deploying microbes that confer tolerance to abiotic stresses

such as drought, salinity, alkalinity, temperature etc. Hence, the plant system is
equipped with unique mechanisms of combating biotic and abiotic stresses via
beneficial microbes (Defense Biome) which tends to ameliorate the ultimate impacts
of these stresses and almost ensures the maintenance of better plant growth and
development. Manipulation of the rhizospheric system is, though an effective
strategy, yet a major challenge for modulating plant growth and development.
Traditionally, this manipulation is done by natural interventions with aim to improve
soil health but with greater understanding and advancement in technology, external
stimuli are now being provided in the form of certain biomolecules by
introducing PGPM or metabolites to make biased/engineered rhizosphere suitable
for desirable results. The soil characteristics and associated agricultural management
practices such as tillage, organic management and crop rotation also shift the soil
microbiome and determine soil quality in terms of plant growth and development.
This book addresses various issues of plant and soil that are to be modulated either
by resident microbes or by their external application. The book covers (1) the role of
microbes in soil and plant health, (2) methods for assessment of microbial diversity
in the rhizosphere, (3) microbes as the driver of nutrient transformation and soil
quality, (4) the role of microbes in bioremediation, and biotic and abiotic stress
management, (5) microbes associated with solubilization and mobilization of
micronutrients for biofertilization and biofortification, (6) signalling in the rhizo-
sphere and (7) commercial aspects of rhizospheric microbes. We expect that the
book would be useful for students, researchers, industrialists, entrepreneurs,
academicians and policymakers to understand the roles of rhizospheric
microorganisms in sustainable agriculture and provide directions for the future
course of action.
Uttar Pradesh/ Chhattisgarh Sushil K. Sharma

Uttar Pradesh Udai B. Singh
Pramod K. Sahu
Harsh V. Singh
Pawan K. Sharma
Contents
1 Microbial Interactions in the Rhizosphere Contributing Crop

Resilience to Biotic and Abiotic Stresses . . . . . . . . . . . . . . . . . . . . 1
Deepti Malviya, Udai B. Singh, Shailendra Singh, Pramod K. Sahu,
K. Pandiyan, Abhijeet S. Kashyap, Nazia Manzar, Pawan K. Sharma,
H. V. Singh, Jai P. Rai, and Sushil K. Sharma
2 Rhizosphere Microbes for Sustainable Maintenance of Plant
Health and Soil Fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Madhurankhi Goswami, Chandana Malakar, and Suresh Deka
3 Dissecting Structure and Function of Plant Rhizomicrobiome:
A Genomic Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Hemant Dasila, Samiksha Joshi, and Manvika Sahgal
4 Plant Root Exudates as Determinant of Rhizomicrobiome . . . . . . 105
V. Balasubramanian, Arunima Sur, Kush Kumar Nayak,
and Ravi Kant Singh
5 Rhizospheric Microbial Community: Ecology, Methods, and
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Amir Khan, Manisha Joshi, and Ajay Veer Singh
6 Signaling in the Rhizosphere for Better Plant and Soil Health . . . 149
Hemant S. Maheshwari, Richa Agnihotri, Abhishek Bharti,
Dipanti Chourasiya, Pratibha Laad, Ajinath Dukare,
B. Jeberlin Prabina, Mahaveer P. Sharma, and Sushil K. Sharma
7 Microbial Transformation of Nutrients in Soil: An Overview . . . . 175
Deep Mohan Mahala, Hemant S. Maheshwari,
Rajendra Kumar Yadav, B. Jeberlin Prabina, Abhishek Bharti,
Kiran K. Reddy, Chiranjeev Kumawat, and Aketi Ramesh
8 Microbial Indicator of Soil Health: Conventional to Modern
Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Dolamani Amat, J. K. Thakur, Asit Mandal, A. K. Patra,
and Kampati Kiran Kumar Reddy
vii
viii Contents
9 Rhizosphere Microbes: Driver for Soil Health Management . . . . . 235

H. K. Patel, R. V. Vyas, A. Ramesh, and J. P. Solanki
10 Ralstonia solanacearum: Biology and its Management in
Solanaceous Vegetable Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
A. Balamurugan, K. Sakthivel, R. K. Gautam, Sushil K. Sharma,
and A. Kumar
11 Seed Endophytes: The Benevolent Existence in the Plant System . . 291
Shrey Bodhankar and Minakshi Grover
12 Exploitation of Plant Tissue Invading Rhizospheric Microbes
as Bio-Fertilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Himani Chaturvedi and Anil Prakash
13 Contribution of Microbe-Mediated Processes in Nitrogen Cycle
to Attain Environmental Equilibrium . . . . . . . . . . . . . . . . . . . . . . 331
Humera Quadriya, Mohammed Imran Mir, K. Surekha,
S. Gopalkrishnan, M. Yahya Khan, Sushil K. Sharma,
and Hameeda Bee
14 Contribution of Zinc-Solubilizing and -Mobilizing Microorganisms
(ZSMM) to Enhance Zinc Bioavailability for Better Soil, Plant,
and Human Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Ramesh Chandra Yadav, Sushil K. Sharma, Aketi Ramesh,
Kusum Sharma, Pawan K. Sharma, and Ajit Varma
15 Fungal Siderophore: Biosynthesis, Transport, Regulation,
and Potential Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Keshawanand Tripathi, Narendra Kumar, Meenakshi Singh,
and Ravi Kant Singh
16 Status of Silicon in Ecosystem, Silicon Solubilization
by Rhizospheric Microorganisms and Their Impact on Crop
Productivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Prakash B. Nagabovanalli, Sabyasachi Majumdar,
and Sandhya Kollalu
17 Diversity and Function of Microbes Associated with Rhizosphere
of Finger Millet (Eleusine coracana) . . . . . . . . . . . . . . . . . . . . . . . 431
Renu Choudhary, Geeta Rawat, Vijay Kumar, and Vivek Kumar
18 Diversity and Community Structure of Arbuscular Mycorrhizal
Fungi in the Rhizosphere of Salt-Affected Soils . . . . . . . . . . . . . . . 453
R. Krishnamoorthy, R. Anandham, M. Senthilkumar, and Tongmin Sa
19 Beta-Glucanolytic Soil Actinomycetes: Diversity
and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Lekshmi K. Edison and N. S. Pradeep
Contents ix
20 Microbial Diversity of Chickpea Rhizosphere . . . . . . . . . . . . . . . . 483

Balram Sahu, Deep Chandra Suyal, Pramod Prasad, Vinay Kumar,
Anup Kumar Singh, Sonu Kushwaha, P. Karthika, Annand Chaubey,
and Ravindra Soni
21 The Rhizosphere Microbiome and Its Role in Plant Growth in
Stressed Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
Bhrigu Bhuyan, Sourav Debnath, and Piyush Pandey
22 Rhizobacteria-Mediated Alleviation of Abiotic Stresses in Crops . . 531
Priyanka Gupta and Manjari Mishra
23 Rhizospheric Microbes as Potential Tool for Remediation of
Carbofuran: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
Mohd Aamir Khan, Abhishek Sharma, Sonal Yadav,
and Satyawati Sharma
24 Trichoderma spp.: A Unique Fungal Biofactory for Healthy Plant
Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
Hesham Ali El Enshasy, Kugan Kumar Ambehabati,
Siti Zulaiha Hanapi, Daniel J. Dailin, Elsayed Ahmed Elsayed,
Dalia Sukmawati, and Roslinda Abd Malek
25 Management of Sclerotium rolfsii Induced Diseases in Crops by
Trichoderma Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
Ratul Moni Ram, Rahul Singh Rajput, and Anukool Vaishnav
26 Biotic Stress Management in Horticultural Crops Using Microbial
Intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
R. Umamaheswari, N. R. Prasannakumar, S. Sriram,
Sushil K. Sharma, M. S. Rao, and M. K. Chaya
27 Commercial Aspects of Biofertilizers and Biostimulants
Development Utilizing Rhizosphere Microbes: Global and Indian
Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
A. John Peter, E. Leo Daniel Amalraj, and Venkateswara Rao Talluri
Editors and Contributors
About the Editors
Sushil Kumar Sharma, National Agriculturally Important Microbial Culture Col-

lection (NAIMCC), ICAR-National Bureau of Agriculturally Important
Microorganisms, Maunath Bhanjan, Uttar Pradesh, India. Present Address: ICAR-
National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
Dr Sushil K. Sharma is currently working as a Principal Scientist (Agricultural
Microbiology) at ICAR-National Institute of Biotic Stress Management, Raipur,
Chhattisgarh, India in the area of microbial resource conservation, secondary
metabolites and antimicrobial peptides for biotic stress management of crops. In
the recent past, he worked at ICAR-National Bureau of Agriculturally Important
Microorganisms, Maunath Bhanjan, Uttar Pradesh, India in the capacity of officer
in-charge/focal point, National Agriculturally Important Microbial Culture Collec-
tion [NAIMCC-International Depository Authority (IDA)-Budapest Treaty Notifi-
cation No. 338]. He has delivered talks in International PGPR Conference in
Medellin, Columbia in June 2012 and subsequently in Asian PGPR Conference,
Tashkent, Uzbekistan in 2019. So far, he had published 110 articles (research,
review, book chapter, book, etc.) in both national and international journals/book/
conferences.
Udai B. Singh, Plant–Microbe Interaction and Rhizosphere Biology Lab, ICAR-

National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan,
Uttar Pradesh, India.
Dr. Udai B. Singh is presently working as Scientist (Senior Scale) in the Plant–
Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agri-
culturally Important Microorganisms, Kushmaur, Maunath Bhanjan, Uttar Pradesh,
India. His specialized area is plant–microbe interactions in the rhizosphere with
special reference to biotic and abiotic stress management/molecular biology/biotech-
nology/plant pathology. He has been awarded Fellow of Society for Applied Bio-
technology, ‘DST Young Scientist’ under Fast-Track Scheme, ‘Young Scientist
Award’ of RASSA, New Delhi, Bharat Shiksha Ratan Award, Scientist of the
Year Award-2019, K.P.V. Menon and Prof. K.S. Bilgrami Best Poster Award for
the Year 2018 by Indian Phytopathological Society, New Delhi and Indian Society
xi
xii Editors and Contributors
of Mycology and Plant Pathology, Udaipur. He has published several research

articles in the national and international journals of scientific reputes, books, scien-
tific magazines, technical bulletins, book chapters and scientific poplar articles. He is
also the editor the journal ‘Indian Phytopathology’ published by Springer Nature.
Pramod Kumar Sahu, Plant–Microbe Interaction and Rhizosphere Biology Lab,

ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath
Bhanjan, Uttar Pradesh, India.
Dr. Pramod Kumar Sahu has completed B.Sc. from IGKVV, M.Sc. (Gold-
Medalist) and Ph.D. (Agricultural Microbiology) from UAS, Bengaluru, India. He
was a DST-Inspire Fellow and was selected in various national level exams includ-
ing ICAR-JRF, ICAR-SRF, ICAR-NET, ARS and GATE. Being a Scientist of
Agricultural Microbiology at ICAR-NBAIM, Mau, India, working on plant–endo-
phyte interaction, consortium of bioinoculants and biological control and has more
than 45 publications, 6 training manuals, 13 extension folders, 3 popular articles and
30 abstracts to his credit. He has won Dr. B. P. Pal’s Prize Gold Medal,
Dr. G. Rangaswamy Gold Medal, Willium Rigo award, several best posters and
best oral presentation awards along with Young Scientist in Agricultural Microbiol-
ogy 2018 award.
Harsh Vardhan Singh, Plant–Microbe Interaction and Rhizosphere Biology Lab,

Bhanjan, Uttar Pradesh, India.
Dr. Harsh Vardhan Singh is currently working as a Principal Scientist (Plant
Pathology) at ICAR-National Bureau of Agriculturally Important Microorganisms,
Kushmaur, Maunath Bhanjan, Uttar Pradesh, India. He is specialized in the areas of
plant–microbe Interactions, biological control, biotic and abiotic stress management.
He has vast research experience in the field of plant pathology with special reference
to temperate crops of Jammu and Kashmir and grasses and fodder crops. He has
served as a Junior Scientist-cum-Assistant Professor in Plant Pathology (2002–2007)
at RARSS, SKUAST-K, Kargil, Programme Coordinator (2007–2009) at KVK,
Poonch, SKUAST-J and Senior Scientist (Plant Pathology) at ICAR-IGFRI, Jhansi
from 2009 to 2018.
Pawan Kumar Sharma, National Agriculturally Important Microbial Culture Col-

lection (NAIMCC), ICAR-National Bureau of Agriculturally Important
Microorganisms, Maunath Bhanjan, Uttar Pradesh, India.
Dr. Pawan Kumar Sharma did his M.Sc. and Ph.D. in Plant Pathology at DR YSP
University of Horticulture and Forestry, Solan, HP. He is working as Principal
Scientist at ICAR-NBAIM at Mau, UP since September 2013. He had been
associated with 14 projects as PI or Co-PI sponsored by various agencies. Currently,
he is working on biological control of wilt and collar rot diseases through liquid-
based formulation of Trichoderma and is also in-charge of National Agriculturally
Editors and Contributors xiii
Important Microbial Culture Collection. He has published 46 research papers in

national and international journals, edited 4 books and authored 1 book.
About the Series-Editor
Naveen Kumar Arora, Fellow of International Society of Environmental Botanists

(FISEB), PhD in Microbiology, is Professor and Head, Department of Environmen-
tal Science at Babasaheb Bhimrao Ambedkar University (a Central University),
Lucknow, Uttar Pradesh, India. He is a renowned researcher in the field of environ-
mental microbiology and biotechnology. His specific area of research is plant–
microbe interactions, particularly plant growth-promoting rhizobacteria. He has
more than 75 research articles published in premium international journals and
several articles published in magazines and dailies. He is an editor of 25 books,
published by Springer. He is a member of several national and international
societies, Secretary General of Society for Environmental Sustainability, in editorial
board of 4 journals and reviewer of several international journals. He is also the
editor in chief of the journal ‘Environmental Sustainability’ published by Springer
Nature. He has delivered lectures in conferences and seminars around the globe. He
has a long-standing interest in teaching at the PG level and is involved in taking
courses in bacteriology, microbial physiology, environmental microbiology, agri-
culture microbiology and industrial microbiology. He has been an advisor to 134
postgraduate and 11 doctoral students. He has been awarded for excellence in
research by several societies and national and international bodies/organizations.
Although an academician and researcher by profession, he has a huge obsession for
the wildlife and its conservation and has authored a book, Splendid Wilds. He is the
President of Society for Conservation of Wildlife and has a dedicated website www.
naveenarora.co.in for the cause of wildlife and environment conservation.
Contributors
Richa Agnihotri ICAR-Indian Institute of Soybean Research, Indore, Madhya

Pradesh, India
E. Leo Daniel Amalraj Prof. TNA Innovation Centre, Varsha Bioscience and
Technology India Private Limited, Jiblakpally, Yadadri District, Telangana, India
Kugan Kumar Ambehabati Institute of Bioproduct Development (IBD),
Universiti Teknologi Malaysia (UTM), Johor Bahru, Johor, Malaysia
A. Balamurugan Department of Plant Pathology, Centre for Plant Protection
Studies, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India
V. Balasubramanian Amity Institute of Biotechnology, Amity University
Chhattisgarh, Raipur, Chhattisgarh, India
xiv Editors and Contributors
Hameeda Bee Department of Microbiology, Osmania University, Hyderabad,

Telangana, India
Abhishek Bharti ICAR-Indian Institute of Soybean Research, Indore, Madhya
Pradesh, India
Bhrigu Bhuyan Department of Microbiology, Assam University, Silchar, Assam,
India
Shrey Bodhankar ICAR- Central Research Institute for Dryland Agriculture,
Hyderabad, Telangana, India
Present address: ICAR-Indian Institute of Oilseeds Research, Hyderabad,
Telangana, India
Himani Chaturvedi Department of Microbiology, Barkatullah University, Bhopal,
Madhya Pradesh, India
Annand Chaubey Banda University of Agriculture and Technology, Banda, Uttar
Pradesh, India
Rajan Chaurasia Institute of Environment & Sustainable Development, Banaras
Hindu University, Varanasi, Uttar Pradesh, India
M. K. Chaya ICAR-Indian Institute of Horticultural Research, Bengaluru,
Karnataka, India
Renu Choudhary Himalayan School of Biosciences, Swami Rama Himalayan
University, Dehradun, Uttarakhand, India
Dipanti Chourasiya ICAR-Indian Institute of Soybean Research, Indore, Madhya
Pradesh, India
Daniel J. Dailin Institute of Bioproduct Development (IBD), Universiti Teknologi
Malaysia (UTM), Johor Bahru, Johor, Malaysia
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti
Teknologi Malaysia (UTM), Johor Bahru, Johor, Malaysia
Hemant Dasila Department of Microbiology, G.B.Pant University of Agriculture
and Technology, Pantnagar, Uttarakhand, India
Sourav Debnath Department of Microbiology, Assam University, Silchar, Assam,
India
Suresh Deka Resource Management & Environment Section, Life Sciences Divi-
sion, Institute of Advanced Study in Science and Technology (IASST), Guwahati,
Assam, India
Ajinath Dukare ICAR- Central Institute of Post-Harvest Engineering and Tech-
nology (CIPHET), Abohar, Panjab, India
Lekshmi K. Edison Microbiology Division, KSCSTE-Jawaharlal Nehru Tropical
Botanic Garden and Research Institute, Thiruvananthapuram, Kerala, India
Editors and Contributors xv
Hesham Ali El Enshasy Institute of Bioproduct Development (IBD), Universiti

School of Chemical and Energy Engineering, Faculty of Engineering, Universiti
City of Scientific Research and Technology Application, New Burg Al Arab,
Alexandria, Egypt
Elsayed Ahmed Elsayed Bioproduct Development Chair, Zoology Department,
Faculty of Science, Kind Saud University, Riyadh, Kingdom of Saudi Arabia
Chemistry of Natural and Microbial Products Department, National Research Cen-
tre, Cairo, Egypt
R. K. Gautam Division of Germplasm Evaluation, ICAR - National Bureau of
Plant Genetic Resources, New Delhi, India
S. Gopalkrishnan ICRISAT-International Crops Research Institute for the Semi
Arid Tropics, Hyderabad, Telengana, India
Madhurankhi Goswami Environmental Biotechnology Laboratory, Resource
Management and Environment Section, Life Sciences Division, Institute of
Advanced Study in Science and Technology (IASST), Guwahati, Assam, India
Minakshi Grover ICAR-Indian Agriculture Research Institute, New Delhi, India
Priyanka Gupta Department of Biotechnology, Maharashtra Education Society’s
Abasaheb Garware College, Savitribai Phule Pune University, Pune, Maharashtra,
India
Siti Zulaiha Hanapi Institute of Bioproduct Development (IBD), Universiti
Manisha Joshi Department of Microbiology, G.B.Pant University of Agriculture
Samiksha Joshi Department of Microbiology, G.B.Pant University of Agriculture
P. Karthika Department of Agricultural Microbiology, College of Agriculture,
Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India
Abhijeet S. Kashyap Plant-Microbe Interaction and Rhizosphere Biology Lab,
Bhanjan, Uttar Pradesh, India
Amir Khan Department of Microbiology, G.B.Pant University of Agriculture and
Technology, Pantnagar, Uttarakhand, India
Mohd Aamir Khan Centre for Rural Development and Technology, Indian Insti-
tute of Technology Delhi, Delhi, India
Yahya Khan M Kalam Biotech Private Limited, Hyderabad, Telengana, India
xvi Editors and Contributors
Sandhya Kollalu Department of Soil Science and Agricultural Chemistry, Univer-

sity of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore, Karnataka,
India
Anup Kumar Singh Department of Agricultural Microbiology, College of Agri-
culture, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India
A. Kumar Division of Plant Pathology, ICAR - Indian Agricultural Research
Institute, New Delhi, India
Narendra Kumar Department of Biotechnology, IMS Engineering College,
Ghaziabad, Uttar Pradesh, India
Vijay Kumar Himalayan School of Biosciences, Swami Rama Himalayan Univer-
sity, Dehradun, Uttarakhand, India
Vinay Kumar ICAR-National Institute of Biotic Stress Management, Raipur,
Chhattisgarh, India
Vivek Kumar Himalayan School of Biosciences, Swami Rama Himalayan Uni-
versity, Dehradun, Uttarakhand, India
Chiranjeev Kumawat SKN Agriculture University, Jobner, Rajasthan, India
Sonu Kushwaha Department of Agricultural Microbiology, College of Agricul-
ture, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India
Pratibha Laad ICAR-Indian Institute of Soybean Research, Indore, Madhya
Pradesh, India
Deep Mohan Mahala ICAR-Indian Institute of Maize Research, Ludhiana,
Punjab, India
Hemant S. Maheshwari Ecophysiology of Plants, Faculty of Science and Engi-
neering, GELIFES-Groningen Institute for Evolutionary Life Sciences, Groningen,
The Netherlands
Past Address: ICAR-Indian Institute of Soybean Research, Indore, Madhya Pradesh,
India
Sabyasachi Majumdar Department of Soil Science and Agricultural Chemistry,
University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore,
Karnataka, India
Present address: College of Agriculture, Central Agricultural University — I,
Kyrdemkulai, Meghalaya, India
Chandana Malakar Environmental Biotechnology Laboratory, Resource Man-
agement and Environment Section, Life Sciences Division, Institute of Advanced
Study in Science and Technology (IASST), Guwahati, Assam, India
Department of Biotechnology, Gauhati University, Guwahati, Assam, India
Roslinda Abd Malek Institute of Bioproduct Development (IBD), Universiti
Editors and Contributors xvii
Deepti Malviya Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-

Uttar Pradesh, India
Nazia Manzar Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-
Mohammed Imran Mir Department of Botany, Osmania University, Hyderabad,
Telangana, India
Manjari Mishra Plant Stress Biology, International Centre for Genetic Engineer-
ing and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India
Prakash B. Nagabovanalli Department of Soil Science and Agricultural Chemis-
try, University of Agricultural Sciences, Bangalore, Karnataka, India
Kush Kumar Nayak Amity Institute of Biotechnology, Amity University
Piyush Pandey Department of Microbiology, Assam University, Silchar, Assam,
India
K. Pandiyan Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-
H. K. Patel Department of Agricultural Microbiology, College of Agriculture,
Indira Gandhi Krishi VishwaVidyalaya, Raipur, Chhattisgarh, India
A. John Peter Prof. TNA Innovation Centre, Varsha Bioscience and Technology
India Private Limited, Jiblakpally, Yadadri District, Telangana, India
B. Jeberlin Prabina Department Of Soil Science and Agricultural Chemistry,
Agricultural College and Research Institute Killikulam, Tamilnadu Agriculture
University, Killikulam, Tamil Nadu, India
N. S. Pradeep KSCSTE-Malabar Botanical Garden and Institute of Plant Sciences,
Kozhikode, Kerala, India
Anil Prakash Department of Microbiology, Barkatullah University, Bhopal,
Pramod Prasad Regional Station, ICAR-Indian Institute of Wheat and Barley
Research, Shimla, Himachal Pradesh, India
N. Prasannakumar ICAR-Indian Institute of Horticultural Research, Bengaluru,
Karnataka, India
Humera Quadriya Department of Microbiology, Osmania University, Hyderabad,
Telangana, India
xviii Editors and Contributors
J. P. Rai Department of Mycology and Plant Pathology, Institute of Agricultural

Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
Rahul Singh Rajput Faculty of Agricultural Sciences and Allied Sciences, Rama
University, Kanpur, Uttar Pradesh, India
Krishnamoorthy Ramasamy Department of Crop Management, Vanavarayar
Institute of Agriculture, Pollachi, Tamil Nadu, India
Aketi Ramesh ICAR-Indian Institute of Soybean Research, Indore, Madhya
Pradesh, India
Ratul Moni Ram Department of Plant Pathology, A.N.D.U.A & T, Kumarganj,
Ayodhya, Uttar Pradesh, India
M. S. Rao ICAR-Indian Institute of Horticultural Research, Bengaluru, Karnataka,
India
Geeta Rawat Himalayan School of Biosciences, Swami Rama Himalayan Univer-
sity Dehradun, Uttarakhand, India
Kiran K. Reddy ICAR- Directorate of Groundnut Research, Junagarh, Gujarat,
India
Manvika Sahgal Department of Microbiology, G.B.Pant University of Agriculture
Balram Sahu Department of Agricultural Microbiology, College of Agriculture,
Pramod K. Sahu ICAR-National Bureau of Agriculturally Important
Microorganisms, Kushmaur, Maunath Bhanjan, Uttar Pradesh, India
K. Sakthivel Crop Protection Section, ICAR -Indian Institute of Oilseed Research,
Hyderabad, Telangana, India
Abhishek Sharma Amity Food and Agriculture Foundation, Amity University,
Noida, Uttar Pradesh, India
Kusum Sharma ICAR- Indian Institute of Pulses Research, Kanpur, Uttar Pradesh,
India
Mahaveer P. Sharma ICAR-Indian Institute of Soybean Research, Indore,
Pawan K. Sharma Plant-Microbe Interaction and Rhizosphere Biology Lab,
Bhanjan, Uttar Pradesh, India
Satyawati Sharma Centre for Rural Development and Technology, Indian Insti-
tute of Technology Delhi, Delhi, India
Editors and Contributors xix
Sushil K. Sharma ICAR-National Bureau of Agriculturally Important

ICAR—National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
Ajay Veer Singh Department of Microbiology, G.B.Pant University of Agriculture
Harsh V. Singh Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-
Meenakshi Singh Department of Botany, Banaras Hindu University, Varanasi,
Ravi Kant Singh Amity Institute of Biotechnology, Amity University
Shailendra Singh Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-
Udai B. Singh Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-
J. P. Solanki Department of Agricultural Microbiology, B A College of Agricul-
ture, Anand Agricultural University, Anand, Gujarat, India
Ravindra Soni Department of Agricultural Microbiology, College of Agriculture,
S. Sriram ICAR-Indian Institute of Horticultural Research, Bengaluru, Karnataka,
India
Dalia Sukmawati Department of Biology, Faculty of Mathematics and Natural
Sciences, Universitas Negeri Jakarta, Jakarta, Indonesia
Arunima Sur Amity Institute of Biotechnology, Amity University Chhattisgarh,
Raipur, Chhattisgarh, India
K. Surekha ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad,
Telangana, India
Deep Chandra Suyal Department of Microbiology, Eternal University, Baru
Sahib, Himachal Pradesh, India
Venkateswara Rao Talluri TNA Innovation Centre, Varsha Bioscience and Tech-
nology India Private Limited, Hyderabad, Telangana, India
Keshawanand Tripathi Centre for Conservation and Utilization of Blue-Green
Algae, ICAR-Indian Agricultural Research Institute, New Delhi, India
xx Editors and Contributors
R. Umamaheswari ICAR-Indian Institute of Horticultural Research, Bengaluru,

Karnataka, India
Anukool Vaishnav Department of Mycology and Plant Pathology, Institute of
Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
Ajit Verma Amity University, Noida, Uttar Pradesh, India
R. V. Vyas Department of Agricultural Microbiology, B A College of Agriculture,
Anand Agricultural University, Anand, Gujarat, India
Rajendra Kumar Yadav Agriculture University, Kota, Rajasthan, India
Ramesh Chandra Yadav ICAR-National Bureau of Agriculturally Important
Amity University, Noida, Uttar Pradesh, India
Sonal Yadav Centre for Rural Development and Technology, Indian Institute of
Technology Delhi, Delhi, India
Microbial Interactions in the Rhizosphere
Contributing Crop Resilience to Biotic 1
and Abiotic Stresses
Deepti Malviya, Udai B. Singh, Shailendra Singh, Pramod K. Sahu,

K. Pandiyan, Abhijeet S. Kashyap, Nazia Manzar, Pawan K. Sharma,
H. V. Singh, Jai P. Rai, and Sushil K. Sharma
Abstract
Rhizosphere is a hot spot where specific kinds of diverse microbial communities

develop under the influence of exudates from plant roots and in turn modulate
growth and development of the plant. Such communities with or without
interactions perform an array of functions, including nitrogen fixation, P, Zn, Si
and K-solubilization, siderophore production, ammonification, hormones produc-
tion, ACC deaminase production, ethylene production, anammox, comammox,
nitrification, denitrification, antagonisms, induce resistance to plant,
C-sequestration, volatile production, secondary metabolites production and
many others that are known to modulate soil and plant health contributing to
the corresponding responses to various stresses of biotic and abiotic nature. The
magnitude of resilience of plant to biotic and abiotic stresses is completely
dependent on types of communities and their interactions. With enhanced knowl-
edge and understanding about rhizosphere, researchers are evaluating various
Deepti Malviya and Udai B. Singh have contributed equally.

All authors contributed in the preparation of manuscript. All authors have approved the final version
of the manuscript.
D. Malviya · U. B. Singh (*) · S. Singh · P. K. Sahu · K. Pandiyan · A. S. Kashyap · N. Manzar ·

P. K. Sharma · H. V. Singh
Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agriculturally
Important Microorganisms, Maunath Bhanjan, Uttar Pradesh, India
J. P. Rai
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
S. K. Sharma
Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agriculturally
Important Microorganisms, Maunath Bhanjan, Uttar Pradesh, India
Present Address: ICAR-National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
# Springer Nature Singapore Pte Ltd. 2020 1

S. K. Sharma et al. (eds.), Rhizosphere Microbes, Microorganisms for Sustainability
23, https://doi.org/10.1007/978-981-15-9154-9_1
2 D. Malviya et al.
approaches to engineer rhizosphere in such way that it enables plant to enhance

the productivity and sustain it while maintaining soil health. This chapter
highlights detailed account of microbial interactions in the rhizosphere with
associated mechanisms that contribute to resilience of plants to stress for better
growth and development.
Keywords
Plant microbiome · Rhizosphere interaction · Biotic stresses · Abiotic stresses ·
PAMPs · MAMPs · Rhizosphere engineering
1.1 Introduction
Plants existed before the arrival of the man on this planet and numerous species of
these serving as source of nutrition and medicine later became the security for man
against starvation and illness. Dependence of man on plants for food dates back to
more than 12,000 years (Singh et al. 2013). Plants are most widely accepted source
of food and nutrition for majority of the species on this planet, including vertebrates,
invertebrates, fungi, bacteria, and even other plants and humans in one way or the
other (Singh et al. 2019a, b). In the beginning, the life of man was that of a hunter
and food gatherer depending on consumption of whatever grew as wild. Later, he
started domesticating useful plant species first by vegetative propagation and then by
planting seeds (Rowley-Conwy and Layton 2011) and thus ensuring continuous
supply of food and eliminating the problem of food vagaries. With growing depen-
dence on plant-based sources of food and rapid increase in human population, the
global demand of plant-based food sources has increased tremendously. This is
reflected by the fact that nearly 1400 million ha of land (12% of the earth surface)
is under cultivation and 80% of the cultivated land area is under some form of food
crops. In spite of this, food production and food security are still the major
challenges before the agricultural scientists and researchers (Agrios 2005; Singh
et al. 2016a, b, 2019a, b). More than 800 million people across the world lack
adequate food and 1.3 billion people live on daily expenses being less than $1. The
availability of food to human population in reasonable quantity is largely governed
by the population density, cultivated land area available for food production, pro-
duction of food per unit area and most importantly, losses caused by biotic and
abiotic factors, including natural calamities (Gahukar 2011). The worldwide annual
yield losses caused by plant diseases and pests are estimated at USD$ 220 billion
(Liu et al. 2020). Among these, available land for cultivation is a limited resource
and that too is shrinking day by day owing to increased urbanization and develop-
mental activities (Singh et al. 2020a, b). Moreover, increase in production from per
unit land area available for food production cannot be proportionate to that in human
population principally on account of pest infestation and abiotic stresses, which our
1 Microbial Interactions in the Rhizosphere Contributing Crop Resilience to. . . 3
crops are exposed to. However, researchers continue their efforts to increase the
productivity every year (Agrios 2005; Singh et al. 2013) to meet the ever-growing
demands for quality food. Apart from biotic stress factors of pest and parasite
infestation, there are several abiotic stresses, viz. soil salinity, unfavourable (higher
and lower than optimum) temperatures, floods, droughts/frequent dry spells, air
pollution, organic contaminants, heavy metals and ultra violet light also take a
heavy toll on field crops (Bray et al. 2000), which contribute to reduction in the
crop productivity.
A number of natural enemies as well as rhizosphere microorganisms have been
identified and used against wide range of pests and pathogens to improve plant
growth, production and crop productivity directly and/or indirectly (Singh et al.
2013, 2016a, b, 2019a, b). Considering all the aspects of productivity enhancement
in food crops, plant-growth-promoting microorganisms (PGPM) are the best
examples of harnessing microbial activities for the purpose (Singh et al. 2016a, b).
Bio-augmentation of a specific PGPM or consortia of compatible PGPM in a specific
niche modulates the microbial community and plays a crucial role in the plant
growth (Sarma et al. 2015; Singh et al. 2016a, b). Colonization of the roots by
certain PGPMs induces systemic responses against subsequent biotic and abiotic
stresses at whole plant level due to a phenomenon called induced systemic resistance
(Singh et al. 2016b, 2019a, b) whereby resistance/tolerance (Singh et al. 2016a,
2020a, b) is induced in the plant. These beneficial microbes initiate localized and
systemic cellular mechanisms in associated plants against pathogens/stress factors.
There is no ambiguity in the fact that plants can use an array of cellular mechanisms
to defend themselves from stresses (Dixon et al. 2002; Harman et al. 2004; Shoresh
et al. 2010; Harman 2011). Root colonization by T. harzianum, apart from improv-
ing root growth and development with improved nutrients uptake and use efficiency,
also contributes to increase in the productivity of crop and its resistance to biotic
stress factors (Harman et al. 2004; Sarma et al. 2015). Strains of Pseudomonas
fluorescens, Trichoderma harzianum, T. viride, T. asperellum and strains of Bacillus
spp. are notable examples. Many researchers have reported that the proteome,
transcriptome and metabolome of plants alter due to the interaction of metabolites
of bacteria secreted into rhizosphere and plant system (Singh et al. 2016a, b; Malviya
et al. 2020). Thus, they re-programme the expression of plant genes leading to
changes in responses of plants to their environment. For this, the beneficial microbial
community in the rhizosphere could be enhanced through the inoculation of
microorganisms externally or bio-augmentation of native microbial community by
creating favourable micro-environment in the rhizosphere and/or incorporation of
organics and nutritional sources externally (Beckman 2000; Sarma et al. 2015). The
concept ‘Defence Biome’ gives the holistic overview of recruitment of microbiome,
plant-microbe interaction under stressed condition (Interactome) and interaction-
dependent modulation of physio-biochemical and molecular mechanisms in the
plant system such as: (1) stress modulate the exudate profile, which directly affects
the microbial community in the specific niche and (2) an increase in the abundance
of beneficial microbiota to compete for resources and space using bio-weapons and
quorum-sensing quenching of specific molecules (Liu et al. 2020). Moreover, abiotic
stresses can increase the community of particular plant-associated microbes via
4 D. Malviya et al.
(1) change in the physico-biochemical properties of niche (pH, EC, nutritional

status, etc.), which favour the specific community, (2) change in the physiology
and immunity, which alter the plant secretome profile and favour particular plant-
associated microbes, and (3) co-increase in the abundance of beneficial as well as
pathogenic microorganisms (Liu et al. 2020).
1.2 Rhizosphere Structure and Function
The plant rhizosphere is the key hot spot for plant-microbe interaction where
attraction of microbes during stressed conditions is mediated by diversified root
exudates and harbour vast microbial diversity being one of the most complex
ecosystems on the Earth (Liu et al. 2020). Rhizosphere harbours complex and
diverse community of microbes, including epiphytes, endophytes, saprophytes,
pathogens and also plenty of beneficial microbes (Avis et al. 2008; Buchholz and
Collins 2013). Plant-growth-promoting rhizosphere microorganisms (PGPM) com-
prising diverse microbial groups are able to promote plant growth and health directly
and/or indirectly. There is preponderance of bacterial genera, viz. Pseudomonas,
Bacillus, Alcaligenes, Azotobacter, Mycobacterium, Arthrobacter, Rhizobium,
Agrobacterium, Flavobacter, Cellulomonas and Micrococcus in the rhizosphere
(Malviya et al. 2020). Plant-associated beneficial microorganisms can have positive
effects on seed germination, seedling vigour, nourishment, plant growth and devel-
opment, disease suppression, and productivity. Plant–microbe interactions play a
key part in crucial ecosystem processes, viz. carbon sequestration, soil aggregation
and nutrient cycling in the rhizosphere ecosystem (Singh et al. 2004). The rhizo-
sphere microbiome is part of a composite food web that utilizes a large amount of
photosynthates released by the plant roots. During the microbial recruitment, roots of
plants generate biochemical signals that cause microorganisms, mostly zoospores of
oomycetes, to move towards the root (Gow 1999; Van West et al. 2003). Further, the
plant genotype, root exudates, border cells and mucilage are major driving forces.
The species of bacterial genera, viz. Bacillus, Azospirillum, Pseudomonas, Strepto-
myces, Klebsiella, Flavobacterium, Azotobacter, Enterobacter, Alcaligenes,
Bradyrhizobium, Mesorhizobium, Rhodococcus, Arthrobacter, Serratia, and
Burkholderia, etc. are known to promote growth of plants (Singh et al. 2016a, b,
2019a, b). Moreover, mycorrhizal fungi are also an important component of the
rhizosphere ecosystem, which are referred to as mutualistic micro-symbionts. They
perform ecosystem services such as nutrient mobilization, enhancement of plant
establishment and nutrient uptake, protection of plant from biotic and abiotic stresses
and also help maintain structure of the soil (Smith and Read 1997). Root exudate/
secretome comprises water-soluble sugars, amino acids and organic acids along with
a small amount of sugar, phosphate esters, hormones, vitamins, phenolics,
flavonoids and small peptides (Uren 2000; Bais et al. 2006). The stressors, viz.
temperature extremes, deficiency of nutrients and/or pathogenic stresses that influ-
ence membrane integrity and improve the efficiency of the exudation process
(Ratnayake et al. 1978; Singh et al. 2020d). The rhizosphere microorganisms utilize
these compounds leading to improvement in microbial biomass and activity in areas
surrounding the roots. This is referred to as rhizosphere effect. ATP-binding cassette

and the key transporters in the roots. These transporters activate and regulate root
exudation, which is an active process and thereby causes phytochemicals transloca-
tion into the rhizosphere (Loyola-Vargas et al. 2007; Badri et al. 2008). Flavonoids
are low-molecular-weight compounds that can mimic quorum-sensing molecules,
thereby influencing the bacterial metabolism (Hassan and Mathesius 2012). In plant
rhizosphere, quorum sensing plays vital role in the recruitment of microbial commu-
nity, establishing root–microbe associations, whether they are beneficial, symbiotic
or pathogenic. Thus, the quorum-sensing system controls the fundamental processes
of bacterial life such as formation of biofilms and motility (Lowery et al. 2008, 2009)
and is expected to influence the quantity and quality of volatile organic compounds.
Further, border-like cells from plant root tips and mucilage, which contains arabino-
galactan proteins in large amounts, are released into the rhizosphere. These proteins
fit in to the hydroxyl proline-rich glycoprotein super family of plant cell wall
proteins (Nguema-Ona et al. 2007, 2012, 2013) and play key roles in various
interaction processes between rhizospheric microbes and plant roots in the rhizo-
sphere ecosystem (Fig. 1.1).
1.3 Microbe-Mediated Mechanisms of Plant Defence to Biotic

Stress
A wide range of biotic and abiotic stimuli continuously challenge plants during
development (Genre et al. 2009). Therefore, in their quest for survival, plants have to
defend themselves against these stresses. Biotic stresses are induced by fungi,
bacteria, viruses, invertebrates and other plants. The effect of pathogenic challenge
and its extent naturally depends on the nature of the organism involved. Biotrophic
pathogens require a living host for their survival, while necrotrophic or hemi-
biotrophic pathogens first kill the host cells and then obtain nutrients from attacked
host cells (Abramovitch and Martin 2004). Natural openings, viz. stomata,
hydathodes, lateral roots, accidental wounds act as portals of entry for the pathogens.
Pathogens can also form appressoria and penetration pegs for direct penetration of
plant surface (Gudesblat et al. 2009; Melotto et al. 2006). Plant in itself is subjected
to stress when it activates its pathogen stress responses and therefore reduction in
yield can be attributed to both the disease and plant defence mechanisms (Heil et al.
2000) employed to counter foil the pathogenic attack or at least reduce its impact on
the plant as a whole, negotiating productivity. Plant mutants have reduced growth
phenotypes or develop disease-like lesions when they show constitutive
SA-dependent responses like PR protein biosynthesis (e.g. cpr mutants) (Alvarez
2000) and their fitness in the field is compromised (Heidel et al. 2004). Plants are
equipped with a multilayered system to recognize pathogenic invaders and trigger
defence responses against colonization (Muthamilarasan and Prasad 2013;
Wirthmueller et al. 2013). Responses of plants to different stressors are complex
and entail alterations at cellular, physiological and transcriptome levels (Atkinson
and Urwin 2012). Plants have arsenals of preformed physical or chemical barriers on
6
D. Malviya et al.
Fig. 1.1 Multi-trophic interactions in the rhizosphere ecosystem define the active rhizosphere effects
their surface, i.e. leaf hair, wax layers, rigid cell walls, antimicrobial secondary
metabolites and others. Induction of endogenous multi-component defence system
occurs when plant recognizes pathogens. If the pathogen overcomes physical
barriers of the plant, the plant recognizes the pathogen by means of its characteristic
chemical signatures, including chitin and flagellin, and then triggers comparatively
more specific biochemical defence responses. This defence response operates in
different plants with various extents of similarity against a common pathogenic
agent. It is operative even in the simple moss where in response to fungal cell wall
extract a peroxidase is produced (Lehtonen et al. 2009). The peroxidase prevents the
pathogenic growth. Plants are also known to produce an array of toxic defence
compounds, which are active against various pathogens. Pathogens suppress plant
defence responses and re-programme the host cell responses to pathogen metabolism
by producing effector proteins that are delivered into the cells. The effectors
produced by pathogens are recognized by the plant eventually leading to activation
of plant defence mechanisms controlled by resistance genes (Dodds et al. 2009). A
very good example of this can be seen in the case of Botrytis cinerea, which, upon
infection in sunflower, shifts the carbohydrate metabolism from hexose production
and alters it towards the mannitol pathway, which is required by the pathogen
(Dulermo et al. 2009).
Rhizosphere microbial populations are affected by plant defences. This happens
either through recruitment of beneficial bacteria or suppression of pathogen prolifer-
ation. Direct and indirect mechanisms are employed by PGPMs to promote growth
of different plants (Harman et al. 2004; Sarma et al. 2015). Processes like atmo-
spheric nitrogen fixation, production of siderophores, solubilization of phosphates,
synthesis and release of plant hormones are involved in direct plant growth promo-
tion. Promotion of plant growth indirectly results in control of diseases through
reduction in harmful effects of pathogens. Various metabolites, viz. cyanide,
antibiotics, antimicrobial peptides, extracellular lytic enzymes, including chitinases,
proteases, β-1,3 glucanases, cellulases, laminarinases cause suppression of
pathogens (Harman et al. 2004). The plant-growth-promoting bacteria produce
1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme (Glick et al. 2007)
in the rhizosphere. This enzyme is involved in stress signalling and negatively
regulates processes that cause elongation of roots. The enzyme ACC deaminase
causes hydrolysis of ACC to ammonia and α-ketobutyrate. Plants release ACC into
the rhizosphere, which is hydrolysed by the bacterial ACC deaminase, thereby
reducing ethylene-mediated repression of root growth. This interface is beneficial
for bacteria, as ammonia and α-ketobutyrate are sources of N and C, respectively.
Plants release ample amounts of secondary metabolites such as terpenes, flavonoids,
glucosinolates, phenylpropanoids, strigolactones (hormones) and antimicrobial
peptides into the rhizosphere. Plant-associated bacteria can trim down the activity
of pathogenic microorganisms by activating the plant to better defend itself, a
phenomenon termed ‘induced systemic resistance’ (ISR) (Shoda 2000; Van Loon
2007). The systemic resistance responses are, depending on the inducing
microorganisms, regulated by the plant hormones jasmonic acid, salicylic acid and
ethylene, foremost to an oxidative burst, the fabrication of secondary metabolites
8 D. Malviya et al.
and cell wall reinforcement. Sometimes, the mechanism of ISR elicited by rhizo-
sphere microorganisms overlaps to some degree with that of pathogen-induced
systemic acquired resistance (SAR).
1.3.1 Phenolics and Plant Resistance
Phenolic compounds are widespread in plants. These are secondary metabolites and
can be defined as a substance, which has an aromatic ring bearing one (phenol) or
more (polyphenol) hydroxyl substituent, including functional derivatives (esters,
methyl ethers, glycosides, etc.). They arise from the shikimate-phenylpropanoids-
flavonoids pathways, producing monomeric and polymeric phenols and polyphenols
(Harborne 1989). Phenolic compounds have antibiotic, anti-nutritional or unpalat-
able properties and, thus, they play a role in plant defence. They are involved in
plant-microorganism, plant–animal relationships and act as antioxidants and metal
chelators. These compounds act as UV light screens and signalling agents between
plant and other organisms in both below and aboveground environments (Wink
1997). Plant phenolics are of two types: preformed (constitutive) phenolics, which
are formed during plant development; and induced phenolics, which are synthesized
in plant in response to infection, physical injury, or upon exposure to abiotic stresses.
Induced phenolics are called phytoalexins (Nicholson and Hammerschmidt 1992;
Hammerschmidt 1999, 2003; Harborne 1999; Hammerschmidt et al. 2001; Dixon
et al. 2002; Sirvent and Gibson 2002; Winkel Shirley 2002). Pre-existing antifungal
phenolics are simple phenols, phenolic acids, flavonols and dihydrochalcones that
are common in plants and are responsible for non-host resistance to filamentous
fungi. They can be referred to as preformed antibiotics due to the fact that enzymes
that are involved in their activation are already present in plant. They are separated
from their substrates through compartmentalization and their rapid activation does
not require transcription of new gene products (Osbourn 1996). Preformed antifun-
gal phenolics are often tissue specific. The flavones, flavonols and other lipophilic
compounds are present in leaf wax at surface of plant and bud exudates or in the
epidermal cells in the cytoplasmic fraction. In healthy plants, preformed antifungal
phenolics are sequestered in conjugated form, usually with glycosidic attachments in
vacuoles or organelles (Wink 1997; Beckman 2000; Nicholson and Hammerschmidt
1992; Morrissey and Osbourn 1999; Katagiri et al. 2002). Biotrophs may avoid the
release of preformed antibiotics by minimizing the damage to the host, whereas
necrotrophs are likely to cause a substantial release of these compounds. Various
flavones and flavanones have been found to be active against Aspergillus sp.,
Botrytis cinerea and Fusarium oxysporum fungi that infect fruits and vegetables
during storage (Weidenbörner et al. 1990).
1.3.2 Induced/Systemic Disease Resistance
Unlike animals, plants are unable to move when they encounter outside stimulus;
therefore, they require more capabilities to cope with stresses and to adapt to
environmental fluctuations. There is a functional continuum between the plant cell
wall and the plasma membrane to mediate extracellular signals between these two,
and this continuum functions as cell wall integrity sensors (Humphrey et al. 2007;
Bouwmeester and Govers 2009; Sahu et al. 2016). After a pathogen overcomes
constitutive defence barriers on the host plant, it might be recognized at the plasma
membrane of plant cell. Pathogen-associated molecular patterns (PAMP) recogni-
tion causes activation of inducible plant defence responses. The PAMP are present in
all microorganisms (Thomma et al. 2001; Singh et al. 2019c, 2020d). Signalling
cascades are triggered by the PAMP perception systems and the recognition of these
cascades activates defence responses in natural plant-pathogen encounters
(Nürnberger and Lipka 2005). Broad-spectrum innate immune responses in the
plant are activated and these responses may be expressed at the site of pathogen
locally or in uninfected tissues of other plant parts. The stomata, hydathodes, lateral
roots or accidental wounds act as portals of entry for the pathogen or it may penetrate
the host directly by formation of specialized structures called appressoria/penetration
peg (Ryan 2000; Gudesblat et al. 2009; Melotto et al. 2006). The ability of the plant
to recognize the pathogen is the first line of defence, which is governed by cell
surface trans-membrane receptors. Two types of molecules can be recognized by
pattern recognition receptors or PRRs. The PRRs are present in plants and animals.
In plants, membrane-bound receptor-like kinases (RLKs) or receptor-like proteins
(RLPs) constitute PRRs (Boller and Felix 2009). Upon entry of pathogen into plant,
the damage-associated molecular patterns (DAMPs) are produced in the plant’s
apoplast. The DAMPs, which include cell wall fragments such as oligo-
galacturonides and cellulose fragments, cutin monomers and peptides such as
systemin, defensin and phytosulphokines, are recognized by PRRs (Albert 2013;
Nühse 2012; Ryan 2000). The pathogen-associated or microbe-associated molecular
patterns (PAMPs/MAMPs) that are conserved microbial structures are also
recognized by PRRs. The PAMPs/MAMPs are vital for fitness and physiology of
pathogen (Newman et al. 2013; Wirthmueller et al. 2013). The PAMPs/MAMPs
comprise peptidoglycan in Gram-positive bacteria, lipopolysaccharides in Gram-
negative bacteria, bacterial flagellins, eubacterial elongation factors (EF-Tu) and
fungal cell-wall-derived glucans, chitins and proteins. The PAMP/MAMP-triggered
immunity (PTI/MTI) response is activated upon perception of PAMP/MAMP and
DAMP by the PRRs causing downstream intracellular signalling events. Mitogen-
activated protein kinases are activated, reactive oxygen species are produced and
transcriptional reprogramming occurs. The net outcome is complex output response
of the plant that precludes growth of microbes (Wirthmueller et al. 2013). In
response to it, pathogens start a counter defence to overcome PTI by expressing
specific elicitors or effector proteins that are referred to as avirulence (Avr) proteins
(Grant et al. 2006). To inject effectors directly into the cytoplasm of plant cell,
pathogenic bacteria use type III secretion mechanisms to cause suppression of
10 D. Malviya et al.
PRR-dependent signalling to facilitate acquisition of nutrients and to ensure dis-

persal of pathogens leading to effector-triggered susceptibility (ETS) (Block et al.
2013; Cui et al. 2013). To counteract the pathogens, plants have co-evolved an
effector-triggered immunity (ETI), which is a second layer of defence that operates
in the plant cell. In ETI, an effector is recognized, leading to encoding of defence
proteins by specific resistance (R) genes. The majority of the R proteins have
nucleotide-binding leucine-rich repeat (NB-LRR). The PTI/MTI and ETI can result
in programmed death of host cell through local activation of a hypersensitive
response (HR), or there can be systemic acquired resistance (SAR) that activates
defences in distal, non-infected parts of plants. The result is increased resistance
throughout the plant (Thomma et al. 2011). Locally induced defence responses are
characterized by a hypersensitive response (HR). The plasma membrane potential
and its ion permeability are changed upon detection of the pathogen by host
resistance (R) genes. There is an increase in the level of extracellular pH and K+,
and influx of calcium and hydrogen ions into the cell. The outward K+ and the
inward Ca2+ and H+ ion flux are dependent and trigger HR, causing formation of
local lesions. Antimicrobial compounds are present in local lesions. Reactive oxygen
species (ROS), produced by the cells undergoing the HR, comprise hydrogen
peroxide, superoxide anions and hydroxyl radicals. Various enzymes, including
copper amine oxidase (which catalyses the oxidative deamination of polyamines
releasing hydrogen peroxide and ammonia), xanthine oxidase, peroxidase, NADPH
oxidase, oxalate oxidase, may be involved in generation of ROS. Partly some of
these cell changes may be due to lipid peroxidation and lipid damage and probably
affect membrane function. There is synthesis of phytoalexin and phenolics and other
compounds in cells around the lesion. Pathogenesis-related proteins (PRs) are
induced and there is deposition of callose and lignin (Singh et al. 2020a, b). These
PR proteins comprise four families of chitinases, one β-1, 3-glucanases, one protein-
ase inhibitors, and one specific peroxidase (Singh et al. 2013, 2016a, b, 2019a, b).
Chitinases and glucanases act on the fungal cell walls. The formation of lignin is
catalysed by peroxidase resulting in the strengthening of the plant cell wall (Lamb
and Dixon 1997; Sticher et al. 1997; Van Loon 1997; Maleck and Lawton 1998;
Mauch-Mani and Metraux 1998; Durrant and Dong 2004). Plants possess stress-
responsive signalling mechanisms, which are mediated by hormonal regulations
(Fig. 1.2). Phytohormones like salicylic acid are involved in both biotic (Vlot et al.
2008, 2009; Dempsey et al. 2011) and abiotic stress adaptation (Kunihiro et al. 2011;
Liu et al. 2012; Drzewiecka et al. 2012).
The signal transduction pathways in plants are regulated by salicylic acid,
jasmonic acid, and ethylene phytohormones. The regulation of these pathways
does not take place in an isolated manner (Fig. 1.2). There is a complex regulatory
network connecting different pathways to help or antagonize the others so as to
ensure the defence response to pathogens. Salicylic acid is responsible for activation
of defence response against biotrophic pathogens. Jasmonic acid and ethylene play a
role in defence against necrotrophic pathogens. There is mutual antagonism between
ethylene/jasmonic acid and salicylic acid pathways (Thomma et al. 2011; Kunkel
and Brooks 2002; Turner et al. 2002; Rojo et al. 2003; Glazebrook 2005; Lorenzo
1
Microbial Interactions in the Rhizosphere Contributing Crop Resilience to. . .
11
Fig. 1.2 Cross-talk and interactions between the jasmonic-acid-dependent and salicylic-acid-dependent pathways in the plants under biotic stress conditions
and Solano 2005; Van Loon et al. 2006). Plants produce ethylene to regulate defence
to pathogens (Chen et al. 2009). Fusarium graminearum infection in wheat causes
production of mycotoxins. To defend itself from the fungus, the wheat produces
ethylene, which stimulates the spread of the mycotoxin around the plant, causing
death of the host. Reduction in the level of ethylene production reduces the spread of
disease. Till date, only a few models have been studied to decipher plant defence
responses. Therefore, we do not know what the true capacity of plants to protect
themselves against pathogens. Through random sequencing approaches of microbial
populations from seawater samples, it became clear that our knowledge of gene
functions existing in nature is very limited (Venter et al. 2004).
1.4 Microbe-Mediated Mechanisms of Plant Defence to Abiotic

Stresses
Environmental stress refers to adverse effects on plant growth and development. At

the ecosystem level, any external constraint that limits productivity below the
genetic potential of the plants may be considered as stress. Stress can be defined
as a set of physical and chemical factors of the environment that are unfavourable for
growth of an organism (Tripathi et al. 2008; Singh et al. 2020a, b). Abiotic stress is
the negative impact of non-living factors on the plants in a specific environment. On
the basis of nature of source, abiotic stress may be of different types such as radiation
(visible, ultraviolet) salinity (salt/NaCl concentration), high temperature (heat
shock), low temperature (cold shock), water deficit (drought, desiccation), excess
water (flooding, anoxia), chemical stressors (pesticides, pollutants), heavy metals,
etc. (Doyon et al. 2010; Rhodius et al. 2012; Meena et al. 2017; Jiang et al. 2017).
1.4.1 Generation of Reactive Oxygen Species and Their Effects

Under Abiotic Stress
When oxygen comes in contact with metabolic systems having unpaired electron, it
transforms into more reactive and toxic forms, commonly referred to as reactive
oxygen species (ROSs). All ROSs are tremendously harmful to plants at higher
concentrations (Meena et al. 2017; Jiang et al. 2017). When the level of ROS
exceeds, the defence mechanisms are activated and a cell is said to be in a state of
‘oxidative stress’. Low concentration of ROS acts as messengers in various phyto-
hormone responses that include closure of stomata, gravitropism of root, germina-
tion of seed, biosynthesis of lignin, programmed cell death, hypersensitive responses
and osmotic stress (AbdElgawad et al. 2016; Sun et al. 2018). Higher concentration
of ROS is responsible for the oxidative stress and is deleterious for the lipid, protein,
and DNA synthesis and function. ROS show the harmful effects at lipid level, such
as chain breakage, which increases the membrane fluidity and permeability. How-
ever, at protein level, ROS affect in various ways such as site-specific amino acid
modification, fragmentation of the peptide chains, and aggregation of cross-linked
reaction products, altered electric charge, and enzyme inactivation, which increase
the susceptibility of proteins to proteolysis (Yamaguchi and Blumwald 2005; Bais
et al. 2006; Liu et al. 2006; Young et al. 2013; Mahmood et al. 2016; Bokhari et al.
2019; Wang et al. 2020). The consequence of higher accumulation of ROS also
affects the deoxyribose oxidation, strand breakage, removal of nucleotides, modifi-
cation of bases, DNA-protein cross-links (Zimmermann et al. 2010; Zhang et al.
2018).
1.4.2 Site of Synthesis of ROS
Chloroplast: PSI: electron transport chain, Fd, 2Fe-2S, and 4Fe-4S cluster, PSII:
electron transport chain, QA and QB, and chlorophyll pigment.
Mitochondria: Complex I: NADH dehydrogenase segment, Complex II: reverse
electron flow to complex I, Complex III: ubiquinone-cytochrome region, and
Enzyme:aconitase,1-galactono-y lactone dehydrogenase.
Peroxisome: Enzyme: xanthin oxidase, membrane: electron transport chain
flavoprotein, NADH and Cyt b, metabolic processes: glycolate oxidase, fatty acid
oxidation, flavinoxidases.
Plasma membrane: Electron-transporting oxido-reductases, and NADPH oxi-
dase, quinone oxidase.
Endoplasmic reticulum: NADPH-dependent electron transport involving
CytP450.
Cell-wall: Cell-wall-associated peroxidase and diamine oxidases.
Apoplast: Oxalate oxidase and amine oxidase.
1.4.3 Type of ROS Synthesized During Abiotic Stresses
Activation of O2 may arise by two different mechanisms: (1) absorption of sufficient

energy to reverse the spin-on-one of the unpaired electrons and (2) stepwise mono-
valent reduction. The ROS may be divided into four major categories:
Superoxide radical (O2˙ˉ): It reacts with double bond-containing compounds,
viz. iron-sulphur (Fe-S) clusters of proteins; reacts with nitric oxide (NO) to form
peroxynitrite (ONOOˉ).
Hydroxyl radical (OH˙): It is extremely reactive with protein, lipids, DNA, and
other macromolecules.
Hydrogen peroxide (H2O2): It oxidizes proteins; reacts with O2˙ˉ in an
Fe-catalysed reaction to form OH˙.
Singlet oxygen (1O2): It directly oxidizes protein and poly unsaturated fatty
acids.
1.4.4 Mechanisms to Scavenge ROS During Abiotic Stresses
1. Presence of systems in cells, which react with reactive forms of oxygen and keep
them at low level, i.e. superoxide dismutase (SOD), catalase, permease, total
peroxidase, proline, etc.
2. Presence of systems that regenerate oxidized antioxidants, i.e. glutathione reduc-
tase (GR), ascorbate (ASA), glutathione (GSH), mono and dihydroascorbate
reductase, etc.
1.4.5 Mechanisms of Plant Defence
Plants possess biochemical defence mechanisms, which reduce damage from ill
effects of biotic/abiotic stressors (Singh et al. 2019a, 2019b, 2020a, b, c). This
mechanism involves the induction of both de novo biosynthesis and rapid accumu-
lation of secondary metabolites, referred to as antioxidants. They have several
alternative mechanisms in the form of defence genes. These genes are consistently
present in plants, irrespective of whether they are resistant or susceptible to those
stresses. Signal transduction in plants in the direction of activating defence genes is
mainly accomplished by several inducers in the plant system (Zong et al. 2009; Zhu
2002, Zhu et al. 2003; Zahra et al. 2018). They have no direct inhibitory effect
against these stresses. The cascade of events that occurs in response to abiotic stress
consists of (1) it should be mobilizing a network of signal transduction pathways and
inducing the expression of sets of downstream genes and (2) It should be
synthesizing specific proteins, and accumulating compatible metabolites such as
specific sugars and proline (Fig. 1.2). The secondary messengers (ABA, ROS, Ca2
+
, inositol phosphates) can alter the levels of intracellular Ca2+, calcium binding
proteins (Colcombet and Hirt 2008). The phytohormone ABA is considered to play a
crucial role in the regulation of cellular responses to abiotic stresses (Danquah et al.
2014). They initiate a protein phosphorylation cascade after interacting with analo-
gous interacting partners. Transcriptome analyses using microarrays and proteomic
studies have given insight into plant signal transduction and gene regulation. Gene
chip and cDNA microarrays along with massive whole-genome sequencing have
enabled identification of new signalling determinants on a whole-genome scale in
response to different various stressors (Popescu et al. 2009). Proteomics approach is
handy in investigating post-translational modifications of the proteins. It can be used
to clone unique genes using differential analysis that will speed up our understanding
of stress-signalling mechanisms in plants (Shinozaki and Yamaguchi-Shinozaki
2007). Further, SNAREs are small, abundant, sometimes tail-anchored proteins,
which are often post-translationally inserted into membranes via a C-terminal
transmembrane domain. These proteins were originally identified as membrane
attached receptors for soluble NSF attachment proteins or SNAPs where NSF is an
NEM-sensitive factor. Plant SNAREs provide crucial physiological responses such
as abiotic stress, gravitropism, pathogen defence and developmental processes such
as autophagy, cytokinesis, morphogenesis idioblast (Lipka et al. 2007). A cDNA
screen in Xenopus laevis oocytes led to the recognition of a tobacco syntaxin,

NtSYR1, which interferes with ABA-triggered potassium and chloride ion fluxes
in both Xenopous oocytes and Nicotiana tabacum guard cell protoplasts. NtSYR1
appears to interact with a tobacco homolog of the Qb + Qc-SNARE, AtSNAP33 and
it plays important role in abiotic stress responses (Geelen et al. 2002). In abiotic
stress tolerance, trehalose is one of the important elicitors in plant defence
mechanisms and induces WRKY6 and other genes, which activate the several
defence cascades in a cooperative manner (Fig. 1.3). It has been shown that trehalose
scavenge ROS under abiotic stresses in a concentration-dependent manner and
protect plants from ill-effects of abiotic stresses (Fernandez et al. 2010; Lunn et al.
2014; Shi et al. 2019).
1.4.6 MAPK Signalling
Plant mitogen-activated protein kinases (MAPK) receive signals from receptors or

sensors and phosphorylate downstream MAPK kinases, which subsequently activate
MAPKs that control the activities and synthesis of transcription factors (TFs),
enzymes, hormones, peptides and antimicrobial chemicals (Zhu et al. 2019; Xu
et al. 2020). The fungi and plants have histidine kinase transduction systems.
These sensors are incorporated into more complex pathways. MAPKs are activated
by ROS. This activation is important for mediation of cellular responses. This
preliminary increase in ROS may be further enhanced by various stresses and
production centres. Transient increases in ROS may start signal transduction
cascades that involve cross-talk with jasmonates (JAs), salicylic acid (SA), ethylene
(ET), abscisic acid (ABA), polyamines and nitric oxide (NO) (de Zelicourt et al.
2016). It can amplify the subsequent cascade through transducer sensors and targets
of ROS-dependent and cell-death-related gene expression. There is also cross-talk
with the plant-pathogen signal transduction pathway, which might rely on detection
of pathogen by the gene-for-gene mechanism and can lead to an opposite effect on
the regulation of production of ROS and ROS-scavenging mechanisms, and on the
activation of programmed cell death (Smékalová et al. 2014). Besides MAPK-
obsessed phosphorylation cascades, other regulatory post-translational
modifications, such as protein oxidation and nitrosylation, might be involved in
ROS-dependent cell death pathways (Thomma et al. 2001; Colcombet and Hirt
2008).
1.5 Endophytes in Biotic and Abiotic Stress Management
In last few decades, there has been a huge development in the endophyte research
with respect to the plant growth and health promotion (Compant et al. 2005; Sahu
et al. 2017a, 2018, 2019a, b, 2020a, b; Singh et al. 2020a). Endophytic microbes are
plant dwellers, which do not cause any harm to their hosts (Schulz and Boyle 2006;
Backman and Sikora 2008). History indicates tremendous potential of endophytic
Fig. 1.3 An overview of microbe-mediated modulation of physio-biochemical mechanisms of

abiotic stress tolerance in plants. CW represents cell wall, PM plasma membrane, POx peroxidase,
OOx oxalate oxidase, AOx Ascorbate oxidase, SOD superoxide dismutase, CAT catalase, APx
ascorbate peroxidase, HR hypersensitive response, IAA Indole acetic acid, MAPK Mitogen-
activated protein kinase, CWR crown root. Overall figure depicts how the plant recruits microbiome
under stressed condition and how the microbiome serves as the first line of defence and maintains
ion homeostasis and plants growth and development under stress conditions
microbes for novel secondary metabolites and other bioactive compounds. In recent
past, endophytes were explored for production of growth hormones, antimicrobial
compounds, stress-alleviating compounds, organic and inorganic acids for nutrient
solubilization, etc. which, in turn, help plants in nutrient uptake, growth promotion
(Sahu et al. 2017b, 2018), nutrient fortification (Singh et al. 2018), biotic (Ting et al.
2010; Thomma et al. 2001; Sahu et al. 2020a) and abiotic stress tolerance (Meena
et al. 2017; Singh et al. 2020b).
New dimensions have been realized in biocontrol of fungal pathogens using
endophytes (Sahu et al. 2020b). Endophytes have advantage of being close to
plant cells to influence and protect plants against pathogens (Compant et al. 2005;
Kloepper and Ryu 2006; Bakker et al. 2013). It is not evident that all endophytes
provide protection against pathogens, but report indicates strongly that the
endophytes are having huge potential to be used as excellent biocontrol agents
(Table 1.1) as they endorse disease tolerance against wide array of plant pathogens
(Berg 2009). Induced systemic resistance (ISR) (Feng et al. 2013; Sahu et al.
2019a, b, 2020a), production of antifungal compounds - iturin, surfactin, fengycin
(Wang and Liang 2014; Sahu et al. 2020b), proteases, chitinases, siderophore
production, competition for nutrients, volatile organic compounds (Sahu and
Brahmaprakash 2018), etc. are few of the major mechanisms of pathogen suppres-
sion by endophytes (Nimnoi et al. 2010; Sahu et al. 2017b). After Wei et al. (1991)
reported ISR for first time by bacteria Pseudomonas fluorescens strain G8-4 against
anthracnose disease of cucumber, massive efforts have been poured to harness it for
suppressing plant pathogens using endophytes (Sahu et al. 2019a, a). ISR against
plant pathogens involves interaction of plants with microbial-associated molecular
patterns (MAMPs) present in beneficial microbes. There are reports of ISR by
endophytes against several pathogens like cauliflower mosaic virus (CMV; Murphy
et al. 2003), Sclerotium rolfsii (Sahu et al. 2019a), Fusarium oxysporum (Chen et al.
1995; Constantin et al. 2019), Rhizoctonia solani (Sahu et al. 2020a), Agrobacterium
tumifaciens (Asghari et al. 2020), etc.
Five fungal endophytes were studied for compatibility with the host (oil palm)
and suppressive ability to Ganoderma in endophyte-calli and endophyte-ramet tests.
Antagonists were found to produce volatile, non-volatile compounds and competi-
tion against Ganoderma boninense (Cheong et al. 2017). Endophyte BTF08 (Peni-
cillium citrinum) was found to enhance calli weight (1013 mg) by promoting its
growth. In vitro screening of endophytes from poplar and willow plants has shown
the efficiency of endophytes in growth promotion, abiotic stress mitigation and
suppression of pathogen. These endophytes were found to have antagonism against
Gaeumannomyces graminis, Rhizoctonia solani, Pythium ultimum and Fusarium
culmorum apart from having different plant growth-promotion activities (Kandel
et al. 2017).
Study of antibiotic marker labelled endophytic bacterium Bacillus subtilis
DZSY21 from Eucommia ulmoides indicated its effective colonization in maize
plant and suppression of southern corn leaf blight (Bipolaris maydis). Production
of antimicrobial compounds surfactin A, surfactin B and fengycin by the endophyte
was detected by MALDI-TOF-MS analysis. Up-regulation of PDF1.2 and
pathogenesis-related genes PR1 and LOX is found as a result of ISR (Ding et al.
2017).
Apart from direct biocontrol activity, endophytes also improve plant yield under
biotic stress by producing plant growth promoting substances like indole acetic acid,
indole pyruvic acid, isopentenyl adenine, gibberellic acid, isopentenyl adenosine,
Table 1.1 Major mode of actions of endophytic biocontrol agents
18
Mechanism of action Endophyte Pathogen Host Reference

ISR Pseudomonas fluorescens Anthracnose pathogen Cucumber Wei et al.
strain G8-4 (1991)
ISR Bacillus pumilus strain Erwinia tracheiphila Cucumber Zehnder
INR7 et al. (2001)
Chitinases Streptomyces plicatus Fusarium oxysporum f.sp. lycopersici and Tomato Abd-Allah
Verticillium alboatrum (2001)
ISR Bacillus subtilis CMV Tomato Murphy
et al. (2003)
ISR B. pumilus strain SE34 and BBTV Banana Zhang et al.
Serratia marcescens strain (2004)
90-166
Siderophore production Streptomyces Fusarium oxysporum f. sp. cubense Banana Cao et al.
griseorubiginosus (2005)
Siderophore production Endophytic actinomycetes Ralstonia solanacearum Tomato Tan et al.
(2006)
ISR Endophytic actinomycetes Erwinia carotovora Arabidopsis Conn et al.
thaliana (2008)
β-1,3, β-1,4 and β-1,6-glucanases, Actinoplanes, Pythium aphanidermatum Cucumber El-Tarabily
phytohormones production Micromonospora, et al. (2009).
Streptomyces
Siderophores and protease Endophytic actinomycetes – Aquilariacrassna Nimnoi et al.
(2010)
Production of antibiotics (NRPS and PKS Streptomyces sp. Pythium aphanidermatum Cucumber Zhao et al.
genes) (2011)
Surfactin and fengycin Bacillus amyloliquefaciens Ralstonia solanacearum Tomato Wang and
BZ6-1 Liang
(2014)
D. Malviya et al.
1
VOCs Endophytes Plant pathogens – Chung et al.

(2016)
2,3-Butanediol Bacillus subtilis Ralstonia solanacearum Pepper Yi et al.
(2016)
Surfactin A, surfactin B, fengycin and ISR Bacillus subtilis DZSY21 Bipolaris maydis Maize Ding et al.
(2017)
Volatile, non-volatile compounds and Penicillium citrinum Ganoderma boninense Oil palm Cheong
competition BTF08 et al. (2017)
Growth promotion, abiotic stress Endophytes Gaeumannomyces graminis, Rhizoctonia Poplar and Kandel et al.
mitigation and suppression of pathogen solani, Pythium ultimum, and Fusarium willow plants (2017)
culmorum
Surfactin A, surfactin B and fengycin Bacillus subtilis DZSY21 Bipolaris maydis Maize Ding et al.
production; up-regulation of PDF1.2 and (2017)
pathogenesis-related genes
ISR, antibiosis Fusarium endophyte Fo47 Fusarium oxysporum Tomato Constantin
et al. (2019)
Antibiosis, ISR, siderophores, ammonia, Bacillus sp. 1PR7a, Sclerotium rolfsii Tomato Sahu et al.
HCN Bacillus sp. 2P2; Bacillus (2019a)
sp. 2PR9b
Antibiosis, ISR, siderophores, ammonia, Bacillus altitudinis GTS-16 Rhizoctonia solani Rice Sahu et al.
HCN (2020a)
Induction of stilbenic phytoalexin; Pseudomonas sp. Sn48 and Agrobacterium tumefaciens Grapevine Asghari
upregulation of PR1, PR2, and PR4 gene Pantoea sp. Sa14 et al. (2020)
expression
Microbial Interactions in the Rhizosphere Contributing Crop Resilience to. . .
19
ammonia, etc. (El-Tarabily et al. 2009; Nimnoi et al. 2010) enhance nutrient uptake
(Malinowski et al. 2000), anti-herbivory substances (Sullivan et al. 2007) and
augment sulphur nutrition (Aziz et al. 2016). With all these effects, endophytes
provide protection against several plant pathogens, which can be harnessed for
sustainable yield enhancement.
Abiotic stress alleviation by endophytes is also well documented (Yu et al. 2019).
Production of stress-reducing compounds by the endophytes is reported as a mecha-
nism for mitigating abiotic stress in plants (Zhu 2002; Schulz et al. 2002). Increased
nutrient assimilation, plant growth, and decreased toxicity to sodium ions by inocu-
lation of Phoma glomerata and Penicillium sp. were reported under salt and drought
stress (Waqas et al. 2012). Production of ACC-deaminase enzyme is one of the key
mechanisms in reducing the harmful effects of stress (Yue et al. 2019). Change in the
expression of stress-responsive genes is another strategy of endophytes that provides
protection against abiotic stresses (Meena et al. 2017; Bilal et al. 2020).
Govindasamy et al. (2020) reported upregulation of drought-responsive genes
sbP5CS2 and sbP5CS1 in the plants treated with bacterial endophytes
Ochrobactrum sp., Microbacterium sp., and Enterobacter sp. against the control
and Escherichia coli-inoculated plants. Down regulation of heavy metal ATPase
gene expression (GmHMA13, GmHMA14 and GmHMA18) and upregulation of
drought-responsive genes (such as GmDREB2) in the Glycine max L. inoculated
with fungal endophytes Paecilomyces formosus LHL10 and Penicillium
funiculosum LHL06 reduced the accumulation of heavy metals in the plants (Bilal
et al. 2020).
1.6 The ‘Holobiome’: Significance of Microbes in Host

Development and Behaviour
In recent past, a series of studies have unveiled that an individual is actually an

arrangement of ‘biomolecular networks’ consisting of visible hosts plus millions of
invisible microbes. These microorganisms have a significant effect not only on the
host development and its behaviour but also on its susceptibility to the diseases and
possibly determine its social interactions with surrounding environment (Suárez
2020). As a whole, the individual behaves as a ‘holobiome’, which is made up of
‘own genome’ and associated ‘microbiome’ (Kim and Lee 2020). A lot more applied
values of this concept are being investigated in clinical microbiology to cure
diseases. With respect to the plants, Leach et al. (2017) have described it as
phytobiome consisting of plants, the organisms associated and the environment.
Next-generation sequencing, metagenomics, metatranscriptomics, metabolomics,
metaproteomics, etc. have made the complex analysis possible to decipher the
impacts of microbiome on the plant system. The importance of the phenomenon
could be understood from the fact that microbiome has been proposed as a platform
for the next green revolution (Čatská et al. 1997; Rodriguez and Durán 2020). The
microbiota and the plants have been co-evolved for maintaining the balance under
different biotic and abiotic stresses. Thus, the investigative studies on ‘holobiome’
could provide novel insights for plant growth and health promotion in the lieu of
second green revolution.
1.7 Rhizosphere Engineering for Stress Management
Improving tolerance towards biotic and abiotic stresses by crop plants remains an
issue that can be solved with molecular tools and techniques. However, in general,
different mechanisms have been evolved by the plant to cope with environmental
stresses (Zhu 2002; Dixit et al. 2015). Plants respond to stresses by accumulation of
compatible osmolytes, metabolites and macro-molecules at cellular level.
Now a days, focus has been given to explore the feasibility of molecular biology
tools and techniques for plant as well as microbes engineering to increase the
tolerance against abiotic and biotic stresses with the enhancement in productivity.
Genetic engineering gives base to transfer one or more genes involved in regulation
and pathways signalling that encodes for the functional and structural defence
compounds (osmolytes and antioxidants). Different techniques, viz. differential
display PCR, suppression subtractive hybridization, serial analysis of gene expres-
sion, DNA microarray and cDNA-amplified fragment length polymorphism, have
been described to identify the genes expressed during different biotic and abiotic
stresses in the plants. Genetically modified stress-tolerant crops, developed by
bioengineering of stress signalling pathways, are the major goal for agricultural
research. Transgenic plant of Arabidopsis thaliana was developed as an
osmotolerant by introduction of proBA genes derived from Bacillus subtilis that
produced higher level of free proline and increased the tolerance against osmotic
stress (Chen et al. 2007). Further, designing of the rhizosphere and plant-associated
microorganism for a specific plant species is an approach to achieve better crop
growth and productivity under stressed conditions (Blount et al. 2012). Advance-
ment in tools for the genetic manipulation like genetic engineering, genome
sequencing, metabolic engineering and synthetic biology helps in design of
microbes as per requirement of desirable traits (Lovley 2012).
1.8 Application of Nano-Bio-Technology
Annual agricultural crop losses through plant diseases are caused by various groups
of microorganisms, including fungi, bacteria, viruses and nematodes that result in
yield reduction, and poor product quality and shelf life. To meet food demand by
global population and changing climate, the need to enhance agricultural productiv-
ity with simultaneous reduction in use of inorganic chemicals for plant stress
management is being underlined. For long-term strategy, novel platforms for crop
disease management are critically needed. However, considering the possible
alternatives, studies on effects of nano-particles on management of plant pathogens
have indicated better prospects in this regard. After application of bio-fabricated
silver nanoparticles, significant reduction in Bipolaris sorokiniana infection was
recorded in wheat plants. Bio-fabricated silver nano-particles at 2, 4 and 10 μg ml 1

concentrations exhibited complete conidial germination inhibition, but in the
absence of bio-fabricated silver nano-particles conidial germination was 100%.
Histochemical studies revealed lignin deposition in vascular bundles induced after
bsAgNPs treatment (Mishra et al. 2014).
Nanoparticle TiO2 with Zn 500 to 800 ppm (μg ml 1 or μg g 1) formulation
exhibited significant reduction in the survival of Xanthomonas sp. strain Xr-1
causing bacterial leaf spot on Rosa ‘Noare’. In non-coated or non-illuminated
controls, there was no reduction of bacterial viability. Light-activated nano-particle
TiO2 with Zn activity was a better option for management of rose diseases (Paret
et al. 2013). The MgO and ZnO nano-particles had an antifungal activity and were
evaluated for Alternaria alternata, Fusarium oxysporum, Rhizopus stolonifer and
Mucor plumbeus. From this study, it was revealed that nano-particles of MgO and
ZnO at highest concentration (0.5 ml) caused maximum inhibition in the spore
germination of Mucor plumbeus, Alternaria alternata, Fusarium oxysporum and
Rhizopus stolonifer followed by 0.3, 0.2 and 0.1 ml concentrations of nano-particles,
whereas least reduction in spore germination was found in untreated control (Wani
and Shah 2012).
Silver nano-particles had shown antifungal activities at various concentrations.
The disease incidence of powdery mildew on cucumber and pumpkin was 53.4,
34.4, 25 and 20% when treated with 10, 30, 50 and 100 ppm silver nano-particles,
respectively. The highest inhibition rate of powdery mildew was observed at
100 ppm silver nano-particles. It was suggested that silver nano-particles were
more effective for microorganisms, which show less sensitivity to antibiotics due
to poor penetration of some antibiotics. Silver nano-particles may disrupt the ion
efflux transport systems, which can cause rapid accumulation of silver ions that leads
to interruption of cellular processes. For example, they produce reactive oxygen
species leading to dysfunction of cells, including damage to proteins, nucleic acids
and lipids. This study revealed that when silver nano-particles were applied
3–4 weeks before disease incidence, even 50 ppm concentration of silver nano-
particles was able to inhibit powdery mildew effectively and in vivo tests showed
significant inhibition of the growth of fungal hyphae and conidial germination
(Lamsal et al. 2011). Jo et al. (2009), upon evaluation, reported that the application
of silver nano-particles in vivo at a 500-ppm concentration significantly reduced the
pathogenicity as well as colony formation of Bipolaris sorokiniana and
Magnaporthe grisea in the plants.
Treatment of Artemisia absinthium-mediated AgNP with concentration of
1.56 μg ml 1 significantly showed zoospore encystment, less spore germination
and reduced the germ tube elongation of Phytophthora parasitica as compared to
untreated control. The silver nano-particle effectively reduced swimming of
zoospores of Phytophthora parasitica. Thus, the silver nano-particle with higher
concentration effectively managed the disease of tobacco plants caused by
Phytophthora parasitica. The 2.5 ppm (μg ml 1 or μg g 1) of silver nano-particles
incubated with Fusarium culmorum spores had significantly reduced the
germinating spores and sprout length compared to control (FC spores in a sterile
water solution), as germinating process is very important for Fusarium culmorum

pathogenesis process in crops (Kasprowicz et al. 2010).
1.9 Conclusion
Rhizosphere has been defined more than 100 years ago and since then a number of
studies have established its role in affecting plant health and its productivity. The
diverse microbial community of plant rhizosphere known as rhizosphere
microbiome extends the functional scope beyond imagination as indicated by studies
across the world. Developments in metagenomics provide detailed pictures of
rhizosphere microbiomes. Root exudates of specific plant recruit specific group of
rhizosphere microorganisms from main reservoir present in soil. A range of direct
and indirect interactions such as plant–plant, microbe–microbe, and plant–microbe
occur in the rhizosphere. Rhizosphere is dynamic in nature and is very much
influenced by its components. Root and soil are complex microbial habitats
harbouring diverse microbial consortia. Understanding, predicting and controlling
the structure and function of the rhizosphere will allow us to harness plant-microbe
interactions and its activities to increase or restore plant ecosystem productivity,
improve plant responses to a wide range of environmental alterations in the function
and mitigate effects of climate change by designing ecosystems. A number of
strategies have been described to alleviate stress-induced adverse effects on plant
growth. Rhizosphere engineering is the process in which manipulation of rhizo-
sphere microorganisms is done for obtaining desired trait(s). This is basically done
by the alteration in root exudation pattern of plant, which can be achieved through
genetic manipulation in the host plant and by natural induction through interventions
in soil. The present chapter helps in understanding of various approaches to manip-
ulate rhizosphere for system sustainability and chemical-free crop production. Effi-
cacy of microorganisms significantly varies with crop and soil. Even reports suggest
that agro-ecological zone may also have had significant impacts in the efficacy of
microorganisms and this also needs to be considered while going for rhizosphere
engineering.
1.10 Future Prospects
The synergistic and complementary mechanisms among microorganisms and of

plant-microbe interactions can be deciphered using model plants grown under
gnotobiotic conditions. Such investigations may advance our understanding of the
phenomenon involved in microbiome-mediated host plant immunity. There is direct
influence of microbial interactions on plants and the host plants are able to affect
microbiome assembly and thus, the selection of a host-microbial association is an
emerging approach to improve plant fitness and productivity. Genetic improvement
of plants, relying on an efficient interaction with beneficial microorganisms and
selection of agricultural practices with less adverse effects on microbiome, needs to
be worked out. Number of omic approaches can be helpful in understanding plant

microbes’ interaction for specific traits and their exploitation. Therefore, scope of
rhizosphere engineering for improved crop performance in future is increasing.
Target-oriented rhizosphere engineering is need of the hour. Numbers of laboratories
are focusing to find out rhizosphere microbes’ interactions with crop plants and
impact of these on crop production and surrounding environment. Protocols may be
developed targeting the selection of a characteristic host phenotype influenced by the
microbiome function, which can then facilitate the transfer of specific trait-
associated microbiome into new plant hosts. Root exudates of plants attract rhizo-
sphere microorganisms the quality of both being interdependent on each other to
varying extents. The qualitative and quantitative alteration in the root exudates
composition is a major approach to remould the rhizosphere microbiome. The
creation of a biased rhizosphere may be novel approach that will involve the
expression of specific genes in transgenic plants to enable roots to produce the
specific nutritional compound that can then be used/recognized by specific beneficial
microorganisms.
Acknowledgements We would like to express our special thanks to Dr. Ruchita Dixit and
Wasiullah for technical assistance in collecting literatures. The authors wish to thanks Dr. Anil
K. Saxena, Director, ICAR-NBAIM, Kushmaur, Maunath Bhanjan, India, for providing technical
support during preparation of manuscript. Our special thanks go to Application of Microorganisms
in Agriculture and Allied Sectors (AMAAS), ICAR-NBAIM, Kushmaur and Indian Council of
Agricultural Research, Government of India, for providing financial support to Udai B. Singh to
carry out the research work.
Declaration of Competing Interest The authors declare that they have no known competing
financial interests or personal relationships that could have appeared to influence the work reported
in this paper.
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Rhizosphere Microbes for Sustainable
Maintenance of Plant Health and Soil 2
Fertility
Madhurankhi Goswami, Chandana Malakar, and Suresh Deka
Abstract
Widespread use of fertilizers in the agricultural system has created a continuous

pressure on the environment and thus seeks for an immediate response.
Biological alternatives are the most feasible and eco-friendly approach for envi-
ronment, agriculture and for the entire mankind. Plant growth promoting
rhizobacteria (PGPR), a group of rhizosphere-colonizing bacteria, is considered
to be the most effective candidate for plant health and soil fertility. Moreover,
implementation of PGPR for sustainable agriculture can also help in reducing
environmental pollution as a result of agricultural run-off, which ultimately
causes groundwater contamination. This chapter will focus explicitly on the
role and potency of the rhizosphere microbes in growth promotion of plants
while reflecting their efficiency in enhancing soil health and soil microbial
diversity.
M. Goswami
Environmental Biotechnology Laboratory, Resource Management and Environment Section, Life
Sciences Division, Institute of Advanced Study in Science and Technology (IASST), Guwahati,
Assam, India
Life Sciences Division, Department of Molecular Biology and Biotechnology, Cotton University,
Guwahati, Assam, India
C. Malakar
Assam, India
Department of Biotechnology, Gauhati University, Guwahati, Assam, India
S. Deka (*)
Assam, India

23, https://doi.org/10.1007/978-981-15-9154-9_2
36 M. Goswami et al.
Keywords
PGPR · Soil health · Soil fertility · Plant growth · Sustainable agriculture
2.1 Introduction
Meeting the global demand for food to feed the growing human population with limited
resources is a major challenge. Conventional agriculture plays a crucial role in meeting
the growing demand for food and making the country self-independent in food
production, but at the same time it is forcing the agricultural system to completely
depend on chemical fertilizers and pesticides (Santos et al. 2012). Undoubtedly, on the
one hand, these chemical fertilizers in agriculture have made the country self-
independent in food production (Mahanty et al. 2017), but on the other hand, it
is imposing several life-threatening impacts on health and environment, such as
development of resistance in phytopathogens and pests, deterioration in soil health
and quality, decrease in crop productivity over the years (Avis et al. 2008). For instance,
initial application of nitrogen fertilizers to the agricultural crop plants increases the nitro-
gen availability in plants, while indiscriminate use of these fertilizers reduces the rate of
biological nitrogen fixation in soil over the years (Vejan et al. 2016). Reports indicate
that long-term usage of chemical fertilizers can cause series of changes in physical,
chemical and biological properties of soil. Moreover, it influences important soil
properties such as soil structure, density, pH and soil water-holding capacity (Divya
and Belagali 2012). To overcome the ill effects of chemical fertilizers, efforts have been
channelized more towards the production of biological-based fertilizers as potent
alternative to agrochemicals. Biological-based fertilizers or bio-fertilizers in the form
of plant growth-promoting rhizobacteria (PGPR) can be one of the reliable alternatives
for sustainable agriculture. PGPR are root-associated bacteria that augment plant
productivity and immunity (Alizadeh and Parsaeimehr 2011). This group of soil
bacteria promotes plant growth either by producing phytohormones or by improving
bioavailability of soil nutrients like iron and phosphorus, effective mobilization and
decomposition of organic matter for easy uptake by plants (Valencia-Cantero et al.
2007). Moreover, PGPR provide food safety and increments soil microbial diversity
with no adverse effects on soil ecosystem. The use of biofertilizers will not only uplift
agricultural productivity and soil health, but also would lessen the problems of ground-
water and soil contamination (Yang et al. 2009). Due to the multifarious traits exhibited
by the rhizosphere microbes, there is a growing body of evidence that demonstrates the
potentiality and efficiency of these microbes in soil ecosystem.
2.2 Rhizosphere: A Hub for Plant Beneficial Rhizosphere

Microbes
The rhizosphere is a part of the soil ecosystem that includes plant roots, soil and the
soil microbiota that are in constant interaction with each other. This positive
interaction not only benefits the plants, but also improves soil fertility and increases
degradation rate of toxic chemicals (Lynch and de Leij 2012). In simpler words, it is
2 Rhizosphere Microbes for Sustainable Maintenance of Plant Health and Soil. . . 37
a densely populated niche in which the roots compete with neighbouring plant roots
for space, nutrition and water and also with the soil microbiome (Ryan and Delhaize
2001). The rhizosphere soil is under the supreme influence of the living plant roots,
as manifested by exudation or deposition of a wide selection of essential low-weight-
molecular compounds affecting the microbial activities occurring within the root
rhizosphere (Hirsch et al. 2003). The intensified microbial activity in the rhizosphere
is due to the nutritional benefit derived by microorganisms from the rhizodeposition
released from the living roots along with the cortical cells and the sloughed epider-
mal hairs. Additionally, there are other factors that control and facilitate the intense
microbial activities occurring at the root surface (Curl and Truelove 2012). The
microbial activity at plant rhizospheric region plays a vital role in overall functioning
of the plant, while assisting the plants in soil nutrient uptake and providing defence
against plant pathogens (Mazzola 2002).
Plant rhizosphere serves as a hub for several microbial cells and for variety
of nematodes and arthropods. The rhizosphere microbial population exhibits a
number of beneficial properties that contribute towards enhanced acquisition of
soil nutrients, tolerance to soil stresses, plant defence against soil-borne pathogens
and regulation of plant immune system. The majority of them are part of a complex
food web that utilizes the maximum nutrients released by the plant. There are several
factors that influence or regulate microbial diversity and activity in the rhizosphere
of plants (Mendes et al. 2013).
2.3 Factors Influencing Rhizosphere Microbiota
The structure and diversity of the rhizosphere microbiome mainly depend on the
plant genotype, developmental stage of the host plant, root exudates secreted by
particular plant roots and basically on the surrounding soil (Dey et al. 2012;
Chaparro et al. 2014). Each of the plant species promotes a unique group of
rhizosphere microbes and with subsequent increase in phylogenetic diversity
between the plant’s species, there occurs a huge diversity in the composition of
rhizosphere microbial assemblages. Thus, even a same plant species with different
genotypes can actively transform the rhizosphere microbial composition. Likewise,
a study by Bulgarelli et al. (2015) with barley plant of same species but with different
genotypes shows that it accounts for approximately 5.7% of variance in rhizosphere
microbial composition. Sugiyama et al. (2012) have similarly reported different
rhizosphere microbial composition with variation in five different Arabidopsis
genotypes. Moreover, they have observed that microbial communities belonging
to the five different genotypes of Arabidopsis thaliana not only vary in their
rhizospheric microbial compositions, but also in their metabolic activity. Berg and
Smalla (2009) have studied the effect of different plant species on the rhizosphere
microbial community and have observed that the rhizosphere microbiome isolated
from various crop plants, vary in their phenotypic, genotypic as well as metabolic
activity. Moreover, existing studies show that plant species richness and plant
functional diversity have a positive impact on the microbial functional diversity,
microbial species richness and microbial catabolic activity. Decrease in plant bio-
mass, due to a drop in plant species richness and functional diversity, poses a strong
effect on the residing microbial communities since microbial diversity and microbial
catabolic activity is linearly related to plant species and plant functional diversity
(Dey et al. 2012).
Studies have shown that the rhizosphere microbiome or rhizosphere microbial
communities differ according to the plant developmental gradient (Chaparro et al.
2014). Micallef et al. (2009) have studied Arabidopsis plant rhizosphere throughout
its developmental stages. They observed that the rhizosphere microbial communities
vary with each developmental stage and the microbial richness and diversity in the
early plant development were more distinct in the bulk soil, which ceases with
succeeding plant age. Similarly, Xu et al. (2009) and Inceoglu et al. (2011) have
studied and analysed the changes in the rhizosphere microbiome of soybean and
potato plants, respectively, with each succeeding developmental stage. Xu et al.
(2009) observed that the rhizosphere microbiome of soybean plants is strictly
influenced by plant development and the density of the microbial communities
was found to be complex during the early developmental stage as compared to the
later stages. Similarly, Inceoglu et al. (2011) demonstrated that the young potato
plants had cultivar dependent rhizosphere microbial communities, but these micro-
bial differences were found to disappear as the plants aged.
Numerous studies have shown that root exudates play a crucial role in shaping the
rhizosphere microbiome. For instance, Zhou and Wu (2012) showed that the addi-
tion of p-coumaric acid to the rhizosphere of cucumber seedlings altered the
rhizosphere bacterial and fungal communities and increased the microbial density
of soil-borne pathogens of cucumber. In another similar study, Zhou and Wu (2013)
observed that addition of vanilic acid completely altered the rhizosphere microbiome
of cucumber plants. Similarly, phenolic compounds present in the root exudates also
have a strong influence on shaping rhizosphere microbial community (Badri et al.
2013). Recent studies have established a positive correlation between phenolic
compounds and the diversity in microbial communities occurring in the rhizosphere
soil. With increase in phenolic content in root exudate, there is significant increase in
rhizosphere microbial communities. Moreover, the influence of phenolics on rhizo-
sphere microbial communities was found to be more pronounced as compared to
other group of compounds that includes sugars, sugar alcohols and amino acids.
This reflects its prominence as specific substrate or signalling molecule for deciding
the composition of the residing rhizosphere microbial communities (Fang et al.
2013; Michalet et al. 2013).
Soil type with the assemblage of its physicochemical parameters determines the
structure and diversity of rhizosphere microbial communities. Several studies deal-
ing with soil microbiome report on the effect of soil type on the structure and
diversity of rhizosphere microbiome. Some of the studies suggested that soil type
or soil texture has a stronger influence on the structure of rhizosphere microbial
community than plant species richness and plant functional diversity (Groffman
et al. 1996). Chiarini et al. (1998) undertook a study to assess the effect of soil type
and cultivar and the growth stage of cultivar on the composition of rhizosphere
microbial community. During their study, they observed that among all the factors,
soil type was the most pronounced factor that affected the microbial density and
community structure. In a similar study, da Silva et al. (2003) observed that soil type,
rather than plant cultivar type, was the prime determinative factor that influences the
rhizosphere community structure. Moreover, soil texture can also modulate or
structure the rhizosphere microbiome by limiting the root exudates. For example,
some amino acids, peptides remain adsorbed to the sand and clay particles in the soil
and remain less accessible to the microorganism than when present in unbound or
free state (Buyer et al. 1999; Kuske et al. 2002). Reports show that fine texture of
soils like clay soil and soil with an increased pH induces a prominent increase in
density of endogenous soil pseudomonads. Presently, studies revealed that of all the
factors associated with soil parameters, soil pH is believed to be the most influential
factor affecting the rhizosphere microbial communities (Fierer and Jackson 2006).
Studies show that a close relationship exists between soil pH and the overall
structure and diversity of rhizosphere microbiome. The reason behind this connec-
tion is the sensitivity of the bacterial cells towards pH as bacterial cells exhibit a
narrow pH growth tolerance (Rousk et al. 2010).
Thus, the dominant effect of soil type among all environmental factors can be
explained by the impact of soil texture on the rhizosphere microbial communities,
which are the primary sources for rhizosphere and rhizoplane colonization
(Manoharachary et al. 2006).
2.4 Interaction between Rhizosphere Microbe and Plant via

Root Exudates
The interaction between the plant roots and the rhizosphere microbiome is a unique
metabolic process by which the plants monitor the changes in the surrounding
environment and react to the same. Root exudates are a class of chemical compounds
secreted by the living plant roots in response to the chemical signals emitted by the
soil microorganisms (Chaparro et al. 2012). The type and composition of the plant
root exudates vary between plant species, ecological niches and even within distinct
roots of a plant (Rovira 1969; Uren 2000; Micallef et al. 2009). Root exudates are
composed of sugars (glucose, arabinose, galactose, fructose, sucrose, xylose, pen-
tose, rhamnose, mannitol), amino acids (all 20 proteinogenic amino acids), organic
acids (acetic acid, succinic acid, malic acid, 1-glutamic acid, l-aspartic
acid, salicylcic acid, shikimic acid, chorismic acid, tartaric acid, gallic acid, ferulic
acid, oxalic acid, citric acid), lignins (catechol, benzoic acid, nicotinic acid, cinnamic
acid, ferulic acid, coumaric acid, chlorogenic acid, pyroglutamic acid, quinic acid),
flavones (naringenin, kaempferol, naringin, rutin, strigolactone), indole compounds
(indole-3-acetic acid, brassitin, sinalexin, brassilexin, methyl indole carboxylate,
camalexin glucoside), protein and enzymes (proteins, lectins, proteases, acid
phosphatises, proxidases, hydrolases and lipase) and stimulators.
Broadly, root exudates have traditionally been grouped into low- and high-
molecular-weight compounds. The low-molecular-weight compounds form the
major part of root exudates and it comprises of amino acids, organic acids, sugars,
phenolics and various secondary metabolites. High-molecular weight compounds
comprises of mucilage and proteins. The composition and concentration of root
exudates vary depending on the surrounding soil, signals received from the environ-
ment and the rhizosphere, age of the plant and also on the environmental stress
conditions. There are various mechanisms that are involved in extrusion of plant root
exudates (for example diffusion, ion channels and vesicle transport), necessary for
exporting compounds to the rhizospheric soil (Bertin et al. 2003; Neumann
and Romheld 2007). The mechanism for the release or secretion of root exudates
into the soil vicinity depends on molecular weight of compounds present in the root
exudates. Low-molecular-weight compounds are released from the roots via passive
diffusion, while the high-molecular-weight compounds are released via active diffu-
sion into the rhizospheric soil (Badri and Vivanco 2009). At the same time, the
secondary metabolites, polysaccharides and proteins are released through
membrane-bound proteins (Weston et al. 2012). Root exudates released by the living
plant roots behave as chemical attractant for soil microorganisms, regulate the soil
microbial communities, cope with herbivores, alter physical and chemical properties
of soil, inhibit the growth of competing plant species and initiate symbiotic
interactions between plant and rhizosphere microorganisms (Estabrook and Yoder
1998; Bais et al. 2006). Carbohydrates and amino acids present in the root exudates
predominantly act as chemo-attractants for a wide range of PGPR isolates. Recent
studies have reported that arabinogalactan proteins (AGPs) play a key role in various
interactions between rhizosphere microbes and plant roots. There are several studies
that report the efficiency of AGPs in rhizospheric interactions attracting beneficial
microbes (Cannesan et al. 2012). In one of the recent studies, it was reported that
AGPs secreted by Arabidopsis root cells and border-like cells affect the colonization
efficiency of Rhizobium sp. which indirectly reflects the potency of AGPs in
recognition and attachment of rhizobia to plant root surfaces (Vicre et al. 2005).
Apart from AGPs, flavonoids, a key constituent of root exudates of legumes, play a
crucial role in initiating root-microbe interactions. Particularly, flavonoids facilitate
interaction of roots with rhizosphere microorganisms, stimulation of rhizobial nod
gene expression, responsible for the synthesis of Nod factors and chemo-attraction
of rhizobia towards the host plant roots (Hassan and Mathesius 2012). Similarly,
benzoxazinoids secreted by the maize roots help in attracting plant beneficial
rhizobacteria towards the plant root surface for colonization (Neal et al. 2012).
Additionally, the plant roots also secrete certain protein compounds as root exudates.
Few of the studies have mentioned that proteins like lectins, secreted as a part of root
exudates, function as defence and recognition factors in symbiotic interactions
(De Hoff et al. 2009). The proteomic analysis of Arabidopsis root by De-la-Pena
et al. (2008) has shown that these roots secrete a wide range of defence proteins such
as chitinases, glucanases and myrosinases during the flowering time. For instance,
Pseudomonas syringae pv. tomato DC3000, pathogen of Arabidopsis plants,
induces the secretion of several defence proteins like peroxidases, glycosyl hydro-
lase family 17, chitinase and glycosyl hydrolase family 18 as root exudates.
2.5 Role of Rhizosphere Microbes in Plant Health
Rhizosphere microbes are the primary unit of soil nutrient cycling and deciders of
soil status, plant health and richness of soil nutrient pool (Meena et al. 2017), They
help in nutrient solubilization, mobilization, mineralization as well as in nutrient
dissolving and uptake by plants (Verma et al. 2010; Meena et al. 2014a, b; Yadav
et al. 2017). The rhizosphere microbes play a dynamic role in aiding the plants in
easy acquisition of N, P or K from rhizosphere soil apart from siderophore produc-
tion, immune modulation, signal transduction and pathogen control.
2.5.1 Nitrogen Fixation
Soil nitrogen (N2) availability fluctuates greatly in both space and time due to a
number of factors like precipitation, temperature, soil type and soil pH. The form in
which the available soil N can be easily taken up by the plants depends strictly on
plant adaption to soil conditions. For instance, plants growing at low pH and basic
soil such as those found in arctic tundra or at mature forests tend to take up
ammonium or amino acids, while plants growing at high pH and more acidic soils
tend to take up nitrate as nitrogen source (Maathuis 2009). In general, N acquisition
by the plant roots depends on the availability of N in the surrounding rhizospheric
soil. Mostly, N in soil is present in organic form and suitable transformation of soil N
is highly essential for enhancing the level of soil fertility (Jetten 2008). According to
the existing literature, the transformation of soil N is basically managed by microbial
processes and it is significantly essential for N fixation for better acquisition by
plants (Adesemoye et al. 2009). The rhizosphere soil microorganisms fix atmo-
spheric N2 in soil and convert it into organic form making it available for the plants.
Moreover, the prevailing rhizosphere conditions favour N2 fixation in soil by
heterotrophic bacteria that utilizes organic compounds as the source of electrons
for the conversion of atmospheric N2 (Dotaniya and Meena 2015).
The process of N fixation is a complex process involving interaction between a
number of bacterial and plant gene products. Legumes form symbiotic associations
with microbes for the purpose of N fixation. Rhizobial symbioses result from
formation of root nodules in the host plants roots. It is a symbiotic association of
diazotrophic microbes with roots of leguminous plants involved in the conversion of
atmospheric nitrogen into ammonia for easy N acquisition by host plants. Specific
substances extruded by the plant roots attract the rhizospheric bacteria towards the
plant enabling a plant-rhizobia symbiotic association. As a result of plant-microbe
symbiosis, infection process starts at the host root system and following infection
flavonoid compounds are released by the host root hairs that induce bacterial
nodulation genes, which result in the synthesis of Nod factors, a group of biologi-
cally active oligosaccharide signals (Lynch and de Leij 2012). Recent studies have
unveiled a wide array of PGPR that includes Azospirillum, Alcaligenes,
Arthrobacter, Acinetobacter, Bacillus, Burkholderia, Enterobacter, Erwinia,
Flavobacterium, Pseudomonas, Rhizobium and Serratia, actively participating in
atmospheric N2 fixation. Among all symbiotic associations, the majority of

biological nitrogen fixation (BNF) occurring in the rhizosphere of terrestrial ecosys-
tem is performed by the well-known symbiotic bacteria belonging to the family
Rhizobiaceae and the leguminous plants (Jones et al. 2007). Similarly, the
non-leguminous plants form mutualistic association with nitrogen-fixing bacteria
(Lynch and de Leij 2012).
About 78% of elemental nitrogen is present in the atmosphere, but it remains
unavailable to the plants. Interestingly, there are no plant species that can convert or
fix atmospheric nitrogen into nitrogenous compounds for making it utilizable by the
plants for its proper growth and development. Thus, the atmospheric nitrogen is
made available to the plants by a phenomenon known as biological nitrogen fixation
that involves few rhizosphere microorganisms that exhibit the ability to fix nitrogen
to ammonia (Gaby and Buckley 2012). In order to achieve enhanced plant growth
and development, nitrogen fixation seems to be an important property. Islam et al.
(2013) have observed that priming of tomato and pepper plants with nitrogen-fixing
bacteria has significantly increased growth parameters in plants that include root and
shoot length, seedling vigour and dry biomass. Similarly, Tien et al. (1979) have
reported that inoculation of Azospirillum brasilense has considerable effect on plant
growth. Seed priming with Azospirillum brasilense resulted in enhanced prolifera-
tion of the root hairs as well as the lateral roots and an increase in nutrient-absorbing
surfaces. These changes have resulted in increased nutrient absorption and, thereby,
plant growth. But there are very scanty reports that have shown an in-depth study
revealing the mechanism involved in growth promotion by nitrogen-fixing bacteria.
Thus, further study is required to explore the potentialities shown by nitrogen-fixing
bacteria in plant growth promotion and increase in crop yield.
2.5.2 Phosphate Solubilization
Phosphate is one of the important macronutrients that promote plant root growth and
hastens maturity in plants. It plays a crucial role in every aspect of plant growth and
development. Soluble form of P in soil is highly limited due to its fixation as
insoluble phosphates (Sharma et al. 2013; Walpola and Yoon 2012; Mehrvarz
et al. 2008). Plants take up P mainly in the form of P anions like HPO24 or H2PO4
depending on the pH of the soil. Although soil contains a large quantity of phos-
phate, both in inorganic and organic form, the maximum portion of soil P remains
inactive and unavailable to plants. This unavailable portion of soil P can be made
available to the plants by a unique group of rhizosphere bacteria. This group of
bacteria is known as phosphorus-solubilizing microorganisms (PSM). PSM
hydrolyse the insoluble organic and inorganic soil phosphorus into simpler soluble
forms, increasing the bioavailability of soil P. A large number of soil
microorganisms, including bacteria, fungi, actinomycetes, algae and arbuscular
mycorrhizae, exhibit the potentiality of P solubilization and mineralization ability.
For instance, several bacterial strains like Bacillus, Paenibacillus, Rhizobium,
Serratia, Rhodococcus and many others exhibit potential phosphorus-solubilizing

ability. Similarly, fungal strains like Aspergillus, Achrothcium, Alternaria,
Cephalosporium, Curvularia, Cunninghamella, Fusarium, Helminthosporium,
Phoma, Phythium, Rhizoctonia, Rhizopus, Sclerotium, Torula, Trichoderma and
Yarrowia also exhibit the inherent ability to solubilize insoluble organic and inor-
ganic soil P (Alori et al. 2017).
According to the existing literature, the principal mechanism of P solubilization is
through production of organic acids, siderophores, protons, hydroxyl ions and CO2.
The acids produced in the periplasmic space by direct oxidation pathway, along with
carboxyl and hydroxyl ions, cause chelation or reduction in soil pH that ulti-
mately results in the release of bound P. Moreover, the drop in pH due to bacterial
organic acid produced within the microbial cell and also acidification of the
surrounding soil causes the release of P ions by substitution of H+ for Ca2+
(Goldstein 1994). According to the theory proposed by Illmer and Schinner
(1995), H+ released as a result of H+ substitution by Ca2+ ions causes P solubiliza-
tion. Cation assimilation by PSM solubilizes soil P either by lowering the soil pH,
chelation or by mineralization (Kalayu 2019). An alternative mechanism for P
solubilization is the release of H+ in the outer space in exchange for cation uptake
through H+ translocation ATPase or via assimilation of NH4+ within microbial cells.
This results in the release of protons that causes the solubilization of P without the
production of organic acids (Sharma et al. 2013). There is another mechanism of
inorganic P solubilization, which is by inorganic acid production or via production
of metal chelators. The efficiency shown by inorganic acids or chelating substances
in P solubilization is less efficient as compared to P solubilization efficiency shown
by organic acids (Alori et al. 2017). Existing literature reports that soil organic
phosphorus constitutes around 30–50% of the total P and is present in the form of
inositol phosphate. Organic P in soil is solubilized by the action of a number of
enzymes and involves the removal of P by dissolution of Ca-P compounds
(Rodriguez and Fraga 1999). Plants cannot directly acquire phosphorus from
phytate; however, the presence of PSM in the rhizosphere compensates the plant’s
inability to acquire phosphate directly from phytate (Richardson and Simpson 2011).
The use of PSB as inoculants simultaneously enhances P uptake by plants and
hence stimulates plant growth and crop yield. Recent studies portray the effect of
PSB on P uptake by plants as well as its role in plant growth and development.
Inoculation of PSB isolates in agricultural lands has a significant impact on plant
growth in terms of field emergence, root length, plant height, nodule dry weight,
nodule per plant, number of seeds and total yield of seeds over control plants (Singh
et al. 2010). There are several pieces of evidence that show the efficiency of PSB on
plant growth and crop yield. Hariprasad and Niranjana (2009) observed that treating
tomato seeds with PSB improved the overall seed quality parameters under in vitro
conditions. Under greenhouse conditions, priming of tomato seedlings with efficient
PSB isolates showed enhanced root and shoot length, dry and fresh weight and
overall phosphorus content of the primed tomato seedlings in comparison to the
control plants. Similarly, Li et al. (2017) have reported that priming of maize
seedlings with efficient PSB isolates has a marked effect on root and shoot length,
root and shoot dry weight, total P and N content of maize seedlings, thereby
enhancing plant growth. Similarly, Wu et al. (2019) observed that priming of
Camellia oleifera Abel. seedlings with two efficient PSB, namely Bacillus
aryabhattai (JX285) and Pseudomonas auricularis (HN038), significantly promoted
the growth of Camellia oleifera plants. Simultaneous inoculation of the strains
significantly increased the plant height and biomass of C. oleifera plants. Moreover,
the study reported the beneficial effect of PSB on the chlorophyll content and
photosynthetic capacity of C. oleifera plants. Similarly, Batool and Iqbal (2018)
observed that potent PSB strains can be further used as bio-inoculants for promoting
growth and yield in plants. They have observed that priming of wheat plants with
efficient PSB strains potentially enhanced different growth parameters in wheat
plants and significantly increased the P content. However, further in-depth study is
required to explore their potentialities as biofertilizer and to study the overall genetic
stability of the particular strains.
2.5.3 Potassium Solubilization
Potassium is one of the important nutrients that play a key role in plant growth,
metabolism and development. It also boosts up plant resistance against a wide
number of diseases, pests and abiotic stresses. Additionally, K helps in stimulating
a set of 80 different enzymes responsible for plant processes. Soil contains a large
amount of K than any other soil nutrients; however, only 1–2% of it is available,
while most of the soil K is unavailable to the plants. The unavailable soil K is made
available by some soil microorganisms known as potassium-solubilizing bacteria
(KSM). The KSM are effective in releasing K from inorganic and insoluble form of
soil K through the process of solubilization. There are evidences that suggest that
soil KSB are highly efficient in transforming insoluble form of K into simpler
soluble K forms that can be easily acquired by the plants. Moreover, recent studies
have reported that KSB can improve plant growth through suppression of plant
pathogens and also improve soil structure. For instance, these microorganisms can
solubilize silicate to release silicon, aluminium and potassium, while secreting
biologically active compounds that enhances plant growth (Meena et al. 2014a, b).
KSB in soil includes both aerobic and anaerobic forms, but majority of the soil KSB
are aerobic and are mostly reported to inhabit the rhizospheric soil in comparison to
non-rhizospheric soil. KSBs are ubiquitous in nature and their number varies from
soil to soil. There are a wide array of soil bacteria that can solubilize soil K, which
includes Acidothiobacillus ferooxidans, Peenibacillus mucilaginosus,
P. glucanolyticus, Arthrobacter spp., Enterobacter hormaechei, Bacillus
mucilaginosus, B. edaphicus and B. circulanscans (Meena et al. 2016a, b). Among
all, Bacillus mucilaginosus, B. edaphicus and B. circulanscans have been reported as
effective K solubilizers (Meena et al. 2015, 2014a, b, 2016a, b).
Microbial solubilization of K involves mobilization and solubilization of insolu-
ble and unavailable forms of K through production of various organic acids. These
acids result in the acidolysis and complexolysis exchange reactions that leads to the
conversion of insoluble form into soluble phosphates, thereby increasing the avail-
ability of the nutrients to the plants (Zarjani et al. 2013; Parmar and Sindhu 2013;
Uroz et al. 2009). KSMs solubilize insoluble K either by lowering the soil pH,
enhancing chelation of cations bound to K or by acidolysis of the surrounding area of
microorganism. The lowering of soil pH is particularly due to the secretion of a wide
range of organic acids that either directly dissolves the insoluble K or chelates both
Al and Si associated with K minerals (Romheld and Kirkby 2010). Thus, the release
of organic acids results in the acidification of microbial cells and the surrounding
environment, which ultimately causes the release of K ions from the mineral K by
protonation and acidification (Goldstein 1994).
Priming of seeds and seedlings with KSM results in significant enhancement of
seed germination, seedling vigour, plant growth and total plant yield. Recent studies
reported that the application of KSM under field-level test crops like wheat, maize
and forage crops could drastically reduce the usage of chemical fertilizers as KSM
could meet plant’s demand for nutrient acquisition (Xie 1998). According to few of
the researchers, inoculation of KSM to agricultural crop fields have resulted in root
growth enhancement and significant increase in the number of root hairs. More-
over, it has also improved the K-use efficiency in agricultural lands, thereby creating
a balance in managing nutrient proportion in soil (Meena et al. 2014a, b; Verma et al.
2010; Basak and Biswas 2010). Basak and Biswas (2010) have reported that priming
of rye plants with KSB isolates boosts the growth of rye plants, increased K content
of plants and its absorption of N and K from soil. Moreover, Zhang and Kong (2014)
reported that inoculation of KSB isolates to tobacco plants promoted the growth of
tobacco plants through mineralization of K compounds in soil, which increased the
total K content in soil, thereby facilitating plant growth. Sheng and He (2006) have
reported that KSB priming of wheat plants not only promoted the growth of wheat
plants, but also enhanced K uptake by plants. Increased nutrient uptake by plants has
been attributed to production of plant growth regulation at plant root interface that
ultimately enhances root development, thereby promoting water and nutrient absorp-
tion from soil. However, further research is essential to evaluate the overall genetic
makeup of the strains and for recommending the bacterial strains for using as
biofertilizers replacing chemical fertilizers.
2.5.4 Iron Chelators or Microbial Siderophore Producers
Iron is the fourth most abundant element in the Earth’s crust and exists in two
oxidation states Fe (III) and Fe (II). It is highly essential for plant growth and
development as it acts as a cofactor for proteins that are involved in a number of
metabolic processes like tricarboxylic acid cycle, electron transport chain, oxidative
phosphorylation and photosynthesis. Moreover, it plays a key role in biosynthesis of
porphyrins, vitamins, cytochromes, antibiotics, siderophores, toxins, pigments, aro-
matic compounds and also in microbial biofilm formation (Saha et al. 2016). Iron is
present in huge quantities in nature, but it is not easily available in the desired state as
it tends to form insoluble complexes in aerobic soils of basic or neutral pH (Ma et al.
2015). Iron is made available to plants by a particular group of rhizosphere

microorganisms via production of certain low-molecular-weight organic compounds
known as siderophores. Siderophores are small metal-chelating agents that primarily
bind or chelate insoluble ferric iron from soil. They are synthesized by both the
bacterial groups under iron-deficient environment in both aerobic and anaerobic
conditions. Siderophore first binds tightly with the insoluble Fe3+ ions and this
complex move across the cell membrane to get inside the bacterial cell with the
help of specific receptor proteins such as FepA [for enterobactin FhuA (for
ferrichrome) and FecA (for ferric citrate)] in E. coli (Braun and Braun 2002; Ma
et al. 2007) and FpvA and FptA in P. aeruginosa (Clement et al. 2004; Greenwald
et al. 2008; Nader et al. 2011; Nader et al. 2007). Recent studies report that the
domain conformational changes of each of the receptors lead to the passage of
substrate into the cells and the energy required for the same is provided by the
Ton B protein complex, which is composed of two other membrane proteins ExbD
and ExbB (Saha et al. 2016). The membrane network is markedly different between
gram-positive and gram-negative microorganisms. In case of gram-positive bacteria,
siderophore-binding proteins, permeases and ATPases are involved in the transport
of siderophore iron complexes through the cell membrane. Similarly, in case of
gram-negative microorganisms, transport of siderophore-iron complex occurs via
involvement of a periplasmic-binding protein, an outer membrane protein and a
cytoplasmic membrane protein belonging to ATP-binding cassette transporter
(ABC-transporter) (Ahmed and Holmstrom 2014). The ferric ions that remain
bound to siderophores move to cytosol, where it gets reduced to ferrous form of
ion and finally gets released from the siderophores. Once siderophore releases iron, it
gets degraded or recycled by excretion through efflux pump system (Saha et al.
2016).
Siderophore-producing plant growth-promoting rhizobacteria play a significant
role in providing iron nutrition to the plants, thereby enhancing plant growth (Parmar
and Chakraborty 2016). There are several pieces of evidence that justify plant
growth enhancement by siderophore-producing microorganisms. Masalha et al.
(2000) undertook a research study to explore the activity of rhizospheric
microorganisms in Fe uptake by plants for suitable growth and development. For
this, the plants were grown in both sterile and non-sterile conditions on loess-derived
loam soil. The plants grown under non-sterile conditions showed good growth with
higher Fe concentrations in the roots, while the plants grown under sterile conditions
showed lesser growth with iron deficiency. They finally concluded that the presence
of microorganisms is crucial for plant growth, since they produce microbial
siderophores that act as iron sources for plants. Pseudomonas strain GRP3A and
PRS9 isolated from the rhizoplane and rhizosphere soil of pea plants were reported
for maximum siderophore production, which significantly helped in seed germina-
tion, induced plant growth and markedly enhanced shoot and root length and dry
weight of maize plants (Sharma and Johri 2003). The same strain GRP3 was found to
promote plant growth by influencing the iron acquisition rate in mungbean plants.
Plants primed with GRP3 strain also increased peroxidase activity and chlorophyll
content in plants (Sharma et al. 2003). Similarly, siderophore produced by an
endophytic Streptomyces sp. isolated from the roots of a Thai jasmine rice plant
induced significant plant growth and enhanced length and biomass of root and shoots
(Rungin et al. 2012). At the same time, the siderophore-producing rhizobacteria can
induce suppression of a wide range of plant pathogens. The siderophores capture Fe
present in the vicinity of the plant roots and thus limit the amount of iron required for
the growth of fungal pathogens causing severe diseases in crop plants (Saha et al.
2016). For instance, Pseudomonas fluorescens strain Mst8.2, Mst 7.4 and MS-3y
showed maximum disease inhibition in wheat plants. The data obtained during the
course of study demonstrated that the mechanism behind the suppression in fungal
disease in wheat plants is primarily due to siderophore production by the particular
strains (Gull and Hafeez 2012). Few of the common siderophore-producing
pseudomonads proposed as potential biocontrol agents against the well-known
soil-borne fungal pathogens include P. fluorescens CHAO (Couillerot et al. 2009)
and P. putida WCS strain (Weller 2007). With the advent of newer molecular
techniques, further research is necessary to understand the beneficial aspect of
siderophore-producing rhizobacteria in the field of environmental microbiology.
2.5.5 Production of Plant Growth Regulators and Secondary

Metabolites by Rhizosphere Microbes
Existing literature on PGPR reveals its potency in synthesis of a wide range of well-
known plant growth regulators that include auxins, gibberellins, cytokinins, abcisic
acid and ethylene. The plant responds to any of the phytohormone present in the
rhizosphere whether synthesized naturally by the rhizosphere microorganisms or
supplemented externally to the soil. These hormones affect cell enlargement, cell
division, root initiation, growth rate, phototropism, geotropisms and apical domi-
nance in plants (Ma et al. 2015; Yadav et al. 2017). Indole-3-acetic acid (IAA) auxin,
synthesized by PGPR and plants, differs only in the biosynthetic pathway and is an
important phytohormone that significantly increases plant growth, stimulates rapid
and long-term responses in plants. Almost 80% of the total rhizosphere microbes are
involved in the production of IAA, and use of these microbes in the agricultural field
will enhance the endogenous IAA content in plants and thereby leaving a remarkable
effect on growth of plants. Auxins produced by the rhizosphere soil bacteria
principally affect the plant root system increasing its size, weight, branching number
and surface area in contact with soil. Additionally, inoculation of auxin-producing
microorganisms stimulates the growth of adventitious roots. IAA is produced by
rhizosphere microbes via two different pathways that include L-tryptophan-depen-
dent and L-tryptophan-independent pathways. In many cases, IAA produced by
rhizosphere microbes is generally produced via L-tryptophan-dependent pathway
utilizing L-tryptophan as the precursor molecule (Jha and Saraf 2015; Goswami et al.
2016). The phytohormone IAA has been directly correlated with the growth promo-
tion of plants (Noel et al. 1996; Tsavkelova et al. 2006). Nain et al. (2012) observed
that priming of cowpea plants with Bacillus sp. RM-2 resulted in significant plant
growth promotion. Further study suggested that the strain exhibited significant plant
growth promoting ability and effective involvement of each particular phytohor-

mone in plant growth promotion. Similarly, Mohite (2013) observed that priming of
plant seedlings with IAA producing strains has profound effect on plant growth. It
enhanced seed germination and induced proliferation of lateral roots and root hairs,
shoot and root elongation and chlorophyll content in plants. There are several other
reports that discuss the growth-promoting effects of bacterial phytohormone IAA on
growth of plants (Khanna and Sharma 2011; Etesami et al. 2015).
Similarly, the phytohormones gibberellins influence a number of plant processes
like germination of seeds, elongation of stems and also setting of flowers and fruits
(Hedden and Phillips 2000). Gibberellins promote root growth and root hair
enhancement, while inhibiting floral differentiation in woody angiosperms,
regulating vegetative and reproductive bud dormancy and delaying senescence in
many organs of plant species (Bottini and Luna 1993; Fulchieri et al. 1993; Reinoso
et al. 2002; Tanimoto 1987). This group of growth regulators is not only produced by
higher plants and fungi, but it is also produced by 7 bacterial species. There are few
reports that suggest that PGPR strains produce gibberellins, but there are only two
Bacillus sp. that have been reported to produce gibberellins that include B. pumilus
and B. licheniformis (Gutierrez-Manero et al. 2001). The pronounced activity of
gibberellins is due to the fact that this group of phytohormones can be translocated
from the roots to the aerial parts of the plant (Goswami et al. 2016). A significant
enhancement in seed germination parameters has been observed in cereals like
sorghum (Raju et al. 1999) and pearl millet (Raj et al. 2003, 2004) on inoculation
of gibberellins producing rhizobacteria. This may be due to the fact, increased
production of gibberellins triggers the activity of specific enzymes like alpha-
amylase that stimulates early seed germination by increasing the availability of
starch assimilation. Apart from growth promotion, gibberellin-producing rhizo-
sphere microbes are involved in induction of stress tolerance in plants. There are
several studies that report the induction of abiotic stress tolerance in plants by
gibberellin-producing microorganisms. Gibberellin-producing Pseudomonas putida
H-2-3 was reported to induce physiological changes on endogenous hormones,
antioxidants and ionic balance in abiotic stress-affected soybean plants to improve
stress tolerance in plants. Additionally, H-2-3 primed soybean plants showed higher
shoot length, plant fresh weight and chlorophyll content as compared to the control
plants (Kang et al. 2014).
Among the secondary metabolites, biosurfactants are the most potent candidates
in enhancing growth of plants, while inhibiting fungal pathogens. Previous research
suggests that biosurfactant-producing plant growth-promoting rhizobacteria enhance
plant growth significantly, while suppressing the growth of a wide number
of phytopathogenic fungi. Sheng et al. (2008) have reported that inoculation of
biosurfactant-producing Bacillus sp. J119 significantly enhanced the growth of
tomato plants. Moreover, they observed that priming of plants with a
biosurfactant-producing bacterial strain enhances root colonization rate, which ulti-
mately promotes growth of plants. Biosurfactants were also reported to enhance
bacterial motility, biofilm formation and root colonization, thereby enhancing plant
protection against a wide range of phytopathogens, while facilitating growth
promotion in plants (Kruijt et al. 2009). But further research is essential for under-
standing the underlying mechanism behind enhanced plant growth promotion in the
presence of biosurfactant.
2.6 Effect of Plant Beneficial Rhizosphere Microbes on Soil

Fertility
Civilization has developed and flourished in areas where soils were fertile, enabling
the human species to meet food needs and other necessities. The fertility of the soil
and its balanced ecology is highly crucial for sustainable agriculture. Agriculture has
been a crucial part of human civilization for thousands of years and still continues to
be an important part of it. Successful agricultural practices result in high yield and for
this purpose soil health plays a crucial role. The soil health is the cumulative
property, which corresponds to agricultural production along with maintenance of
soil native ecosystem. Soil health is reflected in its capability to add to various
agricultural interventions (Kibblewhite et al. 2007). This is highly dependent on
various aspects of soil ecosystem, which is mediated by carbon sequestration,
nutrient cycle of the soil and various management aspects.
Various anthropogenic activities have triggered the depletion of soil health,
thereby decreasing soil fertility and crop yield. Changes in global climate also have
an adverse effect on land quality that results in increased pollution, salinization and
desertification of landmasses. Furthermore, long-term usage of chemical fertilizers
and pesticides for increasing food production has led to a sharp decline in soil
fertility, soil microbial diversity and significant increase in various levels of pollu-
tion. Although there have been various efforts globally to increase soil fertility, more
emphasis was given on green revolution. The quest for biological mediators has been
hailed as an essential part of the revolution. Green revolution aims to enhance soil
fertility and restore soil microbial diversity by remediation of degraded soils
followed by soil enrichment. The soil fertility and plant health is inter-dependent,
which makes plantation an important aspect to maintain soil fertility. A recent study
on rhizosphere has brought attention towards the rhizosphere microbes, which are
inevitable part of the rhizosphere ecology. In this regard, the plant root ecosystem
can be exploited owing to its diverse array of plant beneficial microbes. The
rhizosphere is the most diverse and metabolically active ecosystem that influences
soil nutrition cycle (Zhang et al. 2010). In this regard, along with various bioreme-
diation techniques, rhizoremediation has also been adapted, which implicates the
involvement of various plant beneficial rhizobacteria to enhance soil fertility.
Bio-augmentation of plant rhizosphere with various contaminant-degrading bacteria
as well as plant-beneficial rhizobacteria is an important criterion for soil decontami-
nation, nutrient solubilization for easy plant uptake and enhancing soil fertility and
soil microbial diversity (Divya and Kumar 2011) (Table 2.1).
Table 2.1 Importance of plant beneficial rhizospheric bacteria in remediating soil contaminants,
uplifting soil fertility and plant health
Plant-growth-promoting Significance of microbe-
rhizosphere microbes Test crop plant interaction References
Pseudomonas putida Canola Increased P uptake by Lifshitz
GR12-2 plants, root and shoot et al.
elongation and a significant (1987)
increase in root and shoot
weight
Pseudomonas Chickpea Increases germination Yadav
fluorescens OKC (87%), root length et al.
(108 mm), shoot length (2013)
(122.5 mm), dry shoot
biomass (0.074 g), dry root
biomass (0.075 g) and total
dry biomass (0.149 g) in
comparison to the control
plants
Rhizobium sp. RH4 Germination (86%), root
length (76), shoot length
(91.5 mm), dry shoot
biomass (0.075 g), dry root
biomass (0.070 g) and total
dry biomass (0.146 g) in
comparison to the control
plants
Pseudomonas BA-8, Strawberry Significantly increased total Pırlak and
Bacillus OSU-142 and soluble solids, total sugar Kose
Bacillus M-3 and reduced sugar (2009)
Pseudomonas Highbush blueberry Increases leaf area De Silva
fluorescens Pf5 (748 cm2), number of et al.
leaves (37), increase in (2000)
system diameter (0.40 mm),
dry shoot weight (13 g), dry
root weight (12.7 g)
Pseudomonas Canola Enhances root length and Pallai et al.
fluorescens 6-8 seedling growth (2012)
Bacillus megaterium, Wheat Significantly increases crop Kumar
Arthrobacter yield, total weight and et al.
chlorophenolicus and micronutrient content in (2014)
Enterobacter sp. grains
Klebsiella variicola Tobacco Increases plant dry weight Zhang and
and enhances uptake of Kong
both K and N by the plant (2014)
Enterobacter cloacae Rice Increases root length, Suprapta
number of roots, tillers, et al.
total chlorophyll content (2014)
and macronutrients in leaf,
dry weight of root and shoot
(continued)
Table 2.1 (continued)

Azotobacter Wheat Increases crop productivity Kumar
chrocooccum and et al.
Bacillus subtilis (2014)
Bacillus sp. and Sesame Increases seed yield Jahan et al.
Pseudomonas sp. (2013)
Gluconactobacter Rice Increases plant nutrient Stephen
sp. and Burkholderia sp. uptake and yield parameters et al.
(2015)
Bacillus cereus YL6 Soybean Increases available P in Ku et al.
rhizosphere soil by (2018)
120.16% and plant total P
content by 198.60%
Wheat Increases available P in
rhizosphere soil by 62.47%
and plant total P content by
6.20%
Chinese cabbage Increases available P in
rhizosphere soil by 7.21%
and plant total P content by
78.89%
Acinetobacter (M01 and Tomato Significantly increased root Zhang
M04) and shoot dry weights of et al.
tomato plants. M01 and (2017)
M04 increases shoot dry
weight by 30.5% and
32.6% and root dry weight
by 27.1% and 33.1%,
respectively
Ochrobactrum (M11) Increases shoot dry weight
by 26.2% and root dry
weight by 25.6%
Bradyrhizobium sp., Lupinus luteus Increase in nitrogen fixation Dary et al.
Pseudomonas sp. and along with decreased soil (2010)
Ochrobactrum cytisi heavy metal contamination
of Cu, Cd and Pb
Increase in plant biomass
Pseudomonas Black gram Seed treatment with the Ganesan
aeruginosa MKRh3 isolate MKRh3 (2008)
demonstrated diminished
cadmium toxicity along
with extensive plant growth
and rooting
Bacillus sp. SC2b Brassica napus Enhances biomass and Ma et al.
vigour index in B. napus (2015)
Sedum Increased uptake of Cd and
plumbizincicola Zn from heavy-metal-
contaminated agricultural
(continued)

soil by S. plumbizincicola
when inoculated with SC2b
Cupriavidus taiwanensis Mimosa pudica The metal absorption Chen et al.
TJ208 efficiency of the nodulated (2008)
plant increased by 33, 71,
81% for Cd, Pb and Cu,
respectively
Pantoea sp. Brassica napus Significant removal of Ontañon
phenol from the soil and et al.
enhanced chromium (2014)
accumulation in the hairy
roots of the test crop in
presence of Pantoea sp.
Sphingobium – Enhanced Dams et al.
chlorophenolicum pentacholorophenol (PCP) (2007)
in loamy sandy soil and
decreased PCP
phytotoxicity in plants
grown in soil artificially
contaminated with PCP
Stenotrophomonas Pennisetum 100, 50 and 58% Dubey and
maltophilia MHF ENV pedicellatum cypermethrin degradation Fulekar
22 was achieved in the (2013)
rhizosphere of the host at
concentration of 25. 50 and
100 mg/kg within 72, 24
and 192 h
Ralstonia eutropha, Brassica juncea Biodegradation of phorate Rani and
Pseudomonas by the plant and bacterial Juwarkar
aeruginosa and consortium individually (2012)
Enterobacter cloacae was found to be 38 4%
and 55 4%, respectively.
However in association
with both consortia and test
crop, the biodegradation of
phorate was observed to be
64 5%
Comamonas sp. strain Alfalfa Complete degradation of Liu et al.
CNB-1 4-chloronitrobenzene (2007)
(4CNB) in 1–2 days along
with elimination of 4CNB
phytotoxicity to alfalfa
Rhizobium meliloti Alfalfa Initial concentration of Teng et al.
PAH was decreased to (2011)
51.4% when alfalfa
inoculated with the
bacterium was planted in
PAH-contaminated soil
(continued)

Pantoea sp. strains Italian ryegrass Efficient hydrocarbon Yousaf
ITSI10 and BTRH79 Birdsfoot trefoil degradation was achieved et al.
Pseudomonas sp. strains when the bacterial isolate (2010)
ITRI15 and ITRH76 was used in combination
with the test plants
Rhodoccocus equi, High and low erucic Increased biodegradation of Wojtera-
β-Proteobacterium, acid rapeseed diesel from contaminated Kwiczor
Enterobacter sp., varieties (HEAR and soil with no impact on et al.
Acinetobacter LEAR, respectively) photosynthesis of rapeseed (2014)
calcoaceticus,
Comamonas sp.,
Pseudomonas
alcaligenes
Psedumonas putida Cajanus cajan Seeds sowed in 50 ppm Goel et al.
MTCC Rhizobium DPT toluene-contaminated soil, (2012)
treated with mixed
suspension of both bacteria
was found germinated,
which was otherwise not
observed in seeds sowed in
untreated soil
Microbacterium Wheat Significant increase in Sheng
sp. F10a growth of wheat along with et al.
efficient phenanthrene and (2009)
pyrene removal from
contaminated soil
2.6.1 Carbon Sequestration
The amount of atmospheric carbon has increased in the last few centuries due to
various anthropogenic activities. Mitigating this large pool of atmospheric CO2 by
terrestrial carbon sequestration is a thoughtful alternative. Soil is an immense
reservoir of carbon. The organic components of soil harbour nearly three times the
amount of carbon present in the atmosphere (Gougoulias et al. 2014). Soil organic
carbon (SOC) is found in the form of structural litter input such as root, shoot detritus
and dissolved organic carbon (DOC) such as rhizodeposits and leaf litter leachate
(Sokol and Bradford 2019). The terrestrial carbon cycle is regulated by photosyn-
thesis and respiration, where in the former case the atmospheric carbon is fixed by
various autotrophs and in the latter, the fixed carbon is released into the atmosphere
by various microbial respiration. The reports provided by Lal (2004) have shown
that the depletion of soil quality through various anthropogenic activities has
resulted in loss of 42 to 78 gigatons of carbon, which accounts for 50 to 66% of
historic carbon loss. This loss has been reflected in poor crop yield, which has
affected the nourishment of the inevitably growing human population. Thus, it is
very essential to maintain the soil carbon level, as with increase in 1 ton of soil
carbon, there can be a huge increase in the production of wheat by 20 to 40 kg/ha,

maize by 10 to 20 kg/ha and cow-peas by 0.5 to 1 kg/ha (Lal 2004). In this regard,
rhizosphere microbes can enable carbon sequestration through utilization of DOC,
which would enhance soil health. The soil carbon matter depends on the recently
fixed carbon and its utilization via microbes. However, the carbon sequestration is
not an easy task as it is highly crucial for the microbes to have access to the carbon
that is fixed in the soil ecosystem. In a study, during 25-day incubation experiment
with root exudate from label wheat and maize plant, approximately 36% of 13C
could be retrieved, of which 18.7% and 20.8% of 13C were obtained in the form of
CO2, which was respired by microorganisms (Marx et al. 2007). Vidal et al. (2018),
in one of the studies, observed that rhizosphere microbes utilize photosynthesis-
derived 13C as well as natural soil organic 13C. The atmospheric CO2 concentration
at sub-ambient and elevated concentration was shown to imply an impact on the
colonization of rhizobacteria Pseudomonas simiae WCS417 in Arabidopsis rhizo-
sphere, which was also observed to have an impact on the plant responses towards
the rhizobacteria (Williams et al. 2018). Pinus taeda L., when exposed to an elevated
concentration of CO2, resulted in increased rates of root exudation, which, in turn,
stimulated the microbial metabolism, accelerating the soil organic matter (SOM)
turnover (Phillips et al. 2011). Bacterial species of Gammaproteobacteria and
Firmicutes groups were found to be dominant in dissolved organic matter (DOM)
decomposition in soil obtained from tropical rain forests (Cleveland et al. 2007). The
process of carbon sequestration by rhizosphere microbes is still unclear, but various
studies support that the soil carbon matter is strongly under the influence of various
rhizosphere microbes as they play the role of main decomposers in the soil. It is
feared that if the rhizosphere microbial respiration ceases, it would then take 12 years
of primary production to exhaust the existing atmospheric CO2 (Gougoulias et al.
2014). Co-inoculation of bacteria and cyanobacteria in soil potted with rice variety
Pusa-1460 increased crop yield and organic carbon matter (Prasanna et al. 2012).
Bird et al. (2011) reported that rhizosphere-related priming of SOM decomposition
is influenced by activities of gram-positive bacteria.
2.6.2 Nutrient Mineralization
The involvement of rhizosphere microbes in the geo-chemical cycling of nutrients

such as carbon, nitrogen, phosphorus, potassium and other micronutrients not only
increases the availability of nutrients to plants, but also to the soil microbial
population. Their capacity to mineralize various elements helps in uplifting soil
fertility, which improves plant health in such ecosystem. The appropriate ratio of
various elements required for plant growth and crop yield forms a crucial part of soil
nutrients. The fixing of atmospheric carbon and nitrogen in the soil is dependent on
plant photosynthesis and rhizosphere microbial activity. The soil nutrient cycles are
under tremendous influence of rhizosphere microbes. Rhizosphere priming caused
by soil micro-organisms influence various nutrient cycling processes in soil, thereby
producing fertility effect in the soil. Quantification of microbial-mediated nutrient
fluxes in rhizosphere for 80-year-old tree species in a deciduous hardwood forest,

Indiana, USA, revealed that microbial N cycle may contribute approximately 18% of
total N mineralization (Yin et al. 2014). Soil bacteria belonging to genera Pseudo-
monas, Enterobacter, Bacillus, and fungi Penicillium, Aspergillus solubilize the
total soil P via production of organic acids. It is reported that the activity of alkaline
phosphatase and acid phosphatase increases from 102 to 325% and 205 to 455%,
respectively, in rhizosphere soil (Dotaniya and Meena 2015). Meena et al. (2017)
have reported a significant increase in plant nutrient uptake efficiency when mineral
fertilizers were added along with rhizosphere microbes. Drastic changes were
observed in bacterial communities in desert soil, when long-term farming was
commenced on desert area of Sekem Egypt, which also promoted plants grown in
those regions (Köberl et al. 2011). Thus, the undeniable beneficial impact of
rhizosphere microbes was observed in various soil ecosystems, which not only
affects plant growth, but also improves the soil quality by enhancing nutrient
composition along with its influence on various geochemical cycles of nutrients.
2.7 Rhizodeposition-Mediated Enhancement of Soil Fertility
Rhizodeposition, apart from remediation, is one of the other factors that mediate soil
nutrient cycle (Shimp et al. 1993; Nguyen 2009). The root exudates become
important mediator of C cycle in the rhizosphere microbiome, which, in return,
shapes the soil biota surrounding the rhizosphere (Terrazas et al. 2016; Hartmann
et al. 2008; Paterson et al. 2007). Rhizodeposition varies in various developmental
stages in plants, which can vary from 6% to 21% (Bertin et al. 2003; Rohrbacher and
St-Arnaud 2016). Rhizodeposition of root exudates and other functional metabolites
acts as a source of soil organic carbon (SOC), which helps to maintain soil carbon
sequestration and in maintenance of soil structure (Bronick and Lal 2005). The
influence of rhizodeposition not only ends with its effectivity in determining rhizo-
sphere microbial niche, but it also has importance in biogeochemical properties of
soil (Lambers et al. 2009; Singh et al. 2007). The root exudates are metabolized by
the rhizosphere microbial population, thereby enhancing soil nutrients. Root
exudates released by plants are also reported to enhance the microbial behaviour
to a great extent. It is reported that C-based root exudates can modify the rhizosphere
microbes, thereby inducing their capability to transform heavy metal existing in the
rhizosphere region of the plant. This transformation further helps in detoxification of
heavy-metal-contaminated soil (Seshadri et al. 2015). Degradation of pyrene in
contaminated soil was enhanced from 15% in unplanted soil to 65% when the soil
was planted with wheat (Shahsavari et al. 2015). The root exudates directly or
indirectly affect the biological activity of the rhizosphere microbes facilitating
microbes-associated phytoremediation of soil (Vishwakarma et al. 2017). Moreover,
plant roots are reported to secrete certain molecules, whose structures are analogous
to PAH, thereby facilitating hydrocarbon degradation (Divya and Kumar 2011).
Additionally, various secondary metabolites release as root exudate assists hydro-
carbon degradation via cometabolism and in stimulating various genes responsible
for xenobiotics degradation (Singer 2006; Fletcher and Hegde 1995).

Rhizodeposition-mediated remediation of pyrene-lead co-contaminated soil
(Li et al. 2016), 2,4-dichlorophenoxyacetic acid (Shaw and Burns 2005), phenan-
threne in slender oats (Miya and Firestone 2001) are few of the noteworthy studies
wherein the rhizodeposits seem to enhance the degradation capacity of the microbial
community in the rhizosphere. Various sites of roots are reported to participate in
root exudation of various compounds. The immediate region behind the root tips is
generally considered to be active site of exudation (Badri and Vivanco 2009).
Pearson and Parkinson (1960) reported the secretion of ninhydrin-positive
substances from a region behind the root tips in healthy roots. Hence, exudate-
mediated rhizoremediation is dependent on the sites of active exudation. Biodegra-
dation of phenanthrene was observed to be 86%, 48% and 36%, respectively, at
0–3 mm, 3–6 mm and 6–9 mm distance from the mat of actively exuding roots of
Lolium perenne (Corgié et al. 2003). Rhizodeposition of phenolic root exudate
activates the biodegradation of trichloroethylene by rhizosphere bacteria (Adriano
et al. 2005). Proper knowledge of plant-microbial interaction could trace possible
exploitation of rhizosphere interaction, which can assist elevation of soil fertility.
The influence of rhizosphere microbes not only ends in soil enrichment but also
involves various decontamination processes that help in cleansing various
contaminated sites. This involves rhizoremediation of petroleum-contaminated
sites, pesticide-contaminated sites and also decontamination of heavy-metal-
contaminated sites (Fig. 2.1).
2.8 Rhizoremediation
The association of plant rhizosphere microbes is crucial for plant health. The various
plant growth-promoting traits displayed by the associated microbes play an undeni-
able role in maintaining plant health as well as soil fertility. The rhizosphere is a
connecting link between the plant roots and its surrounding soil, which contains
various rhizosphere microbes contributing significantly towards plant health (Badri
et al. 2009). However, along with the agricultural benefits obtained from plant-
microbe interaction, it has also been found to be an effective medium for soil
remediation, which helps in restoring soil fertility. With more emphasis on green
initiatives to remediate contaminated sites, phytoremediation has gained attention
globally. Although various techniques of phytoremediation, viz. phytoextraction,
phytotransformation, phytovolatilization, rhizoremediation are available, microbe-
assisted remediation is the most effective form of remediation. Rhizoremediation is
an imperative part of phytoremediation. It is the remediation technique that involves
exploitation of both plants and associated rhizosphere microbes for various remedial
strategies. In this regard, plant beneficial rhizosphere microbes play an important
role in uplifting soil quality (Gerhardt et al. 2009). 2–5% of rhizosphere bacteria are
plant growth promoters that are known for their vital role as biofertilizers,
phytostimulators, biopesticides and bioremediators (Antoun and Kloepper 2001;
Bishnoi 2015). This strategy of remediation is one of the most effective and
2
Rhizosphere Microbes for Sustainable Maintenance of Plant Health and Soil. . .
Fig. 2.1 Impact of plant beneficial rhizospheric bacteria in restoring soil fertility and promoting plant growth
57
acceptable forms of bioremediation. Owing to a complex signalling method of the

plant, the rhizosphere microbes are attracted, forming their own rhizosphere
microbiome. The diverse array of microorganisms present in the vicinity of the
rhizosphere influences the impact of rhizoremediation. With its effectivity in
decreasing soil contamination, this plant-microbe interaction in rhizosphere niche
is highly beneficial to the soil as well as towards plant health. Plant-microbe
interaction is mediated by various components released by the roots, known as
root exudates, and the local microbial community, which includes various soil-
inhabitant mutualists and parasite. Moreover, the capability of plant roots to pene-
trate various layers in soil down the earth makes it easier for rhizosphere microbial
communities to reach non-penetrable layers of soil, thereby affecting soil conditions
considerably (Kuiper et al. 2004). In the process of nutrient acquisition, both soil
microorganisms and plants alter soil properties through various metabolic activities
and organic litter deposition, respectively (Jacoby et al. 2017). There are various
reports wherein rhizosphere microbes assisting soil phytoremediation of a wide
range of hydrocarbons and pesticides has been observed. Vetiveria zizanioides
along with one of the rhizosphere-colonizing endosulfan-degrading bacteria showed
better remediation of endosulfan-contaminated soil (Singh et al. 2016). Similarly,
Gordonia sp. S2RP-17 in association with Zea mays was found to remove TPH
significantly, almost by 95.8% from diesel-contaminated soil (Hong et al. 2011).
Although plants possess bioremediation capability of various contaminants, they
lack the metabolic activity that could result in complete degradation of a contami-
nant. The rhizosphere-associated microbes enhance the process of mineralization of
contaminants, which otherwise could not be completely mineralized by plants alone
(Vergani et al. 2017). Thus, agriculture practices and the microbial communities in
the plant rhizosphere are one of the important factors in uplifting and restoring soil
quality.
Among all, hydrocarbon poses the biggest threat to soil quality and fertility and is
a major type of land pollutant. Previous literature establishes the potency and
efficiency of rhizosphere-colonizing microorganisms in hydrocarbon mineralization.
Biological remediation not only helps in decreasing phytotoxicity but also in evapo-
transpiration of hydrocarbon (Khan et al. 2013). Utilizing the plant beneficial
microbes to remediate hydrocarbon contamination is the most efficient cleaning
procedure as these microbes are capable of degrading hydrocarbon of varying
complexity. The hydrocarbon degradation rates and efficiencies vary from strain to
strain within the same species as well as within various species. Various enzymatic
pathways enable the rhizobacteria to degrade and assimilate the hydrocarbon. The
root exudates that help in recruiting beneficial bacteria also play an important role in
enhancing microbe-assisted hydrocarbon degradation. Hou et al. (2015) reported
significant removal of high-molecular-weight aliphatic (C21–C34) and PAH by
Testuca arundinacea L., when inoculated with plant-growth-promoting
rhizobacteria. Positive interaction between the hydrocarbon-degrading microbes
and tropical grass Brachiaria brizantha was observed in diesel-contaminated soil
with significant degradation of the existing contaminants (Mezzari et al. 2011).
There are several studies that report the efficiency of rhizosphere colonizing plant-
beneficial microbes in hydrocarbon decontamination and mineralization in associa-

tion with crop plants (Kumar et al. 2013; Tara et al. 2014).
Pesticide contamination is another inevitable consequence of using pesticides to
get rid of various pests and insects from our crops. These pesticides have now
entered into the food web resulting in various levels of toxicity in the consumers.
There is now a global effort to eliminate the toxic impact of this anthropogenic
practice as a result of which various toxic pesticides in many countries around the
world have been banned. However, the pesticides, which were used in the past, still
persist in the environment, which can still pose threats to the environment as well as
its components. PGPR can be a good candidate in this regard as many of the
rhizobacteria are investigated for their ability to survive the toxic impact of various
pesticides. Enhanced dissipation of lindane was observed in soil planted with
Withania somnifera and inoculated with Staphylococcus cohnii subspecies
urealyticus (Abhilash et al. 2011). Streptomyces sp. M7 was reported to degrade
68% of lindane in lindane-contaminated soil along with better seedling vigour index
of maize plant seeded in same soil (Benimeli et al. 2008). Degradation of herbicides,
namely atrazine, metolachlor and trifluralin, was reported to be enhanced in Kochia
sp. rhizospheric soil compared to non-rhizospheric soil, thereby emphasizing the
potency of rhizosphere microbes in pesticide removal (Anderson et al. 1994). In
reference to pesticide contamination, phytostimulation is one of the significantly
reported remediation processes for efficient biodegradation of various grades of a
pesticide (Velázquez-Fernández et al. 2012). Pisum sativum plants inoculated with
endophyte bacteria were found to degrade 2,4-dichlorophenoxyacetic acid with no
accumulation of herbicide in the aerial tissues (Germaine et al. 2006). The plant rhi-
zosphere is a huge collection of various plant beneficial microbes, which, in turn, is a
large pool of diverse genes. The adaptation of rhizobacteria in root niche is entirely
dependent in its genetic manipulations subjected to various biotic and abiotic factors.
Molecular studies have revealed that incessant pressure of hydrocarbon
contaminants on rhizosphere soil microbiome alters the gene expression pattern
that enables significant degradation of hydrocarbon contaminants. Degradation of
alkanes is mediated by a number of enzymes coded by a range of genes such as
methane monooxygenases (mmo genes), alkane hydroxylases (alkB genes) and
cytochrome P450-type alkane hydroxylases (CYP153 genes) (Afzal et al. 2013)
produced by different rhizosphere soil microorganisms. Siciliano et al. (2003)
reported the expression of three catabolic genes ndoB, alkB and xylE for hydrocar-
bon degradation and mineralization of hexadecane and phenanthrene in rhizosphere
microbes. Expression of alkane monooxygenase (alkB) and naphthalene
dioxygenase IndoB) was reported in endophyte and rhizosphere microbes of
petroleum-hydrocarbon-contaminated sites (Siciliano et al. 2001). Afzal et al.
(2011) reported that there is a positive correlation between the expression of
catabolic genes and hydrocarbon degradation. Catabolic genes alkM, alkB and
xyle that mediate degradation of alkanes and aromatic petrochemical compounds
were reported to be present in various rhizosphere microbes of wild oat (Rajaei et al.
2013). Other than the genomic DNA, extra-chromosomal genes also play an impor-
tant role. Plasmids often carry genes that provide various conditional advantages to
the microbes (Divya and Kumar 2011). Various genes contributing to pesticide
degradation are often reported to be present in plasmids of rhizosphere microbes.
Plasmid of Pseudomonas diminuta was reported to contain parathion-degrading
gene (Serdar et al. 1982). Organophosphate-degrading gene OpdA was obtained
from plasmid of Agrobacterium radiobacter P230 (Horne et al. 2002). Similarly,
parathion-degrading gene degrading gene was also identified in 43-kb plasmid of a
Philippine isolate of Flavobacterium sp. (ATCC 27551) (Mulbry et al. 1986).
Moreover, root exudates are reported to induce certain genes of rhizosphere
microbes, which enable the microbes to cope with contaminant soil, thereby enhanc-
ing degradation rates (Segura et al. 2009). Rainey and Preston (2000) have reported
the presence of various genes in bacteria with unknown functions. The unveiling of
these genes in plant beneficial bacteria through in vivo expression technologies
assists further exploration of PGPR. Certain genes are upregulated only in presence
of certain types of stress, resulting in non-expression of them in normal in vitro
condition. The overall functioning of the important genes can be studied using
promoter trap techniques, differential fluorescence induction and in vivo expression
technology (IVET) (Rediers et al. 2005). Omics-based study has shaped a better
understanding of the effect of rhizobacteria in the remediation of contaminated soil
(Reinhold-Hurek et al. 2015). Various pathways involving the degradation of soil
contaminants via enzymatic as well as genetic regulations are previously reported.
Metagenomics, metatrancrintomics, metaproteomics and genomics are few of the
potential tools that have facilitated in-depth study of the various factors that facilitate
the impacts of rhizobacteria on soil decontamination (Kotoky et al. 2018). The
aforementioned omics-based studies have enabled identification of various genes
involved in the degradation of soil contaminants. Moreover, transgenic studies also
play a crucial role in understanding the hydrocarbon degradation mechanisms. Plants
with various bacterial genes responsible for degradation of various xenobiotics and
hydrocarbon have been investigated for their efficiencies. Sylvestre et al. (2009)
reported that three components of biphenyl deoxygenase and 2,3 dihydroxybiphenyl
dioxygenase are actively produced by transgenic plants, which were critical for
bacterial polychlorinated biphenyl (PCB)-degrading pathway. Transfer of NAH7
plasmid in Pseudomonas putida KT2440 strain enabled efficient expression of Nah
catabolic pathway in vitro and in situ, leading to complete mineralization of naph-
thalene (Fernández et al. 2012). Brazil et al. (1995) reported successful expression of
bph gene in sugarbeet using recombinant P. fluorescens F113pcb strain. Mueller and
Sachs (2015) reported about microbiome engineering, which can be deployed to
enhance plant and animal health. Similar approach can be initiated to strengthen the
rhizosphere microbes, which, in turn, can strengthen the plants as well as soil health.
2.9 Conclusion
Constant anthropogenic exploration of soil has depleted the global soil fertility to
such an extent that there is an urgent requirement to re-establish the soil health. Loss
of carbon as well as other nutrients from the soil due to various agricultural practices
has resulted in decreased soil fertility. Use of various fertilizers and pesticides to
increase crop yield has further worsened the condition of soil. Excessive use
of fertilizers without considering the rhizosphere microbes has resulted in alteration
of nutrient cycles. With advancement in green biotechnology, the beneficial impact
of plant beneficial bacteria has attracted various researchers to explore their
potentialities in agricultural sector. The plant-growth-promoting rhizobacteria are
reported to be associated with various agricultural as well as ornamental plants.
These microbes provide enormous benefits to the plants by making various nutrients
available to the plants, providing protection from wide range of phytopathogens and
secreting various plant beneficial hormones. The beneficial impacts of rhizosphere
microbes are not only limited to the plants, but also are extended towards soil health.
Various root exudates released by the living plant roots help in developing the
rhizosphere microbial ecology, which further increases carbon sequestration in soil
and maintains the nitrogen cycle. This ultimately results in a significant increase in
soil nutrients. The rhizosphere microbial niche is a rich pool of various genes with
high catabolic efficiencies towards various forms of hydrocarbon as well as
pesticides. Molecular approaches to identify such genes and their effect have
allowed scientists around the globe to understand the benefits rendered by the
rhizosphere microbes. Genetic modification of various plants, wherein insertion of
microbial catabolic genes has shown a better tolerance to various contaminants.
Advancement in omics study has provided information regarding the rhizosphere
ecology, which can be further exploited for re-establishing the soil ecology. The
rhizosphere is a complex ecology with constant signalling between roots and the
rhizosphere microflora. Thorough study of rhizosphere ecology is required to better
understand the mutual relation between the microbes and the plant root to utilize it
for the betterment of the environment.
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Dissecting Structure and Function of Plant
Rhizomicrobiome: A Genomic Approach 3
Hemant Dasila, Samiksha Joshi, and Manvika Sahgal
Abstract
Rhizomicrobiome is the most diverse and highly dynamic environment primarily

influenced by root exudates of the host plant. It is a hotspot region for total
microbial activities that imparts a beneficial effect on plants by enhancing nutrient
availability and suppressing the growth of invading phytopathogens. Numerous
efforts have been undertaken to unmask the total microbial diversity and interac-
tive mechanisms occurring at the rhizosphere. Communication within the rhizo-
sphere microbiome via signalling molecules provides a basic model for
interactions, but there is a need to explore important events at the molecular
level. Dissecting the rhizomicrobiome environment with classical (culture isola-
tion techniques, biochemical characterization, and 16S rRNA gene sequencing)
and modern approaches (metagenomics, metatranscriptomics, proteomics, and
metabolomics) in combination with bioinformatic tools (BLAST, KEGG, Swiss-
Prot, etc.) has unravelled some of the important aspects of microbial interactions
at rhizosphere. The role of molecular and bioinformatics tools becomes very
important as they cover microbial responses at every stage, e.g. at DNA, RNA,
and protein levels. These classical and modern approaches along with bioinfor-
matics tools are a great attempt to unfold hidden secrets in rhizomicrobiome with
respect to plant health.
Keywords
Rhizomicrobiome · PGP · Bioinformatics · Classical approach · Modern approach
H. Dasila · S. Joshi · M. Sahgal (*)

Department of Microbiology, G.B. Pant University of Agriculture and Technology, Pantnagar,
Uttarakhand, India

23, https://doi.org/10.1007/978-981-15-9154-9_3
74 H. Dasila et al.
3.1 Introduction
Rhizosphere refers to the volume of soil influenced by roots. It is a highly dynamic

and complex environment where biogeochemical cycles of elements are affected by
an interaction between microorganisms at the root interface (Peterson 2013). Rhizo-
sphere is associated with numerous microbes. Rhizosphere-associated
“microbiome” is known as rhizomicrobiome (Berendsen et al. 2012). It is considered
as plants’ second genome because it directly influences plant health and agriculture
productivity under normal and stressed conditions (Philippot et al. 2013).
Rhizomicrobiome is associated with phytohormone production, nitrogen fixation
(Kathiravan et al. 2013), and biocontrol activity against phytopathogen (Yin et al.
2013) under abiotic and biotic stress. Soil rhizomicrobiome composition varied in
healthy and diseased plants and also acts as an indicator of the future disease
outcome in plants. The initial composition of rhizomicrobiome predetermined
whether the plants will succumb or survive disease in the future (Wei et al. 2019).
The nature of rhizosphere colonizing bacteria, as either attractant or repellent,
depends on the plants’ development stage (Peiffer et al. 2013; Chaparro et al.
2014). The dominant and common inhabitants of the rhizomicrobiome include
plant growth-promoting rhizobacteria (PGPR) (Ayyadurai et al. 2007; Sekar and
Prabavathy 2014). The soil type and microbial community in the rhizospheric region
are influenced by plant root exudation profile (De Angelis et al. 2009). Organic
carbon released by the plants results in a significant increase in total microbial
activity within rhizomicrobiome. Soil microorganisms are chemotactically dragged
towards plant root exudates as they provide them a carbon-rich environment (Jones
et al. 2009; Mendes et al. 2013). A recent study of total DNA community analysis
provides strong evidence that microbial interaction at the rhizosphere is plant
species-specific (Rout and Southworth 2013). The rhizosphere microbiome of
wheat, rapeseed, and maize each carries different bacterial communities (Bakker
et al. 2013). Even within a bacterial group, certain species, for example, fluorescent
Pseudomonas spp. is plant species-specific. Biotic and abiotic stresses, anthropo-
genic effects and climatic variation are key determinants in plant–microbe associa-
tion and affect the population dynamics of rhizosphere microbes (Bulgarelli et al.
2012; Lundberg et al. 2012). Rhizomicrobiome provides specific nutrition and
protection to the plants from invading phytopathogens by producing enzymes or
secondary metabolites ( Philippot et al. 2013; Turner et al. 2013).
3.2 Rhizomicrobiome
Rhizomicrobiome comprises of nutrient-rich environment that serves as a junction

for nutrient exchange between soil, plants, and microbes. The density of microbial
cells in the rhizosphere is much greater as compared to that of plant cells. The
constituent microflora of rhizomicrobiome can affect seed vigour, seed germination,
nutrition, productivity, plant growth, and development and in disease occurrence
(Mendes et al. 2013). It is dynamic and diverse and comprises micro-organisms at
3 Dissecting Structure and Function of Plant Rhizomicrobiome: A Genomic Approach 75
different domains, viz., fungi, bacteria, algae, archaea, nematodes, protozoa, viruses,
and microarthropods (Buée et al. 2009). Amongst these, domain bacteria is the most
dominating followed by fungi, actinomycetes and other groups such as
Actinobacteria, Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes
(Buée et al. 2009; Turner et al. 2013; da Rocha et al. 2013).
3.3 Root Exudate–Rhizomicrobiome Relationship
Root–microbe interaction was first reported in the wheat rhizosphere (Foster and
Rovira 1976). Root–microbe interaction includes the exchange of signals that initiate
all the physiological and metabolic activities in the rhizosphere. Root–microbe
interaction is influenced by both root exudates and microbial components. Plant
root exudation pattern governs the structure of rhizomicrobiome (Peter 2011). The
density and diversity of rhizospheric bacteria vary significantly from that of bulk
soil. There is considerable chemical diversity in root exudates which include
monosaccharides (glucose, mannose, and fructose) disaccharides (maltose), amino
acids (aspartate, glutamate, asparagines, glutamine, arginine, and cysteine), organic
acid (benzoic, ascorbic, and acetic acid), and a few high molecular weight com-
pound, such as enzymes, fatty acids, auxins, gibberellins, nucleotides, tannins,
terpenoids, alkaloids, polyacetylenes, and vitamins (Gunina and Kuzyakov 2015;
Hayat et al. 2017). Although both C and C4 plants release root exudates during
photosynthesis, the pattern varies. In C3 plants, dominant sugars are ribose, maltose,
and mannose (Vranova et al. 2013), whereas ribitol and inositol in C4 plants (Nabais
et al. 2011).
3.3.1 Signal Exchange Between Rhizomicrobiome and Plant Roots
3.3.1.1 Rhizomicrobiome-Mediated Phytohormone Production

Phytohormones are one of the key players in plant growth and development. The
phytohormones act as molecular signals in response to environmental stress that
otherwise becomes lethal or limits plant growth (Fahad et al. 2015). Important
phytohormones are gibberellins, cytokinins, and ethylene. Rhizosphere-colonizing
bacteria such as Azotobacter sp., Bacillus subtilis, Pantoea agglomerans, Pseudo-
monas fluorescens, Paenibacillus polymyxa, Rhizobium sp., and Rhodospirillum
rubrum produce gibberellins or cytokinins, whereas Achromobacter, Acinetobacter,
Agrobacterium, Alcaligenes, Azospirillum, Bacillus, Burkholderia, Enterobacter,
Pseudomonas, Ralstonia, Rhizobium, and Serratia produce ACC deaminase (Kang
et al. 2010). The enzyme ACC deaminase regulates the rhizosphere ethylene level.
Phytohormone ethylene affects plant growth and development in various ways
which include root initiation, promoting fruit ripening, promoting leaf abscission,
inhibiting root elongation, stimulating seed germination, promoting lower wilting,
and initiating the synthesis of inducible factors that regulate other plant hormones.
Lower the ethylene level, more the plant growth. It has been reported that
76 H. Dasila et al.
co-inoculation of ACC deaminase positive Pseudomonas sp. and R. leguminosarum

resulted in improved nodule dry weight, nodule number, grain yield, straw yield,
fresh biomass, and nitrogen content in grains of lentil (Iqbal et al. 2012).
3.3.2 Signalling: Microbe to Plant
Rhizomicrobiome comprises bacterial strains that produced secondary metabolites

and volatile organic compounds (VOCs). Priming of VOC producing bacterial
strains can improve stress tolerance and promotes plant growth development. For
example, polyamines play important physiological roles in plants. Polyamine helps
in higher biomass production, elevated photosynthetic capacity, and altered root
architecture in Arabidopsis (Zhou et al. 2016). Bacillus megaterium strain BOFC15
is reported to secrete a spermidine that in turn induces polyamine production in
Arabidopsis.
3.3.3 Signalling: Plants to Rhizomicrobiome
Plants harbour the mutualistic, parasitic, and commensal microorganisms. Legumes

are the best-studied examples of signalling from the plant to a microorganism. The
exchange of signals between plant and rhizobia gives rise to nodule formation
(Oldroyd 2013). Flavonoids (2-phenyl-1,4-benzopyrone derivatives) are the first
plant signals exchanged with its rhizobial symbionts which further induced nod
genes in rhizobia (Redmond et al. 1986) to secrete lipochitooligosaccharides (LCOs)
molecules known as Nod factors. The nod factors are central signalling molecules
that initiate the nodulation process in plants (Lerouge et al. 1990). Plant perceives
these LCOs signal via its tyrosine kinase receptor present at the root epidermis.
Tyrosine kinase initiates signalling cascade which finally leads to nodule develop-
ment (Limpens et al. 2015).
3.4 Communication in the Rhizomicrobiome
3.4.1 Signal Molecules
Quorum sensing (QS) is the dominant mechanism through which rhizobacteria

detect and respond to within the rhizosphere environment (García-Contreras et al.
2015). QS signals belong to different classes of chemical, viz., fatty acids, N-acyl
homoserine lactones, and alcohol (Venturi and Keel 2016). A few gram-negative
bacteria like Burkholderia spp. and Stenotrophomonas maltophilia reportedly pro-
duce diffusible signal factor (DSF) which is chemically cis-2-unsaturated fatty acids
(Ryan et al. 2015) whereas rhizosphere-associated proteobacterial strains such as
Erwinia, Pseudomonas putida, Pseudomonas chlororaphis, Pseudomonas syringae,
Serratia, and Ralstonia produce and respond to the N-acyl homoserine lactone
(AHL) type of QS signal (Ferluga et al. 2008). However, ascomycetes mostly secrete
alcoholic QS signals (Benocci et al. 2017).
3.4.2 Communication Within Rhizomicrobiome: Operational

Molecular Machinery
A communication event within the rhizosphere microbiome involves activation and

inactivation of genes, induction, and repression of genes to the various signals.
Various signals involved in rhizosphere communication include pattern recognition
receptors (PRRs), legume-rhizobia system, induced systemic resistance (ISR), ABC
(ATP-binding cassette), and multidrug and toxic compounds extrusion (MATE)
transporters. Pattern recognition receptors (PRRs) are microbe-specific receptors
that recognize different molecular signals. PRRs further bind to microbe-associated
molecular patterns (MAMPS). PRRs induce responses in the form of plant elicitors’
peptides when encountered with non-beneficial bacteria (Lee et al. 2018; Ruiz et al.
2018). These responses trigger a signalling cascade that activates certain transcrip-
tion factors, defense-related genes, reactive oxygen, and nitrogen species (Ipcho
et al. 2016). The legume-rhizobia system has been extensively used to uncover
molecular determinants for symbiosis (McCormick 2018; Wood and Stinchcombe
2017). In rhizobia, nod transcription factors (NF), i.e. lipochitooligosaccharides
(LCOs), are activated via release of flavonoids from legumes. These nod factors
are responsible for rhizobial host specificity (Behm et al. 2014). The LCOs activated
via rhizobia–legume interaction promote plant growth and health. Jasmonic acid
released by plants trigger induced systemic resistance (ISR) to counter pathogenesis
(Zamioudis and Pieterse 2012). The connection between transporter proteins and soil
phytopathogens has not been extensively studied, but in a few studies, the presence
of MATE transporters is detected in rice roots. MATE transporter proteins promote
the release of phenolic compounds into the xylem (Baetz and Martinoia 2014).
In Arabidopsis (Garcia et al. 2004) out of 129 genes encoding ABC transport
proteins, 25 were involved in root secretions. The expression level of these genes
in root cells were higher (Badri et al. 2008). Both ABC and MATE transporter
proteins are capable of releasing root phytochemicals into the rhizosphere. MATE
transporter proteins are also connected with the release of citrate that confers
aluminium resistance in plants (Stukkens et al. 2005). Overexpression studies
involving genes responsible for the synthesis of SA (salicylic acid) and camalexin
(provides disease resistance against nematodes) in Arabidopsis (Youssef et al. 2013)
reveal that knockout of the above genes results in low accumulation of camalexin
which later results in increased probability of nematode attacks (Iven et al. 2012).
PsoR protein from Pseudomonads plays an important role in the regulation of
antimicrobial genes that are responsible for biocontrol. PsoR proteins are regarded
as a subfamily of LuxR proteins (Gonzalez and Venturi 2013).
78 H. Dasila et al.
3.5 The Direct Role of Rhizomicrobiome in Plant Growth

Promotion
Rhizomicrobiome promotes direct plant growth via the production of plant

hormones and solubilization of mineral phosphates and essential nutrients. Most of
the organic nitrogen and other essential nutrients retained by the PGPRs result in
reduced dependence on fertilizers.
After nitrogen (N), phosphorus (P) is the second-largest nutrient that is crucial for
plant growth and development. It plays a key role in major metabolic pathways such
as macromolecular biosynthesis, photosynthesis, respiration, and energy transfer
mechanism (Li et al. 2017). This leads to increased demand for phosphate fertilizer.
However, the use of phosphate fertilizer is not only expensive but also damaging to
soil micro-environment. Phosphorus can cause eutrophication of lakes resulting in
reduced soil fertility. To meet the high demand for phosphorus in an eco-friendly
way, phosphorus-solubilizing bacteria (PSB) are now becoming a central point of
the research. About 95–99% of phosphate present in the soil is insoluble,
precipitated, and is in an immobilized form (Ahemad and Kibret 2014). Different
rhizosphere-associated microbes such as bacteria and fungi have phosphorus solubi-
lization potential. PSB constitutes 1–50% of the whole microbial component of
rhizosphere followed by fungi which constitute only 0.1–0.5% (Satyaprakash et al.
2017). Rhizosphere-associated bacterial genera Arthrobacter, Bacillus, Beijerinckia,
Burkholderia, Enterobacter, Erwinia, Pseudomonas, Rhizobium, Rhodococcus, and
Serratia are known phosphate solubilizers. Phosphate solubilizing fungi isolated
from the rhizosphere of haricot bean, faba bean, cabbage, tomato, and sugarcane
belong to genera Aspergillus, Penicillium, and Fusarium. Amongst them, Aspergil-
lus and Penicillium solubilized 728.77 μg mL1 and 514.44 μg mL1 phosphorus,
respectively (Elias et al. 2016).
Nitrogen-based fertilizers are being widely used to fulfil crop nitrogen requirements
(Santi et al. 2013). An alternative environment-friendly approach is crop inoculation
with potential biological nitrogen-fixing plant growth-promoting rhizobacteria
(Gaby and Buckley 2012). Nitrogen-fixing rhizobacteria include Azoarcus,
Acetobacter, Azospirillum, Burkholderia, Azotobacter, Cyanobacteria,
Enterobacter, Pseudomonas, and Gluconacetobacter (Bhattacharyya and Jha 2012).
3.5.3 Potassium Solubilization
Potassium (K) is the third major essential macronutrient for plant growth and
development. Soil usually contains a low amount of potassium and most of the
potassium present in the soil are found as silicate minerals and insoluble rock
(Parmar and Sindhu 2013). Nowadays, potassium deficiency has become a major
constraint in crop production. Potassium deficiency symptoms include low yield,
poorly developed roots accompanied by slow seed growth rate and small seed size.
Several species present in the rhizosphere microbiome have the potential to solubi-
lize potassium rock by the production of organic acid, and it provides an alternative
source of potassium for plant uptake (Kumar and Dubey 2012). Bacterial strains
Acidothiobacillus ferrooxidans, Bacillus edaphicus, Burkholderia, Paenibacillus
sp., Pseudomonas, and Bacillus mucilaginosus (Liu et al. 2012) are responsible for
potassium solubilization.
3.5.4 Siderophore Production
Iron is one of the most important micronutrients for almost all organisms of the
biosphere. In soil, iron is present as a ferric ion (Fe+3) that is sparingly soluble and
not easily assimilated either by plants or bacteria (Ma 2005). Rhizobacterial strains
have evolved specialized mechanisms for iron assimilation, i.e., production of low
molecular weight iron-chelating compounds referred to as siderophores (Arora et al.
2012). Depending upon functional groups, siderophores are divided into three main
families, namely catecholate, hydroxamate, and carboxylate. To date, 500 different
types of siderophores are known, out of which 270 have been structurally
characterized (Cornelis 2010). The study of radiolabeled ferric siderophores as a
sole iron source confirmed that plants can assimilate labelled iron by several
rhizobacterial strains, i.e., Aeromonas, Azotobacter, Bacillus, Burkholderia, Pseu-
domonas, Rhizobium, Serratia, and Streptomyces sp. (Sujatha and Ammani 2013).
3.6 Indirect Plant Growth Promotion
Phytopathogenic microorganisms are a major threat to sustainable agriculture. The

phytopathogens deplete soil fertility, disturb soil ecology, damage groundwater
quality, and directly influence crop yield. Bacteria constituting rhizomicrobiome
contribute to soil fertility and promote plant growth indirectly by suppressing the
phytopathogens (Tariq et al. 2014).
3.6.1 Antibiosis
Antibiotic production is one of the most powerful weapons that microorganisms use
to eliminate phytopathogens. It is one of the most studied biocontrol mechanisms
80 H. Dasila et al.
(Shilev 2013). Majority of rhizobacteria produce antibiotics. Pseudomonads have

been reported to produce numerous antibiotics, for example,
2,4-diacetylphloroglucinol (DAPG), amphisin, phenazine, oomycin A, pyoluteorin,
kanosamine, oligomycin A, pyrrolnitrin, tropolone and cyclic lipo-peptides (Loper
and Gross 2007). Bacillus, Streptomyces, and Stenotrophomonas sp. produce anti-
biotic compounds xanthobaccin and zwittermicin A (Compant et al. 2005). Antibi-
otic compounds, such as polyketide and lipopeptide, produced by Bacillus
amyloliquefaciens seem to elucidate biocontrol activity against soil-borne pathogens
(Ongena and Jacques 2008). At times, a few phytopathogens might develop resis-
tance against antibiotics. Thus, it becomes very difficult to implement these
rhizobacteria as biocontrol agents in the field. The problem is overcome by the use
of microbial strains producing more than one antibiotic (Glick 2012). There are
various examples for use of antibiotic-producing strains as biocontrol agents, for
example, 2, 4- DAPG (2, 4-diacetylphloroglucinol) positive strain is used in soil
treatment for biocontrol of wheat disease caused by fungus Gaeumannomyces
graminis var. tritici (de Souza et al. 2003). Bacterization of wheat seeds with
P. fluorescens prevents it from phytopathogen attack due to the production of
antibiotic, phenazine-1-carboxylic acid (PCA) (Weller 2007). In addition to the
antibiotic production, some rhizobacterial strains are also capable of producing
a volatile compound, HCN (Hydrogen cyanide) to suppress phytopathogenic inva-
sion, e.g. HCN- mediated biocontrol of tobacco black rot disease was done by
Pseudomonas fluorescens CHAO strain (Sacherer et al. 1994). HCN along with
DAPG released by Pseudomonas fluorescens LBUM 223 strain promotes the bio-
control of tomato Canker disease caused by Clavibacter michiganensis (Lanteigne
et al. 2012).
3.6.2 Lytic Enzymes
Enzymatic activity is another important mechanism adopted by rhizobacterial bio-

control agents. The enzymes involved in biocontrol activity include dehydrogenases,
chitinases, lipases, β-glucanase, phosphatases, and proteases (Joshi et al. 2012:
Hayat et al. 2012). These enzymes attack pathogens by excreting cell wall
hydrolysing enzymes. These enzymes play a very important role in protecting the
plant from a biotic stress. The important phytopathogenic fungi that are suppressed
through the action of hydrolytic enzymes include Botrytis cinerea, Fusarium
oxysporum, Rhizoctonia solani, Phytophthora sp., and Pythium ultimum (Nadeem
et al. 2013).
3.6.3 Induced Systemic Resistance (ISR)
ISR is a physiological state of an enhanced defensive property that is elicited in

response to specific stimuli and boosts plant innate defenses against biotic factors
(Avis et al. 2008). Rhizobacteria play an important role in providing systemic
resistance to plants against a broad spectrum of phytopathogens. Jasmonic and

ethylene signalling within plants is a classic example of induced systemic resistance
that elevates the host immune system in response to a vast variety of pathogens
(Glick 2012). Bacterial components that can stimulate induced systemic responses in
host include flagella, siderophores, homoserine lactone, lipopolysaccharides, DAPG
along with volatile compound, 2, 3-butanediol, and acetoin (Doornbos et al. 2012).
3.7 Endophytic Root Microbiome
Endophytes are those microbes (bacteria or fungi) residing inside plant tissues that
do not cause negative effects on plant growth (Coombs and Franco 2003). Distribu-
tion of endophytes throughout the plant is based on several factors, such as their
colonization ability and allocation of plant resources. Based on their plant-inhabiting
lifestyle, endophytes are classified into three main categories: (a) Obligate
endophytes: derived from seeds and unable to survive in soils (b) Facultative
endophytes: widely exist in soil, which can colonize and infect plant in favourable
situations, and (c) Passive endophytes: lack colonization ability and can only infect
via wounds or cracks on the plant (Fig. 3.1) (Hardoim et al. 2008). Most of the
endophytes with plant growth promotion traits belong to a facultative group. Root
endophytes often colonize and penetrate the epidermis at sites of lateral root emer-
gence, below the root hair zone and in root cracks (Zakria et al. 2007). Structure and
composition of root endosphere microbiome is found to be less diverse as compared
Endodermis
Epidermis
Root Cracks
Endosphere/Endophyte
Root
Hair
Rhizosplane
Microbiome
Phloem
Xylem
Rhizosphere
Microbiome
Colonization Types
Passive Soil Bacteria
Facultative Obligate
Fig. 3.1 Bacterial distribution and colonization patterns of endophytes in the plant root
82 H. Dasila et al.
to rhizosphere or bulk soil microbiomes (Liu et al. 2017) which indicate roots as
effective habitat filters, restricting community membership to progressively and
more narrowly defined lineages as environments deviate from soil to roots
(Bulgarelli et al. 2012).
The dominant endophytic bacterial communities in plant roots mainly belong to
phyla Proteobacteria followed by Actinobacteria, Firmicutes, and Bacteroidetes.
Other bacterial phyla commonly found in the root endosphere, but in fewer fractions,
are Armatimonadetes, Cyanobacteria, Chloroflexi, Verrucomicrobia, Nitrospirae,
and Planctomycetes (Edwards et al. 2015). Members from Archaea, Acidobacteria,
and Gemmatimonadetes are either absent or rarely present in the root endosphere
(Sessitsch et al. 2012). The dominance of phyla was plant-specific, whereas the
abundance is influenced by soil cultivation history. Proteobacteria, Actinobacteria,
Firmicutes, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Chloroflexi were
the most abundant phyla associated with grapevine roots (Samad et al. 2017).
Correa-Galeote et al. (2018) demonstrated Proteobacteria, Firmicutes, and
Bacteroidetes as predominant phyla inside the maize roots. On exploring the rice
root microbiome, members from Rhizobiaceae, Bradyrhizobiaceae,
Streptomycetaceae, and Comamonadaceae family were found in abundance
(Edwards et al. 2015). Fungal endophytic communities in roots are mainly
dominated by Polyporales, Russulales, and Agaricales (Sokolski et al. 2007).A
few endophytes may form mycorrhiza-like structures. For example, Leptodontidium
formed peloton-like structures on roots of Dendrobium nobile, an orchid, and
induced positive effect on various plant growth parameters (Hou and Guo 2009).
Some common root endophytes are DSEs (dark septate endophytes), hyaline hyphal
endophytes, or hyphomycetes endophytes. DSEs occur in roots of Rosmarinus
officinalis along with arbuscular mycorrhizal fungi (AMF) and in Pinus halapensis,
with ectomycorrhizal fungi (Girlanda et al. 2002). Similarly DSEs occur with AMF
in Pedicularis roots (Li and Guan 2007). In a mutualistic association between
Heteroconium chaetospira (DSE) and Brassica campestris, endophytic fungi
received carbon from the plant and supplied nitrogen to it (Usuki and Narisawa
2007). Aquatic hyphomycetes are commonly found in riparian and coastal
ecosystems. For example, multiple species of aquatic hyphomycetes were isolated
from roots of riparian plants including grasses (Cupressaceae) and Pteridophytic
trees (Sridhar and Bärlocher 1992).
Endophytes exert various beneficial effects on plant via plant growth hormones,
nitrogen fixation, increased nutrient uptake, and inhibition of phytopathogens. In
addition, they provide diverse bioactive secondary metabolites (Santoyo et al. 2016),
and therefore are used as bio inoculants. Several factors influence the structure and
diversity of the endophytic community. These include plant species and genotype,
agricultural practices, environmental conditions, seasonal variation, geographical
location, soil type, and host plant nutrient status (Hardoim et al. 2015). Spatiotem-
poral patterns have been documented in endophyte communities of certain plant
species.
With the availability of new molecular approaches, such as high throughput
sequencing (e.g., 454-pyrosequencing) (Lundberg et al. 2012), microarray multiplex
technology (Gao and Tao 2012), and nucleic acid-based stable isotope probing
(Radajewski et al. 2000), discovering and characterizing the endophyte community
structure and dynamics has become a reality. A study of potato root extracts by
pyrosequencing showed that five of the 10 most common genera were reported for
the first time as potato endophytes (Manter et al. 2010). With the help of SIP-rRNA
analysis, the diversity within primary bacterial consumers of plant-derived carbon
has been explored, and new endophytic phylotypes have be en identified
(Vandenkoornhuyse et al. 2007). Although endophytes have been detected in all
plant parts, roots are the first to the recruit them.
3.8 Classical Approach to Study the Microbiome
Classical approach to get insight into microbial community associated with rhizo-
sphere involves isolating and culturing microbes using specific nutrient media and
growth conditions depending on the target organisms and host environment.
Obtaining a pure culture of an organism is required for detailed studies of its genetics
and physiology. Moreover, it will give actual microbial composition and its func-
tional implications on performance of host plant.
3.8.1 Culture-Dependent Methods
These are the methods used for isolation and morphological, physiological, and
functional characterization of microbes from an environmental sample.
3.8.1.1 Isolation and Culturing

Classically, the microbial community analysis has relied on culturing techniques
which include isolation of microbes through enrichment or serial dilution methods
followed by spread or pour plating on specific medium and subsequent determina-
tion of viable counts (Fig. 3.2). Variety of selective (e.g., the bile salts in MacConkey
agar) or non-selective (e.g., nutrient agar or plate count agar, etc.) media are
available for maximum recovery of different microbial species. The specific growth
media for all the three domains, i.e., archaea (standard growth media, chemically
defined medium, and halophilic medium), bacteria (Kings’ B agar, ammonium
mineral salt, Antarctic bacterial medium, Jensen N2-free agar, T3 agar, R2 agar,
tryptic soya agar, and yeast mannitol agar), and eukarya (fungus) (Czapek Dox agar,
Rose Bengal agar, Sabouraud dextrose agar, and potato dextrose agar) have been
described (Yadav et al 2008). The culturable approach has revealed a diversity of
microorganisms associated with various soil quality parameters, such as organic
matter decomposition and disease suppression. Through this approach, analysing
large number of highly diverse samples is very time consuming. Considering the
‘great plate count anomaly’ which states that microscopic bacterial counts are
considerably higher than the equivalent total viable counts (Torsvik and Ovreas
84 H. Dasila et al.
Fig. 3.2 Different culturable and unculturable approaches for characterization of microorganisms
2002), it has been estimated that only 1% of the total bacteria can be readily
cultivated in vitro.
Significant efforts have been made in past years to advance culturing methods for
unculturable species which accounts for 99% of the actual bacteria present. Certain
bacteria have fastidious growth requirements, for example, N-acetyl muramic acid
for Tannerella forsythia; Pyridoxal or L-cysteine for Abiotrophia spp.; and
Granulicatella spp. (Vartoukian et al. 2010).Whereas others require specific oxygen
concentration, nutrient level, a humic acids, and signalling molecules in growth
medium (Stevenson et al. 2004). It has been observed that addition of signalling
molecules, for example, cyclic AMP (cAMP), 5-amino-acid peptide, or acyl
homoserine lactone, improves the cultivability of unculturable soil bacteria (Janssen
2008). Use of Gellan gum instead of agar resulted in enhanced bacterial growth and
10-fold higher Colony-forming unit (CFU) counts (Kamagata and Tamaki 2005).
Furthermore, beneficial bacterial interactions are also found to be useful in in vitro
cultivation of unculturable bacteria only when these bacteria are co-cultivated with
helper strains (Nichols et al. 2008). When the cell free extract or spent culture
supernatant obtained from helper strain was added to culture media growth of
species, such as Sphingomonas spp., Psychrobacter spp., Catellibacterium spp.,
and Symbiobacterium spp. was stimulated (Nichols et al. 2008). Recently a novel
strategy, SMART (selective medium design algorithm restricted by two constraints),
was developed to design selective media using non-natural material and containing
two selective agents: a carbon source and an antimicrobial agent. This strategy has
been used for designing selective media for culturing Acidovorax avenae,
Pectobacterium carotovorum, Burkholderia glumae, Xanthomonas campestris,
and Ralstonia solanacearum (Kawanishi et al. 2011).
3.8.1.2 Functional Characterization

One of the most widely used method for assessing gross functional diversity is
community-level physiological profiles (CLPP) (Konopka et al. 1998). This
approach is facilitated by the use of a commercial taxonomic system, known as
the BIOLOG® system that is based on utilization of different carbon sources
(Garland 1996). Utilization of each substrate is detected by the reduction of a
tetrazolium dye, which results in a colour change that can be quantified with spec-
trophotometer. Advantages of CLPP include ability to differentiate between micro-
bial communities, relative ease of use, reproducibility and production of large
amount of data describing metabolic characteristics of the communities. Prior to
BIOLOG, several commercial kits utilizing phenotypes for determination of bio-
chemical properties of microbes were also available (Busse et al. 1996). Further-
more, microbes can be checked for various functional traits (Fig. 3.2) like nitrogen
fixation (Boddey et al. 1995); phytohormones production (Bric et al. 1991), zinc
solubilization (Fasim et al. 2002), ammonia production (Cappuccino and Sherman
1992), siderophore production (Schwyn and Neilands 1987), HCN production
(Bakker and Schippers 1987), and phosphate solubilization (Pikovskaya 1948)
both qualitatively and quantitatively. Although many new molecular approaches
have been applied for studying the biodiversity and role of microorganisms in the
environment, classical approach is still useful to assess metabolic potential of diverse
microbial communities and provide detailed understanding of their metabolism and
functions.
3.8.1.3 16S rRNA Gene Sequencing

16S rRNA molecule is the most common house-keeping genetic marker used for
studying bacterial phylogeny and taxonomy. It is 1500 bp in length. Its function has
not changed over the time, hence random sequence changes in 16S rRNA are
accurate measure of the evolution (Christensen et al. 2001). 16S rRNA genes may
not be identical in all organisms and comprised of both variable and conserved
regions (Tortoli 2003). The presence of variable regions does not affect the use of
16S rRNA for bacterial identification and in finding the close relationship at the
genus or species level (Garrity and Holt 2001). Universal primers usually comple-
mentary to the conserved regions present at the beginning of the gene and at either of
540bp region or at the end of the whole sequence (1550bp) are used for amplification
and sequencing. The sequence of the variable region in between is used for the
comparative taxonomy (Relman 1999). Sometimes sequencing of entire 1500bp
region is required to distinguish between particular taxa or strain (Sacchi et al.
2002). Since initial 500bp region is slightly more diverse, it provides adequate
86 H. Dasila et al.
differentiation for identification and also provides bigger percent difference between
strains. The applications of 16S rRNA gene sequence are as follows:
Identification of Novel Pathogen

In every publication regarding description of new prokaryote, 16S rRNA gene
sequence is important. 16S rRNA gene sequence analysis provides accurate identifi-
cation at the species level and can also clarify their clinical importance (Clarridge
et al. 1999). Other techniques for the identification include the most common DNA–
DNA hybridization technique which is considered as standard for proposing new
species. Depending upon DNA–DNA hybridization, which provides reassociation
kinetics, the genetic definition of a species is quantifiable, i.e., (1) 70% DNA–
DNA relatedness and (2) 5 C or less ΔTm for the stability of heteroduplex molecules.
Identification of Unculturable Bacteria

Universal primers against 16S rRNA gene region are used to derive a sequence for
uncultured bacterium.
3.9 Modern Approach
3.9.1 Functional Genomics
Functional genomics is the study of how genes, genomes, proteins, and metabolites
contribute to particular phenotype. Functional genomics comprise the study at DNA
(genomics), gene (transcriptomics), protein (proteomics), and metabolite
(metabolomics) level. Functional genomics unfolds the interaction between genes
and proteins. It combines data derived from various molecular process related to
DNA sequence, gene expression, and protein function. Further analysis requires
proper modeling for molecular dynamics and interactive study that regulate gene
expression, cell cycle progression, and cell differentiation. Various tools used for
functional genomics are as follows:
3.9.2 DNA Microarray
It is a molecular technique that comprises thousands of microscopic DNA probes

(spots) attached to solid surface, such as silicon chip or glass. Labelled ssDNA or
RNA fragment from a sample of interest are hybridized under stringent conditions.
The amount of nucleic acid content extracted from an original sample is directly
proportional to the amount of hybridization detected in microscopic probes spots.
3.9.3 Next-Generation Sequencing
In next-generation sequencing (NGS), DNA template is fragmented, attached to

adaptors followed by amplification in PCR, and then immobilized on beads where
identical cluster of DNA are formed. These cluster DNA fragments are ready for
nucleotide incorporation followed by washing and detection, where the read length
is directly proportional to the number of cycles. Prominent and widely used next-
generation sequencing (NGS) platforms are Roche 454 platform (Roche Life Sci-
ence), Illumina and Applied Biosystem SOLiD platform (Shendure et al. 2005).
3.9.4 Mass Spectrometry
Mass spectrometry analyses the samples through generation of multiple ions which
separate according to charge-to-mass ratio. Three main components of mass spec-
troscopy include an ion source (convert gas phase into simple ions), mass analyser
(separates sample ions under influence of electromagnetic field), and a detector.
Most recent and advanced mass spectrometer available nowadays is Orbitrap, is
versatile, dynamic in range, has high accuracy and high resolution which enable its
use in proteomics and metabolomics application. In Orbitrap, ions are trapped in an
orbital motion around the spindle, and the image obtained from these trapped ions
are detected which are further converted into mass spectrum by using Fourier
transform. Orbitrap provides tandem mass spectra of compounds which help in
elucidation of analyte structure thus resulting in identification of trace-level
components from the complex mixture (Makarov and Scigelova 2010).
3.9.5 Transcriptomics
Transcriptomics is profiling the abundance of transcript under various conditions in

different cell types. These RNA sequences (Transcript) also enable to analyse exon/
intron and transcript boundaries which result in some novel transcript discovery. In
addition, transcriptomics can also be used for profiling noncoding RNA, ribosome-
associated mRNA, and has potential to unfold the regulation at transcription and
post-transcriptional level. The advantage of RNA-Seq over DNA microarray is that
it is independent of species of interest and thus can be implemented for all.
3.9.5.1 Transcriptomics and Agriculture

Transcriptomics approach provides a feasible tool to study microbial community
associated with different plants (Molina et al. 2012; Sheibani-Tezerji et al. 2015).
Metagenome and genomic analysis enumerate presence or absence of genes, but
transcriptomics provides expression level of specific genes under different micro-
environment conditions. Thus, it became an important key to understand the
microrhizobiome association with host plants. Profound analysis of differentially
expressed genes in host plants helps in elucidating mechanism of association
88 H. Dasila et al.
between plants and rhizomicrobiome. Dual RNA sequence profiling provides a

better resolution of expressed genes in both partners (in symbiosis) at the same
time. Symbiotic association between Azospirillum brasilense and wheat plants has
been monitored through study of upregulation of genes associated with the cell cycle
progression and nutrient acquisition (Camilios-Neto et al. 2014).
3.9.6 Proteomics
Proteins are considered to be most important functional unit of cell. Sometimes

transcript level does not necessarily correlate with the quantity of protein then,
proteomics is used. Proteomics also unravel the mechanism of complex post trans-
lational modifications. Different techniques are used to study the protein content of
the cell. The 2D-gel electrophoresis is used to evaluate the protein content. The
technique involves the separation of protein first by mass then by the charge present
on polypeptide. Another relatively most used proteomics approach is GeLC-MS, in
which one-dimensional gel electrophoresis (SDS-PAGE) proteins are first separated
and then each gel lane is divided into equally sized section from which proteins are
digested and separated by liquid chromatography and finally analysed by mass
spectroscopy (MS). More recently, a high-throughput technology,
e.g. multidimensional protein identification technology (MudPIT), has been devel-
oped. It provides an efficient way to identify large number of proteins from complex
ones. In MudPIT, proteins are digested into peptides followed by separation through
two-dimensional chromatography based on hydrophobicity and charge and are
subsequently analysed by MS.
3.9.6.1 Proteomics and Agriculture

Proteomics analysis emphasized identification of novel physiological stress-related
proteins that are expressed in most complex environments of rhizomicrobiome.
Total protein can be extracted from the rhizomicrobiome either by direct or indirect
lysis method (Maron et al. 2007). Direct lysis method involves total protein isolation
from rhizomicrobiome that are expressed under natural and stress conditions. The
protein fingerprints thus obtained are further subjected to production and analysis of
those metabolites which are necessary for the plant protection. Indirect method
includes isolation of total proteins from cultured rhizosphere endophytes that are
expressed under stress condition. The protein fingerprint thus obtained was further
subjected to 2D-gel electrophoresis (Bhuyan et al. 2015).
3.9.7 Metabolomics
Metabolomics deals with the detection and characterization of exudates that are
released by microorganisms in the rhizomicrobiome. Tracing of metabolite directly
correlates with cellular phenotype rather than genes or proteins. Metabolomics
targets the complex cellular metabolism and characterization of new cellular
pathways that can later be used for drug delivery system like in cancer. The most
widely used tools in metabolomics involve liquid chromatography coupled with
mass spectroscopy (MS) and NMR (Nuclear magnetic resonance). The main advan-
tage of NMR is easy sample preparation and high reproducibility.
3.9.7.1 Metabolomics and Agriculture

Metabolomics is an emerging field and its application in agriculture field is still
under progression. Several factors are limiting the study of root exudates. These
include limitation in tracing specific exudates; sensitivity error in current
metabolomics platforms; and difficulties in tracking the temporal and spatial dynam-
ics of exudates at relevant scales (Van Dam and Bouwmeester 2016). But question
remains the same that how metabolomics help in analysing a complex system such
as rhizomicrobiome? GC-MS and LC-MS along with NMR (Nuclear magnetic
resonance) spectroscopy are most widely used technologies for analysing
metabolites in the biological samples (Dunn and Ellis 2005). At present, extraction
and identification of root exudates is still the major challenge in rhizosphere
metabolomics (Pandey and Mann 2000). It requires separate processing of microbe’
and plant fractions. The final product is formed upon their interactions, and use of
final metabolites allows to evaluate the functional community present in soil.
3.9.8 Interactomics
It is the study of networking and interaction at molecular and physiological level. It

involves protein–protein interaction, e.g., functional interaction found in signal
transduction, metabolic pathways and transcriptional regulation and many important
biological functions. Dissecting these interactions plays a vital role in understanding
the interactive pathways that forms the basis of many cellular processes. Recent
interactomics’ techniques involve tandem affinity purification (TAP) which involves
two steps of high specificity affinity purification. In combination with other tools,
such as phage display and protein microarray, these technologies help in understand-
ing the interactive protein networking.
3.9.9 Metagenomics
Metagenomics is defined as an analysis of the complete microbial genome from any

environmental niche (Zeyaullah et al. 2009). It gives a comprehensive view of the
species composition, genetic diversity, evolution, and interactions of microbial
communities with its natural environment (Simon and Daniel 2011). The
metagenomic approach can either be functional (Rabausch et al. 2013) or
sequence-based (Soni et al. 2012), which can be used for targeting different aspects
of a microbial community. The sequence-based approach is a reliable tool to study
the structure of the uncultivated microbial population through 16S rDNA based
analysis, whereas the metabolic or functional potential of a microbial community can
90 H. Dasila et al.
be analysed by using functional metagenomics. Thus, functional metagenomics can

be considered as a major tool for identifying and characterising novel gene families
from rhizosphere metagenome. Metagenomics is not only helpful in determining
bacterial diversity but also useful in exploring the fungal population in the rhizo-
sphere (LeBlanc et al. 2015). Many large-scale metagenomics projects have been
undertaken to investigate various aspects of the microbial composition, e.g. Human
Microbiome Project, International Census of Marine Microbes, and Earth
Microbiome Project. Bioinformatic software are required to handle a large amount
of data generated from different next-generation sequencing platforms, such as
454 pyrosequencing and illumina-based sequencing. Some important and widely
used software available for such metagenomic analysis include Quantitative Insights
into Microbial Ecology (QIIME), mothur, CARMA, and MEGAN (Escobar-Zepeda
et al. 2015). Software namely Illumina reads and PacBio reads have been recently
developed for short and very long sequence analysis, respectively. Moreover, a
connection between taxonomic classification from meta-profiling and metabolic
information is established through PICRUSt software (Langille et al. 2013). Despite
the immense importance of microbes in the rhizosphere, very little information is
available about their diversity and functions at a community level. Metagenomics
has an advantage over culture-dependent approaches to investigate the community
structure and exploit the functionalities of microbiome as all of them are life-forms
based on DNA as a carrier of genetic information. Furthermore, sequence-based
metagenomic studies reported that rice rhizosphere has the abundance of
Actinobacteria, Firmicutes, Acidobacteria, Planctomycetes, and Proteobacteria
(Bhattacharyya et al. 2016), whereas rhizosphere of wheat mainly showed associa-
tion with phyla Chloroflexi and Planctomycetes (Naz et al. 2014) and
Actinobacteria, Firmicutes, Archaea, Proteobacteria, virus, fungi, and unclassified
taxa (Hernandez-Leon et al. 2012). The soybean rhizosphere was colonized by
γ-Proteobacteria, Actinobacteria, and Ascomycetes (Bresolin et al. 2010). Similarly,
in the sugarcane rhizosphere, Proteobacteria, was dominant followed by
Acidobacteria, Bacteroidetes, Firmicutes, and Actinobacteria (Pisa et al. 2011).
Comparative assessment of bacterial communities associated with shisham rhizo-
sphere through Illumina sequencing showed a dominance of Proteobacteria and
Firmicutes at Lachhiwala and Tanakpur (healthy forest stands), whereas
Acidobacteria at Pantnagar forest (diseased stand) (Joshi 2018). Different manage-
ment practices, for example, conventional tillage and no-tillage, affected the func-
tional potential of soil microbial communities of soybean and wheat fields (Souza
et al. 2013). Chhabra et al. (2013) used a functional metagenomic approach to study
mineral phosphate solubilization genes in the rhizosphere of barley, which had not
received phosphate fertilizer for the last 15 years. Metagenomic approach was used
to investigate microbiome of tap root of sugar beet for various plant-growth-promot-
ing traits. A high number of genes coding for ACC deaminase, ß-1,3-glucanases,
siderophore production, and phosphate solubilization were detected, whereas genes
involved in IAA production or nitrogen-fixation were rare or not found at all
(Tsurumaru et al. 2015). Metagenomic technology has been successful at all scales;
it has been used to study single genes, pathways, organisms, and communities.
Approaches that involve massive sequencing to capture entire communities will

likely become more common with further advances in sequencing technology.
3.9.9.1 Metagenomics and Agriculture

Metagenome study provides an excellent idea about how different microbial com-
munity present in rhizomicrobiome help in degradation of toxic compounds that are
accumulated in soil. The abundance of Bacteroidetes, Firmicutes, Cyanobacteria,
and Actinobacteria in wheat rhizosphere was found to be associated with the
degradation of organic soil pollutant, e.g., benzoates, naphthalene, carbazoles,
phenols, xenobiotics, and biphenyls (Singh et al. 2018).
3.9.10 Metatranscriptomics
Messenger RNA (mRNA) is a short-lived intermediate between DNA and protein.

Transcription of mRNA is a highly regulated and energy-expensive process, but
mRNA is the most powerful weapon to study cellular responses with respect to
change in environmental conditions (Moran et al. 2013).
Using high-throughput sequencing techniques such as Illumina,
metatranscriptomics offers a non-PCR-based method for looking at transcriptional
activity occurring within a complex and diverse microbial population at a specific
point in time. However, curation and annotation of these complex data has emerged
as a major challenge. Metatranscriptomics uses short sequence reads. The short
sequence reads can be either individually aligned directly to external reference
databases (hereafter “assembly-free”) or assembled into longer contiguous
fragments (contigs) for alignment (hereafter “assembly-based”). Poulsen et al.
(2013) used an assembly-based approach to analyse gut microbiome. Whereas, the
assembly-free approach has been used in analysis of lactic acid bacterial strains
(Jung et al. 2013), aligning short reads to reference genomes. An open source
pipeline developed by Martinez et al. (2016) to analyse metatranscriptomics datasets
that align short reads directly to a protein database before annotation.
92 H. Dasila et al.
Sample collection
RNA extraction
cDNA libarary preparation
Assigning transcript to gene
de-novo assembly
Absolute versus relative abundance-normalization
Metatranscriptomics Workflow
3.9.10.1 Metatranscriptomics and Agriculture

Metatranscriptomics plays an important role dissecting the microbial community and
their functional aspects. It also plays an essential role in evaluating rhizobacteria-
induced systemic resistance in plants (De Vleesschauwer and Hofte 2009). For
example, Pseudomonas fluorescens strains promote resistance in tomato leaf curl
virus (Sangeetha et al. 2010). Metatranscriptomics study allows researcher to com-
pare and modulate bacterial gene expression profiles of samples treated with signal-
ling molecules, e.g. ethylene, jasmonic acid, salicylic acid and abscisic acid, and
control plants from the rhizosphere to monitor defense response. The expression
analysis of coi1, npr1, cpr5, pad4, eds5, jin1, jar1, and etr1 genes help in evaluating
the responses that arise during signalling cascade (Jones and Dangl 2006; Kazan and
Schenk 2007). Metatranscriptomics analysis of microbial communities associated
with rhizosphere help in evaluating the responses that arise during signalling
cascade.
3.9.10.2 Metatranscriptomics Tools

Compared to other omics approaches, metatranscriptomics is less common.
Metatranscriptomics reads are generally analysed and mapped to specialized
databases (NCBI) by using specialized alignment tools such as, Bowtie 2, BLAST,
and BWA. Results are then annotated using Swiss-Prot, KEGG, GO, and COG. The
most recent technique used in the metatranscriptomics analysis is stable isotope
probing (SIP) which targets specific transcriptomes of aerobic microorganism.
Second strategy is to assemble the small reads of transcriptomes into longer
fragments called contigs (Celaj et al. 2014). Several software packages such as
Trinity (Grabherr et al. 2011), Oases (Schulz et al. 2012), AbySS, Trans-Abyss,
MetaVelvet (Namiki et al. 2012), Scriptures, and Cufflinks (Guttman et al., 2010;
Trapnell et al. 2010) are used to analyse contigs. Amongst these, Trinity is sensitive
across broadest range of gene expression levels. Li and Dewey (2011) developed
RNA-Seq by Expectation Maximization (RSEM), a quantitative approach for
transcriptome analysis. In RSEM, inputs are taken from reference or standard
transcriptome or assembly (analysed from Trinity) along with RNA-Seq reads that
are generated from the samples which result in normalizing transcriptomics
abundance.
3.9.10.3 Factors Affecting Metatranscriptomic Analysis

The parameters that affect the analysis of data generated through metatranscriptomic
analysis are as follows
Read length: Normalization of gene length during retrieving of data from a
shotgun sequencing because sequencing data reads will be directly proportional to
the length of the gene.
RPKM/FPKM (reads per fragment per kilo base per millions reads)
ðcDNA reads obseved per geneÞ 10

This can be calculated by RPKM ¼
ðgene lengthÞ ðtotal number of reads in cDNA libraryÞ
cDNA/ DNA ratio: It is used to measure the number of transcript present in each
gene copies.
3.9.10.4 Computational Analysis of Metatranscriptomic Data

Dataset of metatranscriptome contains millions of RNA sequenced reads. To analyse
such a high informative data, a special tool is required, and several comprehensive
tools have also been developed in last few years, e.g. HUMAnN51 and
MG-RAST52. These tools along with specialized bioinformatic tools, e.g.GEM54
and BOWTIE53, are used for mapping; Trimmomatic 55 tool is used for quality
filtering and Cuff Duff56 tool is to analyse differential gene expression.
3.9.11 Comparative Metatranscriptomics
Comparative metatranscriptomics deals with the comparative study of the rhizo-

sphere microbial communities associated with different plant types in the same or
different set of environments. It measures community-wide comparative gene tran-
scriptional behaviours, an overview of total metabolic potential, microbial commu-
nity composition, and transcriptionally active taxa. It plays a very important role in
the analysis of different mutants growing under the same set of condition, e.g.,
comparative study of rhizosphere, microbiome of an avenacin-deficient mutant, and
wild type oat sad1 under same field conditions (Haralampidis et al. 2001). Compar-
ative metatranscriptomics is very useful in determining rhizosphere microbial
communities that are expressed under seasonal variation and stress condition,
e.g. differential gene expression of day/night pattern of microbial assemblage
94 H. Dasila et al.
(<5μm) from the surface of the water in Hawaiian ocean (Poretsky et al. 2009).
Comparative metatranscriptomics study is precise and often comes out with hidden
information, and this information sometimes gives insight into microbial evolution,
ecology, and adaptation, e.g., combined metaproteomic and metagenomic analysis
have been conducted to reveal the speciation and evolution in Acid Mine Drainage
(AMD) biofilms (Denef et al. 2010).
3.9.11.1 Comparative Metatranscriptomics and Agriculture

Comparative meta-transcriptomics approach explores disease resistance mechanism,
e.g. analysis of endophyte infected and endophyte-free plants lead us to better
understanding of endophyte-mediated disease resistance and plant-growth-promot-
ing properties. Comparative study of differential gene expression in
rhizomicrobiome-associated endophytes within and outside host plants unravels
the major factors involved in maintaining the relationship between host plant and
rhizomicrobiome (Johnson et al. 2004).
3.9.11.2 Applications
1. Comparative community analysis: Community analysis can be done through
omics approaches but comparative abundance or pattern of microbial community
can precisely be done via comparative metatranscriptomics.
2. Cellular-level resolution: Comparative metatranscriptomics aid in cellular level
gene expression that are expressed under different environment conditions, and
there is high resolution for proteins’ expression that are being expressed under
different conditions.
3. Exploring ecosystem: Comparative metatranscriptomics help in exploring
estimated number of taxa, signature proteins, and potential biomarkers in a
different ecosystem, e.g. activated sludge, ocean water, soil, surface water, etc.
The plant rhizosphere microbiome is recognized as a hot spot for functional micro-
bial activity. Dissection of rhizosphere microbial community through classical and
modern approaches helps in the elucidation of unexplored microbial interactions
occurring in the rhizomicrobiome region. These interactions are highly complex and
essential in shaping the rhizomicrobiome and influencing plant health. The rhizo-
sphere is dynamic. Therefore, it is more challenging to adopt advanced molecular
tools such as metatranscriptomics, next-generation sequencing, and genomics to
understand the structure and function of a microbial community in the rhizosphere.
These approaches provide an insight into the activities of the rhizosphere in real-
time.
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Plant Root Exudates as Determinant
of Rhizomicrobiome 4
V. Balasubramanian, Arunima Sur, Kush Kumar Nayak, and
Ravi Kant Singh
Abstract
The rhizosphere, a microecosystem, harbours numerous organisms such as bac-

teria, fungi, actinomycetes, and nematodes. The definite and diverse community
that can grow or survive in the rhizosphere is called rhizomicrobiome. The
community and activity of microorganisms could be enhanced and decided by
the respective plants by the special molecules called root exudates. The plant root
exudates facilitate molecular communication in and from root to root and root to
microorganisms. The exudates not only attract the microorganisms, but also
suppress the growth of unfavorable microbes. The plant exudates have been
facilitated to establish the relationship with the plant’s root system as mutualism
or antagonism or neutralism. These roots release molecules that play a vital role in
communication and maintain the microbial community in the rhizosphere. For
example, some molecules are also involved in bioremediation process by absorp-
tion of heavy metals. Overall, this chapter concentrates on transport and release of
plant root exudates into rhizosphere and how the exudates influence the specific
and diversified microbial growth and their adaptation in microecosystem of
rhizosphere.
Keywords
Bioremediation · Plant-microbes interaction · Rhizomicrobiome · Rhizosphere ·

Root exudates
V. Balasubramanian · A. Sur · K. K. Nayak · R. K. Singh (*)

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, India
e-mail: rksingh@rpr.amity.edu

23, https://doi.org/10.1007/978-981-15-9154-9_4
106 V. Balasubramanian et al.
4.1 Introduction
The root system is an important organ of the plants. Roots are responsible for
holding of the plant to the soil, water absorption and extract nutrients from the
soil, circulation of nutrients and water to stem, storage of food, vegetative reproduc-
tion, and competition with other plants and soil binding. Additionally, roots perform
special roles, including capability to produce, accumulate, and release various
chemical molecules into the rhizosphere (Flores et al. 1999; De-la-Peña et al.
2008). Those molecules are referred to as plant root exudates. The exudates attract
and repel the microbial communities as well as enhance or suppress the growth of
other plants near the accessible area (Estabrook and Yoder 1998; Bais et al. 2001;
De-la-Peña et al. 2010). About 5–21% of the photoproduced carbon transported to
root system is released as exudates (Marschner 2011). This is one of the ways to
maintain the carbon level in the soil ecosystem by the plant. Exuded molecules of
roots are majorly classified into low- and high-molecular-weight exudates. The
composition and complexity of the biochemicals have not been studied so far.
Amino acids, organic acids, phenolic and simple sugar compounds, and other
secondary metabolites are classified under low-molecular-weight exudates, whereas
proteins and polysaccharide mucilage are high-molecular-weight exudates.
Rhizosphere is a root-surrounding microecosystem formed under the influence of
root exudate chemicals. The communication occurs from root to root and root to
microbes with positive and negative interactions (Fig. 4.1). The stimulation of
environmental factors includes abiotic and biotic factors that regulate and control
the production of exudation of chemicals from the root system (Aulakh et al. 2001;
Fig. 4.1 Representation of positive and negative interaction from root to root and root to microbes
4 Plant Root Exudates as Determinant of Rhizomicrobiome 107
Chaparro et al. 2013; Van Dam and Bouwmeester 2016). For example, the chemical
composition of root exudates of Arabidopsis thaliana comprises 30 compounds of
primary (Chaparro et al. 2013; Badri et al. 2013) and 103 as secondary metabolites
(Strehmel et al. 2014). The exuded chemicals from the plant root have their specific
functions such as make low-level soil nutrients available to plants (phosphorous);
absorption and accumulation of heavy metals; and accommodating the supportive
indigenous microbial population, including rhizobial and endophytic bacteria or
mycorrhizal fungi surroundings of root (Jones et al. 2004; Bouwmeester et al.
2007; Badri and Vivanco 2008; Faure et al. 2009).
In addition, the influence of neighbouring plant species also determines the
chemical composition of exudates (Badri and Vivanco 2008) through direct or
indirect communication with other plant root system (Van Dam and Bouwmeester
2016). The production of exudating chemicals expression depends on the influence
of specific microbes and also root’s exuding chemicals varied plant to plant (Herz
et al. 2018) and their performance (Wilsone et al. 1999; Gubsch et al. 2011; Freschet
et al. 2015). The specific size of the leaf area is associated to growth rate of the plant
and biomass of the plant related to release of large amount of carbon in the form of
exudates (Lambers et al. 2008). In case of unavailability of nutrient, the biomass and
surface of the root system increased due to the transport of nutrients from the soil to
plants by absorption with the help of root exudate molecules. Silva Costa Moscôso
et al. (2018) reported that the root organic carbon molecules, physiological variables
of the plant and shoot dry matter are determined by the photosynthesis of the
irrigated rice cultivars. Rhizomicrobiome has horizontal relationship with plants
and is involved in balancing of hormones, defense system, and metabolism of plants
(Berendsen et al. 2012). The physiology and balance of hormones are performed
through controlled tuning of level of plant hormones by rhizomicrobiome
(Ravanbakhsh et al. 2018). The chemical communication of root and
rhizomicrobiome determines the structure and specific microbial population in
rhizosphere (Bulgarelli et al. 2012; Mendes et al. 2013; Bai et al. 2015). The exuded
root chemicals are involved in important defense processes against the plant patho-
genic fungi, oomycetes, bacteria, viruses, nematodes, and root-feeding arthropods
(Baetz and Martinoia 2014).
This chapter discusses the mechanism of transport and release of exudates from
root system, how the exudates assist and influence the specific rhizomicrobiome and
plant’s growth. The plant root exudates consist of various chemicals and how these
exudates are communicating with microorganisms in soil and the mechanism of
exudation chemicals from the root system into the soil.
4.2 Chemical Composition of Root Exudates
Root exudates contain a variety of components, viz. sugar molecules, amino acids,
phenolic compounds, etc. Sugar molecules include ribose, glucose, arabinose,
sucrose, fructose, raffinose, rhamnose, galactose, mannitol, pentose, xylose, etc.
Among these chemicals, glucose is the predominant molecule released into soil
(Toal et al. 2000). All common amino acids along with aminobutyric acid,
homoserine, L-hydroxyproline, and mugineic acid are present in exudates. Citric
acid, p-coumaric acid, ferulic acid, gallic acid, piscidic acid, protocatacheuic
acid, oxalic acid, tartaric acid acetic acid, L-aspartic acid, caffeic acid, chorismic
acid, L-glutamic acid, isocitric acid, p-hydroxybenzoic acid, malic acid, salicylic
acid, shikimic acid, sinapic acid and succinic acid are phenolic compounds and
organic acids are present in the root exudates (Seal et al. 2004; Rentz et al. 2005).
The growth of surrounding plants and soil microbial population are enhanced by the
chemicals, especially some of the phenolic compounds exuded from roots
(Steinkellner et al. 2007; Evidente et al. 2009). Various chemicals are released to
the soil through root system and provide the signals to attract or repel the diversified
population/community surrounding the root (Table 4.1).
4.3 Transport Mechanism of Exudate Chemicals Releasing

from Root System
The chemicals are exuded from root as primary and secondary metabolites into the
soil by various mechanisms. The primary metabolic molecules (PMM) are sugar
molecules, amino acids, and organic acids. Flavonoids, phenolic compounds, and
allomones are considered as secondary metabolic molecules (SMM). Both PMM and
SMM are released by different mechanisms.
4.3.1 Exudates-Releasing Mechanism from Root
4.3.1.1 Movement of Exudate Chemicals to Root Tips

Releasing of exudate chemicals from root is a complicated process that is initiated by
the movement of carbon (C) to roots and its release from roots to soil. The movement
of C from the organ in which it is synthesized occurs through phloem by the widely
recognized Munch’s pressure-driven mechanism of phloem flow (Münch 1876 B
1930). This mechanism involves movement of exudate chemicals by turgor pressure
created by concentration gradients between destination and produced organs
(De Schepper et al. 2013). The theory has been substantiated by the recent
experiments done by Knoblauch et al. (2016). Ross-Elliott et al. (2017) described
the process of exudate chemicals unloading to the root tips from the phloem. The
unloading processes happen via plasmodesmata in a combined way of diffusion and
mass flow observed in Arabidopsis. A specific “funnel plasmodesmata” is involved
in this process and the tissues are connected with protophloem through the proteins
and small-exudate chemicals solutes are passed into the phloem-pole pericycle
(Fig. 4.2). Large-exudate chemicals of proteins are released by batch unloading to
and blocked at phloem-pole pericycle, whereas small-exudate chemicals solutes pass
out of the phloem-pole pericycle without any restriction and are dispersed to adjacent
cells by diffusion with the help of connection of plasmodesmata (Ross-Elliott et al.
2017).
Table 4.1 Chemical composition of exudates released from root system

Types of
components Name of the compounds References
Carbohydrates Arabinose, glucose, galactose, fructose, Jones and Darrah (1995),
sucrose, pentose, rhamnose, raffinose, Lugtenberg et al. (1999), Toal
ribose, xylose, and mannitol et al. (2000)
Amino acids All amino acids with aminobutyric acid, Seal et al. (2004)
homoserine, L-hydroxyproline, & mugineic
acid
Organic acids Acetic acid, succinic acid, malic acid, Seal et al. (2004)
salicylic acid, shikimic acid, isocitric acid,
chorismic acid, sinapic acid, caffeic acid, p-
hydroxybenzoic acid
Phenolic Gallic acid, tartaric acid, ferulic acid, Rentz et al. (2005)
compounds protocatacheuic acid, p-coumaric acid,
mugineic acid, oxalic acid, citric acid, and
piscidic acid
Flavonols Naringenin, kaempferol, quercitin, Steinkellner et al. (2007),
myricetin, naringin, rutin, genistein, Evidente et al. (2009)
strigolactone, and their substitutes with
sugars
Derivatives of Catechol, benzoic acid, nicotinic acid, Badri and Vivanco (2008)
lignin phloroglucinol, cinnamic acid, gallic acid,
ferulic acid, syringic acid
Anthocyanins Cyanidin, delphinidin, pelargonidin, and Bais et al. (2006)
their substitutes with sugar molecules
Indole Indole-3-acetic acid, brassitin, sinalexin, Bais et al. (2006)
compounds brassilexin, methyl indole carboxylate,
camalexin glucoside
Fatty acids Linoleic acid, oleic acid, palmitic acid, Bais et al. (2006)
stearic acid
Sterols Campestrol, sitosterol, stigmasterol Bais et al. (2006)
Allomones Jugulone, sorgoleone, 5,7,40 -trihydroxy-30 , Bais et al. (2006)
50 -dimethoxyflavone, DIMBOA, DIBOA
Proteins and PR proteins, lectins, proteases, acid Narasimhan et al. (2003)
enzymes phosphatases, peroxidases, hydrolases,
lipase
4.3.1.2 Release of Exudate Chemicals from Root Tips to Soil

Exudate chemicals can be released easily by the symplastic pathway that helps to
release the exudates into soil, for which it must transport to apoplast via plasma
membrane. Gas and smaller-exudate chemicals (e.g., glycerol, urea) are freely
allowable by plasma membrane, whereas polar uncharged molecules such as glucose
and charged molecules (ions) cannot cross plasma membrane (Yang and Hinner
2015). The movement of these molecules has been explained by Sasse et al. (2018).
Special structured transmembrane proteins in lipid bilayer are predominantly utilized
for the crossing of charged and polar molecules through membrane without
disturbing the fatty acids of phospholipids. The specific and efflux carriers are
Fig. 4.2 Root structure and areas of root exudation. The upper figure represents the longitudinal
section of a root. Tissues are indicated in different colors for the different zones of the root (listed at
the bottom). The two circles focus on two distinct zones, a differentiated versus an undifferentiated
area, to show the presence of a Casparian strip and low abundance of plasmodesmata in the
differentiated area (left circle), and the presence of funnel plasmodesmata in the undifferentiated
area (right circle). The square represents a cross section close to the meristematic area where root
exudation is the highest. The lower figures represent a schematic representation of solute movement
sites from phloem unloading to the soil environment, either in the differentiation zone (a) or in the
undifferentiated root tip (b). (a) Solutes move through both the symplastic and apoplastic pathways,
but then they are reuptaken into the cytoplasm as the Casparian strip limits the apoplastic pathway.
Only the cortex and epidermis are responsible for the flux of metabolites into the apoplast and
consecutively into the soil (root exudation). Cortex and epidermis represent the major control point
for root exudation. (b) At the phloem-unloading site, both symplastic and apoplastic pathways are
used. Because of the lack of a Casparian strip, solutes can move out of the root (root exudation)
through both the apoplastic and the symplastic pathways. (Reproduced with permission from
Frontiers in Plant Science (Open Access), (Canarini et al. 2019))
used to transfer the amino acids, sugar, and organic acids, and these may be carried
out by the exudation flux fine tuning that happened via the up or down gene
regulation in gene expression or during the process of post-translational modification
(Badri et al. 2008). The PMM transports on efflux in membrane have been analyzed,
especially UMAMIT transporters (Okumoto and Pilot 2011; Moe 2013; Besnard
et al. 2016; Dinkeloo et al. 2018), GDU transporters (Pratelli et al. 2010), and CAT
transporters (Yang et al. 2010) are helpful to transport of amino acids; likewise,
SWEET transporter family (Williams et al. 2000; Chen et al. 2015; Manck-
Götzenberger and Requena 2016) for sugar molecules. Organic transporters such
as ALMT and MATE transporters are involved in the transport of malate and citrate,
respectively (Meyer et al. 2010; Mora-Macías et al. 2017).
These types of transporters are not energy-dependent H+ pumps (ATP) and
antiports (H+) transporters (Fig. 4.3), whereas MATE transporters for citrate required
H+-coupled antiport activity (Meyer et al. 2010) and ATP is utilized by
ATP-Binding Cassette (ABC) transporters for SMM transport (Badri et al. 2009).
Furthermore, PMM transporters are predominantly passed via transmembrane car-
rier proteins by either substrate-specific or gradient in concentration through high-to-
low concentration from intracellular to extracellular. Gene expression of SWEET
and UMAMIT transporter’s synthesis and regulation to facilitate the sugar and
amino acids into soil have not been explored so far. Moreover, not much information
about the gene expression is available and it depends on the edaphic and environ-
mental factors and nutrient demands of plants. Recently, Chen et al. (2015) have
reported that SWEET transcription was induced by pathogen to increase the efflux of
glucose into apoplast of root system. The malate exuded by ALMT in root was
enhanced by Al3+ toxicity or P deficiency in the plant (Ma et al. 2001; Kochian et al.
2005; Mora-Macías et al. 2017). Characterizing the efflux carriers involved in
exudation of prominent PMM into the soil will help to regulate the release of root
exudate chemicals and enhance the yield in crop plants (Sasse et al. 2018).
4.4 Plant-Plant Interactions via Exudate Chemicals Release
Release of exudate chemicals by root occurs in two ways—first includes secretion of

compounds with known function, whereas other involves secretion with unknown
function such as defense (Bais et al. 2004). In parched and semiparched
communities, both negative and positive relations among plants have been noticed
(Holzapfel and Mahall 1999). It can be antagonistic and synergistic (Fig. 4.3).
Negative plant-plant interactions are also called allelopathy and they are the exudate
chemicals mediated by plant-plant interference. By this process, plants secure
themselves over their competitors. These types of toxic compounds are termed as
allelochemicals (Muller 1969). Those plants, which are potent producers of
phytotoxins, can compete successfully by reducing growth, inception, or survival
of susceptible neighbours. However, their mode of action, nature of exudate
chemicals secreted varies from plant to plant species. It is clear from various
experiments that phytotoxins can interfere with metabolite production, hamper
photosynthesis, induce negative effect in respiration, and membrane transport in
susceptible plants (Einhellig 1995). Moreover, it is known that it interferes in
germination and cell mortality also.
Researchers have identified various types of compounds present in root exudates
such as Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and
2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) from wheat Triticum aestivum
Fig. 4.3 Summary of the main exudation mechanisms through the plasma membrane at the root
tip. The top panel represents active transport mechanisms, either primary active (direct consumption
of ATP) or secondary active (e.g., coupled to H+ pumps that actively consume ATP). The bottom
panel represents passive transport mechanisms, which allow diffusion following electrochemical
gradients. Red arrows represent movement of solutes against their electrochemical gradient, while
green arrows represent movement following their electrochemical gradient. On the right side of the
figure, examples of membrane transporters allowing movement of primary metabolites are
provided. (Reproduced with permission from Frontier in Plant Science (Open Access), (Canarini
et al. 2019))
(Wu et al. 2000), Juglone from Juglans nigra, black walnut (Jose and Gillespie
1998), and 8-hydroxyquinoline from Centaurea diffusa (Vivanco et al. 2004).
Studies revealed that some compounds share common factors like aromaticity,
common functional groups like presence of ketone group, whereas they differ also.
Plants secrete toxins at different concentrations, For example, Centaurea maculosa
and C. diffusa produce high concentrations of allelochemicals. In contrast to this,
Juglans nigra produces low level of phytotoxins (Pal Bais et al. 2002).
4.4.1 Factors Influencing the Secretion of Exudate Chemicals

for Plant-Plant Interaction
The release of root exudate chemicals showed positive or negative communication

between plant and rhizomicrobiomes, which depends on the type of microbial
population/community (Inderjit 2001; Wardle et al. 2007). Root-exuded chemicals
can either increase or decrease the availability of nutrients present in soil through
changing the biological processes in soil and its chemistry. The released root exudate
chemicals into rhizosphere restrict the nutrient availability during the plant’s com-
petition, although the effects on nearest plants through resources availability in soil
via liberation of exudate chemicals impact on properties of soil leads to negative or
positive effects. The availability of limited literature and studies on interaction
between plant and plant is a barrier for complete understanding of plant-root-
liberating chemicals. The characterization of metabolites and genes responsible for
the liberation of chemicals by root system especially for the interaction of plants is
important to control the growth of weed plants.
4.5 Interaction Between Plants and Microbes through Root

Exudate Chemicals
4.5.1 Root Exudates as Rhizomicrobiome Determinants
Soil is a heterogeneous, diverse, and sporadic system with poor nutrients and organic
sources (Nannipieri et al. 2003). It is made of both organic and inorganic layers
produced by concerted action of biotic and abiotic factors (Gobat et al. 2004).
Organic substances usually formed in soil are mainly of plant products. It is clear
that the plant remains are degraded by microorganisms by the process of biodegra-
dation and biodeterioration. As a matter of fact, more than 80% of reactions in soil
are mediated by microorganisms (Coleman et al. 2004). In agroecological systems,
microbes especially bacteria play a vital role in improving soil quality and, hence,
enhance the fertility. The microbial activity in soil is depending on the growth of
microbial population and root-exuded chemicals (Nannipieri et al. 2003). The
accessibility of ecological status is determined by pH, mineral composition, salinity,
nutrient availability, etc. Nowadays, researchers are studying about soil, plant, and
microbes as triangular interactions with different parameters, including soil condi-

tion and inter/intra interaction between plants and microbes.
Recently, plants have been acknowledged as meta-organism with well-defined
microbiome and a symbiotic relationship with microorganisms (Ho et al. 2017). The
plant and microorganisms interactions have been identified, but detailed mechanism
is still not clear. As plants are unmovable, they are showing more involvement to
establish relationship with rhizosphere microbes and by various mechanisms
through root exudates that regulates their interactions (Oldroyd 2013). In plants,
roots constantly manufacture, collect, and secrete broad range of signaling molecules
into soils (Jones and Dangl 2006). The portion of soil surrounding the root is called
rhizosphere, which is controlled by root. This zone is abundant in nutrients as
compared to bulk soil, as amassing of broad range of organic substances and
deposition of those substances occurs there (Ligaba et al. 2004). Researchers even
claimed that the specific types of microbial population/community in rhizosphere are
hundred times more as compared to bulk soil but with less diversity (Faure et al.
2009). It has been observed that plant species select particular rhizosphere microbial
communities and perform defined interactions (Ciccazzo et al. 2014). It is also
important to have aggressive colonization, which depends on microbes and rhizo-
sphere competence (Jjemba and Alexander 1999). Sometimes, change in microbial
community leads to feedback, which, in turn, can be of two types, positive and
negative feedback. The positive plant-soil-microbial interaction strengthens the
relationship of microbial community, whereas negative interaction leads to replace-
ment of plants and so initiates recolonization of microbes in the rhizosphere
(Ho et al. 2017).
Plant-associated microbes help plants to cope up with both biotic and abiotic
stress. Abiotic stresses are temperature, pH, salinity, etc. Biotic stress includes
interaction with pathogens, which leads to damage to the plant system. Microbes
help plants to resist unfavorable conditions such as heavy metals, drought, and
insufficient nutrients. The interactions sometimes become neutral to beneficial
depending on the type of organisms, viz. pathogenic or symbiotic involved (Glick
1995).
It is not surprising that microbial population and communities vary from species
to species of plants (Innes et al. 2004; Batten et al. 2006), even within the genotypes
of same species (Kowalchuk et al. 2006) and it also differs in stages of plant
development (Mougel et al. 2006; Xu et al. 2007). The total biomass of the plant
is measured with the plant population or improved organic total carbon and exudate
chemicals released into soil.
Mostly flavonoids molecules are phenylpropanoid derivatives that act as signal-
ing molecules and are largely involved in these relationships of specific plants with
microbes. About 4000 molecules of flavonoids have been analyzed as signaling
exudates from legumes plant system into soil to attract nitrogen-fixing bacteria to
make root nodules (Perret et al. 2000). Glycine max (Soybean) produce
iso-flavonoids such as genistein and daidzein that act as signaling molecules,
which actively induce Bradyrhizobium japonicum’s nod genes to infect the host
plants, whereas these molecules suppress the transcription of nod genes of
Sinorhizobium meliloti but needs luteolin for their nod genes activation for expres-
sion (Zhang et al. 2007). These specific signaling molecules are important to
differentiate the host legumes plants from others. Signaling molecules released
into soil assist the rhizobia to reach the host plant by chemotaxis (Bais et al.
2006). Rudrappa et al. (2008) have demonstrated that L-malic acid is an exudate
chemical released by root for the signal to rhizobacteria to make root infection for
nodule formation. Steinkellner et al. (2007) explained changing flavonoid level
influences the Fusarium infection with host plants. Mostly, the iso-flavonoids are
analyzed in the family of leguminous plants.
Zeng et al. (2003) have studied the inducing effect of Brassicaceae plants on
ectomycorrhizal fungal germination, which led to infecting host plant. Arabidopsis
thaliana and Medicago truncatula can maintain indigenous soil fungi, but cannot
maintain nonindigenous population of fungi in their rhizosphere. These actions are
highly accomplished and regulated by the exudate chemicals released by the root.
When these root exudate chemicals were added with specific fungal population
developed root system in vitro, the qualitative and quantitative results of both
in vitro and in vivo plants grown in soil were similar (Zhu et al. 2009). Plant-
associated microbes are beneficial as they increase the nutrient content, enhance and
stimulate root and shoot growth by producing hormones like indole acetic acid by
4,3,1-aminocyclopropane-1-carboxylate (ACC) deaminases (Glick 1995).
Organic acids and amino acids are also produced through root exudates. A study
in banana has revealed that organic acid from root exudates plays a major role in
PGPR colonization. It has also been identified those organic acids from banana root
exudates help in interaction with Bacillus amyloliquefaciens. Fumaric acid helps in
biofilm formation, whereas malic acid promotes chemotactic response in the banana
(Yuan et al. 2015). Rice root exudates have amino acids like histidine, proline,
glycine, etc. and contain carbohydrates like glucose, mannose, galactose, and
glucuronic acid. It promotes chemotactic response in endophytic bacteria Coryne-
bacterium flavescens and Bacillus pumilus (Chaturvedi et al. 2016). The amount of
sugar secretion might affect infection by plant pathogens. It has been proved that
Arabidopsis vacuolar sugar transporter SWEET2 actually protects plant from
Pythium infection. The vanillic acid and p-coumaric acid are the exudate chemicals
released by the root of cucumber into soil, which can alter the microbial population
in rhizosphere of cucumber (Chen et al. 2015).
Some of the exudate chemicals act as a bacterial mimic molecule (quorum-
signaling molecules) to alter the communication between bacteria in rhizosphere
(Gao et al. 2003). This is a kind of communication among the bacteria facilitated by
the small signaling molecules generally called as N-acyl homoserine lactone
(AHLS) in Gram-negative bacteria and peptide produced in Gram-positive bacteria
(Walker et al. 2003; Mathesius and Watt 2010). These exudate chemicals play a vital
role in controlling bacterial population density by regulating the genes. For example,
exudate chemicals of Pisum sativum (Pea) is an AHLS mimic molecule, which
increases the growth of specific bacteria strain and suppresses the growth of others
(Knee et al. 2001).
Strigolactones (SLs) are carotenoid-derived terpenoids, a modulating plant hor-

mone involved in plant development, and play a major role in the symbiotic
association with arbuscular mycorrhizal fungi (AMF). Furthermore, these molecules
facilitate the harmful interaction with some plant bacterial and fungal pathogens and
root parasitic plants (López-Ráez et al. 2017). Importantly, the researcher described
the regulation of SLs production that enhances the beneficial relation and suppresses
the harmful relationship with pathogens or root parasitic plant. Xie et al. (2015)
explained that SLs are moving within the plants from root-to-shoot communication
during the plant and microbe’s interactions.
More than eighty percent of terrestrial plants have association with the AMF (Van
Der Heijden and Sanders 2002). These beneficial association contribute to uptake of
nutrients by the plants and a few indirect advantageous relationships such as
increasing plant immune system against herbivores and pathogens in some of the
plants (Bennett et al. 2006; Pozo and Azcón-Aguilar 2007; Vannette and Rasmann
2012; Cameron et al. 2013). As an obligate fungus, AMF’s survivability depends on
their capability to quickly establish synergetic association with the root of plants.
The exuded compounds from the plant roots before the infection are important for
germination of AMF spores and extension of hyphal growth in rhizosphere soil to
infect the root (Giovannetti et al. 1996). Flavonoids released to rhizosphere by root
make significant change in the AMF to convert nonsymbiotic to presymbiotic form,
i.e., it supports AMF spore germination and hyphal development for the infection of
plant root hairs (Besserer et al. 2006). SLs have been identified to induce the
proliferation of Gigaspora rosea and help in germination of spores in Glomus
claroideum and G. intraradices and these roots exudates enhance the branching of
hyphae in G. margarita (Akiyama et al. 2005).
4.5.2 Root Exudates as Defense Chemicals against Soil-Borne

Pathogens
Rhizomicrobiome is associated with the root tip that produces mucilage and border
cells, which play central role in interaction between plant and microbes (DeAngelis
et al. 2009). These border cells are released into rhizosphere as single cells or as
border-like cells (remain together to each other cells). These processes happen
depending on the organization of root meristematic cells. The release of higher
amount of border cells occurs by the pathogen attack and stimulates the mucilage
production by root tips and border cells. Mucilage consists of proteins with the
function of antimicrobial activity; also DNase released extracellular defense against
certain bacteria and fungi (Watson et al. 2015; Martha et al. 2016). The survival of
the border cells depends on the number of cells released from root tip cells; for
example, the single-border cell is able to live in rhizosphere for a month with starch
in soil, whereas border-like cells survive 2 weeks only and are produced by
Arabidopsis.
Root exudate chemicals are involved in plant defense system against the plant
pathogens (Neal et al. 2012). Proteins released through roots have the potent
function to recognize the nonpathogenic and pathogenic bacteria (Wen et al. 2007;
De-la-Peña et al. 2008). For example, lectins function as defense factors and help to
establish the mutual relationship (De Hoff et al. 2009). Defense enzymes such as
chitinase, glycosyl hydrolase family 17 and 18, and peroxidases are released by the
A. thaliana against Pseudomonas syringae (De-la-Peña et al. 2008). With the help of
rhizomicrobiome, it is now clear that defense mechanism of plants is elucidated and
it reduces plant pathogenic infection even after the attack of pathogens, which
induces systemic resistance in plants (van Loon et al. 1998). Another aspect of
interaction is production of secondary metabolites like antibiotics and antimicrobial
peptides. The low-molecular-weight antibiotic secreted has a negative effect on plant
pathogens. For example, antibiotics such as phenazines, DAPG, and pyrrolnitrin
have proved a boon for controlling plant diseases in crop plants (Raaijmakers et al.
2002).
Chemicals of root exudates contribute a lot in belowground plant defense.
Phytoanticipins and phytoalexins are two important antimicrobial chemicals.
Phytoalexins are antimicrobial compounds synthesized by plants against the micro-
bial infection and are also called as plant antibiotics. Phytoalexins inhibit the growth
of microbes and they are synthesized by de novo anabolic pathways after elicitation
of pathogen’s infection. Therefore, the pathogens induction is required for the
production of phytoalexins by inducing the transcription and translational activities.
The mechanism of induced response also includes the production and transfer of
antimicrobial compounds to the site of infection (Grayer and Kokubun 2001).
Phytoanticipins are compounds, which are defensive in nature. They are pro-
duced by root hair just before plant encounters biotic stress. Van Etten et al. (1994)
first coined the term phytoanticipins as low-molecular-weight chemical that inhibits
the growth of plant pathogens. Phytoanticipins are present in plant cells before and
after the microbial infection. These antimicrobial compounds are sometimes found
in surface of the plants and some are impounded in vacuole as efficient compounds
or organelles. It is released by the hydrolyzing enzymes after the microbial infection
in plants. Therefore, the pre-existing phytoanticipins are liberated by enzyme action
and are not synthesized by de novo pathways like phytoalexins (Osbourn 1996).
The plant defense system produces chemicals and releases into soil as exudates to
control the plant pathogens. Lanoue et al. (2010) have demonstrated that Hordeum
vulgare (Barley) root releases chemicals, which contain five phenylpropanoids
potent to inhibit F. graminearum (soil-borne fungal pathogen). Among them, de
novo synthesis and t-cinnamic acid secretion were remarkably noted by labeling
experiment. The seedlings of Oryza sativa (rice) biosynthesize momilacton A and
diterpene derivate released through root system as antimicrobial compounds
(Toyomasu et al. 2008; Kato-Noguchi et al. 2008). Moreover, this compound is
accumulated in leaf and inhibited the growth of blast fungi in rice leaves (Hasegawa
et al. 2010). Hairy root culture of Coleus blumei released antimicrobial compound
rosmarinic acid via root against β-cryptogein during in vitro pathogen mimic
experiment. In this experiment, β-cryptogein acts as an elicitor, which is normally
produced by oomycetes during plant infection (Bais et al. 2002; Vuković et al.
2013). The same kind of in vitro studies have been performed on Arabidopsis (Badri
et al. 2008) and hairy roots of Catharanthus roseus (Ruiz-May et al. 2009), which
induces the gene transcription for the synthesis of defense molecules, viz. salicylic
acid (SA), nitric oxide (NO), and methyl jasmonate (MeJA) that are released into
medium by root (Badri and Vivanco 2008).
Benzoxazinoids are released by the Zea mays (maize) as a defense chemical at the
time of emergency condition via crown and lateral roots specially to inhibit the
pathogen growth on site at which temporarily more vulnerable or in developmental
stages (flowering) where the chance of pathogenic infection is high (De-la-Peña et al.
2010). This is a kind of precautionary process in which the antimicrobial compound
is synthesized and secreted through root without the stimulation of pathogens
(Steeghs et al. 2004; Woong et al. 2004). Phytoalexin (camalexin, indole
glucosinolates, and salicylic acid) produced by Arabidopsis as root exudates
imparted resistance against Phytophthora capsici (Wang et al. 2013), and the
phytoalexin also controlled P. cinnamomic (Rookes et al. 2008). Pisum sativum
(pea) roots secrete pisatin, an isoflavonoids derivative, which is a potent antimicro-
bial compound of legumes (Wu and VanEtten 2004; Weisskopf et al. 2006). Pisatin
is released by specific tissue root tip by the induction of pathogens (Cannesan et al.
2011).
Tryptophan-derived secondary metabolites such as indole derivative camalexin
or glucosinolates are another potent antioomycete exudate released via root in soil by
Arabidopsis (Schreiner et al. 2011; Wang et al. 2013). Some of the molecules
involved in biosynthesis of camalexin have been recognized at the gene level. The
fungal pathogen infection leads to release of camalexin by roots of Arabidopsis. The
fungal infection determined the required quantity of production and accumulation of
camalexin (Bednarek et al. 2005; Iven et al. 2012).
4.5.2.1 Root Exudates as Volatile Compounds

Largely, researchers have discovered that the volatile compounds are responsible to
repel the insect pests, but recent research showed evidence that the root also released
the volatile compounds against plant pathogens (Matsui et al. 2006). The derivatives
of terpenoids are the chemicals involved in below- and above-ground defense
system of plants and exuded as root chemicals (Toyomasu et al. 2008; Kato-Noguchi
et al. 2008; Schmelz et al. 2011; Vaughan et al. 2013). It has been believed that as
nonvolatile terpenoids alone are involved in defense and released into soil
(Toyomasu et al. 2008; Kato-Noguchi et al. 2008), though the recent studies
revealed that the released volatile compounds in soil participated in defense directly.
These volatile chemicals were previously known as attractant for indirect defense of
plants against natural enemies (Rasmann et al. 2005; Hiltpold et al. 2011; Robert
et al. 2012; Hiltpold and Turlings 2012). The volatile chemical, monoterpene
1,8-cineole, is released by hairy root of Arabidopsis as defense compound f during
the pathogen interactions (Steeghs et al. 2004; Chen et al. 2004). Interestingly,
in vivo experiment demonstrated that the GR24 is a synthetic analog of strigolactone
that controls phytopathogenic fungi released into growing medium. This compound
is exuded directly into soil (Dor et al. 2011) or indirectly attacks the natural enemies
by defense pathways of hormone modification (Torres-Vera et al. 2014).
4.6 Conclusion and Future Perspective
In the context of modern agricultural biotechnology, rhizomicrobiome has been

recognized to play an important role in agricultural ecosystems because it is respon-
sible for healthy soils, which support better plant health and good crop yields (Zorner
et al. 2018). Rhizomicrobiome-based studies may overcome the problem of exces-
sive uses of pesticides and chemical fertilizers in near future without compromising
food quality and crop yield. Variation in population of rhizomicrobiome is found to
be diverse in geographic and physiochemical environments of soil (Khan et al.
2020). Thus, there is a need of comprehensive studies for collection of data related
to soil microbial population, which would help us to develop relationship pattern
between soil and microbial-crop ecology. In the near future, such studies would open
an opportunity to develop predictive tools through which researchers can manage
various applications of microbial ecology in agriculture to improve the crop yield
and productivity. In addition, information of genes, which are responsible for the
expression of signaling molecules or control quorum-sensing phenomenon, plays
crucial role in the development of rhizosphere microenvironment that includes
signaling from root to root, root to microbes, and vice versa. There is little informa-
tion about the regulation of about those genes involved in signaling and quorum-
sensing activity. Thus, it becomes very important to investigate the factors involved
in down- and upregulation of these genes. These kinds of studies would enable us to
control the growth of microorganisms in rhizosphere and help in the improvement of
crop health as well as its yield.
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Rhizospheric Microbial Community:
Ecology, Methods, and Functions 5
Amir Khan, Manisha Joshi, and Ajay Veer Singh
Abstract
Soil is a primary natural resource that maintains ecosystem functioning, water

balance, and supports plant growth. Soil is also a habitat for diverse microflora
that is very crucial to soil sustaining activities. Microbial ecology is the study of
microbial community dynamics and its functioning in the environmental system.
Diversity in any ecological niche is the major descriptor for community structure
which decides the dynamics and functioning of a community. Conventionally,
microbiome characterization is based on culture-dependent techniques, but due to
the insufficiency of appropriate culture media most of the microorganisms are
unable to grow; therefore, for microbial community analysis culture-dependent
techniques have proven less appropriate. However, over the last few decades,
culture-independent techniques are being practiced to assess microbial
communities and these approaches appear more satisfactory because they are
more advanced and able to determine almost all genomes acclimatized in a
variety of environmental samples. Moreover, microbes present in ecological
niche participate in the development and maintenance of multiple functioning
of ecosystems which include pedosphere development, litter decomposition,
nutrient cycling, climate regulation, plant growth promotion, and sustainability
maintenance. Therefore, in the present era, the characterization of microbiome is
very important. This review provides a wider understanding of the functioning of
microbial communities and methods of their assessment in the soil.
Keywords
Microbial diversity · Microbial function · Rhizosphere · Metaomics · PGPR
A. Khan · M. Joshi · A. V. Singh (*)

Department of Microbiology, College of Basic Sciences and Humanities, GBPUAT, Pantnagar,
Uttarakhnad, India

23, https://doi.org/10.1007/978-981-15-9154-9_5
128 A. Khan et al.
5.1 Introduction
Current agriculture system is focusing on sustainable agricultural techniques with a

view to ensure high-quality yield and environmental health. Therefore, scientists
aspire to get insight into the microbial community that could benefit the agricultural
system. The uppermost layer of the pedosphere is rich in microbial diversity such as
bacteria, fungi, and actinomycetes. This immense microbial diversity has vital
importance in the functioning of the agro-ecosystem, pedosphere development,
nutrient mobilization, and plant growth promotion. Microbes acquire their nutrition
from the plants and in return solubilize nutrients and shield them from pathogens,
thereby improving their growth and productivity. Microbes also contribute to the
cycling of nutrients, organic matter deposition, and maintaining soil structure. Soil
microflora changes with changes in the environmental and soil physiological
conditions. Therefore, alterations in the microbial communities in response to soil
characteristics and environmental fluctuations and their functioning in all ecological
conditions have engendered an interest toward the assessment of soil microbial
community index. Besides the plant diversity, characterization of soil microbial
diversity and assessment of their functioning is a new approach. But this approach
is riddled with several problems, the major problem being the inability to assess all
the microbial diversity by classical microbial approaches owing to 99% of microbes
being uncultivable due to failure to provide appropriate culture mediums/conditions.
However, in the previous decades, various culture-independent approaches have
attracted and provided a better understanding of the microbial community of soil.
Constant improvements in molecular techniques have opened the doors for identifi-
cation of the whole structure of microbial community and understanding the linkages
among diversity. Besides, sequencing techniques have also contributed to advance-
ment in microbial diversity analysis. The present manuscript focuses attention on
microbial diversity present in the soil, methods of their assessment, their fluctuations
with various factors, and their functional capacity in promoting plant growth and
maintaining ecological functions.
5.2 Microbial Communities in Rhizosphere
In the region below ground, plants and microbes can have a mutualistic relationship,
i.e.. benefit each other. Microbes take nutrients from plants for their metabolic
activities and in turn assist plants in their growth by various activities such as nutrient
mobilization, soil development, and plant protection. Microbial community present
in the rhizosphere region can be classified as fungi, bacteria, actinomycetes,
protozoa, and nematodes. Among these classes, the bacterial and fungal classes
are most studied; also the rhizosphere effect (R/S ratio) for bacterial population is
higher in comparison to other microflora present in soil.
5 Rhizospheric Microbial Community: Ecology, Methods, and Functions 129
5.2.1 Bacteria
Bacteria are very small and advanced biological entities that can adapt in any kind of
surrounding conditions. As per Olanrewaju et al. (2019), the structure of rhizosphere
bacterial community is dependent on root exudates of distinct plant species which
vary with plant age, soil type, root system, and location. Rhizosphere inherits a wide
range of bacterial diversity which promotes plant growth and favors its development.
Collectively, the bacteria which accelerate plant growth and support its development
are called Plant Growth Promoting Rhizobacteria (PGPR). A study conducted on
legume rhizosphere to characterize different bacterial communities present revealed
that the prominent rhizobial microflora consists of Bacillus, Pseudomonas, Coryne-
bacterium, Alcaligenes, and Serratia (Rawat and Mushtaq 2015). Apart from these
genera, the most prominent bacterial population in rhizosphere region includes
species of Rhizobium, Mycobacterium, Azospirillum, Acinetobacteria, Azotobacter,
Agrobacterium, etc. Since these plant growth promoting rhizobacteria are also
capable of eliciting bio-control and inducing growth promoting activities in plants
(Fig. 5.1), they can be exploited as a tool to overcome food security and can aid in
maintaining productive agricultural sustainability to fulfill increasing food demand
(Kumar et al. 2017).
Microbial diversity based on location, functional role, and plant growth promot-
ing activities forms the basis of classification of PGPR. Based on their location, it is
of two types, i.e., intracellular plant growth promoting rhizobacteria (iPGPR) and
extracellular plant growth promoting rhizobacteria (ePGPR). The ePGPRs are most
prominently found in the rhizosphere or are in proximity to root cortex, but not
inside the cell (Fig 5.1). Depending on the distance between them and the root
cortex, the ePGPRs are further subdivided into three divisions, i.e., root surface
colonizers, ones living in close proximity, but not in contact with the root cortex, and
the ones occupying the intercellular space of root cortex. Bacillus and Pseudomonas
species usually reside in cortical intercellular spaces and in soil. The iPGPR can
reach inside the plant cells and cause nodule formation. Frankia and Rhizobium are
prominent iPGPRs that aid in nitrogen fixation. Apart from conferring resistance
against biotic factors, these bacteria also elicit stress tolerance in plants. Yang et al.
(2008) reported that Arabidopsis thaliana was more tolerant to drought on treatment
with Paenibacillus polymyxa. Similar results were obtained for tomato (Sola-
num copersicum L.) and pepper (Capsicum annuum L.) when these crops were
treated with Achromobacter piechaudii ARV8. These strains increased plant height,
shoot dry weight, and improved nodule number in drought stress conditions,
indicating synergistic effects. During exposure to elevated temperature, Pseudomo-
nas aeruginosa strain AMK-P6 showed induction of Heat Shock Proteins (HSP);
likewise during low temperature conditions, i.e., cold stress conditions, bacteria
responded by inducing production of cryoprotectant proteins. Genus Pseudomonas
is well documented for its growth promoting activities in plants during water stress
conditions (Bilinski et al. 2019).
Based on functional role, PGPRs are categorized into bioprotectants
(biopesticides and biocontrol agents), biofertilizers, phytostimulators, and
130
Fig. 5.1 Microbial communities associated with plant rhizosphere

A. Khan et al.
rhizoremediators. Bacillus species (e.g., B. subtilis, B. vallismortis,

B. amyloliquefaciens, B. cereus, etc.), Streptomyces species, and Pseudomonas
species (e.g., P. aureofaciens, P. fluorescens, P. cepacia, and P. aeruginosa) are
used as biocontrol agents (BCAs) owing to the significant role played by them in
controlling plant pathogens by production of antibiotic compounds such as
pyrrolnitrin, phenazine, hydrogen cyanide, and 2,4- diacetylphloroglucinol. They
also retard pathogenic bacterial growth by the mechanism of quorum quenching.
Bacillus species produce an enzyme N-acetyl homoserine lactonase which
degrades N-acetyl homoserine lactone, which acts as an important signaling mole-
cule that aids in cell-cell communication. Pseudomonas spp. are potent iron
scavengers; they inhibit pathogenic bacterial growth by competing them for iron
availability. For example, substances produced by Pseudomonas such as
pyoverdines have high binding affinity for iron in contrast to other bacterial micro-
flora present in rhizosphere. Hence, they compete with pathogenic bacteria for iron
availability and provide protection against diseases by suppressing their growth. The
PGPR can trigger plant growth through indirect or direct means during stress
conditions. Indirect means include their biocontrol action by virtue of which they
provide plants protection from pathogens and, hence, confer disease resistance.
Whereas direct means are liberation of stress phytohormones such as abscisic acid
(Cohen et al. 2015), jasmonic acid (George et al. 2016), and salicylic acid (Beneduzi
et al. 2012) and stress signaling enzymes like 1-aminocyclopropane-1-carboxylate
(ACC) deaminase (Mahmood et al. 2016a, b).
5.2.2 Fungi
Fungal diversity is prevalent in all type of soil. It can be determined through

monitoring the presence of fruiting bodies in the environment. Diversity in fungi
varies from macro-fungi (mushrooms) to microscopic fungi (unicellular yeasts). Due
to limited isolation techniques, it is very complicated to determine the absolute
number of fungal species in soil. Hawksworth and Lücking (2017) estimated the
presence of 2.8–3 million fungal species in the soil but the exact figure of species still
remains unfolded. The most characteristic genera present in soil are Penicillium,
Fusarium, Cephalosporium, Mucor, Aspergillus, Trichoderma, Alternaria, etc.
Rhizosphere inherits both pathogenic and symbiotic fungi. Arbuscular mycorrhi-
zal fungi (AMF) belonging to order Glomerales live in symbiotic association with
roots of plants. This symbiotic association can be seen in terrestrial plants in
agricultural as well as natural ecosystem, encompassing hydrophytes, halophytes,
xerophytes, ferns, angiosperms, and gymnosperms. AMF are also widely exploited
for its role in bioremediation and biofertilization. Their positive impact is enhanced
by the presence of some specific interactions (microbe-microbe). The study by Vivas
et al. (2003) confirmed the positive impact of microbe-microbe interaction through
inoculation of AMF along with Bacillus sp. which enhanced mycorrhizal activity in
nutrient deficient soils. These symbiotic fungi improve plant diversity, productivity,
and biomass by providing more abiotic stress tolerance (salinity or drought
132 A. Khan et al.
tolerance) and nutrient acquisition. Additionally, they also enhance soil quality by
heavy metal immobilization and modulate rhizo-deposits, thus affecting microbial
activity (Lenoir et al. 2016). An iron containing protein secreted by arbuscular
mycorrhizal fungi, “glomalin” determines its advantageous ability (Vlcek and
Pohanka 2020). This iron containing protein also takes part in scavenging elements
hazardous to plant health and in turn increases chances of survival in polluted soils.
Apart from playing a role in toxic element sequestration, glomalin enhances soil
penetration by air and water by stabilizing soil aggregates, thus also conferring
erosion resistance. Biocontrol activities of these fungi have also been reported.
They confer protection to plants against biotic stress such as infection caused by
parasitic nematodes. They suppress nematodes in regard to nutrients and space and
bring out induced systemic resistance (ISR) in plants during pathogen attack
(Schouteden et al. 2015). Arbuscular mycorrhizal fungi positively influence plant
invasion and facilitate seedling establishment. Studies indicate that co-inoculation of
different arbuscular mycorrhizal species helps to increase plant-plant facilitation.
This facilitation is ecologically significant as it helps establishment of woody plants
on semiarid regions (Montesinos-Navarro et al. 2012). Plants share common mycor-
rhizal network among them. In such cases, arbuscular mycorrhizal acts as a mediator
to partition and transfer phosphorus, nitrogen, water, and carbon from resource-
abundant plants (source) to resource-scarce plants (sink) (Walder et al. 2016). This
translocation aids plant growth by providing resources during stress conditions and
speedy recovery from water crises (Babikova et al. 2013). Glomus and Rhizophagus
irregularis species are most widely studied in natural ecosystems (Tisserant et al.
2013).
Fungal biocontrol agent Trichoderma spp. has significantly contributed to plant
protection. It competes with plant pathogens through several mechanisms, namely,
induced resistance, competition, parasitism, and antibiosis (Zhang et al. 2016). Other
species of Trichoderma have also been reported to enhance plant growth by produc-
ing growth factors and aiding in nutrients uptake by solubilizing them (Li et al.
2015).
5.2.3 Others
Bacterial and fungal population covers a major biomass in rhizobiome; other

organisms residing in rhizobiome are protozoa, oomycetes, and nematodes.
Nematodes are eukaryotic, free living worms mainly known for their pathogenic
action in plants. However, they also exhibit biocontrol properties by suppressing the
growth of additional pathogens present on rhizosphere (Kenney and Eleftherianos
2016). Nematodes belonging to genera Heterorhabditis and Steinernemaanong
restrict the growth of many pests and pathogenic insects in rhizosphere and, hence,
are potent biocontrol agents. These entomopathogenic nematodes (EPN) are
employed in agriculture for pest management. Root exudates such as secondary
metabolites attract the nematodes and also act as selection criteria for EPNs over
other pathogenic nematodes (Hiltpold et al. 2014). Several reports indicate that
commercially produced EPNs are used as potent biocontrol agents against insect
herbivory present at plants (Lacey and Georgis 2012).
The other organism residing in rhizobiome besides nematodes is protozoa. It is a
unicellular organism dependent on microbes for food supply. Protozoa enhance
organic matter release and supply ample amount of nitrogen to plants who otherwise
have limited to access nitrogen. The grazing activities of protozoa also aid in fast
translocation of photosynthates from source to roots. Meanwhile, they also build up
a connection with arbuscular mycorrhiza and enhance nutrient supply (Koller et al.
2013).
5.3 Factors Affecting Microbial Communities
The ongoing research indicates that the biochemical and ecological processes in
rhizosphere region are interceded by interactions taking place between soil
microorganisms and plants, which in turn also affect the microenvironment biologi-
cally, chemically, and physically. For the preservation of biodiversity and ecological
sustainability, the contribution of rhizobiome and plants is significant in bio-
geochemical transformation, nutrient cycling, biomass turnover, and energy utiliza-
tion during photosynthesis in terrestrial ecosystem (Weyens et al. 2015). At root-soil
interface and plant roots, microbial colonization is facilitated by various root
exudates (Voges et al. 2019). An estimate about the total genome of microbial
communities at rhizosphere indicates that it is greatly huge than the genome of
the plant itself, thereby also called the plant’s second genome (Berendsen et al.
2012).
Colonization of root vicinity by microbes triggers rhizosphere processes that
involve various biological and physico-chemical turnovers such as uptake of water
and minerals by the host plant, release of gases such as CO2, and production of
various chemical compounds (Philippot et al. 2013). The biological and physico-
chemical processes, along with various biotic and abiotic factors, affect the popula-
tion and metabolic activity of these microorganisms residing in rhizosphere. Many
soil microorganisms remain in dormant state in conditions of carbon(C) limitation in
normal. Good growth of plants results in an increment of soil organic matter by 20-
to 30-fold around the area of root secretions. This hike in organic input boosts
microbial metabolism and activity which in turn enhances the turnover of soil
organic matter (Xiaoping et al. 2019). Growing roots usually support fast growers
such as bacteria, while mature roots aid slow growers like actinomycetes and fungi.
Mature roots secrete less quantity of cell lysate and mucilage due to a lack of
emerging lateral roots and border cells. During transpiration, inward salt and mineral
movement and outward diffusion lead to generation of chemical gradient near roots
and create a diverse range of microbial habitats. Compounds with low molecular
weight like sugars, organic acids, amino acids, and flavonoids are easily utilized by
microbes and their availability plays a major role in maintaining the dynamics of
microbial community in soil.
134 A. Khan et al.
5.3.1 Biotic Factors
Plant genetic makeup plays a major role in the selection of rhizosphere microbial
flora because the genetic and physiological control of plant decides the amount and
variety of compounds secreted as exudates by roots. Studies on horticultural crop
plants such as Medicago truncatula (Kisiel and Kępczyńska 2016), Arabidopsis
thaliana (Bulgarelli et al. 2012, 2013) and non-cultivated plant species like
arbuscular mycorrhizal association demonstrate that different plant species grown
in same soil have variable root biome. In nutshell, these studies indicate that the
quality and quantity of secreted root exudates by plant root affect microbial popula-
tion in the soil.
Root structure of plants also affects oxygen pressure and availability of nitrogen
and carbon which influence various transformation processes in soil by inhabiting
microorganisms. This may also result in a shift of microbial community as the
microbial population varies according to sources and availability of carbon and
nitrogen. Additionally, various growth stages of plant and roots alter physical and
chemical properties of the soil such as organic content, mineral content, salinity
levels, water potential, and pH. The plant genotype influences rhizosphere microbial
community which in turn has an effect on plant health, i.e., its growth and survival.
Hence, it is likely that co-evolution exists between plants and microbes. The root-
microbial associations can be symbiotic or non-symbiotic. Symbiotic associations or
symbiosis is a highly evolved interaction, while the non-symbiotic association is less
specific. However, the extent of specificity and co-evolution still remains
unanswered.
The symbiotic associations are more prone to evolve host specificity. The legume
rhizobia interactions are bound by range restrictions. Single rhizobium species is
constrained to single specific plant genus or group of genera, in contrast to the
condition that a single plant may host many microbial strains of identical species.
The non-symbiotic association can be plant genotype-specific or plant species-
specific. The variations in rhizosphere microbial communities at various develop-
mental stages of plants have not been much widely studied. However, alteration in
microbial community structure due to the shift from seedling, mature plant to the
senescence stage has been well studied.
5.3.2 Abiotic Factors
The role of microbes in nutrient recycling in soil ecosystem is well known. This
versatile microbial population serves plants in various forms, but it has to cope up
with many types of abiotic factors that affect them adversely. Temperature, soil pH,
soil topology, soil texture, humidity, use of insecticides, and pesticides are some of
the factors which influence microbial community structure, their growth, and their
physiology. Studies on the climate change effects on soil environment also con-
firmed their influence on soil environment including soil microbiome through
indirect and direct pathways. Microbial community structure varies depending on
temperature range. Therefore, an increased seasonal temperature affects microbial

growth and physiological activities in soil. For example, experimental warming in
grassland soils depicts increase in bacterial biomass during spring and winter (Belay-
Tedla et al. 2009). Furthermore, it has also been observed that bacterial biomass is
negatively affected by warm weather during summer and early rain fall (Liu et al.
2009), suggesting that warming situations may have seasonal effect on microbial
community of soils. The prolonged exposure to increased temperature may result in
water stress conditions, which in turn affect both plant and microbial community by
influencing plant-microbe interaction and major nutrient cycles. Studies focusing on
precipitation manipulation indicate that rainfall had little impact on grassland micro-
bial community (drought conditions being an exception). Additionally, under labo-
ratory conditions, it was found that rainfall frequency had no change in composition
of microbial community. The soil health is also another key factor which also
influences its microflora. The deficiency or abundance of minerals and organic
acids influences soil microbial population. The activity and growth of
microorganisms are markers of soil health because they are sensitive toward the
changes in temperature, nutrition, texture, pH, soil water content, etc. Moreover,
microbial biomass and microbial diversity are often used as important parameters for
soil quality diagnosis. A study to demonstrate the influence of soil texture on soil
microbial populations demonstrated that clay-loam and silty-clay-loam soils harbor
the highest bacterial population, while the lowest level of bacterial population was
detected in sandy-loam and silt-loam soils. The study also showed that soil texture
had no significant effect on fungal population. This difference in harboring bacterial
population among varied soils (in texture) may be because of nutrient and water
holding capacity of soils (Kaviya et al. 2019). Sandy soil has the least water holding
capacity, while clay loam has th highest water holding capacity; hence it can hold
water and nutrients for a longer time and house a diverse variety of microbes
(Hamarashid et al. 2010). Besides, soil pH is also one of the critical parameters for
determining soil microbial community. For instance, a study performed by Silva-
Sánchez et al. (2019) stated that increased pH values caused decrease in fungal
growth in stark contrast to increment in bacterial growth. Continuous utilization of
pesticides and insecticides such as endosulfan and profenofosmake them persistent
in soil for prolonged time. They are used worldwide (use of endosulfan is currently
banned) for control of various insects or pests in several crops. The bioaccumulation
of these insecticides affects soil enzymes like soil dehydrogenase which is produced
mostly by all kinds of soil bacteria and hence negatively influences microbial
population dynamics (Nasreen et al. 2015).
5.4 Methods of Studying Microbial Ecology in Rhizosphere
Rhizosphere looks like a microbial cloud around the root, where vast diversity and
an infinite number of microorganisms (Bacteria, Fungi, and others) reside. About
1011 cells and 30,000 species of microorganisms dwell in a gram of soil (Berendsen
et al. 2012). It is very difficult to isolate and identify each bacterium from this huge
136 A. Khan et al.
microbial diversity present in tiny amount of soil. Currently practicing conventional

techniques are generally less efficient to identify whole microbial community of
interest at one time. Therefore, understanding about the number and diversity needs
advanced, broad-range, specific, and appropriate tools to cover whole diversity.
Some of the determination methods are described in this section.
5.4.1 Traditional Approaches
Enumeration and determination of microbial community residing in the rhizosphere

have been in practice since many decades. Culture-dependent techniques used
initially rely upon the isolation, culturing, and identification of microorganisms.
They are time-consuming, expensive, laborious techniques and have a narrow range.
These techniques include collection of rhizospheric soil samples followed by dilu-
tion and pour plating of diluted sample to grow the cultures. Subsequently, single
colony is isolated to develop pure culture. Afterward, various traditional culture-
dependent techniques such as plate count, carbon source utilization profiling, fluo-
rescence microscopy, and fatty acid methyl ester (FAME) analysis are used to
estimate and identify the microbial communities in the rhizosphere. There are a
specific set of identification protocols developed based on microscopic, biochemical,
and other tools which identify the signature behavior of a specific microbial class.
5.4.1.1 Plate Count Analysis

Plate count is a very common method used for the study of microbial communities in
the rhizosphere. The procedure includes dilution of 1 gm of rhizosphere soil,
followed by pouring on to different media and incubating the plates in order to let
the bacterial colony grow. Each colony stands for one colony forming unit. Subse-
quently the colonies are counted to estimate the microbial population in the sample
in terms of colony forming units per gram of soil. A large number of media are
available to grow the microorganisms according to their physiological and
nutritional requirements. But this technique is applicable for only laboratory-
cultivable organisms. Moreover, it focuses only on the study of microorganisms of
a small fraction of soil sample.
5.4.1.2 Carbon Source Utilization Profile

Carbon source utilization profile technique is also referred to as community-level
physiological profiling or biochemical identification system (API or Biolog) (Gar-
land and Mills 1991). This microbial identification system is based on the patterns of
carbon source utilization by the microbes. Community Level Physiological Profiles
(CLPP) is a broad term covering various studies undertaken using a number of
assays. However, currently the term CLPP is used exclusively to refer to the data
collected using BIOLOG microplates. In this method, the samples are directly
inoculated into 96 Biolog micro plates, where each well is supplied with a different
C source and a dye tetrazolium (Redox dye). On inoculation of sample in the well,
NADH is produced as a result of cell respiration. It leads to reduction of tetrazolium
dye to formazan, as a result of which a color change occurs within each well which is
detected through photometer. This approach enables intensive sampling across
temporal and spatial scales, owing to low manpower requirements. However, it
requires incubation of 2–7 days for color response in individual wells.
5.4.2 Molecular Approaches
The traditional methods are used to study only the culturable microorganisms.
However, a major fraction of the soil microorganisms are non-culturable, which
makes it difficult to interpret the overall community of the soil. The molecular-based
techniques for studying rhizospheric microbial communities are based on culture-
independent methods (Fig. 5.2). These techniques do not involve the time-
consuming processes of isolating and culturing the bacteria. This method mostly
includes the lysis of bacterial cells in soil system followed by extraction of nucleic
acid and analysis of targeted gene sequence to get the genetic information of the
organisms.
5.4.2.1 PCR Independent Approaches

Mole % G + C: A percentage of Guanine + Cytosine (mole % G + C), which forms
the base composition of DNA of microorganisms, has been used as a tool for
taxonomical classification for years. G + C content of microorganisms differs from
one another and taxonomically related groups are known to be different only by
Fig. 5.2 Culture-dependent and independent techniques to analyze microbial community

dynamics
138 A. Khan et al.
35–63% (Nüsslein and Tiedje 1999). This difference in G + C content of the DNA is
used to measure bacterial diversity. With this technique, DNA profile is produced
and relative abundance of DNA as a function of G + C content can be indicated.
Since G + C content of various different taxonomic groups may be similar, this
technique does not give a clear species-level resolution. G + C analysis is devoid of
PCR biases and since all the extracted DNA is involved in analysis, all the major and
rare members of microorganisms population get uncovered.
DNA–DNA Hybridization: It is also known as DNA–DNA re-association. This
technique involves making comparison between whole genomes of many different
organisms to calculate overall genomic similarities amongst them. This technique
involves the following steps: (i) extraction of DNA from environmental samples,
(ii) its purification, (iii) denaturation, (iv) and re-annealing. The hybridization rate
varies with similarity between DNA sequences. DNA–DNA re-association generally
ranges from 25 to 75 %. Two strains having 75% or more than 75% of re-association
capability will belong to the same species, whereas reassociation between 50 and
75% demonstrates that the strains belong to same genera, but different species.
Reduction in the rate of DNA re-association is observed with increase in complexity
of microbial diversity. The kinetics involved in re-association of DNA reflects the
variations among the sequences present in the environment, thereby reflecting the
diverse microbial communities in the environment.
DNA Microarrays: In this method, a target DNA and a probe (both complemen-
tary, single stranded regions of DNA) are allowed to hybridize. Either one of the two
is labeled with fluorescent dyes like Cy3 and Cy5. High-resolution scanning is used
to detect the hybridization events by detecting the labelling of the bound comple-
mentary target. Presence of thousands of highly specific DNA sequences in a single
array makes the use of this tool quite precious in bacterial diversity analysis
(DeSantis et al. 2007).
Reverse Sample Genome Probing (RSGP): RSGP is another molecular technique
for analyzing microbial community. RSGP comprises four steps: (1) genomic DNA
isolation, (2) cross-hybridization of DNA fragments to test the hybridization per-
centage, (3) genome arrays preparation on solid support, and (4) defined mixture
from the total community DNA is then used for random labelling. Thus, the RSGP is
a valuable approach for low diversity conditions, but assessment of highly diverse
community by using this approach is difficult (Agrawal et al. 2015).
5.4.2.2 PCR-based Techniques

Amplified Ribosomal DNA Restriction Analysis (ARDRA): Microbial diversity rely-
ing on DNA polymorphism can be studied using this technique. Presence of repeti-
tive component of nuclear ribosomal DNA (rDNA) having preserved coding and
variable non-coding segments forms the basis of this technique. PCR amplification is
performed for coding and non-coding regions. At first, environmental DNA is
obtained followed by its amplification. The amplified PCR product is then generally
digested using tetra cutter or hexacutter restriction endonucleases. The DNA
fragments get separated on gels (agarose or polyacrylamide). Variations among the
bands depict the diverse microbial communities structure present in the sample
(Singh et al. 2012; Franco-Duarte et al. 2019).
Random Amplification of Polymorphic DNA (RAPD): In this technique, short
primers (about 10 bases) are selected randomly and amplified by using PCR. These
primers bind non-specifically with complementary nucleotides in the genome
(Franco-Duarte et al. 2019). The amplicons are then separated on gels. The banding
patterns obtained on gel distinguish organisms on the basis of the presence or
absence of bands (polymorphism).
Ribosomal Intergenic Spacer Analysis (RISA)/Automated Riobosomal intergenic
Spacer Analysis (ARISA): RISA and ARISA are both riobosomal-based fingerprint-
ing techniques to study the microbial diversity. These techniques involve the
amplification of intergenic spacer regions present between the 16S and 23S ribosome
subunits by PCR, followed by its denaturation and separation under denaturing
conditions on polyacrylamide gel. Heterogeneity of the length and sequence of
IGS region is used to differentiate bacterial strains. Fully automatic edition of
RISA, known as ARISA, uses a fluorescence-labeled forward primer. Laser
detectors are used to detect ISR fragments, thereby determining the microbiome
structure.
Denaturing Gradient Gel Electrophoresis (DGGE)/Temperature Gradient Gel
Electrophoresis (TGGE): In this technique, 16S rDNA is utilized to analyze the
microbial diversity in environmental samples to determine community structure,
enriched microorganisms at specific sites, population shift, screen clone library, etc.
It includes: (i) extraction of DNA from samples, (ii) amplification of the 16S and 18S
rDNA with universal primers, (iii) denaturation followed by movement on poly-
acrylamide gel. On account of the melting behavior of double-stranded DNA, they
get separated on polyacrylamide gel prepared with gradient concentration of urea
and formamide. Molecules having diverse sequence cease their migration at different
points in the gel. After this, DNA bands are visualized using ethidium bromide,
SYBR Green I, or silver staining. The number of bands present in the gel
demonstrates the number of species present in the sample and these could be
identified by the sequencing of such gene (Salam and Varma 2019).
Terminal Restriction Fragment Length Polymorphism (T-RFLP): This technique
is generally used for examining complex microbial communities structure based on
recognition sequence of restriction enzymes in 16S rDNA. This method was
invented by Liu et al. (1997). In this method, a fluorochrome molecule like
TAMARA, HEX, or 6-FAM is used to label 5' end of either one or both the primers.
Then amplicon mixture gets digested with one or more restriction enzymes. After-
ward, digested fragments get separated in DNA sequencer either by capillary
electrophoresis or by polyacrylamide electrophoresis, and the size of different
terminal fragments is detected using fluorescence detector. Only the fluorescent
labelled terminal fragments are detected due to the use of dye. This makes
T-RFLP different from RFLP or ARDRA where all the separated fragments of
DNA are visualized.
Single-Strand Conformation Polymorphisms (SSCPs): This method involves
detection of differences in the sequence of single-stranded DNA (ssDNA). The
140 A. Khan et al.
process involves i) target DNA amplification, ii) denaturation of PCR product

(double-stranded), and iii) resolution of sample using non-denaturing poly-acrylam-
ide gel electrophoresis (PAGE). According to their sequence, ssDNA fragments fold
up in their three-dimensional structure during electrophoresis. These folded second-
ary structures cause differences in running speed of the fragments. SSCP is used to
differentiate same size DNA molecules of PCR product, but having different nucle-
otide sequences caused by PCR product, making this method a promising tool for the
analysis of microbial community at the ribosomal gene level (Nema 2019).
Metagenomics: Metagenomics is at the forefront of environmental microbiology
research in recent years. It is being widely used to study microbial community
compositions in a variety of environmental samples from terrestrial, soil, marine,
freshwater, gut microbiota, and rare and extreme habitats including deep sea floors
and acid mines. Omics approaches, i.e., metagenomics, metaproteomics,
metatranscriptomics, and meta-metabolomics, can provide an integrated view of
community dynamics and their functioning in the environmental samples. A
“metaomic” study mainly endeavors to categorize a group of microbial organisms,
their genes, metabolic functions, or pathways for their characterization of microbial
community of environmental samples. Metagenomic refers to whole-community
sampling of DNA which encodes all the genetic information obtained from all
individuals present within the sample. It is basically based on sequencing and
studying complete genomes of environmental microbiota similar to the whole
genome sequencing of a bacterial culture. Metagenomic and metatranscriptomic
particularly are used to investigate the genetic diversity and composition within
microbial communities using culture-independent sequencing technologies, i.e.,
Next Generation Sequencing (NGS) (Bonk et al. 2018). Nowadays, the term NGS
is used for various advanced sequencing techniques such as Illumina, SoLiD, Iron
torrent, and Roche.
5.5 Functions of Microbial Community in Rhizosphere
5.5.1 Nutrient Mobilization
Plants obtain nutrients from the soil and convert them into complex molecules of
carbohydrates, proteins, and all essential compounds. The three primary nutrients
that plants require are nitrogen (N), phosphorus (P), and potassium (K). Calcium
(Ca), magnesium (Mg), and sulfur (S) are the secondary nutrients, while boron,
chlorine, copper, iron, manganese, molybdenum, and zinc are the micro-nutrients.
Plants are largely dependent on soil microbes to make these nutrients accessible.
Microbes secrete acids, enzymes, and hormones which are useful for promoting
plant growth and also increasing the bioavailability of nutrients by mobilization of
key nutrients, i.e., phosphorus, potassium, and iron.
5.5.1.1 Nitrogen Fixation

Nitrogen (N) is required for the growth and metabolic activities of the plant.
However, its availability is limited for the plants because of its abundance in
diatomic form (N2). Plants can take nitrogen only in the form of nitrate or ammonia.
However, some microorganisms known as diazotrophs, are able to fix atmospheric
N2 in the form of ammonia (NH3) and undergo normal metabolic processes. These
microbes mainly include Cyanobacteria, Proteobacteria, Archaea, and Firmicutes.
Some of the microorganisms of genera Azoarcus and Azotobacter are present in
large amounts in both the rhizosphere and bulk soil. However, there are some other
species of bacterial genera Herbaspirillum and Azospirillum which are able to
colonize only in rhizosphere region. Nitrogen fixation process in rhizosphere
involves infection of roots and induction of root nodules formation. Root nodule
formation involves establishment of a highly specific interaction between micro-
organism and plant. These interactions occur at various stages in which exchange of
signals between the bacterium and plant occurs. Bacteria have a complex enzyme
nitrogenase that is crucial for them to fix N2. This enzyme system contains two
separate elements: (1) dinitrogenasereductase (Fe-protein) which has high reducing
powers and serves as an electron donor and (2) dinitrogenase metal cofactor, which
is a substrate reducing component that accepts the energy of electrons and converts
N2 molecule into ammonia. The following equation shows chemical reaction of
microbial N fixation-
N2 þ 8 Hþ þ 8e þ 16MgATP ! 2NH3 þ H2 þ 16MgADP þ 16Pi
5.5.1.2 P Mobilization
Phosphorus (P) is an essential nutrient for plants, but due to its active chemistry,
available phosphorus gets converted into unavailable fraction in the soil. Most of the
soils have inorganic or organic P in immobilized or unavailable form (Parveen et al.
2018; Singh et al. 2018). Microbes help in P mobilization by direct as well as indirect
methods. In the direct methods, P is solubilized by reducing the pH of external
medium by proton extrusion and production of low molecular weight organic anions
like citric acid, succinic acid, α-ketogluconic acid, gluconic acid, and oxalic acids.
After this process, ligand exchange occurs, where these anions get exchanged with
P. Hydroxyl and carboxyl groups present in these acids chelate the cations bound to
phosphate, thereby making them soluble. Organic P compounds are also hydrolyzed
by production of phosphatases or phytases. Besides, there are numerous indirect
methods of P mobilization that microbes follow. These are: (i) Respiration
by-product CO2 of these microbes dissolves in water present in the soil pores,
resulting in formation of carbonic acid, which decreases the pH of mycorrhizosphere
and solublizes P. (ii) During assimilation of NH4+, microbes release proton (H+)
which lowers soil pH and available P gets solublized (Singh et al. 2018).
142 A. Khan et al.
5.5.1.3 Potassium Solubilization

Potassium (K) is required for soil fertility and growth of plant. In most of the soils
proportion of K ranges from 0.04 to 3%. Ninety-eight percent of this is bound within
phyllosilicates structures. Acidolysis and complexolysis are the two main exchange
reactions undertaken by microbes to solubilize potassium. During acidolysis,
succinic, citric, gluconic, α-ketogluconic, and oxalic acids are produced by
microbes, thereby decreasing the surrounding pH. In addition to this,
microorganisms can also chelate Si4+, Al3+, Fe2+, and Ca2+ ions associated with K,
thereby releasing K ions from the mineral (Rashid et al. 2016). Subsequently,
microbes also discharge H+, inorganic and organic acids in order to acidify their
own cells, and the surroundings of potassium (K) minerals. These processes play a
crucial role in mobilizing the unavailable forms of K salts present in soil and making
them available in the soil rhizosphere.
5.5.1.4 Fe Sequestration
Iron is a key micronutrient required by plants and predominantly exists as ferric (Fe3
+
) form in nature. It is not readily available for plants, microbes, or other biota in
aerated soil. Additionally, ferrous (Fe2+) present in soil also gets oxidized to Fe3+,
thereby limiting amount of iron available for assimilation by plants or microbes.
Therefore, a fierce rivalry exists among bacteria, fungi, and plants in the rhizosphere
to meet their iron requirement. Under such conditions, few specific strains of bacteria
synthesize siderophores. These are low molecular mass proteins that deploy high
attraction to chelate and solubilize iron from insoluble compounds (Khan et al.
2019). Uptake of these complexes by the cell membrane of both Gram positive as
well as Gram negative bacteria can reduce siderophore bounded Fe3+ to Fe2+, which
is guided by membrane standing or free extracellular ferric-chelate reductases. Later,
cell links its inner and outer membrane and expels these ions from the siderophores.
This mechanism is known as “gating.” Thus, siderophores solubilize iron from
unavailable minerals or organic compounds in iron limiting conditions.
5.5.2 Plant Protection
The rhizospheric region consists of a mixed population of beneficial and detrimental

microorganisms. Microbes produce certain enzymes and antibiotics to prevent other
rhizosphere colonizers, including pathogens. Some rhizobacteria have microbial
associated molecular patterns (MAMPs) like flagellin at their cell surfaces, which
are also found to be effective in inducing systemic defense response against
pathogens. Antibiotics secreted by the rhizobacteria and fungi act as elicitors for
the defense response. Examples of a few other chemicals that elicit defense response
are surfactin, a lipoprotein that is a secretion of Bacillus subtilis (Ongena et al. 2007);
Massetolide, a biosurfactant that is derived from P. fluorescens (Tran et al. 2007);
2, 3-butanediol, a volatile organic compound formed by Bacillus spp. (Yi et al.
2016). Proteins like endo-chitinase released by Trichoderma spp. are also known to
improve plant defense. Products of avirulence genes such as Avr, which are
produced by the phytopathogens and beneficial microbes, are also known to be

associated with immune system of the plants. They specifically elicit the hypersen-
sitive responses in plants encompassing the corresponding resistance (R) gene.
Besides, Trichoderma species are also well known for production of some secondary
metabolites that induce defense response activity mainly by eliciting the expression
of pathogenesis-related (PR) proteins on plants.
Signal-transduction pathways regulate plant-microbe interactions which allow
plants to prioritize defense responses at the time of stress. Two distinct kinds of
resistance induced by these pathways are (i) systemic acquired resistance (SAR) and
(ii) induced systemic resistance (ISR). SAR is mediated by salicylic acid, which gets
produced as soon as infection occurs. While ISRs are JA- and ethylene (ET)—
dependent. These get activated by beneficial microbes capable of producing antimi-
crobial compounds, siderophores, O-antigen of lipopolysaccharides (LPS), and
salicylate. Various mechanisms of ISR are: (1) Developmental mechanism that
promotes the plant growth, (2) Physiological mechanism that reduce the expression
of symptoms, (3) Environmental mechanism, which is related to microbial
antagonists in the rhizosphere, and (4) Biochemical mechanism, which is associated
with induction of phytoalexins, PR proteins, and “priming” of defense responses.
PGPR that elicit ISR in one plant species might not do so in another, proving their
specificity. Suppression of plant diseases by microbes is eco-friendly and provides a
sustainable approach to plant disease management. Such beneficial microbes have
the potential to enhance the innate immunity level of plants by enhancing the
antioxidant status of the plant, by reprogramming defense-related enzymes,
modulating the quorum sensing activities, and activating phenylpropanoid pathway,
resulting in production of phenolics and lignin deposition (Lyu et al. 2019).
5.5.3 Decomposition
Decomposition involves the conversion of complex organic molecules of dead

material into simpler organic and inorganic molecules by either physical breakdown
or biochemical transformation. Decomposition is often carried out by soil
microorganisms. The process of decomposition is quite complex. Abiotic and biotic
drivers interact together to bring physical and chemical changes in the substrate. The
course of decomposition is greatly influenced by temperature and moisture, along
with the kind of substrate and the community and diversity of microorganisms
involved. The following interactions facilitate microbe mediated decomposition-(i)
Microbial decomposers have facilitative interactions among themselves. For exam-
ple, bacterial penetration into leaf tissue is assisted by bacteria where both degrade
specific structural polymers into smaller molecules that get assimilated conse-
quently. (ii) They might divide resources when different species have complemen-
tary enzymes that can degrade a variety of plant polymers. Even in this case, activity
patterns among species might vary. A series of events occur in the process of
decomposition. Firstly, the cell wall gets degraded leading to release of carbon
contained in the litter. The cell wall consists of various components which vary in
144 A. Khan et al.
the degree of recalcitrance. The water-soluble molecules like amino acids and sugars
which are easily accessible to fastidious microorganisms degrade at quicker pace;
(ii) the cellulose, hemi-cellulose, and pectin; and (iii) lignin and other aromatic
compounds. Cellulose, the most abundant natural polymer, is composed of repetitive
units of 1,4-linked D-glucose. It has linear chains held together with hydrogen bonds
creating crystalline structure and amorphous regions that grant rigidity to cell walls
(Rytioja et al. 2014). The hemicellulose is a branched polymer mainly constituting of
xylans, xyloglucans, and mannans (Rytioja et al. 2014). Lignin is the most recalci-
trant of all the polymers present in the litter. Lignin is a complex aromatic substance,
which provides the plant cell wall with rigidity and strength. It functions as an
adhesive between the fibers of polysaccharides.
Easily soluble compounds like starch, amino acids, and sugar are lost rapidly due
to leaching and microbial activities during decomposition. Lignin and cellulose
which are left in the litter are acted upon by specific microbial taxa. The rate of
decay of these substances is comparatively very slow. Fungi are mainly responsible
for litter and wood decomposition and, up to lesser extent, bacteria (Tlaskal et al.
2016). Bacterial communities are known to change successively during the process
of litter decomposition. Most abundant taxa found over the entire process are
Proteobacteria, Actinobacteria, and Bacteroidetes (Purahong et al. 2016). Generally,
phyllosphere bacteria play their role only during the initial stage of litter decompo-
sition. Subsequently, they are replaced with the taxa that produce proteolytic and
cellulolytic enzymes (Frigobacterium and Sphingomonas) (Tlaskal et al. 2016). In
the later stages, Bradyrhizobium, Burkholderia, and Streptomyces begin their role.
Fungal communities are also known to undergo a distinct succession of taxa in the
process of decomposition of available biopolymers. The pioneer fungal taxa mostly
involved in the initial stage of the decomposition of both litter and wood are
endophytes (Purahong et al. 2016). In the later stages, i.e., the second phase, fungi
of the phyllosphere are taken over by taxa that produce enzymes like endo-cellulases
and endoxylanases and are able to use cellulose. In the later phase, increasing
fractions of Basidiomycota which are capable of degrading lignin and humic acids
are found. This group of fungi primarily produce manganese peroxidase, laccase,
and lignin peroxidase during this phase.
5.6 Conclusion
Agro-ecosystem is endowed with different microhabitats where massive numbers of

microorganisms reside. These microorganisms perform various functions in their
native environment and affect the associated entities positively as well as negatively.
A huge number of microorganisms have been reported that play a vital role in soil
development, plant growth promotion, and protection. Diverse microorganisms
execute various functions through a variety of mechanisms. Diversity and function-
ing of an ecosystem change with the fluctuations in native environmental conditions.
Metaomics studies could be useful to understand these changes in a better way.
Moreover, these techniques reduce culture dependency for analysis and are helpful
in the exploitation of uncultivable microorganisms in order to maintain

sustainability.
Soil dwelling micro-organisms possess a variety of features to promote plant growth.

Soil is a repository of an infinite and largely unexploited microorganisms for plant
growth promotion and environmental sustenance. Therefore, study of microbial
diversity and functional profile of microbes in their natural habitat is important.
Shift in microbial diversity depicts the fluctuation in native environment; hence, it
can be an indicator for spatial and temporal changes in the environment. Further
microbiome analysis can improve our insights about the links between the function-
ing of ecosystem and the shift in diversity. Moreover, research in this aspect should
also determine the threshold level of diversity that can maintain the ecosystem
balance. In addition, future studies need to be focus on meta-omics in order to utilize
beneficial genes and enzymes of un-culturable microorganisms.
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Signaling in the Rhizosphere for Better
Plant and Soil Health 6
Hemant S. Maheshwari, Richa Agnihotri, Abhishek Bharti,
Dipanti Chourasiya, Pratibha Laad, Ajinath Dukare,
B. Jeberlin Prabina, Mahaveer P. Sharma, and Sushil K. Sharma
Abstract
The rhizosphere is a hot spot for bidirectional plant-microbe interaction that

occurs through the exchange of signals between both the partners. Due to the
sessile nature of plants, it utilizes crying-for-help strategies to combat various
biotic and abiotic challenges by producing a wide array of root exudates and root
volatile compounds in the rhizosphere. Whereas microbe releases various micro-
bial volatile organic compounds (MVOCs), phytohormones, quorum-sensing
compounds, and establish a relationship with plants. Such signal compounds
determine the structure, abundance, and richness of the rhizomicrobiome
(rhizobiome). We know the role of specific plant-microbe interactions, e.g.,
Rhizobium and legumes, mycorrhizae with roots of higher plants, and some
plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting
fungi (PGPF) with plants.
H. S. Maheshwari (*)
Ecophysiology of Plants, Faculty of Science and Engineering, GELIFES-Groningen Institute for
Evolutionary Life Sciences, Groningen, The Netherlands
ICAR-Indian Institute of Soybean Research, Indore, Madhya Pradesh, India
e-mail: h.s.maheshwari@rug.nl
R. Agnihotri · A. Bharti · D. Chourasiya · P. Laad · M. P. Sharma
A. Dukare
ICAR-Central Institute of Post-Harvest Engineering and Technology (CIPHET), Ludhiana, Punjab,
India
B. J. Prabina
Department of Soil Science and Agricultural Chemistry, Agricultural College and Research
Institute Killikulam, Tamil Nadu Agriculture University, Vallanadu, India
S. K. Sharma
ICAR-National Institute of Biotic Stress Management (ICAR-NIBSM), Raipur, Chhattisgarh, India

23, https://doi.org/10.1007/978-981-15-9154-9_6
150 H. S. Maheshwari et al.
Nevertheless, several unknown signals and their role in specific interactions

have yet to be understood. In this chapter, (i) we will discuss the role of plant-
microbe signaling in shaping the rhizomicrobiome population, (ii) chemo-
attraction of beneficial PGPR and PGPF (iii), the role of microbe-plant signaling
in priming and eliciting the induced systemic response (ISR) and systemic
acquired resistance (SAR), (iv) modification of host plant gene expression, and
regulation of hormones (salicylic acid (SA), jasmonic acid (JA), and ethylene
(ET)) and their role in biotic and abiotic stress management, and (vi) intra- and
inter-specific signal exchanges of quorum sensing (QS) molecules, volatile
organic compounds, and microbial phytohormones are described. We will also
discuss the role of plant and microbial signal in rhizosphere functioning and
sustainability as an alternative solution for increased plant fitness.
Keywords
Rhizosphere signaling · Root exudates · Microbial volatile organic compounds

(MVOCs) · Microbial phytohormones
6.1 Introduction
Understanding chemistry and biology of the underground

rhizomicrobiome-associated microorganisms (bacteria, fungi, actinomycetes, and
protists) has paramount significance in developing strategies for plant growth pro-
motion. Plant-associated beneficial microorganisms release diverse signal
compounds, e.g., phytohormones, quorum-sensing compounds, antimicrobials, and
volatile organic compounds (VOCs), which influence plant growth and develop-
ment. Similarly, the plant also mediates interaction with its associated microbes by
secreting various low- and high-molecular-weight compounds called root exudates
and root volatile compounds. The underground chemical environment determines
the plant-microbe interactions or vice versa, growth and development of a plant,
disease suppression of pathogens, and induced-systemic tolerance (IST) in the
plants. In order to understand the role of signaling, this chapter has been categorized
into three sections, in particular, signaling between plant to microbe, microbe to
plant, and microbe to microbe (Fig. 6.1). The role of microbial exo-metabolites as
plant growth stimulants brought modulations in the plant’s physiological, morpho-
logical, biochemical, and molecular state has been discussed. Furthermore, the role
of plant root exudates in shaping up the microbial communities and associated
factors have also been elucidated. Since the rhizosphere is the dynamic environment
where most of the functional microbial processes are taking place. Hence the
priming with beneficial microbes likely to elicit induced systemic resistance (ISR)
and IST in the plants in overcoming abiotic and biotic stresses.
Similarly, plant root exudates or signals also determine the shape of microbial
communities, ecology, and gene expression. Since the rhizosphere is the niche for
both beneficial and deleterious microorganisms, that means both friend and foe are
6 Signaling in the Rhizosphere for Better Plant and Soil Health 151
Fig. 6.1 Signaling in plants for better plant fitness and soil health (Modified from Venturi and Keel
2016)
living together. Priming with beneficial microbes likely elicits induced systemic
resistance (ISR) in the plants against pathogenic microorganisms. Hence, the role of
microbial signals in overcoming abiotic and biotic stresses in the plants and selective
recruitment of beneficial microorganisms needs to be studied.
6.2 Rhizosphere Communication: Plant to Microbe Signaling
Rhizosphere constitutes a narrow soil zone near the vicinity of the roots, where
intense microbial diversity and activity occur due to exudation of a wide array of root
exudates. Rhizosphere communication is mediated by various root exudates
(a cocktail of both low and high molecular weight), volatile organic compounds
(VOCs), and plant hormones like strigolactones (SLs), which are released from the
root for chemoattracting beneficial microorganisms and repelling deleterious patho-
genic microorganisms. These compounds determine the composition of
microorganisms in the rhizosphere and the fate of plant health and its role in
alleviating biotic and abiotic stresses. All symbiotic interacting microbes have to
overcome the plant immune barriers and physical barriers for any interactions. Plant
immune gets activated upon interaction with microbial signature molecules termed
as a microbe, or pathogen-associated molecular pattern (MAMP)/PAMPs triggered
immunity (MTI) (Mhlongo et al. 2018). It is due to bacterial flagellin,

lipopolysaccharides, chitin, EF-Tu, and peptidoglycan. Another line of defense
termed as ETI gets activated due to plant resistance proteins (Macho and Zipfel
2014). These two MTI and ETI are complementary and induced in the early stages.
Some bacterial pathogens can overcome the immune system by type III secretion
systems (Deslandes and Rivas 2012). Another response is damage-associated molec-
ular patterns (DAMPs) that get activated in the plants from damaged parts like
necrosis or dead cells, and signaling cascade is initiated (Choi and Klessig 2016).
The physical plant barriers include suberin, lignin, waxes, and callose produced by
the plant during metabolism (Mhlongo et al. 2018). Here, in the following section,
we discuss the role of root exudates and volatile compounds in plant-microbe
interactions.
6.2.1 Root Exudates as a Signal
About 20–40% of photosynthetically fixed carbon compounds are released as root

exudates from plant roots and these exudates play a vital role in recruiting specific
beneficial microorganisms for the better plant’s survival and protection of the plants
from several environmental and biotic stresses (Olanrewaju et al. 2019). Root
exudates chemo-attract beneficial microorganisms from the diverse microbial popu-
lation dwelling in the bulk soil, and, thereby, plant compensates their photosynthates
lost. These attracted microorganisms assist plants in nutrient acquisition and combat
against several environmental and biotic stresses (Badri and Vivanco 2009; Meier
et al. 2017). The root exudates, categorized as low-molecular and high-molecular-
weight compounds, are released from the plant’s root collectively termed as
rhizodeposition. The sugars, organic acids, amino acids, phenolic, and secondary
metabolites are the low-molecular-weight compounds that are passively transported.
In contrast, mucilage and proteins are classified as the high-molecular-weight
compounds, which are released actively from the roots by different transporters
(Badri and Vivanco 2009). The composition of secreted root exudates is mainly
relying on host genotype, the growth stage of the plants, root morphology, environ-
ment, and plant nutrient transporters, which result in determining the configuration
of the microbial community structure in the plant rhizosphere (Sasse et al. 2018).
Root exudates can mediate positive interactions with mycorrhiza and recruit PGPR
to increase plant fitness (Hernández and Chailloux 2004). Root exudates may also
mediate negative interactions like allelopathy, competition, parasitism, and herbiv-
ory for killing phytopathogens in the soils (Hayat et al. 2017).
Certain specific compounds released during root secretion, e.g., flavonoids for
meditating rhizobial interactions with legumes (Abdel-Lateif et al. 2012),
strigolactone 5-deoxystrigol for AM fungal branching (Akiyama et al. 2005),
L-malic acid for chemoattracting PGPR Bacillus subtilis in the Arabidopsis roots
(Rudrappa et al. 2008), and amino acids and sugars for several other PGPRs present
in the soils (Somers et al. 2004) are well-known examples of different functions
mediated through signaling. The other examples of signaling through root exudates
are: (1) interaction with microorganisms in a multitrophic way simultaneously, such

as chemoattracting both rhizobia and PGPR for improving nodulation parameters
(Guiñazú et al. 2010) and chemoattract nematode which acts as a carrier of rhizobia
for increased nodule formation in the legumes (Horiuchi et al. 2005); (2) facilitating
the interaction of PGPR with mycorrhizal fungi to increase the colonization effi-
ciency of AMF in plant roots (Hernandez and Chailloux 2004); (3) arabinogalactan
proteins (AGPs) of root exudates are a highly diverse family of glycoproteins
commonly found in plants. These AGPs chemoattracts the beneficial
microorganisms such as PGPR, PGPF, and biocontrol agents and repel disease-
causing pathogenic microorganisms (Xie et al. 2012), (4) banana root releases
various compounds, namely, fumaric acid, oxalic acid, and malic acid, which are
involved in bacterial chemotaxis activity and induction of biofilm of Bacillus
amyloliquefaciens NJN-6 in the banana roots (Yuan et al. 2015); (5) the cucumber
root infected by wilt fungus enhances the secretion of fumaric acid and citric acid in
which citric acid acts as a chemical attractant while fumaric acid acts as a stimulator
for biofilm formation by Bacillus amyloliquefaciens SQR 9 (Liu et al. 2014).
Further, Arabidopsis thaliana plants attacked with foliar pathogen Pseudomonas
syringae DC 3000 activates plant defense response by releasing L-malic acid in the
root exudate, which chemoattracts Bacillus subtilis FB17 bacterium in their root
surface and induces biofilm formation (Rudrappa et al. 2008). Hence, this revealed
long-distance interplant signaling helping in the colonization of P. syringae DC
3000 triggering induced systemic response (ISR) and protecting the plant against this
pathogen. The maize root produces heteroaromatic secondary metabolites
benzoxazinoids, mainly flavonoids synthesized from tryptophan-dependent
pathways. These metabolites are involved in shaping bacterial and fungal genera
in the rhizosphere of the maize (Cotton et al. 2019). Similarly, root secretes iron-
mobilizing phenolic compounds, coumarin scopoletin compounds due to transcrip-
tion factor MYB72 in Arabidopsis-Pseudomonas simiae WCS417 interaction, and
these compounds inhibit the growth of Verticillium dahlia and Fusarium oxysporum
pathogenic fungi in the rhizosphere (Stringlis et al. 2018).
6.2.2 Plant Volatile Organic Compounds (PVOCs) as a Signal
Besides root exudates, plants also produce volatile organic compounds (VOCs),
which can quickly diffuse in the below-ground distantly in the soil micro- and
macro-pores to protect plants against herbivores, pests, and phytopathogens. These
VOCs can stimulate the migration of distantly placed beneficial bacteria during the
biotic stress conditions. Plant roots infected with pathogen Fusarium culmorum in
Carex arenaria result in the alteration in the composition of plant VOCs, which in
turn recruit specific bacteria and fungi with antimicrobial properties as a plant
defense mechanism (Schulz-Bohm et al. 2018). Under biotic stresses, the plant-
mediated VOCs signal other healthy plant tissues to help in promoting beneficial
bacteria and developing shields in the rhizosphere zone to protect them against the
invading pathogens and herbivores. The positive allelopathy in neighboring plants
and the underlying mechanisms need further investigation (Liu and Brettell 2019).
This possible mechanism performed on the effect of plant-volatile compounds on the
interruption of bacterial quorum sensing (QS) revealed that certain volatile
substances have an inhibitory role, whereas some have a stimulatory function in
the QS, indicating a possible alternative for controlling plant pathogens (Ahmad
et al. 2015). The Arabidopsis plant infected with foliar downy mildew pathogen
Hyaloperonospora arabidopsidis alters the root microbiome composition by
chemoattracting consortia of three bacteria, namely, Xanthomonas,
Stenotrophomonas, and Microbacterium, which act synergistically, induce the
defense system, and promote a healthy plant stand.
6.2.3 Strigolactones
Strigolactones (SLs) term was first coined by Butler (1995) to designate a compound
exuded from roots of different plant species that induce germination of Striga
(witchweed). Strigolactones (SLs) are the carotenoid-derivatives plant hormones,
which play a pivotal role in beneficial plant-microbe interactions (AM fungi with
roots of plants, and rhizobia-legume symbiosis), as well as in interaction with
phytopathogenic microorganisms. The beneficial role of SLs is to enhance
(i) spore germination and hyphal branching processes in AM fungi, and (ii) initiate
nodulation in rhizobial symbiosis, and (iii) interaction with plant defense-related
phytohormones like salicylic acid, jasmonic acid, and ABA during pathogen devel-
opment (López-Ráez et al. 2017). The detrimental role is in the case of stimulating
seed germination of Striga, Phelipanche, Alectra, and Orobanche weeds and
involved in disease development with many pathogenic bacteria and oomycetes
fungi (López-Ráez et al. 2017). The SLs synthesis is regulataed by auxin and
phosphate concentrations available to the plants and also helps in nutrient availabil-
ity to the plants (Andreo-Jimenez et al. 2015). The SLs are also involved in the
modification of root system architecture during phosphorus deficiency in
Arabidopsis plants and enhances lateral root branching. Moreover, it inhibits shoot
branching and thereby, increasing the root/shoot ratio (Andreo-Jimenez et al. 2015).
The phosphate-starvation and rhizobia LCOs trigger SLs synthesizing gene MtD27
in rice (van Zeijl et al. 2015). However, the response of SLs in the case of controlling
or promoting pathogenic microorganisms in plants is inconsistent and varies with
different pathogen-plant system.
6.3 Rhizosphere Communication: Microbe-to-Plant Signaling
Microorganisms produce various signals like microbial volatile organic compounds

(MVOCs), phytohormones, metabolites, and various enzymes involved in
controlling phytopathogens and regulating quorum-sensing compounds in the rhizo-
sphere. Beneficial microorganisms produce MVOCs, which incite ISR induction in
the plants against phytopathogens, phytostimulation (auxin homeostasis, and sugar/
ABA signaling), and tolerance (IST induction and sodium ion homeostasis)
mechanisms. Furthermore, secondary metabolite compounds produced by various
Gram-negative bacterial biocontrol agents (BCAs) are, namely, diacetyl
phloroglucinol (DAPG), siderophore, HCN, pyrroles, pyrrolnitrin, phenazines, quin-
olone derivatives, phenylacetic acid, etc.. In contrast, Gram-positive bacterial bio-
control agents produce iturins, bacillomycin, fengycins, surfactins, and various lytic
enzymes. Similarly, various PGPF and mycorrhizal fungi also produce secondary
metabolites that kill phytopathogenic microorganisms and help in increasing plant
health (Navarro et al. 2019). Moreover, the quorum-sensing compounds produced
by microorganisms play a role in modulating root system architecture (RSA). Hence,
these microbial signals play a role in rhizosphere modification like increased rooting,
triggering host plant immunity, and increasing yield (Ortíz-Castro et al. 2009).
Recently, a new concept termed as systemically induced root exudation of
metabolites (SIREM) published. The colonization of specific microorganisms in
the root zone triggers the systemic exudation of specific compounds. Here, coloni-
zation of specific bacteria in tomato root triggers exudation of acyl sugars
insecticides, organic acids hydrocinnamic acid derivatives, and steroidal glycoalka-
loid compounds. When Bacillus subtilis 3610 and Pseudomonas fluorescens
SBW25 were introduced in root individually, it resulted in exudation of acyl sucrose
and ferulic acid hexose, respectively. It means rhizomicrobiome can change the
exudation profile of primary as well as secondary metabolites by systematically
altering plant metabolism. SIREM explains long-distance communication of
rhizomicrobiome with the above-ground plant parts (Korenblum et al. 2020).
6.3.1 Microbial Volatile Organic Compounds (MVOCs) as a Signal

Molecule
Many soil bacteria and fungi are known to release certain volatile organic
compounds, which are low-molecular-weight (<300 Da) lipophilic and odorous
compounds (<C15) having low boiling temperatures, and having high vapor pres-
sure (0.01 kPa at 20 C). These MVOCs can quickly evaporate and diffuse over short
and long distances in the root vicinity. The MVOCs are chemically related to
alkenes, alcohols, benzenoids, ketones, pyrazines, sulfides, and terpenes classes
(Kanchiswamy et al. 2015). Such compounds have typically implicated in the
microbe-microbe, plant-microbe, and diverse intra-/inter-kingdom interactions
(Schulz-Bohm et al. 2018). Many microbes, namely, Bacillus, Pseudomonas,
Alternaria and Fusarium, produce MVOCs which are involved in enhancing the
root and plant biomass and modulate phytohormones for the better plant health
(Bailly and Weisskopf 2012). The MVOCs, in dole produced by many soil bacteria,
are involved in lateral roots formation and promote shoots biomass via auxin in a
controlled pathway in Arabidopsis plants (Bailly et al. 2014). The MVOCs secreted
by many Bacillus species are isolated from lemon grass rhizosphere identified
ketones, aldehydes, and alcohols, which modulate the root-system architecture and
increase plant biomass in the Arabidopsis seedlings (Gutiérrez-Luna et al. 2010).
During the field evaluation of two MVOCs, 3-pentanol and 2-butanone for antago-
nistic activities against Pseudomonas syringae pv. lachrymans causing angular leaf
spot disease in cucumber and anaphid Myzus persicae, it was found that these
volatile compounds significantly reduced the aphid population and increased the
number of natural enemies such as the lady bird beetle Coccinella septempunctata
(Song and Ryu 2013). Contrary to the growth promotion response of MVOCs, their
adverse effects on plant fitness have also been reported in certain studies. For
example, hydrogen cyanide (HCN) produced by bacteria induces oxidative stress
phytotoxicity in Arabidopsis plant tissue (Blom et al. 2011). Similarly, some volatile
compounds produced by bacterial genera Bacillus sp., Serratia, and
Stenotrophomonas, and many soil-borne pathogenic fungi retard Arabidopsis seed-
ling biomass (Vespermann et al. 2007). The use of MVOCs in agriculture for
managing phytopathogen or insect-pest may be a sustainable alternative to toxic
agrochemicals in the field for higher crop production. We have briefly mentioned the
role of various microorganisms and their MVOCs for plant growth promotion by
alleviating biotic as well as abiotic stresses in Table 6.1.
6.3.2 Microbial Phytohormones as a Signal
The plant’s roots recruit a diverse variety of soil microorganisms by releasing a wide
array of root exudates, which produce phytohormones like auxin, cytokinins,
gibberellin acid, and abscisic acid (ABA). The PGPR convert tryptophan into
indole-3-acetic acid (IAA) via various pathways (el Zahar Haichar et al. 2014) and
by non-tryptophan-dependent pathway (Prinsen et al. 1993); similarly,
aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor released from
plant roots are cleaved by the PGPR-mediated ACC deaminase enzyme and thereby
alleviating the abiotic stresses (el Zahar Haichar et al. 2014). The notable example of
multiple plant growth–promoting hormones (IAA, GA, zeatin, ET, and ABA)
producing bacteria is the Bradyrhizobium japonicum strain USDA110, E109, and
SEMIA5080 (Boiero et al. 2007).
6.3.2.1 Auxin
PGPR-mediated auxin biosynthesis plays a dominant role in the formation and
development of lateral roots and root hair and modification of root system architec-
ture (RSA) (Vacheron et al. 2013). Auxin biosynthesis in microorganisms occurs by
many pathways, and more than 80% of rhizospheric bacteria can synthesize indole-
3-acetic acid (Khalid et al. 2004). Many PGPR, bacterial and fungal endophytes,
including ectomycorrhizal fungi, produce auxin, and its transport and signaling alter
IAA homeostasis in the plants (Sukumar et al. 2013). The IAA acts as a mutual
signaling molecule and Phyto-stimulant in Azospirillum and plant interaction
(Lambrecht et al. 2000). The biocontrol agent Pseudomonas fluorescens, which
controls black-pepper foot rot caused by Phytophthora capsici, is involved in the
root proliferation mediated by auxin and gibberellic acid (Paul and Sarma 2006). The
root colonization by Trichoderma viride in Arabidopsis occurs via an exchange of
Table 6.1 Mechanisms of MVOCs in plant growth and mitigate biotic- and abiotic-stresses
Sr
no Microorganisms MVOCs Effect References
1. Pseudomonas 2R,3R-butanediol Induces ISR in tobacco Han et al.
chlororaphis 06 against Erwinia carotovora (2006)
sub sp. carotovora SCC1
2. Bacillus subtilis 2,3-butanediol, and Increases the surface area of Ryu et al.
GBO3 acetoin the leaf in Arabidopsis (2003)
Increase plant biomass of Xie et al.
the Arabidopsis (2009)
Induction of jasmonate and Kwon et al.
salicylate acid pathway, and (2010)
ROS scavenging by Zhang et al.
antioxidant enzymes in (2008)
Arabidopsis
Alleviate salinity by
downregulating sodium
transporter HKT1
expression in root and Na+
homeostasis in the
Arabidopsis
3. Bacillus subtilis FB17 Acetoin Trigger ISR by SA and ET Rudrappa
dependent pathways against et al. (2010)
Pseudomonas syringae
pv. tomato DC 3000 in
Arabidopsis
4. Pseudomonas simiae Induces IST for tolerating
AU salinity.
Reduction in the uptake of
Na+ and accumulation of K
+ and P.
5. Pseudomonas strains HCN and Antagonistic against Hunziker et al.
1-undecene Phytophthora infestans by (2015)
compounds inhibiting hyphal growth
and sporulation.
6. Pseudomonas Antagonistic against Cordero et al.
fluorescens MGR 12 Fusarium proliferatum (2014)
7. Colombians Various MVOCS Fungal inhibition of Garbeva et al.
fungivorans Ter331 phytopathogenic genera (2014)
and Fusarium, Pythium,
Collimonas pratensis Chaetomium, and Mucor
Ter91
8. Trichoderma viride Isobutyl alcohol, The plant had larger leaves, Hung et al.
isopentyl alcohol, higher plant biomass (45%), (2013)
and root biomass, increased
3-methylbutanal, lateral roots, earliness in
flowering and chlorophyll
content (58%) as compared
to the control-treated plant
(continued)

Sr
no Microorganisms MVOCs Effect References
9. Streptomyces 2-methyl Antagonistic activity against Cordovez
pentanoate and root-rot pathogen et al. (2015)
1,3,5-trichloro-2- Rhizoctonia solani
methoxy benzene
10. Bacillus 3-pentanol Xanthomonas axonopodis Choi et al.
amyloliquefaciens pv. vesicatoria in pepper (2014)
IN937a plants.
Up regulating the PR-related
protein CaPR1, CaPR2, and
Ca protease inhibitor2
(CaPIN2) against bacterial
spot and cucumber mosaic
virus
11 Bacillus sp B55 Dimethyl-disulfide Improving sulfur nutrition Meldau et al.
and increases the growth of (2013)
the tobacco seedlings
12 Pseudomonas 13-Tetradecadien- Plant growth promotion of Park et al.
fluorescens SS101 1-ol (13-TDD), tobacco plants (2015)
2-butanone,
2-Methyl-n-1-
tridecene (2-MT)
13 Enterobacter 2, 3-butanediol Controlling northern leaf D’Alessandro
aerogenes blight caused by et al. (2014)
Setosphaeria turcica in
maize
14 Bacillus species Ketones, Modify root-system Gutiérrez-
aldehydes, and architecture (RSA) and Luna et al.
alcohols increased plant biomass of (2010)
Arabidopsis seedlings
IAA-related indole, which facilitates the induction of salicylate or jasmonate/ethyl-

ene pathways against phytopathogens (Salas-Marina et al. 2011). Bacterial indole
functions as an intercellular signaling compound because it is produced at specific
growth stages during the stationary phase and accumulates extra cellularly (Lee and
Lee 2010). Indole is vital for bacterial spore formation, virulence, biofilm formation,
and plasmid stability, which makes indole a signal compound (Lee and Lee 2010).
For example, Paenibacillus polymyxa BFKC01 produces IAA, which stimulates
pathways responsible for auxin signaling in plants and also augments lateral root
development. It also enhances iron nutrition in plants by activating Fe deficiency-
induced transcription factor1 (FITI 1) and helps in increasing photosynthetic activity
in alkaline soils (Zhou et al. 2016). Similarly, Bacillus amyloliquefaciens FZB42
produces IAA hormone via tryptophan-dependent pathways, which can produce a
fivefold increase in the IAA for plant growth, whereas knockout mutants cannot
produce (Idris et al. 2007).
6.3.2.2 Gibberellins and Cytokinin

The gibberellic acid-producing Leifsonia soli SE134 and Pseudomonas monteilii
promote growth and development in many vegetable and cereal crops (Kang et al.
2014; Pandya and Desai 2014). Gibberellic acid and brassinosteroids are involved in
the legume-rhizobial and arbuscular mycorrhizal fungal (AMF) symbiosis in plants
(McGuiness et al. 2019). However, the presence of gibberellic acid induces a
negative impact on mycorrhiza colonization. This inhibition is mediated via
DELLA protein at the arbuscular initiation stage (Foo et al. 2013). Conversely, the
brassinosteroid hormones help in the root development processes like root hair
formation, root cellelongation, lateral root initiation, regulation of root gravitropic
response, AMF symbiosis, and root nodulation in legume crops (Wei and Li 2016).
The low concentration favors root development, but higher concentration retards
developments.
Bacilli are known to produce cytokinins that stimulate plants to lower the root:
shoot ratio by increasing shoot biomass to protect plants from drought stress
(Hussain and Hasnain 2009). Inoculation of Bacillus subtilis in Platycladus
orientalis seedlings has improved shoot growth under drought stress as well as in
normal watered conditions (Liu et al. 2013). Further, cytokin in producing bacterium
Bacillus subtilis IB-22 activates the deposition of amino acids in the wheat rhizo-
sphere, stimulating colonization by beneficial bacteria (Kudoyarova et al. 2014).
6.3.2.3 Ethylene
Plants under abiotic stresses accumulate a large amount of stress ethylene (Paul et al.
2017). The ethylene modulation is a holobiont process as it is influenced by a
combination of both plant and associated microbes, indicating an ecological and
evolutionary role in the plants (Ravanbakhsh et al. 2018). Plant-associated
microorganisms modulate the ethylene signaling by producing
1-aminocyclopropane-1-carboxylate (ACC) deaminase and ACC synthase enzymes.
The ACC deaminase cleaves plant ACC, an ethylene precursor, into alpha-
ketobutyrate and ammonia, thereby reducing ethylene level inside the plants.
Whereas ACC synthase enzyme can increase ethylene accumulation in the plants
(Glick 2014). These associated microbes both upregulate and downregulate ethylene
by integrating with plant ethylene response factors (ERFs), which leads to complete
or partial inhibition of the ethylene pathway (Ravanbakhsh et al. 2018). It shows that
plants and associated microorganisms are co-evolved, and plants are dependent on
their partners (Ravanbakhsh et al. 2018). The bio-priming of seeds with
ACC-deaminase producing microbes imparting abiotic stress resilience may be an
alternative strategy to alleviate abiotic stress in plants (Gupta and Pandey 2019). The
ACC-deaminase positive Variovorax paradoxus 5C-2 increases shoot K+: Na+ ratio
improves photosynthetic parameters, and ion homeostasis in salinity stress (Wang
et al. 2016). Here, we mention some of the microorganisms and their hormones in
plant-microbe interaction in Table 6.2.
Table 6.2 Role of microbial hormones in microbe-plant interactions

Sr
No Microorganisms Hormone Effect References
1. Paenibacillus illinoisensis Auxin Increases plant root Kudoyarova
IB 1087 and Pseudomonas biomass and higher et al. (2017)
extremaustralis IB-К13- phosphorus content in the
1А wheat plants.
2. Pseudomonas moraviensis Auxin Increases nitrogen, Ul Hassan
phosphorus, and potassium and Bano
content. Increase in IAA, (2019)
ABA, and GA contents.
3. Pseudomonas fluorescens Cytokinin Biocontrol activity against Großkinsky
G20-18 Pseudomonas syringae in et al. (2016)
Arabidopsis
4. Bacillus amyloliquefaciens Cytokinin Changes RSA and promote Asari et al.
UCMB5113 growth in Arabidopsis (2017)
5. Azospirillum lipoferum Abscisic Drought stress alleviation Cohen et al.
acid (ABA), in maize (2009)
IAA, and
Gibberellins
6. Bacillus licheniformis IAA, ABA, Minimization of water loss Salomon
Rt4M10 and and GAs by promoting ABA et al. (2014)
Pseudomonas fluorescens biosynthesis in grape
Rt6M10
7. Rhodococcus sp. P1Y ABA Decreasing plant ABA Belimov
and Novosphingobium utilizing content and increases plant et al. (2014)
sp. P6W microbes growth
6.3.3 Microbial Exometabolites as Signaling for Suppression

of Plant Disease Caused by Pathogens
Microbial biological control agents (MBCAs) using beneficial rhizobacteria are an

ecological and sustainable approach for the control of many soil-borne plant-
pathogens (Dukare and Paul 2018; Köhl et al. 2019). Various PGPR and PGPF
produce diverse secondary metabolites during metabolisms like antibiotics, hydro-
lytic enzymes, volatile biocidal compounds, siderophores, and detoxification
enzymes, which act as allelochemicals signal compounds, which kill pathogenic
microorganisms and protect plants (Saraf et al. 2014). Many lytic enzymes like
chitinase, glucanase, protease, and cellulases are produced by many biological
control agents, which degrade the cell wall of pathogens and protect plants (Dukare
et al. 2013, 2019). Under iron-deficient conditions, PGPR and PGPF produce low-
molecular-weight (500–1000 Da) compounds termed as iron-chelator or
siderophores that convert Fe+3 into Fe+2 inside the cell, and in that way, due to
lack of iron, phytopathogens growth is retarded in the rhizosphere (Neilands 1989).
The beneficial PGPR and PGPF mainly produce hydroxymates and catecholate type
of siderophores for sequestering iron for plants and lower the disease-causing
potential of phytopathogens. The Trichoderma protects the plants from various
pathogenic microorganisms and acts as a biocontrol agent. It kills harmful pathogens
by utilizing various mechanisms as (i) competition for space and nutrients by
colonization in root and, thereby, starving pathogens, (ii) antibiosis through produc-
ing various antibiotic compounds, (iii) mycoparasitism via secreting cell wall lytic
enzymes (chitinase, cellulases, b-glucanases, and proteases), (iv) production of
siderophore and various secondary metabolites, (v) eliciting plant defense via
ethylene production and hypersensitive responses, (vi) plant growth enhancement
via the production of growth hormones and nutrient (P) solubilization (Abdel-Lateif
2017; Kumar 2013). The role of important biocontrol agents (BCAs) and their
secondary metabolites for disease management are briefly discussed in Table 6.3.
6.3.4 Role of Microbial Signal in the Elicitation of Induced Systemic

Resistance in Plants
Many beneficial plant- and soil-associated bacterial species such as Pseudomonas

and Bacillus trigger plant defense response against pathogens upon root coloniza-
tion, which is referred to as an induced systemic resistance (ISR) (Kloepper et al.
1992). The key ISR signals of PGPR are siderophore, lipopolysaccharides (fucose
and rhamnose), flagellins, and antibiotics (phenazine, phycocyanin, and DAPG),
which activate ISR reaction in the plant’s tissue (Bakker et al. 2003). Moreover,
numerous plant defense enzymes such as phenylalanine ammonia-lyase (PAL),
peroxidases, polyphenol oxidase, superoxide dismutase (SOD), catalase, ascorbate
peroxidase, chitinase, glucanase, and proteinase inhibitors have also been produced
(Annapurna et al. 2013). Likewise, pathogens induce systemic acquired resistance in
the plant that leads to the accumulation of pathogenesis-related protein (PRs)
mediated via a salicylic acid pathway. Therefore, the combination of SAR and ISR
could be a viable option to manage diseases caused by bacteria, fungi, nematodes,
and protists (Van Loon and Bakker 2005). When the plants are infected with a
necrotrophic pathogen, insect, herbivores, and abiotic stresses, they activate
jasmonic acid and salicylic acid pathway as a defense response (Carvalhais et al.
2015). Under such circumstances, the hormone-mediated alteration of the root
exudates composition activated, which selectively chemo-attracts the functionally
unique bacteria. The altered microbiome assists in the absorption of nutrients and
water as well as help plants to protect against the invading pathogens. In this way,
altered microbiome acts as a soil-borne legacy, and facilitates in improving the plant
fitness for succeeding crops in the same area (Bakker et al. 2018). One notable
example of triggering the defense response in the plant is Arabidopsis where the
foliar pathogen-infected arabidopsis plant triggers the formation of salicylic which
selectively chemoattracts the colonization of specific root bacterial communities as a
defense mechanism for higher plant growth (Lebeis et al. 2015).
Table 6.3 Biocontrol agents and their secondary metabolites for disease suppression
Sr. Secondary
no Microorganisms metabolites Effect References
1. Bacillus alvei NRC-14 Lytic enzymes Against Fusarium Abdel-
oxysporum in tomato Aziz
(2013)
2. Pseudomonas putida Siderophore Induces ISR in Meziane
WCS358 and Arabidopsis against et al.
pseudobactin Pseudomonas syringaepv. (2005)
tomato
Against Colletotrichum
lindemuthianum in bean
and Botrytis cinerea in
tomato and bean
3. Bacillus subtilis Hydroxymate Suppress Fusarium Patil et al.
CTS-G24 type of oxysporum f. sp. ciceris (2014)
siderophore and
Macrophominaphaseolina
pathogen in chickpea
plants
4. Bacillus fortis AGS162 Phenylacetic Elicitor of SAR against Akram
acid (PAA) Fusarium wilt by et al.
phenylpropanoid pathways (2016)
5. Bacillus sp. Non-ribosomal Fira et al.
peptides, (2018)
polyketide,
bacteriocins,
and siderophore
compounds.
Circular
lipopeptides
namely,
surfactin,
fengycin, and
iturin
6. Pseudomonas 2, 4-diacetyl Control soil-borne Lohitha
fluorescens phloroglucinol pathogens like Sclerotium et al.
(2, 4-DAPG), rolfsii in groundnut (2016)
and phenazine-
1-carboxylic
acid (PCA)
7. Pseudomonas Pyrrolnitrin and Controlling tomato late Park et al.
chlororaphis O6 phenazines blight fungal infestation (2011)
8. Bacillus subtilis GB03 2R,3R- Against bacterial pathogen Ryu et al.
and Bacillus butanediol Erwinia carotovora subsp. (2004)
amyloliquefaciens Carotovora by eliciting
IN937a ISR in Arabidopsis
9. Paenibacillus polymyxa Long-chain Elicits ISR in Arabidopsis Lee et al.
E681 volatile plants against (2012)
compound Pseudomonas syringaepv.
tridecane maculicola ES4326
6.4 Rhizosphere Communication: Microbe-to-Microbe

Signaling
Quorum sensing (QS), a population density–related biological communication phe-

nomenon, is governed by chemical signals between cells or from one cell to another
upon reaching a critical density. In this phenomenon, both Gram-negative and
Gram-positive bacteria interact with each other through auto inducer N-acyl
homoserine lactone (AHL) and short oligopeptides molecule, respectively. Beyond
acyl-HSLs, cell-cell communication can be multilingual by using different dialects.
Gram-negative bacteria have other QS molecules like aryl homoserine lactones(aryl-
HSL), photophores (PPYs), dialkylresourcinols (DARs), and 2-heptyl-3-hydroxy-4-
quinolone (PQS) for cell-cell communications (Brameyer et al. 2015). The bacterial
genus Bradyrhizobium and Rhodopseudomonas employ aryl-HSLs (p-coumaroyl-
HSL) for colonization in the roots and environmental signaling (Schaefer et al.
2008). The type of AHL is classified based on the number of carbon atoms (4C-
18 C) and the presence of either hydroxyl or carbonyl group at C-3 atom or presence
of double bond in the acyl chain. The QS mechanism helps in plant hormone
synthesis, stress response, defense response, and flavonoid synthesis. AHL-incited
resistance in plants helps in the initiation of signaling cascade MAP kinases and
WRKY-type transcription factors against many biotrophic and hemibiotrophic
pathogens. The AHL primed plants synthesize oxylipins and salicylic acid, which
activate plant defense response like deposition of callose, phenolic acid, and closure
of stomata (Schikora et al. 2016). The Sinorhizobium meliloti explicit AHL,
3-oxo-C14-homoserine lactone at one μM concentration increases the nodule num-
ber in legume plant Medicago truncatula via an ET-dependent pathway (Veliz-
Vallejos et al. 2014). Similarly, Sinorhizobium meliloti, capable of producing AHL,
oxo-C14-HSL, protects tomato from the late blight and barley from the powdery
mildew fungus Blumeria graminis f. sp. hordeii (Hernández-Reyes et al. 2014). The
AHL, oxo-C14-HSL primed Arabidopsis plants acquire resistance through callose
deposition, synthesizing phenolic compounds, and lignifying their cell wall against
pathogen Pseudomonas syringae. AHL priming also leads to the closure of stomata
by increased production of oxylipins and salicylic acid (Schenk et al. 2014). Two
AHL-releasing bacteria Serratia liquefaciens MG1 and Pseudomonas putida IsoF
when colonized systemically induce SA- and ET-dependent pathways against the
leaf pathogen Alternaria alternata in tomato plants. The priming of winter wheat
seeds with bacteria-producing N-hexanoyl-L-homoserine lactone (C6-HSL) has
significantly improved seed germination and growth at an early stage. The priming
increases dry plant biomass, grain yield, and quality under both in vitro and field
conditions. Thus, these QS signal molecules can act as phyto-stimulators and
minimize the use of plant protection chemicals (Moshynets et al. 2019).
6.5 Some Examples of Microbial Signal in Plant-Microbe

Symbiosis
6.5.1 Rhizobium-Legume Interactions
The Rhizobium is a symbiotic nitrogen-fixing bacteria present in bulk soil, which

colonizes root nodules of the legume plants for biological-nitrogen fixation (Clúa
et al. 2018). Bacterially produced lipo-chitooligosaccharide (LCO), signals termed
as Nod factors (NFs), and specific flavonoids compounds provided by legume
partners mediate the rhizobia and legume plant interactions. The signaling
compounds govern the specificity by secretion of flavonoids, isoflavonoids, and
rhizobial nodulation protein D (Nod D) factors (Wang et al. 2018). The specific NFs
are required for crosstalk, nodule development, and host phytohormones homeosta-
sis. Root hairs sense the rhizobial NFs signals through receptors, which induce
calcium spiking and activation of cytokinins and gibberellic acid hormones, and
their downstream signaling pathways. These hormones further modulate the levels
of auxins, ABA, and ethylene inside the plants (Buhian and Bensmihen 2018). Other
signals involved in the interaction are capsular polysaccharides, extracellular
polysaccharides (EPS), receptor kinases (Leucine-rich repeat), and secretion system
(T3SS. T4SS and T6SS) (Nelson and Sadowsky 2015). On the other hand, the plant
signal also autoregulates the number of nodule formation by the formation of CLV3/
ESR-related (CLE), which transfers the signal from root-to-shoot and thereby
inhibits nodulation (Reid et al. 2013).
6.5.2 Signaling in Mycorrhizal Symbiosis
Arbuscular mycorrhizal fungi (AMF) forms symbiotic interaction with the roots of
more than 80% higher plants. Under phosphate starvation, the plant releases
strigolactones (SLs), which stimulates the AM symbiosis by the stimulation of
hyphal branching during the pre-symbiotic growth phase of the fungus (Zwanenburg
et al. 2016). Symbiosis is mediated between plant-produced strigolactones and
mycorrhizal fungi produced lipochito-oligosaccharides. In-plant root and AM
fungi symbiosis, AM-fungi Nod/Myc factor give the signal to activate secondary
messenger molecules like POLLUX, CASTOR, or DMI. After activation of these
messengers, cytoplasm becomes hyperpolarized, and permeation of K+ ion occurs,
which leads to Ca2+ spiking. Now, Ca2+ ions bind with CCaMK and further signal
transfer to the CYCLOPS gene, which regulates the mycorrhiza colonization process
(Venkateshwaran et al. 2012).
Symbiosis involves the three-tiered response of AM host plant as i) Perception:
AMF host plant perceives Myc-LCO by its receptor kinases SYMRK/DMI2, ii)
Transmission: enzyme reductase converts DMI2 to DMI1 and calcium spiking
occurs, and iii) Transcription: Conversion of DMI3 into RAM 1 takes place in the
presence of calcium and DELLA (MacLean et al. 2017). AMF helps in the uptake of
nitrogen, phosphorus, and many other macro- and micro-nutrients from the soil
through extra-radical mycelium (ERM) and exchange nutrients through intra-radical

mycelium (IRM) in a specialized tree-like structure termed as arbuscules. AMF
fungi, in turn, receive sugars, root exudates, and fatty acids as a source of carbon
from the plant (Luginbuehl and Oldroyd 2017). In general, these fungi help plants to
grow well and reduce nutrient losses from soils by enhancing nutrient acquisition in
plants (Cavagnaro et al. 2015; Sharma and Adholeya 2004).
6.6 Manipulation of Rhizospheric Signals for Better Plant

Fitness and Soil Health
The rhizospheric signals can be manipulated via three methods (i) microbiome-based
method, (ii) plant-based method, and (iii) meta-organism-based method to enhance
biological functions required for nutrient recycling, disease suppressiveness, and
tolerance against biotic and abiotic stresses (Quiza et al. 2015). Many microbiome-
based approaches are currently being advocated. Firstly, the inoculation of beneficial
PGPR, AMF, microbial-endophytes, and nutrient fixers or solubilizers in the rhizo-
sphere are being practiced to have better plant functions through greater nutrient
availability, phytohormones production, induction of ISR, and IST (Chaparro et al.
2012). Secondly, to transfer desired genes for performing specific functions through
horizontal gene transfer by the application of recombinant strains (Ryan et al. 2009).
Thirdly, disrupting microbial communities by application of physical and chemical
forces like tillage and agrochemicals for facilitating the management of the desired
composition of resident beneficial microorganisms (Bakker et al. 2012). Plant-based
approaches involve the use of breeding or selecting cultivars, which release desired
root exudates compounds for chemoattracting beneficial microorganisms and repel-
ling pathogenic microorganisms (Bakker et al. 2012). Additionally, altering the
genetic makeup of plants for producing specifically desired root exudate compounds
may impart higher nodulation, siderophore, biocontrol substances, and bioremedia-
tion of heavy metal or toxic compounds (Sharma et al. 2013). Genetic modification
in the plant to alter the composition of root exudate compounds, which selectively
chemoattract beneficial bacteria from bulk soils. Altered root exudates may modify
soil quality like soil pH for tolerance against heavy metals toxicity to roots (Ryan
et al. 2009). Similarly, genetically altered transgenic plants release a range of
quorum sensing and quorum quenching compounds that interfere with communica-
tion between pathogens thus hinder the growth of pathogens (Zhang et al. 2015).
Meta-organisms-based approaches besides improving the health of plants and soil
through the addition of fertilizers and organic inputs (Mazzola 2007), the use of
agronomic practices involving growing non-host crops in crop rotation will help in
the suppression of growth and development of pathogens in the soil. Additionally,
crop rotations may induce suppressive soil formation by altering soil biodiversity,
involving in recycling nutrients, and improving the physicochemical properties of
soils (Ryan et al. 2009).
6.7 Conclusion
The critical outcomes of rhizosphere signaling are the recruitment of beneficial

microorganisms in the rhizosphere or endosphere involved in the uptake of nutrients
and conferring tolerance against several biotic and abiotic stresses. These recruited
microbes modulate both underground and above-ground plant parts to enhance plant
health and fitness. Identifying unknown key microbial signals and integrating or
amending them into the rhizosphere can help in improving plant productivity and
soil health by facilitating the desired rhizospheric manipulation. This will help to
increase beneficial PGPR populations and to reduce pathogenic and deleterious
phytopathogens in the soil. Advanced analytical techniques such as liquid
chromatography-mass spectrophotometry (LC-MS), gas chromatography-mass
spectroscopy (GC-MS), and capillary electrophoresis-MS (CE-MS) should be
employed for the identification of plant and microbial signals. Coupling multi-
omics strategies such as metatranscriptomics, metaproteomics, and metabolomics
result in root exudate responses, which may reveal the specific role of plant genes
responsible for the synthesis of signaling compounds. This will help in understand-
ing the signaling pathway events and critical factors involved. These microbial
signals can also alter soil chemistry (temperature, pH, water content, and nutrient
status) to enhance soil health. Hence, microbial signal modulation of the rhizosphere
may be a safer low-cost, environmentally friendly strategy to encourage better plant
growth, production, and productivity and improve the soil ecosystem.
Acknowledgments Hemant S. Maheshwari acknowledges the Ministry of Agriculture and Farmer

Welfare, Govt of India and Director, ICAR-Indian Institute of Soybean Research, Khandwa Road,
Indore-452001, India for providing financial assistance for pursuing doctoral studies at the Univer-
sity of Groningen, the Netherlands (ICAR-NS-IF-2017), under the supervision of Dr. J.T.M
Elzenga (Professor, Ecophysiology of Plants Lab, University of Groningen, the Netherlands).
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Microbial Transformation of Nutrients
in Soil: An Overview 7
Deep Mohan Mahala, Hemant S. Maheshwari,
Rajendra Kumar Yadav, B. Jeberlin Prabina, Abhishek Bharti,
Kiran K. Reddy, Chiranjeev Kumawat, and Aketi Ramesh
Abstract
The indiscriminate as well as imbalanced application of inorganic fertilizers and

climate change associated land degradation negatively affected the soil’s physi-
cal, chemical, and microbiological characteristics, thereby affecting global food
production. In soils, various microbial communities are involved in nutrient
transformations, thereby determining the mobilization, and fixation capacity of
nutrients impacting crop growth and development. Some bacterial and fungal
genera are involved in a number of mechanisms such as organic acid production,
proton extrusion, critical enzyme production that enables the transformation of
unavailable forms of nutrients into available forms. Furthermore, the definitive
D. M. Mahala
ICAR-Indian Institute of Maize Research, Ludhiana, Punjab, India
H. S. Maheshwari (*)
Ecophysiology of Plants, Faculty of Science and Engineering, GELIFES-Groningen Institute for
Evolutionary Life Sciences, Groningen, The Netherlands
e-mail: h.s.maheshwari@rug.nl
R. K. Yadav
Agriculture University, Kota, Rajasthan, India
B. J. Prabina
Agricultural College and Research Institute, Killikulam, Tamil Nadu Agricultural University,
Vallanadu, Tamil Nadu, India
A. Bharti · A. Ramesh
K. K. Reddy
ICAR- Directorate of Groundnut Research, Junagarh, Gujarat, India
C. Kumawat
SKN Agriculture University, Jobner, Rajasthan, India

23, https://doi.org/10.1007/978-981-15-9154-9_7
176 D. M. Mahala et al.
role of different soil abiotic and biotic components also determines the fate of the
nutrient transformation and its availability to the plants by affecting the microbial
community structure as well as diversity. Taking sustainability into consideration,
the exploitation of microbial inoculants to increase the availability of essential
plant nutrients could be a viable alternative option for increasing food productiv-
ity without compromising soil quality. In this chapter, we discussed the role of
diverse microbial communities in nutrients transformation of major (nitrogen,
phosphorus, potassium, sulfur) and minor elements (iron, manganese, and cop-
per), their mechanism, and their key roles in plant and soil health, and the
conditions favouring the availability of nutrients are elucidated in detail.
Keywords
Nutrient transformation · Mobilization · Solubilization · Mineralization · Soil

quality · Macro- and Microelements
7.1 Introduction
To meet the food, fuel, and fodder requirements of the burgeoning population,
agricultural practices are linked with indiscriminate use of chemical fertilizers and
pesticides that pollute water, air, soil and is a major cause of concern for human and
animal health (Sharma and Singhvi 2017). The world’s requirement of agriculture
fertilizers by nutrient was 107.9 million metric tons in 2011–12, and its requirement
is expected to be 189 million metric tons by 2020–21 (https://www.statista.com/
statistics/438930/fertilizer-demand-globally-by-nutrient). Furthermore, the nutrient
use efficiency (NUE%) of chemical fertilizers like nitrogen (30–50%), phosphorus
(15–20%), potassium (70–80%), sulfur (8–10%), and other micronutrients (2–5%) is
very low. It is mainly as a consequence of nutrient leaching, surface runoff due to
soil erosion, nutrient fixation, immobilization, and gaseous losses (Parihar et al.
2019). The imbalanced fertilization also causes soil salinity, accumulation of toxic
heavy metals, eutrophication of water bodies, accumulation of nitrate and phosphate
in drinking water, nitrosamine formation, and release of toxic nitrogen and sulfur
gases as greenhouse gases leading to global warming (Savci 2012; Coskun et al.
2017; Parihar et al. 2019).
Furthermore, the rampant use of fertilizer and pesticides negatively impacts
global food production by altering the natural soil microflora structure and composi-
tion, diversity and ultimately affecting soil quality and nutrient availability (Prashar
and Shah 2016; Mandal et al. 2020). Excessive nitrogen fertilizer causes soil acidity,
whereas chemical fungicides and insecticides negatively impact soil health (Bitew
and Alemayehu 2017). Therefore, the inclusion and increasing the population of soil
nutrients transforming microorganisms can help in maintaining sound soil ecology
and also an agricultural intervention that aid in the proliferation of beneficial
microorganisms can improve soil quality leading to a sustainable, environmental-
7 Microbial Transformation of Nutrients in Soil: An Overview 177
friendly, low-cost approach with the replacement of agrochemicals and fertilizers

(Ahmad et al. 2018; Sammauria et al. 2020).
The nutrient flow and cycle in an ecosystem involves the integration of elements
presents in the atmosphere as gases, unavailable insoluble minerals in rocks and
soils, readily available chemical nutrients, and organically bound nutrients that are
present in living autotrophs heterotrophs, and dead organic matter (Likens et al.
1981). The nutrient cycle is mediated by physical, biological, and microbiological
agents. The diverse soil microorganisms such as bacteria, actinobacteria, fungi,
algae, and protozoa are involved in soil nutrient transformation of major and
minor elements. Furthermore, soil microbial communities’ composition determines
the soil’s physical and chemical properties (Wani et al. 2015). These microbes are
key players in soil organic matter decomposition, solubilization, mineralization, and
humification in the soil (Billings and Ziegler 2005). The soil organic matter, such as
plant litter, sloughed roots, and mucilages, and dead and decaying organisms
consists of a complex organic polymer of cellulose, hemicellulose, lignin, and pectic
acid. Soil microorganisms decompose these materials into inorganic nutrients and
humus and are termed as mineralization and humification, respectively. The released
nutrients further get chelated as organo-metal-complexes or leached through the soil
or immobilized or become available to the plants (Sahu et al. 2017). During organic
matter decomposition, various soil microbial enzymes, such as amylase,
arylsulfatase, ß-glucosidase, cellulases, chitinase, proteases, dehydrogenase, urease,
and phosphatases, are involved and thereby play a pivotal role in sustaining soil
health and fertility (Sahu et al. 2017). The soil nutrients are classified as
macronutrients (nitrogen, phosphorus, potassium, sulfur, calcium, and magnesium)
and micronutrients (chlorine, iron, manganese, zinc, molybdenum, selenium, boron,
and copper) and its availability determines crop growth, productivity, and quality of
agricultural produce for human and animal consumption. These are absorbed in ionic
forms by the plant roots via root interception, mass flow, and diffusion mechanisms.
The nutrient cycle aims at transforming elements into an available form, storage of
elements, ecosystem functioning, the flow of substances, and linking both living and
non-living components of the ecosystem. In this chapter, we highlighted the impor-
tance of soil microorganisms in soil nutrient transformation (nitrogen, phosphorus,
potassium, sulfur, and micronutrients like iron and zinc), their mechanisms, and their
role in plant and soil health (Table 7.1).
7.2 Nutrient Transformation of Macronutrients
7.2.1 Nitrogen Transformation
Nitrogen is a pivotal cellular component of chlorophyll pigment, amino acids, and

other important cellular biomolecules, including ATP and nucleic acids of the plants
and determines crop productivity (Kuypers et al. 2018). In managed ecosystems,
plants meet their nitrogen demand through exogenous application of mineral N
Table 7.1 Soil conditions inducing nutrient deficiencies and availability for plants
Plant
available
form of Condition favouring
Nutrient nutrient availability Condition favouring deficiency
N NH4+, NO3 Less in acidic pH having Leaching of soil in heavy precipitation,
lesser than 5 and in an low organic matter content in the soil;
alkaline pH crop residue burnt sites
P H2PO4, Optimum pH 6.5–7.2 Acidic and alkaline soil
HPO42
K K+ 2:1 Smectite-dominated Sandy soil, lime application, intensive
black soil cropping system
Ca Ca2+ Near neutral to alkaline Acidic, alkaline, or sodic soils
pH
Mg Mg2+ Near neutral to alkaline Acidic, alkaline, or sodic soils
pH
S SO42 Available pH range Organic matter content deficient soils;
4.0–7.5 application of nitrogenous and
phosphatic fertilizers having no Sulfur
content, and crop residue burning
Fe Fe2+ Acidic pH Higher pH, high calcareous soils
Zn Zn2+ Acidic pH Light texture soil, high pH, high
calcareous soils, eroded soil, low
organic matter, lowland rice soil
Mn Mn2+ Acidic pH High pH, high calcareous soils
Cu Cu2+ Acidic pH Sandy soils, organic soils, liming
soil rich in Zn, Fe, and Mn content
B H3BO3, pH range 5.0–6.5 Acid light texture soil low organic
H2BO3, matter, high calcareous soils, dry
HBO32, weather
BO33
Mo MoO42 Higher pH and less Ca Podzolized soils and well-drained
activity calcareous soils
sources. However, the nitrogen use efficiency (NUE) of applied chemical nitrogen
fertilizer is less than 50% due to leaching, volatilization loss, denitrification, and
surface-runoff. These losses are undesirable because of environmental concerns and
the high cost of production (Coskun et al. 2017; Beeckman et al. 2018; Xin et al.
2019). In natural soils, 98% of the nitrogen is found in-bound in organic forms,
which cannot be easily accessible by the plants except for some amino acids and
peptides (organic molecules) (Näsholm et al. 2009). Despite the high abundance of
nitrogen in the atmosphere, plants cannot utilize it directly due to the nitrogen
molecule’s triple-bonded nature. So, nitrogen fixation and mineralization are crucial
processes for the release of N for plant nutrition. The nitrogen cycle, which is a
repetitive cycle of processes by which nitrogen moves through the atmosphere, soil,
water, and plant can be divided into mineralization, immobilization, nitrification, and
denitrification, as shown in Fig. 7.1. The soil microorganisms convert nitrogen from
7
Microbial Transformation of Nutrients in Soil: An Overview
Fig. 7.1 Nitrogen transformation in the soil-plant-atmospheric continuum

179
3 to +5 oxidation numbers. The nitrogen cycle mainly includes the transformation

mentioned below (Robertson and Groffman 2007; Kuypers et al. 2018).
1. Nitrogen mineralization in which organic nitrogenous materials are decomposed

and converted to simpler inorganic compounds that can be assimilated directly by
plants and microbes.
2. Nitrogen immobilization in which available nitrogen compounds get locked
temporarily into unavailable forms (priming).
3. The oxidation of nitrogen into a molecular form that escapes into the atmosphere.
4. The fixation of gaseous nitrogen in the terrestrial ecosystem through chemical
nitrogen fixation (industrial fixation), biological nitrogen fixation (BNF), rain,
and lightning.
Soil microorganisms mediate the nitrogen transformation in the soil. The micro-
bial diversity, activity, and abundance are impacted by global climate change and
also associated parameters such as temperature, precipitation patterns, and carbon
dioxide (Ollivier et al. 2011). Furthermore, the nitrogen deposition into the soil by
the atmosphere and chemical fertilizers increases the net or gross mineralization,
nitrification, and immobilization rates (Cheng et al. 2019). The nitrogen transforma-
tion also varies between the agricultural and forest ecosystem. The agricultural soils
have a higher rate of nitrification, gross ammonification, and a lower immobilization
rate than forest soils (Xu and Xu 2015). Other drivers that determine the nitrogen
transformation is the cropping pattern. The conversion of the maize-soybean system
into the grass field altered the nitrogen mineralization, gross nitrification, ammo-
nium, and nitrate immobilization processes, thereby increasing the supply of soil
nitrogen (Li et al. 2018).
Additionally, the cropping system also influences nitrogen transformation and is
well documented in maize-soybean intercropping. It showed enhancement of
ammonifiers, nitrifiers, and denitrifiers. It also increased nitrogen use efficiency by
reducing volatilization loss and N2O emission (Chen et al. 2019).
Furthermore, the toxic elements like cadmium also decreased the microbe-
mediated soil nitrogen transformation activities. These effects were more pro-
nounced in upland rice soil system due to alteration in soil bacterial and archaeal
composition. Similarly, the extent of soil degradation also determines the minerali-
zation and nitrification rates by decreasing the soil microbial biomass nitrogen
(Singh et al. 2007). The agrochemical application adversely affected nitrogen trans-
formation processes like urea hydrolysis, nitrification, and denitrification, as is
evident in the case of a commonly used fungicide, such as carbendazim and
chlorothalonil (Ding et al. 2019). Similarly, the application of pesticides in soil
affects nitrogen transformations, such as ammonification, nitrification, and
biological nitrogen fixation by negatively affecting microbial activity and biomass
(Zhang et al. 2016).
The invasion of weed Praxelis clematidea in the soil increases the soil nitrogen
transformation rate by increasing the microbial biomass nitrogen in tropical savanna
(Wei et al. 2017). The availability of oxygen also affects the transformation by
altering the microbial diversity and composition. In the anoxic condition of low-land
paddy, the nitrogen transformation is mediated by the Deltaproteobacteria group of
microorganisms, mainly Geobacter and Anaeromyxobacter (Masuda et al. 2017).
The inoculation of microbial nitrogen transformants also influenced the process. The
inoculation of nitrogen transforming microbial strains into compost reduced the
microbial diversity and favoured dominant genera, thereby making the environment
favourable for nitrogen fixation and increasing the nitrogen content (Yang et al.
2019). The fungal endophyte Phomopsis liquidambari was found to decompose the
below-ground rice straw and affect nitrogen transformation processes and thereby
acting as a priming effect (Sun et al. 2019). We explained the different nitrogen cycle
events separately below.
7.2.1.1 Nitrogen Mineralization and Immobilization

The conversion of the organic form of nitrogen into inorganic form by heterotrophic
soil microorganisms are termed as nitrogen mineralization. In contrast, the transfor-
mation of readily available inorganic form into bound organic form is termed
immobilization (Lin et al. 2016). Diverse soil microbial communities are involved
in the release of nitrogen from the soil organic matter. Primarily, soil organic matter
consists of cellulose, hemicellulose, pectin, and lignin that are decomposed by
various soil microorganisms such as bacteria, fungi, and actinobacteria. The depo-
lymerization of complex nitrogen polymers (nucleic acid, protein, and chitin) into
simpler forms are mediated by specific microbial exoenzymes (Killham 1994). The
important exoenzymes for cellulose and hemicellulose decomposition are
endoglucanases, endoxylanases, mannosidases, β-xylosidases, β-glucosidases, and
carbon-binding modules (López-Mondéjar et al. 2016). Whereas, for lignin decom-
position, key enzymes involved are laccase peroxidases, lignin-modifying enzymes,
and many auxiliary enzymes (Janusz et al. 2017). Saprophytic bacteria and fungi
mediate the extracellularly or intracellularly proteolysis of nitrogenous compounds.
After depolymerization, organic nitrogen breaks down into amino acids and is
further deaminated to form inorganic ammonia, and this is termed as ammonifica-
tion. Similarly, the organic nitrogenous compounds of microbial origin such as
chitin and peptidoglycan get depolymerized into respective monomers through
microbial enzymes such as exo- and endo-chitinases and chitin-modifying enzymes
such as chitodextrinases, chitin depolymerase, chitin deacetylases, chitosanases, and
N-acetyl-beta-glucosaminidases into the D-amino acids and amino sugars (Howard
et al. 2003; Hu et al. 2018). The key drivers in soil nitrogen mineralization depend
upon soil organic carbon (SOC), total nitrogen content, microbial biomass carbon
(MBC), and microbial biomass nitrogen (MBN) (Booth et al. 2005). Moreover, soil
temperature, soil moisture, and soil pH also play a significant role in mineralization
and immobilization processes (Kai et al. 1973). During the composting process,
bacterial communities are predominant and play a significant role in the mineraliza-
tion of organic nitrogen into ammonium ions. The availability of the nitrogen is also
dependent on carbon/nitrogen (C/N ratio), either native or applied organic sources,
moisture availability, and pH value (Zhou et al. 2018; Zhu et al. 2019b). The diverse
plant communities have higher nitrogen mineralization rates and dissolved nitrogen
content than monoculture cropping (Mo et al. 2016). The C: N ratio plays a critical
role in both mineralization and immobilization processes. When C: N ratio is >25:1,
immobilization occurs, whereas <25:1 mineralization occurs. The soils that are
having acidic pH < 5.0, the nitrogen mineralization is mediated by heterotrophic
fungi, as acidic pH favours fungal population, while under neutral or slightly
alkaline conditions, bacterial community predominates (Rousk et al. 2009). The
various agricultural management practices like crop residue retention, organic
manure, tillage operations, fertilizer, and green leaf manuring alter soil microbial
communities that determine nitrogen mineralization and plays a role in nitrogen
availability to the plants. The dry soils favour nitrogen mineralization by heterotro-
phic nitrifiers (Angus et al. 2006; Phillips et al. 2015).
Organic nitrogen ! Inorganic nitrogen ðAmmoniaÞ
Proteins constitute a major component of soil organic nitrogen. They are

degraded by extracellular enzymes, such as proteinases and peptidases, that are
being released by soil microorganisms, and proteases are exudated by plant roots
that degrade the proteins into amino acids (Vranova et al. 2013). The proteolysis or
protein depolymerization is dependent on moisture, temperature, soil pH, enzymes
sorption on clay and sesquioxides, parent material, and land use (Noll et al. 2019).
The important microorganisms involved are Clostridium histolyticum,
C. sporogenes, Proteus sp., Pseudomonas sp., and Bacillus sp. These are more
active with proteolytic activity and degrade proteins. Peptidases are widespread
among many soil microorganisms (Bagyaraj and Rangaswami 2007).
Proteinases peptidases
Proteins ! Peptides ! Amino acids
The amino acids obtained through protein degradation are further acted by a
different group of microorganisms in which the amino groups from the amino acids
are displaced, and this phenomenon is called deamination. This reaction is com-
monly brought out by Proteus sp., Clostridium sp., Micrococcus sp., etc. In aerobic
conditions, amino acids are deaminated to organic acids, alcohol, or aldehyde
(Coyne and Coyne 1999).
R CH NH2 COOH þ H2 O ! RCHOH:COOH þ NH3

Organic acid
R CH NH2 COOH þ H2 O ! RCH2 OH þ CO2 þ NH3
Alcohol
R CH NH2 COOH þ H2 O ! RCHO þ HCOOH þ NH3
Aldehyde
Under anaerobic conditions, amino acids are converted to ammonia and volatile
fatty acids (Greenwood and Lees 1956).
CH3 :CH2:CHNH2 :COOH ! CH3 CH ¼ CH:COOH þ NH3
For nucleic acid decomposition, DNA is attacked by deoxyribonucleases and

RNA by ribonucleases, which are together called nucleases into mononucleotides by
hydrolysis. The key intermediate formed is uric acid, and soil microorganisms
further degrade it to urea and allantoin. The soil microorganisms like Bacillus
spp., Pseudomonas spp., Brevibacterium spp., Arthrobacter spp., Cephalosporium,
Mycobacterium spp., and Aspergillus spp. produce nucleases. From the
mononucleotides, the phosphate group is separated and gets converted to purine or
pyrimidine base still linked to sugars. Finally, the purine and pyrimidine are
separated from the sugars (Antheunisse 1972; Sylvia et al. 2005).
Hydrolysis
Nucleic acid ! Nucleotides
! PO4 þ Sugar þ Nitrogen bases
Furthermore, the nitrogen bases, the purines, and pyrimidines are acted upon by
microbial enzymes. The ammonia thus released is either utilized directly by plants
and soil microorganisms or released into the atmosphere or under favourable
conditions oxidized to form nitrates. The urea thus obtained from the degradation
of nucleic acid is absorbed by plants. Some are converted to ammonia by ureases.
The application of organic and inorganic nitrogenous compounds to the soil alters
the chitinolytic and ureolytic bacterial communities that are involved in mineraliza-
tion by secreting extracellular enzymes (Ouyang and Norton 2020). The key
ureolytic microorganisms are Acidobacteria, Proteobacteria, Bacteroidetes,
Cyanobacteria, and Verrucomicrobia that are identified based on 16S rRNA gene and
Illumina sequencing (Oshiki et al. 2018).
7.2.1.2 Nitrification
The biological transformation of ammonia into nitrite and further to nitrate is
mediated by chemoautotrophic, heterotrophic bacteria and fungi, and comammox
bacteria. In autotrophic nitrification, the first step of conversion of ammonia into
nitrite is done by chemoautotrophic ammonia-oxidizing bacteria (AOB), such as
Nitrosomonas, Nitrosococcus, and Nitrosospira, and ammonia-oxidizing archaea
(AOA), such as Nitrosotalea and Nitrososphaera. The second step involving the
conversion of nitrite to nitrate is mediated by Nitrobacter, Nitrococcus, Nitrospina,
and Nitrospira (Purkhold et al. 2000; Leininger et al. 2006). Heterotrophic nitrifica-
tion is mediated by many soil heterotrophic bacteria and fungi, which oxidizes both
inorganic and organic nitrogen compounds (Hayatsu et al. 2008). Furthermore,
recently two Nitrospira species were reported to have both the genes ammonia
monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO) that catalyse
both steps of nitrification and are termed as comammox bacteria (Daims et al. 2015).
The chemoautotrophic nitrifications are more common than heterotrophic. Ear-
lier, it was thought that nitrification normally occurs between pH 7.0 and 7.6 and
completely stops below pH 4.0. However, recent developments showed that even
below pH 3.0, nitrification occurs and is governed by heterotrophic fungi, and even
above pH 13.0 also nitrifiers are active in the soil. It showed that soil pH has the least
influence on nitrification processes (Weber and Gainey 1962; De Boer and
Kowalchuk 2001). However, another study showed that soil pH plays a significant
role in gross autotrophic nitrification and N2O emission in the agricultural soils (Zhu
et al. 2019a). The nitrification rate is higher with high temperatures and neutral to
alkaline soils. It also showed that inorganic fertilizers’ applications increase nitrate
content and are prone to leaching losses compared to organic nitrogen amendments
(Thangarajan et al. 2015).
The addition of nitrogen inhibitors reduces nitrogen losses by inhibiting nitrifica-
tion rates and preventing N2O emission (Tao et al. 2018). The addition of biochar
in the composting increases nitrate-nitrogen (Liu et al. 2017b). Similarly, a decrease
in soil moisture results in the inhibition of the first step of the nitrification process in
semiarid regions (Tatsumi et al. 2019). Another critical factor is temperature; the
heterotrophic nitrification increases with an increase in temperature from 5 C to
25 C and further decreases with further increase in the temperature. In chemoauto-
trophic nitrification, the rate increased up to 35 C to 45 C. The higher temperature
increases the soil nitrogen availability by nitrogen mineralization in the subtropical
forest having acidic soils (Dan et al. 2019). During composting, the nitrification
process occurs under high temperatures during the curing stage. The conversion of
gaseous ammonia into nitrate takes place in a two-step process mediated by various
chemoautotrophic and heterotrophic microorganisms and is linked to the acidifica-
tion of compost (Cáceres et al. 2018). Similarly, the plant releases different high and
low-molecular-weight organic compounds from the roots that chemoattracts specific
soil microbial communities involved in nitrogen transformation. Some compounds
such as sorgoleone, sakuranetin, Brachialactone, Methyl 3-(4- hydroxyphenyl pro-
pionate, and 1,9-Decanediol are inhibitors of nitrification, which generally block
AMO and HAO (Coskun et al. 2017).
NH3 þ 1½ O2 ! NO2 þ 2Hþ þ H2 O þ 66 kcal mediated by Nitrosomonas sp:
NO2 þ ½ O2 ! NO3 þ 18 kcal mediated by Nitrobacter sp:
To prevent nitrate leaching losses, various strategies are advocated, such as

application of ammonium carrying fertilizer over nitrate-based, development of
plant genotypes with greater NUE, and use of balanced, split-doses, and slow-
releasing nitrogenous fertilizer (Ahmed et al. 2020).
7.2.1.3 Denitrification
The conversion of plant-available nitrate into atmospheric gaseous form is
microbially mediated and is termed as denitrification or dissimilatory nitrate reduc-
tion (Van Spanning et al. 2005). Under microaerophilic or anaerobic conditions, the
denitrifiers use nitrate and other oxidized forms of nitrogen as an electron acceptor
instead of molecular oxygen. The reductive process that involves the conversion of
nitrate to atmospheric N2 gas occurs through a series of enzymes, namely nitrate
reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous
oxide reductase (Nos). These enzymes are involved in the conversion of nitrate to
nitrite (NO2-), nitrite to nitric oxide (NO), nitric oxide to nitrous oxide (N2O), and
nitrous oxide to dinitrogen (N2), respectively (Hochstein and Tomlinson 1988)
(Fig. 7.1). This is an undesirable process mediated by many heterotrophic bacteria
like Thiobacillus denitrificans, Pseudomonas denitrificans, and Paracoccus
denitrificans (Zumft 1997). Additionally, some fungi. Fusarium sp. Gibberella sp.
Cylindrocarpon tonkinense (ascomycetes), and Trichosporon cutaneum-
basidiomycetes and archaea are also involved in denitrification (Hayatsu et al.
2008). Moreover, some nitrogen fixers such as Bradyrhizobium, Azospirillum
(Rösch et al. 2002), and AOB (Nitrosomonas and Nitrosospira) are also involved
in the denitrification process (Shaw et al. 2006).
The nitrogen addition to tropical forests increases the N2O emission rates from
the soil; conversely, phosphorus addition did not affect it. However, under wet
seasons, the interaction between the fertilizer enhances the N2O emission by
increasing microbial activity (Wang et al. 2014). For reducing the N2O emission
in the soil system, Bradyrhizobium carrying nitrous oxide reductase enzymes genes
on the nos gene cluster are advocated in soybean (Sanchez Gomez and Minamisawa
2019). Additionally, molybdenum application to the soil decreases soil pH and
denitrification activities and increases plant biomass, and nitrate nitrogen in the
wheat rhizosphere and was recently documented (Wen et al. 2019).
Another route for conversion of nitrite and ammonium into gaseous nitrogen
directly is performed by microorganisms belonging to the phylum Planctomycetes
termed as anammox (anaerobic ammonium oxidation). The main bacterial genera
involved in this process are Brocadia, Kuenenia, Scalindua, and Anammoxoglobus
(van Niftrik and Jetten 2012). The conversion occurs inside a unique compartment
anammoxosome consisting of a unique lipid ladderane (Jetten et al. 2009). This
mechanism does not involve aeration and requirement of any electron donors. The
possible pathways for the conversion are below.
2NH4 þ þ3O2 ! 2NO2 þ 4Hþ þ 2H2 O, and NH4 þ þ NO2 ! N2 þ 2 H2 O
The denitrification processes are favoured by nitrate, soil temperature (80 F to

100 F), wet soils, and organic carbon availability such as compost, manure, cover
crops, and crop residues. Furthermore, topsoil denitrification rates are higher due to
the availability of organic carbon and higher microbial activities. Other factors that
affect localized denitrifications are fertilizer rate and timing, cover crops, and irriga-
tion. As denitrification is an undesirable process, it can be overcome by proper soil
drainage, nitrification inhibitors, irrigation methods, especially drip and sprinkler
methods.
7.2.1.4 Biological Nitrogen Fixation (BNF)

The biological nitrogen fixation (BNF) is the conversion of atmospheric triple-
bonded inert nitrogen into ammonia (NH3), an enzymatic reduction catalysed by
some prokaryotic nitrogen-fixing soil microorganisms called diazotrophs.
Diazotrophs are widespread in marine and terrestrial ecosystems, either as free-
living or in symbiotic associations with different plants. Nitrogen-fixing prokaryotes
Fig. 7.2 Biochemistry of PYRUVATE + CoA ATP N2

nitrogen fixation
NifH
NifD NifK
NifJ NifF
NifK
NifH NifD
ACETYL CoA + CO2 2NH3+H2

ADP
can be (1) Free-living, aerobic-Azotobacter, (2) Free-living, anaerobic-Clostridium,

sulfate reducing bacteria (SRB), such as Desulfovibrio and some archaea-like
methanogens, (3) Facultative anaerobes-Bacillus polymyxa, Klebsiella pneumoniae,
Escherichia intermedia, (4) Oxygenic photosynthetic bacteria-many heterocystous
and non-heterocystous cyanobacteria, (5) Anoxygenic photosynthetic bacteria-
Rhodospirillum sp., and (6) Associative or endophytic colonizing nitrogen-fixer-
Azospirillum, Azoarcus, Herbaspirillum, Burkholderia, Gluconobacter, and
Acetobacter. These diazotrophs can form nodules or non-nodule former (Mus
et al. 2016). Nodules are formed by rhizobia and are specific to legumes and are
cross inoculation groups. Similarly, an actinobacteria genus Frankia forms a nodule
with eight different woody plant families and three different orders. The plant
families involved in actinorhizal symbioses are Betulaceae, Casuarinaceae,
Coriariaceae, Dastiscaceae, Myricaceae, Rhamnaceae, Elaeagnaceae, and Rosaceae
(Van Nguyen and Pawlowski 2017). The nodulating root bacteria can be classified as
classical rhizobia, which includes Rhizobium, Allorhizobium, Mesorhizobium,
Azorhizobium, and Sinorhizobium. Moreover, novel rhizobial genera such as
Aminobacter, Cupriavidus, Burkholderia, Devosia, Methylobacterium,
Herbaspirillum, Ochrobactrum, Microvirga, Shinella, and Phyllobacterium
(Selvakumar et al. 2013) help in N fixation. The non-nodule forming belongs to
plant growth promoting rhizobacteria (PGPR) group of microorganisms. In all these
associations and symbioses, symbiotic bacteria fix the atmospheric N2 and provide it
to plants and utilize carbon as an energy source that can be termed as symbiotic
interaction.
It has been estimated that total amount of N fixed biologically on an annual basis
is in the order of 55 Tg N (about 65% of the total input of N) (Crews and Peoples
2005), and it is apparent that BNF contributes significantly to agricultural production
throughout the world. The first nitrogen-fixing soil microorganism was an anaerobe
Clostridium pasteurianum discovered by Sergei Winogradsky in 1983. Similarly,
the first free-living aerobic nitrogen fixer, Azotobacter chroococcum, was isolated by
Beijerinck in 1901. Diazotrophs catalyse atmospheric nitrogen fixation by reduction
of nitrogen into ammonia by an enzymatic complex called nitrogenase. Nitrogenase
is a complex, di-protein, or two-component metalloenzyme, dinitrogenase reductase
(Fe-protein), and dinitrogenase (Mo-Fe protein) are detailed in Fig. 7.2. During the
catalysis process, electrons are transferred from Fe-protein (reducing protein) to the
Mo-Fe-protein (catalytic protein) (Hu and Ribbe 2013; Lee et al. 2019). For every
single electron transfer, two ATP molecules are hydrolysed. Therefore, for fixing a
unit of N2, a total of 16 ATP is required (Igarashi and Seefeldt 2003). Nitrogenase
enzymes catalyse this bioconversion in diazotrophic organisms. Nitrogenase is an
extreme oxygen (O2)-sensitive enzyme. It gets irreversibly inactivated even at low
O2 concentrations (Lee et al. 2019). In the symbiotic or endophytic relationships,
bacteria colonize the host plant root system and form a structure called a nodule,
which protects the nitrogenase from exposure to oxygen.
Overall reaction : N2 þ 8e þ8Hþ þ 16ATP ! 2NH3 þ H2 þ 16ADP þ 16Pi
7.2.1.5 Mechanism of Nodule Formation and Nitrogen Fixation

The mutual communication between specific rhizobia and legume partners is
regulated by the quorum-sensing mechanism (Hirsch and Fujishige 2012). The
symbiotic event is initiated under nitrogen starvation, and BNF is carried out
under a specialized structure called a nodule. The rhizobial partner provides nitro-
gen, which in turn receives photosynthates from the plants. The recognition events
between partners are highly specific. The plant produces chemo-attractants
flavonoids compounds that interact with nod D of bacteria that activates transcription
of nod A, B, and C and produces specific lipo-chitooligosaccharides (LCOs), termed
as nod factors. The formation of LCOs initiates symbiosis signalling events that help
in the deformation of root hairs, curling, rhizobial infection, nodule organogenesis,
and biological N-fixation (Oldroyd et al. 2011).
7.2.2 Phosphorus Transformation
About 95–99% of phosphorus present in the soil are in the fixed form and is
unavailable to plants. It is vital for plant growth and development as it is an integral
component of nucleic acid (RNA and DNA), adenosine triphosphate (ATP), and
phospholipids of the cell membrane. The nutrient use efficiency of phosphatic
fertilizers is low in the soils due to sorption on clay lattices, aluminium and iron
oxides, primary (apatites), and secondary phosphorus minerals (Ca, Fe, and Al
phosphates). In low pH soils, phosphorus in the soil gets fixed in the form of iron
and aluminium phosphate. In contrast, in high pH soils, phosphorus is bound or
precipitated as calcium and magnesium phosphate (Pierzynski et al. 2005). Further-
more, available phosphorus gets immobilized into organic polymers like phytin,
nucleic acids, and phospholipids. The factors favouring phosphorus sorption are
highly weathered soils like oxisols and ultisols, high clay content, low and high pH,
and high temperatures. Factors that decrease phosphorus sorption are anions like
carbonates, sulfates, molybdate and silicates, organic matter, and flooding. The
plant-available forms of phosphorus are dihydrogen phosphate (H2PO4) and
hydrogen phosphate (HPO42-). The phosphorus mineralization and immobilization

are largely determined by carbon: phosphorus (C: P) ratio in the soil. When C: P
ratio < 200:1, net mineralization occurs, and conversely C:P ratio > 300:1, the net
immobilization of phosphorus occurs. In most agricultural soils, a large portion of P
is in fixed form, and therefore to meet the crop demand, P needs to be supplemented
through external phosphorus application. Non-judicious and indiscriminate use of P
fertilizers not only increases the cost of production but also has a negative impact on
soil health. Repeated and indiscriminate use of chemical P fertilizer inhibits the
substrate-induced respiration of microbes (Bolan et al. 1996). It reduces microbial
respiration and metabolic quotient (qCO2) (Thirukkumaran and Parkinson 2002),
thus affecting soil fertility.
The fixed unavailable forms of inorganic and organic phosphorus are solubilized
or mineralized and is a sustainable and eco-friendly through phosphate solubilizing
microorganisms (PSMs) (Krishnaraj and Dahale 2014; Saeid et al. 2018; Rafi et al.
2019). The application of chemical fertilizers, along with PSMs, is advocated for
increased phosphorus assimilation by plants. PSMs solubilize insoluble inorganic
phosphorus and mineralize the organic phosphorus (lecithin, phospholipids nucleic
acids, phytin, etc.). The key bacterial PSMs involved in P solubilization and
mineralization are Bacillus, Pseudomonas, Azotobacter, Paenibacillus,
Enterobacter, Agrobacterium, Erwinia, Serratia, Thiobacillus, Mycobacterium,
Micrococcus, and Bradyrhizobium. Fungal PSMs are Aspergillus, Penicillium,
Trichoderma, Alternaria, Chaetomium, Saccharomyces, Torula, Fusarium, and
Curvularia, etc. (Ingle and Padole 2017; Kalayu 2019). Similarly, the first report
of archaea as a P solubilizer was isolated from high pH, high temperature, and
salinity soils, namely, Natrinema sp. strain IARI-WRAB2 and Halococcus
hamelinensis strain IARI-SNS2 and are found to be P solubilizer by releasing
various organic acids (Yadav et al. 2015). The endophytic bacteria, namely
Aneurinibacillus sp., Lysinibacillus sp., and Bacillus sp. isolated from banana, act
as both P solubilizer and P mineralizer (Matos et al. 2017).
In the P cycle, phosphorus solubilization, mineralization, and immobilization are
essential reactions. The PSMs employ mechanisms such as lowering the soil pH by
releasing various organic and inorganic acids (Saeid et al. 2018), chelation, produc-
tion of H2S, and competing with phosphorus ions for adsorption sites. Among these,
the release of low molecular weight organic acid is known to be a predominant
mechanism of P mineralization. The PSMs releases organic acids such as citric,
gluconic, oxalic, 2-ketogluconic, malic, succinic, fumaric, tartaric, propionic, acetic,
glyoxylic, isobutyric, isovaleric, itaconic, lactic, and aspartic acid (Alori et al. 2017).
Apart from that, the PSMs during metabolism also release the CO2, NOx, SOx that
interact with water to form carbonic acid, nitric acid, and sulfuric acid, respectively.
The hydroxyl and carboxyl groups of released acids get chelated with the cations
bound to the phosphate and convert insoluble phosphate to soluble form
(Mohammadi 2012; Mardad et al. 2013; Kalayu 2019) (Fig. 7.3). The PSMs are
involved in mineralization of unavailable organic forms into available forms through
enzymatic action. Enzymes such as phosphatases and phytase convert phosphorus
into a plant-available form. The organic phosphorus compounds are nucleic acids
Fig. 7.3 Microbial-mediated transformation of phosphorus and potassium in the soil
(DNA, RNA), phospholipids, phytic acid, and polyphosphates. Many

microorganisms produce various acid and alkaline phosphatases that mineralize
organic to inorganic forms. The organisms involved in mineralization are Bacillus
subtilis, Arthrobacter, Streptomycetes, Aspergillus, and Penicillium. Yeasts are also
involved in the transformation of phosphorus by producing phytases that mineralize
organic phosphorus, especially when P is in limited condition (Nakamura et al.
2000). Furthermore, the soluble phosphorus may also be immobilized through
assimilation into microbial nucleic acids, phospholipids, or other protoplasmic
substances.
The use of PSMs such as Pseudomonas plecoglossicida, and Pantoea cypripedii
along with rock phosphate enhanced the yield of wheat and maize by increasing
available P, total P, organic carbon, and enzymatic activities (Kaur and Reddy 2014).
The three Bacillus species (B. megaterium, B. subtilis, and B. cereus) added to
poultry bone, fish bones, and ash was found to produce organic acid which decreased
the pH and solubilized phosphorus, and Bacillus megaterium along with fish meal
combination was better in P solubilization (Saeid et al. 2018). Similarly, PSM
inoculation increased the phosphate content in tricalcium phosphate-enriched com-
post through a decrease in pH and enhanced organic acid production (Wei et al.
2018). Two promising P solubilizers, namely, Pseudomonas sp. MR7 and
Acinetobacter sp. MR5, produced gluconic acid and possess a glucose dehydroge-
nase gene that promoted plant growth in rice (Rasul et al. 2019). Fungal isolates
Trichoderma koningiopsis (NBRI-PR5) produce organic acids, alkaline phosphatase
enzymes for tricalcium phosphate solubilization, and accumulate polyphosphate
under high pH and drought stress (Tandon et al. 2019). The two fungal and bacteria,
namely, Aspergillus niger and Acinetobacter, respectively, are found to solubilize
calcium phosphates (Li et al. 2019). The conditions favouring phosphorus solubili-
zation are temperature (25–30 C), warm, humid climate, aeration, organic matter,
crop rotation, compost addition, and soil pH (6–7.5) (Alori et al. 2017). The increase
in soil organic carbon content and soil pH also enhances phosphorus availability
(Nobile et al. 2020). Similarly, drought stress associated with a decrease in soil pH
helps in the solubilization of calcium phosphates and increases phosphorus avail-
ability in temperate forest soil (Zhang et al. 2020). Moreover, the cultivation of
leguminous trees enhances phosphorus availability and could be used to reclaim
weathered soils (Aleixo et al. 2020).
7.2.3 Potassium Transformation
Potassium (K) is another essential macronutrient needed in a large quantity after

nitrogen and phosphorus for plant growth. Approximately 80–90% is present in the
unavailable form (Bhattacharya et al. 2016; Haro and Benito 2019). Therefore,
potassium-solubilizing bacteria (KSB) can replace chemical fertilizer and enhance
soil quality and crop yield (Dong et al. 2019).
In soil, K is present in four different pools- (1) unavailable K (K bound within
minerals, i.e., feldspar and mica), (2) fixed- K/captured K (within clay minerals),
(3) exchangeable K, and (4) soil solution K (readily available K). The exchangeable
K is found to be in equilibrium with soil solution K. The exchangeable K and soil
solution K are plant available pools of the K, but the size of these two pools is very
small. The size of the exchangeable K pool is 1–2%, and the soil solution pool is
0.1–0.2% of the total K in the soil. To reduce the chemical K fertilizer needs and
meet the plant K requirement, microorganisms can be used as biofertilizers.
Diverse soil microorganisms are known to solubilize insoluble and unavailable
forms of potassium into available forms, thereby increasing soil solution K concen-
tration (Keshavarz Zarjani et al. 2013 and Sahu et al. 2018). Some selected
potassium-solubilizing bacteria (KSBs) are Bacillus edaphicus, B. mucilaginosus,
B. circulans, Acidithiobacillus ferrooxidans, Burkholderia, Pseudomonas,
Paenibacillus, and nitrogen-fixing rhizobia. Potassium solubilizing fungi (KSFs),
namely Aspergillus terreus and mycorrhizal fungi, are also involved in K solubili-
zation (Meena et al. 2014a Sattar et al. 2018). These KSMs (Table 7.2) employ
various mechanisms such as lowering the soil pH, acidolysis, production of various
organic and inorganic acids, chelation, exchange, and complexolysis by dissolving
potassium minerals such as orthoclase, illite, mica, feldspar, biotite, and muscovite
(Etesami et al. 2017) (Fig. 7.3). The organic acids intermediates of the Krebs cycle of
the soil microorganisms, namely oxalic, citric, succinic, tartaric acids, etc., are
responsible for potassium solubilization (Sahu et al. 2018). Apart from this, some
of the KSMs also release malic, coumaric, syringic, ferulic acids and have been
reported to solubilize soil K (Sahu et al. 2018; Setiawati and Mutmainnah 2016).
Microbial decomposition of soil organic matter produces ammonia and H2S. These
by-products, in turn, are oxidized to form strong inorganic acids such as nitric and
sulfuric acids. The H+ ions displace K+ from the cation exchange complex in the soil.
The polymers such as exopolysaccharides (EPS), lipopolysaccharides, and biofilms
produced by the KSM also play a significant role in K solubilization. These EPS
Table 7.2 Potassium-solubilizing bacteria and fungi

Microorganisms References
Acidithiobacillus thioxidans Calle-Castañeda et al.
(2018)
Bacillus edaphicus, B. mucilaginosus, and Arthrobacter sp. Keshavarz Zarjani et al.
(2013)
Bacillus megaterium, Bacillus horikoshii, Bacillus Verma et al. (2015)
amyloliquefaciens, Exiguobacterium antarcticum, Achromobacter
piechaudii, Stenotrophomonas maltophilia, Klebsiella sp.
Paenibacillus mucilaginosus, Bacillus muciloginosus AS1153 Liu et al. (2012)
B. pseudomycoides Pramanik et al. (2019)
Janthinobacterium sp. Rajawat et al. (2019)
Bacillus megaterium and Bacillus subtilis Anjanadevi et al. (2016)
Arthrobacter, Cladosporium sp., Enterobacter hormaechei (KSB-8), Meena et al. (2016)
Bacillus circulans, Aspergillus terreus, Glomus intraradices, Glomus
mosseae, Ectomycorrhizal fungi
Bacillus mojavensis JK07 Feng et al. (2019)
Agrobacterium tumefaciens, B. mucilaginosus, B. circulanscan, and Meena et al. (2015)
B. edaphicus
Bacillus circulans XD-K-2 Shen et al. (2016)
Ensifer adhaerens L18 Peng et al. (2017)
Bacillus methylotrophicus DD-1 Liu et al. (2020)
enhance the dissolution rate of feldspars by making complexes with a framework of

ions into the solution (Welch and Vandevivere 1994). Similarly, biofilm formation
increases the retention time of water and enhances the potassium weathering by
creating conducive micro-environment around the microbial cells for weathering
(Meena et al. 2014b) The potassium-solubilizing ability of microbes depends on
factors such as the source of potassium, pH and oxygen concentration (Etesami et al.
2017), and the type of carbon source (Bhagyalakshmi et al. 2012).
7.2.4 Sulfur transformation
Sulfur is a secondary nutrient that is required for all life forms. In agricultural soils,
sulfur exists mainly (95%) in organic forms as ester sulfates or carbon-bonded form
(Kertesz and Mirleau 2004). The sources by which sulfur gets added to the soil are
through organic matter decomposition of plant and animal residues, weathering of
sulfur-containing minerals, and atmospheric deposition (Saha et al. 2018). The sulfur
is categorized into organic and inorganic sulfur. The organic sulfur is present as ester
sulfates (C-O-SO3) in the form of choline sulfate, sulfated polysaccharides, and
carbon bonded sulfur (C-S) in the forms of amino acids such as methionine, cysteine,
and sulfo-lipids (Neptune et al. 1975). The inorganic forms like SO2, SO3, SO4, and
H2SO4 are common in soil and are added through various biochemical processes. All
these forms of sulfur are cyclically metabolized in the soil to have an eco-balanced
environment (Fuentes-Lara et al. 2019).
The soil microorganisms mediate four key sulfur transformation processes. They
are oxidation of inorganic sulfur compounds, reduction of sulfates and other anions
to sulfides, microbial assimilation, and immobilization through organic matter
decomposition. (Vidyalakshmi et al. 2009; Anandham et al. 2011).
1. Microbial oxidation of reduced inorganic sources of sulfur-like sulfide, sulfite,

and thiosulfate to sulfate is mediated by a diverse group of microbes ranging from
chemo- and photo-lithoautotrophic sulfur oxidizers (Ghosh and Dam 2009) such
as Thiobacillus ferrooxidans and T. thiooxidans (Zhi-Hui et al. 2010), the green
sulfur bacteria (Chlorobium and Chromatium), purple sulfur bacteria
(Rhodopseudomonas) and filamentous bacteria such as Thiothrix, Thioploca,
and Beggiatoa. The heterotrophs that mineralize S are Bacillus, Arthrobacter
Pseudomonas Aspergillus, Alternaria tenuis, Myrothecium, and Penicillium that
are involved in sulfur oxidation.
The sulfur-oxidizing pathway differs from microbes to microbes. The facultative

chemolithotrophic, alphaproteobacteria employ the Paracoccus sulfur oxidation
pathway with the Sox enzyme system (Friedrich et al. 2005). The photo-lithotrophic
sulfur oxidizer such as Allochromatium vinosum employs a branched thiosulfate
oxidation pathway in which truncated Sox and reverse dissimilatory sulfite reductase
enzymes are involved (Hensen et al. 2006). Similarly, Tetrathiobacter kashmirensis,
a chemolithoautotrophic beta proteobacterium, employs a tetrathionate intermediate
pathway (S4I) to oxidize thiosulfate (Ghosh et al. 2011). The oxidation of sulfur
occurs faster in warm moist soil with high organic matter content and alkaline soil
than in acidic soil (Germida and Jansen 1993; Zhao et al. 2015). In extremely acidic
conditions, the sulfur-oxidizing species of the genus Acidithiobacillus such as
A. thiooxidans, A. albertensis, A. ferrooxidans, A. ferridurans, A. ferriphilus are
dominant and play a significant role in sulfur biogeochemical cycling (Zhang et al.
2019). The distribution of such acidophiles in sulfur-enriched niches plays a major
role in the acidification process, particularly in natural water bodies (Colman et al.
2018).
Oxidation is a key process in sulfur transformation since SO42, which is the
available plant form and depends upon oxidation of the completely mineralized form
of sulfur in the soil. The sulfur oxidizer Proteus mirabilis BUFF14 bacterium
isolated from cow dung increased the plant growth and yield of Foeniculum vulgare
crop (Dhiman et al. 2019). The bacterium Thiobacillus thiooxidans could reclaim
saline-alkali soil (pH 7.5 to 8) because of its capacity to release sulfuric acid from
hydrogen sulfides and many other sulfides (Bao et al. 2016). Similarly,
Acidithiobacillus spp., an acidophilic SOB, was involved in sulfur transformation
in acid soil (Wang et al. 2019). Some portion of the available sulfur gets assimilated
into organic compounds by microbes (Fuentes-Lara et al. 2019) apart from utiliza-
tion by plants.
Fig. 7.4 Microbial-

influenced sulfur oxidation
and reduction reactions
2. The elemental sulfur that is made available as sulfate S+6 is further microbially
reduced to hydrogen sulfide. Reduction of sulfate into H2S by sulfate-reducing
bacteria (SRB) such as Desulfotomaculum, Desulfomonas, and Desulfovibrio is
termed as dissimilatory sulfate reduction, and this transformation is not desirable
with regards to agricultural perspective (Mori et al. 2018) (Fig. 7.4).
Sulfate-reducing bacteria (SRB) are obligate anaerobes and have desulfurases and
bisulfate reductase enzymes for sulfate reduction. The H2S produced is further
transformed into elemental sulfur by various green and purple-sulfur bacteria. The
bacterium Desulfurivibrio alkaliphilus can reduce sulfate and dissimilatory nitrate
reduction processes in the soil (Thorup et al. 2017). Sulfate-reducing microbes are in
abundance in marine and wetland ecosystems (Pester et al. 2012; Bowles et al.
2014).
The assimilatory sulphate reduction (ASR) process utilizes reduced sulfur
compounds for the synthesis of cellular biomolecules (Bolten et al. 2010), whereas
in the dissimilatory sulphate reduction process microbes utilize sulfur, sulfite, sulfate
or elemental sulfur as terminal electron acceptor (Grein et al. 2013) for energy
generation, with the involvement of dissimilatory sulfite reductase (DsrAB) and
adenosine 50 phosphosulfate reductase (AprAB) enzymes under anaerobic condition
(Rabus et al. 2015). The DsrAB genes are distributed among the Acidobacteria and
Chloroflexi phyla (Anantharaman et al. 2018). The elucidation of the involvement of
Dsr C protein with the dissimilatory sulfite reductase (DsrAB), the key enzyme in
DSR, provide bio-energetics insights as the activity of these proteins are linked to
chemiosmotic coupling (Venceslau et al. 2014). Under anaerobic conditions, nitro-
gen and sulfur cycles are coupled, and this process was first elucidated by
Fdz-Polanco et al. (2001a, b) and is termed as sulfate-reducing ammonium oxide
(SROA). The SROA constitutes direct oxidation of ammonium into di-nitrogen,
oxidation of ammonium to nitrite, and ammonium to nitrate (Fdz-Polanco et al.
2001a, b). The Anammoxoblobus sulfate and Bacillus benzoevorans have been
reported as SROAs (Liu et al. 2008; Cai et al. 2010).
The organic sulfur compounds like sulfur-containing amino acids (cysteine, and
methionine), proteins, lipids, and biotin are either ester sulfates (C-O-SO3), or
carbon-bonded S (C-S bond) and get microbially mineralized in soil (Ghani et al.
1992) The key enzymes involved are sulfatases and rhodanese. The rate of organic
sulfur transformation depends on the cropping system. It is influenced by the
microbial biomass present in the soil (Castellano and Dick 1991), and the influence
is attributed to the amount and composition of the root exudates. The bacterium
Pseudomonas putida S-313 could mineralize ester sulfates for sulfur nutrition in
plants (Kertesz and Mirleau 2004). Under high-temperature terrestrial environment,
the thermophilic Firmicutes (Bacillales) group of microorganisms are involved in
organic sulfur compounds mineralization into sulfate (Santana et al. 2016).
3. Immobilization is a reduction process, where inorganic sulfate compounds are

converted into sulfate esters and then to carbon bound sulfur inside the living
organisms (Kertesz and Mirleau 2004). The most important cellular constituent
that contains sulfur is the amino acids and immobilized sulfur that are assimilated
into cellular organic matter by covalent bonding (Strickland et al. 1987). The
primary factor that decides sulfur immobilization is the C:S ratio, wherein it is the
dominant factor in soils with C: S ratio of 50:1 and above.
7.3 Microbial Transformation of Micronutrients
7.3.1 Nutrient Transformation of Zinc
Zinc is an essential micronutrient needed in minute quantities (5–100 mg kg 1) for

plant development. It is necessary for the biosynthesis of auxins, carbohydrates,
cytochromes, nucleotides, and chlorophyll in the plants (Singh et al. 2005). How-
ever, the availability of zinc is reduced in high pH soils, arid soils, low organic
matter, high magnesium to calcium soils, bicarbonate content, phosphorus, and iron
in the soil (Goteti et al. 2013). Moreover, zinc is converted into an unavailable form
such as zinc hydroxide in soils with high pH, as zinc carbonate in calcium-rich alkali
soil, as zinc phosphate in near neutral to alkali soil, and as zinc sulfide in low oxygen
environment like flooding. The applied zinc fertilizers are readily converted into
insoluble forms (Alloway 2008a, b), thereby decreasing the bioavailability of zinc
for plant uptake.
The zinc-solubilizing microorganisms (ZSMs) are Bacillus, Pseudomonas
fluorescens, P. striata, Azospirillum, Gluconacetobacter diazotrophicus, Serratia
marcescens, S, liquefaciens, Burkholderia cenocepacia, Burkholderia sp.,
Acinetobacter sp., and Bacillus thuringiensis (Kumar et al. 2019). The fundamental
mechanisms are through the production of organic acids, such as gluconic acids, and
inorganic acids, such as H2SO4, HNO3, and H2CO3 that acidify the rhizosphere and
chelate that sequester zinc cations (Kumawat et al. 2017). Other mechanisms are the
production of siderophore and exopolysaccharide and via modification of root
system architecture (Hussain et al. 2018). An endophyte diazotroph
Gluconoacetobacter diazotrophicus PAL5 supplemented with glucose produced
5-ketogluconic acid, which solubilized ZnO (Saravanan et al. 2007). Similarly, the
Bacillus aryabhattai strain MDSR7 and MDSR 14 solubilize insoluble zinc

compounds by reducing the pH due to the secretions of organic acids. These two
strains promote plant growth of soybean and wheat crops. Moreover, they also
improve soil health by increasing microbial respiration, microbial biomass carbon,
β-glucosidase, and dehydrogenase activity (Ramesh et al. 2014). Furthermore, many
Bacilli strains isolated from the soybean rhizosphere, namely, Bacillus anthracis,
B. cereus, B. thuringiensis, B. subtilis, and B. tequilensis were found to be zinc
solubilizer and increased the soybean yield (Khande et al. 2017). Another phospho-
rus and zinc solubilizer strain, Bacillus sp. strain AZ 17, and Pseudomonas sp. strain
AZ 5 produce organic acid and IAA that increase plant biomass nodulation, phos-
phorus, and zinc uptake, and yield of chickpea grown in the rain-fed area (Zaheer
et al. 2019). The Aspergillus niger fungus isolated from maize rhizospheric soils was
found to produce organic acids, such as oxalic, citric, malic, fumaric, and succinic,
that acidify the soil and help in the solubilization of insoluble tricalcium phosphate
and zinc phosphate (Bakri 2019). The bacterium Pseudomonas sp. RZ 1 isolated
from the rice phyllosphere produces acetic acid and gluconic acid and solubilizes
insoluble zinc (Jaivel et al. 2017). The ZSMs were isolated from the cultivated
agricultural field from Coimbatore, Tamil Nadu, India, from eight different crops.
They recovered Enterobacter aerogenes (ZSB-13), Mycobacterium brisbanense
(ZSB-10), Xanthomonas retroflexus (ZSB-23), Stenotrophomonas maltophilia
(ZSB-1), and Pseudomonas aeruginosa (ZSB22) based on gluconic acid production
and IAA production and screened qualitatively (Sunithakumari et al. 2016). The
ZSMs isolated from maize rhizosphere and tested qualitatively and quantitatively for
zinc and phosphate solubilization. They found three efficient bacterial strains,
namely Bacillus aryabhattai (S10 and ZM 31) and Bacillus sp. (ZM 20). These
isolates promoted maize plant growth due to multifarious plant growth-promotion
activities and could be used in Zn biofortification (Mumtaz et al. 2017). The
Acinetobacter and Burkholderia strain solubilized Zn, thus promoted plant growth
and yield in rice (Vaid et al. 2014). Similarly, another ZSB, the Bacillus megaterium,
solubilized zinc and enhanced plant growth and yield in Capsicum annuum (Bhatt
and Maheshwari 2020). The siderophore-producing and zinc-solubilizing bacterium
Pseudomonas japonica was found to increase plant growth in maize (Eshaghi et al.
2019). However, the toxicity of higher levels of zinc to the plants was also reported.
The Serratia sp. was found to be zinc tolerant and had multiple plant growth
promotion potential and could be used for higher Zn-containing areas (Kour et al.
2019).
7.3.2 Nutrient Transformation of Iron
The iron is the fourth most abundant element in the earth’s crust and is predomi-
nantly available in ferric form (Fe+3) (Radzki et al. 2013; Sahu et al. 2018). The iron
availability is limited in problematic soils such as calcareous, saline, sandy, and
alkaline soils (Alloway 2008a, b). Iron exists in Fe2+ and Fe3+ forms, but the former
is an available form to the plants, and the latter is unavailable. The iron gets oxidized
to Fe3+ form with phosphate, carbonates, and oxide/hydroxide in the soil. The iron
oxidation can take place under aerobic, microaerophilic, and anaerobic environment.
The bacterial iron oxidizer was enlisted by Singh et al. (2018), depending upon
oxygen, pH, and energy requirement are as follows. The acidophilic, aerobic iron
oxidizers are Thiobacillus, Leptospirillum ferrooxidans, Acidithiobacillus
ferrooxidans, Metallogenium, and Sulfolobus.
2Fe2þ þ 0:5O2 þ 2Hþ ! 2Fe3þ þ H2 O 6:5 kcal=mol:
In neutral pH under aerobic conditions, the key iron oxidizer is namely

Gallionella ferruginea, Sideroxydans paludicola, Pseudomonas, Crenothrix
polyspora, Leptothrix ochracea, Siderocapsa geminate, and Pedomicrobium. Simi-
larly, the anoxygenic photosynthetic bacteria, such as green-sulfur bacteria, purple-
sulfur, and purple-non-sulfur bacteria under a reduced oxygen environment, are also
involved in iron oxidation. The key bacterial genera are Chromatium, Rhodobacter,
Rhodovulum, and Thiodictyon. Similarly, iron-oxidizing neutrophilic bacteria that
respire on nitrate as electron acceptors are Escherichia coli, Gallionellaceae sp.,
Paracoccus denitrificans pd. 1222. Iron oxidizers’ important implications in the
oxidation of toxic heavy metals or metalloid in the bioleaching process for
recovering useful elements and sewage and sludge treatment (Singh et al. 2018).
10Fe2þ þ 2NO3 þ 24H2 O ! 10FeðOHÞ3 þ N2 þ 18Hþ
4Fe2þ þ HCO3 þ 10H2 O þ light ! 4FeðOHÞ3 þ ðCH2 OÞ þ 7Hþ
In iron reduction, hydrogen, short and long fatty acids, sugars, amino acids, and
aromatic compounds act as electron donors. Key iron reducers are
Enterobacteriaceae (Escherichia, Serratia, Proteus, and Vibrio), Clostridiae,
Bacillaceae, sulfate-reducing bacteria (Desulfovibrio), Achromobacter, Paracoccus,
Shewanella oneidensis, Stenotrophomonas, and Geobacter sp. (Fig. 7.5). The iron-
reducing bacteria Shewanella oneidensis MR-1 plays a role in the immobilization of
dissolved cadmium at low concentration (Yuan et al. 2018). Furthermore, iron
reduction helps reduce the methylation of arsenic, thereby decreasing the arsenic
uptake by the rice plants (Xue et al. 2020).
Additionally, the soil microorganisms produce low-molecular-weight, iron-
chelating compounds under iron-deficient conditions (<10KD) siderophore, which
chelates iron and zinc and increases the availability to the plants and deprived the
same to the phytopathogen. The siderophore is of hydroxamates, catecholates,
carboxylates, and mixed type (Neilands 1981).
The bacterium Pseudomonas protegens Pf-5 produces two types of siderophore
pyoverdine and enantio-pyochelin for the iron chelation and phytopathogen inhibi-
tion in the soil (Drehe et al. 2018). The siderophore-producing bacterium Pseudo-
monas aeruginosa strain RSP5 enhances the iron availability in the soil and maize
plants (Sah et al. 2017). Two siderophore-secreting bacteria Paenibacillus
illinoisensis YZ29 and Bacillus sp. DZ13 plays a role in iron absorption in the
Fig. 7.5 Microbial-mediated iron and manganese transformation
peanut in alkaline in calcareous soil (Liu et al. 2017a). Similarly, a nitrogen-fixer soil
bacterium Azotobacter chroococcum synthesizes three different types of
siderophores: vibrioferrin, amphibactins, and crochelin A and play a role in iron
availability (Zhang et al. 2019).
7.3.3 Nutrient Transformation of Manganese
Manganese is the fifth most dominant metal element on the earth’s surface. It is
required in minute quantity for photosynthesis and superoxide dismutase enzyme
activity in the plants (Nealson et al. 1988). The manganese transformation in soil
depends on soil pH, oxygen availability, temperature, and soil organic matter content
of a given soil environment (Sparrow and Uren 2014). The Mn+2 oxidation rate is
higher under high pH soils, high temperatures, and elevated O2 concentrations (Uren
2013). However, recently many anoxygenic photosynthetic bacteria such as
Chlorobium under anaerobic conditions have been found to be oxidizing manganese
(Daye et al. 2019).
In acid soils, manganese is present in the bivalent state (Mn2+), whereas in natural
and alkaline soils, it occurs in the trivalent or tetravalent state (Mn4+), which are not
readily available to the plants.
The trivalent and tetravalent oxidation state forms insoluble oxides and adsorbs
many metals and radionuclides such as cadmium, copper, cobalt, and nickel.
Whereas bivalent oxidation states also form insoluble minerals and are required in
trace amounts in the plants. However, it is toxic at a higher concentration (Nealson
2006). Therefore, the oxidation state of the element is the primary factor affecting its
absorption by the plants. Many heterotrophic bacteria and fungi mediate the manga-
nese oxidation in the soil. The common bacterial Mn2+ oxidizer includes Bacillus,
Azotobacter chroococcum, Leptothrix discophora, Pedomicrobium sp.,
Rhodobacter, Arthrobacter, Hypomicrobium, Caulobacter, Pseudomonas
chlororaphis, Proteus, Chromobacterium, Vibrio, Escherichia coli,
Oceanospirillum, Corynebacterium, Enterobacter, Flavobacterium, and
Citrobacter (Tebo et al. 2005, Nealson 2006, and Wang et al. 2017a, b) (Fig. 7.5).
The possible mechanisms of manganese oxidation are direct and indirect. The
indirect mechanisms include free radical formation (hydrogen peroxide, hydroxyl
radical, and superoxide formation), via modification of redox potential by oxygen
production, increasing soil pH, and via chelators (organic chelators and manganese
oxides). In contrast, the direct mechanisms for manganese oxidation are through
oxidases enzymes and manganese binders such as bacterial cell components such as
glycocalyx and proteins (Nealson 2006).
The principal fungal genera involved in Mn oxidation are Metallogenium,
Phanerochaete chrysosporium, Cephalosporium, Helminthosporium, and
Curvularia (Tebo et al. 2005). On the other hand, most common manganese reducers
are Bacillus, Clostridium, Pseudomonas, Micrococcus, Acinetobacter, and
Arthrobacter (Ghiorse 1988; Gounot 1994). A facultative anaerobic bacterium
Shewanella putrefaciens is involved in the reduction of Mn and Fe oxides in anoxic
condition (Nealson and Myers 1992). The manganese reduction occurs in acidic and
near-neutral pH in the soil.
Mn2+ + 1/2O2 + H2O ! MnO2 + 2H+

MnO2 + 4H++ 2e ! Mn2+ +2 H2O
7.4 Role of Mycorrhiza in the Nutrient Transformation Cycle
The arbuscular mycorrhizal fungi (AMF) are associated with about 80% vascular
plants and make nitrogen and phosphorus nutrients available to the plants and takes
carbon in the form of lipids from the plants (Wang et al. 2017a, b). The AMF
employs two pathways for phosphorus absorption. First, through direct nutrient
absorption in the root epidermis and root hairs, second via arbuscules or hyphal
coiling, which provide bi-directional nutrient exchange site (Smith and Andrew
Smith 2011). These two pathways operate independently and coordinate due to the
involvement of different nutrient transporters and cell types: (1) mobilization and
acquisition by forming extensive extraradical mycelium (ERM) and (2) transporta-
tion of absorbed nutrients across the fungal–root interface (arbuscules or intracellular
coils) (Ferrol et al. 2019). The highly coiled mycelium termed as extraradical
mycelium absorbs inorganic phosphorus through fungal importers. The transfer of P

occurs in the form of negatively charged polyphosphate granules, which finally
export through intraradical mycelium (IRM). The unique phosphate transporter
system and proton ATPases associated with different hosts were listed by (Wang
et al. 2017a, b). This transport system is an energy-dependent system that involves a
series of protonation and deprotonation accompanied by conformational changes,
which creates pores for import and export of phosphate ions (Karandashov and
Bucher 2005). It was observed that AMF-dependent P transporter dominates the
direct transfer pathway since transporter involved in direct transporter is suppressed
(Volpe et al. 2016). The hyphal branching pattern is specific to the genus, since
Glomus species form profuse branching and anastomosis vicinity to the root,
whereas Scutellospora genus forms long-distant hyphae and less anastomosis and
can go beyond for nutrient uptake (Parniske 2008).
In addition to this, AMF produces large quantities of thermostable, recalcitrant,
hydrophobic glycoprotein termed as glomalin-related soil protein (GRSP) or
glomalin, which help in carbon sequestration, soil aggregate formation, sequestra-
tion of toxic elements such as copper, cadmium, lead, and manganese present in soil
(Gonzalez-Chavez et al. 2004). The glomalin acts as soil glue and play a role in soil
aggregation and chelate with inorganic phosphorus. The AMF outcompete existing
nitrifiers and upregulate the nosZ gene and downregulate the nirK gene, thereby
preventing N2O emissions in the nitrogen cycle. Additionally, the AMF reduces the
nutrients use efficiency by preventing losses through surface runoff, leaching,
denitrification, immobilization, and volatilization losses, further preventing environ-
mental pollution (Babalola and Igiehon 2017; Parihar et al. 2019). The AMF plays a
role in increasing NUE of nitrogen fertilizer by 54% by preventing leaching losses
under different water regimes in tomato plants (Bowles et al. 2018).
Uptake of calcium, magnesium, potassium, and phosphorus increases from soils
deficient in these nutrients in AMF-colonized maize plants, thereby increasing the
biomass of maize plants (Liu et al. 2002). The AMF also provides zinc and nickel
elements to the soybean and lentil plants (Jamal et al. 2002). AMF plays a vital role
in the nitrogen and phosphorus cycle by reducing the losses in terms of leaching and
gaseous N2O fluxes and increases the uptake of both the nutrients in the plants.
Thereby, they increase the N and P use efficiency in the plant-soil ecosystem (Singh
2012 and Bender et al. 2015). AMF extensive extraradical hyphae mobilize immo-
bile nutrients in the soil such as copper, zinc, manganese, and iron, which cannot
absorb directly by the plant root due to low diffusion rate and formation of nutrient
depletion zone created by plant root. However, nutrient uptake is determined by
already existing nutrient content, other nutrient elements, soil type, plant type, and
fungal genera involved. Absorption of copper and zinc depends upon their content in
the soil, whereas manganese and iron depend upon oxidation and reduction.
Similarly, under saline conditions, AMF reduces the uptake of Na+ and Cl and
increases the absorption of magnesium (Sathiyadash et al. 2017). AMF-mediated
zinc nutrition in plant meta-analysis showed that soil texture has a primary
modulator role and positively impacts shoot, root, and fruit. Whereas, for the
shoot, soil pH, and soil Zn concentration and fruit, soil phosphorus content
influences AMF-mediated Zn concentration (Lehmann et al. 2014). The Glomus
intraradices fungus has zinc transporter GintZnT1, which plays a role in zinc
homeostasis and providing Zn nutrition to plants (González-Guerrero et al. 2005).
Similarly, in soybean, plants showed higher dependency on mycorrhiza. When both
mycorrhizal and non-mycorrhizal inoculated plants were analysed, the N, P, K, Mg,
and Ca contents were significantly increased in mycorrhizal inoculated plants. The
role of AMF in other micronutrients such as copper, manganese, and iron uptake and
transfer in crops was studied through meta-analysis (Lehmann and Rillig 2015).
The ectomycorrhizal fungi (ECM) are involved in nutrient mobilization by
producing extracellular enzymes such as peptidases and proteinases, which
hydrolyse the organic nitrogen compounds and convert them into simpler amino
acids, which is further taken up by fungi (Shah et al. 2013). Moreover, the secretion
of many extracellular phosphomonoesterase and phosphodiesterase enzymes by
ECM help in mobilizing soil nutrients. The enzyme phosphodiesterase can mobilize
phosphorus, a structural part of nucleic acids (Nall 2010). Many reports showed that
the ECM could produce siderophores, which bind and form complexes with oxalate
and iron that increases the uptake of potassium by the symbiont. The reducing agents
produced by the ECM increase the acquisition of ions form stable oxides, such as
MnO2, which enhances plant nutrient availability (Lindahl et al. 2001, 2007). The
soil conditions favouring the availability and deficiency of the soil nutrients are listed
in Table 7.1.
7.5 Conclusion and Future Directions
Productive and durable life of the soil is attained if the biotic and abiotic
components, organic matter, microbial biomass, minerals, air, and water are
complementing and supplementing each other with the self-regulating natural mech-
anism. The nutrient flow and cycling of nutrients in an ecosystem involve the
integration of elements present in the atmosphere as gases, unavailable insoluble
minerals in rocks and soils, chemically readily available nutrients, and organically
bound nutrients present in living autotrophs and heterotrophs and dead organic
matter. The soil microorganisms play a vital role in the nutrient transformation of
the soil and its availability to the plants. These microorganisms mediate mineraliza-
tion, solubilization, and transforms into various forms. Many efficient
microorganisms for major and minor nutrients are screened, identified, and exploited
in agriculture. However, there is a need for regional-based microbial strains for better
performance. Furthermore, microbial strains should be isolated from unexplored
sites. Identifying microbes with multi-nutrient transformation capabilities with min-
imum dependence on soil organic matter for food could be another potential area for
soil plant-health microbiologists.
Furthermore, microbial strains having additional abiotic and biotic stresses toler-
ance potentials have always been advantageous for plant growth. Additionally,
omics tools such as metagenomics, metatranscriptomics, metaproteomics, and
metabolomics should be employed to trap the unculturable diversity of
microorganisms, which could be a better nutrient transformer than known culturable
isolates. Additionally, looking for fungal and bacterial consortium rather than any
individual strains should be focused. Simultaneously, their efficient method of
delivery for better performance in the soil for plant fitness should be optimized.
Furthermore, a particular focus on the development of suitable carriers or
formulations increases the microbes’ shelf life.
Acknowledgments The authors are grateful to the editorial team for giving critical comments for
substantial improvement in the quality of this chapter.
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Microbial Indicator of Soil Health:
Conventional to Modern Approaches 8
Dolamani Amat, J. K. Thakur, Asit Mandal, A. K. Patra, and
Kampati Kiran Kumar Reddy
Abstract
Increasing population pressure and rapid urbanization have put added pressure on
land for higher food production per unit area for ensuring food security, which
has led to intensive agriculture resulting in deterioration in soil quality. To protect
the soil from degradation and for its sustainable management, soil quality assess-
ment strategies are required. Hence, monitoring of soil quality is important and
can be achieved through assessment of physical, chemical, and biological
parameters of soil. Among the different soil quality parameters, biological
parameters are more responsive to changes in management practices. With the
accessibility of new tools and techniques, assessments of microbiological
parameters have become more rapid, reproducible, and informative. The shift in
conventional methods like microbial biomass carbon and soil respiration to
modern tools like molecular methods and high-throughput enzyme assay
techniques not only gives more information about what is happening in an
ecosystem, but also helps to formulate a strategy for sustainable land management
for future.
Keywords
Microbial indicator · Soil health · Microbial biomass · Soil enzyme · Microbial
diversity
D. Amat (*) · J. K. Thakur · A. Mandal · A. K. Patra

ICAR—Indian Institute of Soil Science, Bhopal, Madhya Pradesh, India
K. K. K. Reddy
ICAR—Directorate of Groundnut Research, Junagadh, Gujarat, India

23, https://doi.org/10.1007/978-981-15-9154-9_8
214 D. Amat et al.
8.1 Introduction
Soil sustains civilization; hence, it is imperative to restore and sustain the soil health
for a healthy civilization. Being an important natural resource, soil health is funda-
mental for sustainable food production, the very basic need of all organisms (Awale
et al. 2017). Different ecosystem services rendered by soil include soil aggregation,
increased retention and availability of water, nutrient cycling, microbial diversity
and functions, detoxification, filtration, etc. Soil health represents the physical,
chemical, and biological conditions of soil which have important role in agricultural
productivity in the long term with little environmental impacts. It gives an overall
view of performance of soil. Soil health cannot be assessed directly, rather it can be
ascertained by the measurement of important soil properties, soil organic matter, and
soil fertility status (Arias et al. 2005).
The terms “soil health” and “soil quality” have been used interchangeably in the
literature. Soil health has been defined by different researchers in different ways. In a
broad ecological sense, soil quality has been defined as “the capacity of a soil to
function within the ecosystem boundaries to sustain plant-animal productivity,
maintain or enhance water and air quality and support human health and habitation”
(Karlen et al. 1997). Doran et al. (1996) defined soil quality as “The capacity of a soil
to function within ecosystem boundaries to sustain biological productivity, maintain
environmental quality, and promote plant and animal health.” Similarly, Doran and
Safley (1997) integrated into sustaining nature of the soil functions or services and
defined soil quality as “the continued capacity of soil to function as a vital living
system, within ecosystem and land use-boundaries, to sustain biological productiv-
ity, promote the quality of air, water and maintain plant, animal and human health.”
Singer et al. (2000) related the term soil quality to agricultural productivity and soil
fertility. The characteristics of a healthy soil are: it should be stable, resistant to
stress, biologically more diverse, and should have higher internal nutrient cycling
(Elliott and Lynch 1994). It should also retain and release water and nutrients
properly, encourage and sustain proper root growth, maintain soil biotic habitat,
respond to management practices, and resist from soil degradation (Manna et al.
2013). Similarly, the characteristics of deteriorated soils are: prone to erosion,
extremely low or high soil pH, high salt content, compact subsoil and poor physico-
chemical properties, low organic matter content and soil biomass carbon, and
decreased diversity and activity of soil fauna (Saha et al. 2012).
Soil health assessment helps in the evaluation of suitability of a soil to carry out
selected functions and its resilience. Researchers, growers, and land managers give a
relative value to soil health using different qualitative and quantitative indicators
(Awale et al. 2017). For example, soil compaction may lead to the loss of soil
structure, restricted air and water infiltration, and poor development of root, making
the soil less productive compared to a well-structured soil. Here, the fitness of the
soil for proper root growth (soil function) can be judged by assessing penetration
resistance and bulk density (soil health indicator) of soil with the use of standard
methodology. Estimation of soil physical, chemical, and biological properties that
are sensitive to changes due to management practices provides knowledge about the
8 Microbial Indicator of Soil Health: Conventional to Modern Approaches 215
different soil processes. Compared to physical and chemical parameters that undergo
slow changes to management practices (Ghosh et al. 2020), soil biological and
biochemical parameters are prone to little variations in soils and could give accurate
information on variation of soil quality (Paz Ferreiro and Fu 2016). Sensitivity of
soil health indicators varies in different agro ecosystems. Hence, selection of most
sensitive soil health indicators is required with respect to spatio-temporal variations.
From a large dataset of microbiological soil health indicators, selection of group of
indicators that are most sensitive forms the ‘Minimum Data Set’ (MDS) which can
be obtained using ‘Principal Component Analysis’ (PCA), cluster analysis, multiple
regression analysis, multiple corelation analysis, etc. (Patra et al. 2015). The chapter
focuses on importance of microbiological soil health indicators and different
techniques available for its assessment with inclusion of some modern techniques.
8.2 Indicators of Soil Health
Assessment of soil health needs the selection of soil health indicator for evaluation of
its status (Arias et al. 2005). Soil health indicators are properties of soil that can be
determined to provide facts regarding the capacity of the soil to render essential
environmental services. Among the different indicators, those which are quick to
respond to the management practices are the most appropriate ones (Arshad and
Martin 2002). These indicators are usually related to a particular soil property and
are of two types, i.e., stable and dynamic. Soil texture, its type, and depth are
considered as stable properties. Soil-forming factors like climate, topography, parent
material, and organisms influence the stable soil properties which undergo little
change with different management practices. Similarly, dynamic properties, soil pH
and organic matter, can change with land use and management practices over short
period of time.
Many soil physical, chemical, and biological parameters (Table 8.1) have been
suggested as useful soil quality indicators (Arshad and Martin 2002; Anderson 2003;
Schloter et al. 2003; Winding et al. 2005).
Soil quality characterization requires the selection of the soil properties that are
most sensitive to changes in management practices (Yakovchenko et al. 1996). It has
been proved that microbiological processes can respond to disturbance and alteration
in land use systems within short period of time compared to other soil properties. In
addition, these are directly related to the ecosystems processes like recycling of
nutrients and soil aggregation (Doran and Zeiss 2000; Rillig 2004). Hence, these
properties serve as early and sensitive indicators of changes due to management
practices such as addition of fertilizers, tillage, crops, and environmental conditions
(Kandeler et al. 1999).
216 D. Amat et al.
Table 8.1 Potential physical, chemical, and biological parameters for soil health assessment
Physical Chemical Biological
Soil color Organic C and N Soil respiration
Aggregate stability Particulate organic matter Potential mineralizable nitrogen
Water infiltration Active carbon Microbial
biomass
Bulk density pH Soil enzymes
Penetration Base saturation and cation exchange Earthworms
resistance capacity
Water holding Electrical Crop condition, and root growth
capacity conductivity
Runoff and erosion Heavy metals Weed and disease pressure
Rooting depth
Source: Awale et al. (2017)
8.2.1 Microbiological Indicators of Soil Health
Microorganisms play a main role in soil organic matter (SOM) decomposition,

degradation of agricultural pollutants, nutrient recycling, formation of stable soil
aggregates, disease suppression, and plant health improvement. Because of the
important role played by microorganisms in many soil processes, they have the
potential to provide a unified measurement of soil health which cannot be achieved
by measurement of physical or chemical properties of soil alone (Nielsen and
Winding 2002). They are more sensitive to the environmental stresses compared
to higher organisms due to close relations with the surroundings and because of
higher surface area to volume ratio. Sometimes changes in population of microbes or
their activity occur before the occurrence of detectable changes in the physical and
chemical properties of soil, and therefore, give an advance indication of improve-
ment in soil characteristics or an early alert for its degradation (Pankhurst et al.
1995). Due to the availability of large number of techniques for assessment of
microbiological processes and enzymes involved in nutrient transformation in soil,
these are used as microbial indicators. In addition to that, microbial processes are
directly responsible for the final outcome of an ecosystem functioning (Stenberg
1999).
Various reasons for using microbial indicators for soil quality evaluation are as
follows (Stenberg 1999):
• They play key role in the organic matter degradation and recycling of nutrients.
• They are more sensitive to the modification in the soil environment.
• Their activities in soil are affected by the all factors that regulate the degradation
and transformation of nutrients.
• They are of dynamic in nature and are closely associated with the microenviron-
ment of soil.
• They show quick response to natural disturbance and environmental pressure

because of short doubling time, intimate relation with the surroundings, and
higher surface area to volume ratio.
• Sustaining of soil fertility and many soil functions are mainly regulated by the
decomposition activity of microbes (Anderson 2003).
8.2.1.1 Characteristics of a Good Microbiological Indicator

Brookes (1993) provided different criteria for the selection of microbial indicators
for the monitoring of soil pollution. The indicators need to be:
• measured correctly and with more precision covering a variety of soil conditions
and soil types
• easy and inexpensive because many analyses have to be carried out
• helpful in accurate determination of pollutant effect in comparison to a control
• more sensitive to show pollution, and at the same, time it should also be robust
enough not to provide false results
• valid scientifically based on current and reliable scientific knowledge used in two
or more sets for making the evaluations more robust.
• apart from above characteristics, it should also show cropping and history of
management in long-term studies and support soil physicochemical properties
(Jordan et al. 1995).
• consistent with basic ecosystem processes in soil and chemical and physical
indicators of soil health (Sharma et al. 1999)
All the microbial indicators of soil health can be categorized into three groups,
i.e., microbial biomass, microbial activity, and microbial diversity (Fig. 8.1)
Fig. 8.1 Microbial Indicators of Soil Health

218 D. Amat et al.
8.2.2 Microbiological Indicators of Soil Health
8.2.2.1 Microbial Biomass

The living constituent of soil comprises of soil organisms (invertebrates and
microorganisms) and living roots. In the biological component of soil, microbial
biomass represents the entire microbial population of soil treated as an entity
(Powlson 1994) and consists of protozoa, fungi, bacteria, actinomycetes, etc. With
regard to metabolic activities, biomass comprises of fungi and bacteria as the
dominant group of soil microorganisms (Anderson and Domsch 1973). The soil
flora and fauna play a crucial role in recycling of nutrients by degradation of organic
matter and in keeping a well-structured soil by formation of soil aggregates, humus,
and providing better aeration. Soil particles aggregations are mostly carried out by
filamentous group of fungi and actinomycetes. Apart from this, extracellular
metabolites, like polysaccharides, lipids, and proteins released by microorganisms,
act as cementing agents and gums for stabilization of aggregates (Gupta and
Germida 1988). Blue-green algae improve soil nutrient status (nitrogen and organic
carbon) in soil of arid region as they can fix N2 and CO2 from the atmosphere. Most
of the detoxification and decomposition processes in soil are carried out by
microorganisms and are facilitated by the pulverizing and burrowing actions of
invertebrates (Van Gestel and Van Straalen 1994). Microbial biomass constitutes a
major pool of nutrient that is perpetually used in the growth of micro and
macrophytes. Soils having high fraction of microbial biomass have the capacity to
store and cycle nutrients in the soil system to a greater extent (Anderson and Domsch
1980). Giacometti et al. (2012) used microbial biomass carbon as microbiological
indicator for evaluation of long-term effect of fertilizer and manure on soil quality
and found that it was a valuable indicator of long-term soil quality changes.
Determinations of microbial biomass can be carried out by both direct and indirect
approaches. Direct approach includes dilution plating, staining techniques along
with epifluorescence microscopy, and automated image analysis (Bloem et al.
1995, 2003). An indirect approach includes DNA measurement, chloroform fumi-
gation incubation and extraction method, fluorescence in situ hybridization
(FISH) etc.
Direct Method
(a) Enumeration by plating
Microbial populations such as bacteria, fungi, and actinomycetes are enumerated by

serial dilution of soil samples and different media specific for the growth of
microorganisms. Choudhary et al. (2018) found that microbial populations varied
with different cropping systems. Though this method is easy, it has limitations like
less than one percent of the microbes can be cultivated using the presently available
media.
(b) Enumeration using fluorescence microscopy

Fluorescence microscopy along with computerized image analysis can determine

bacterial count in soil, cell volumes, and enumeration of dividing cells. In this
technique, a known quantity of water or homogenized soil suspension is put on a
known area of a microscopic slide, fluorescent dye is used to stain the
microorganisms, and numbers are counted using a microscope. Biomass and volume
can be determined using lengths and widths of the recorded image (Bloem et al.
1995). Fluorescent dyes like fluorescein isothiocyanate (Hasebe et al. 1984),
SYTO9, DAPI (40 ,6-diamidino-2-phenylindole) (Johnson and Criss 2013), and
propidium Iodide are used in this method.
Indirect Methods
(a) DNA measurement
Total microbial DNA extraction from soil and its quantification is an easy and
practicable method for quantification of microbial biomass (Girvan et al. 2004).
Nonetheless, further work is required for correlating measurements of DNA with
respect to soil types. Using quantitative PCR, total number of bacteria in soils under
different agricultural management practices was determined (Bach et al. 2002).
Cluzeau et al. (2012) quantified copy numbers of 16S rRNA gene to measure the
size of the bacterial community with real-time PCR. By extraction and quantification
of total soil microbial DNA, Munoz et al. (2017) determined microbial biomass.
(b) Chloroform fumigation method
Jenkinson and Powlson (1976) introduced chloroform fumigation incubation

(CFI) method which is a physiological method for quantification of microbial
biomass. As CFI is not suitable for the soil with low pH and high amount of
degradable organic matter, Vance et al. (1987) developed chloroform fumigation
extraction (CFE) method, a modification of CFI. These two methods along with
substrate-induced respiration (SIR) (Anderson and Domsch 1978) are the most
widely used methods for microbial biomass estimation. In chloroform fumigation
method, the vapors of chloroform are utilized to kill the microbes in the soil samples
and quantity of dead biomass is determined either by quantifying the amount of CO2
released (CFI) or extraction followed by quantification of extractable carbon (CFE)
after fumigation. CFE helps in analyzing microbial nitrogen as well as carbon.
(c) Substrate-induced respiration
Substrate-induced respiration (SIR) method is based on the principle that at

increasing levels of glucose (easily degradable substrate), the initial rate of respira-
tion of a soil is directly proportional to the biomass. The assumptions in this method
are (i) initial rate of respiration in glucose-incorporated soil is equal for different
segments of the microbial population and (ii) measurements of microbial biomass by
chloroform fumigation incubation method are correct. Substrate-induced respiration
measures the evolution of CO2 from soil after adding optimal concentration of
220 D. Amat et al.
glucose. In this method, metabolically active portion of the microbial biomass is

estimated by measuring the change in the initial soil respiration rate after addition of
an easily degradable substrate like glucose. Rate of respiration is correlated with
microbial biomass carbon determined by chloroform fumigation incubation method
(Anderson and Domsch 1978) and then SIR is converted to microbial biomass
carbon using calibration factors.
This method provides rapid and direct information for comparison of microbial
biomass in various soils. But, the limitation is that it is not possible to quantify the
biomass directly. However, using this method, partitioning of biomass can be done
into fungal and bacterial components by use of specific inhibitors. It has been
observed that contributions of bacteria and fungi to soil respiration are 78% and
22%, respectively (Anderson and Domsch 1973).
(d) Microbial ATP content
Measurement of microbial ATP content in soil for estimation of microbial

biomass was introduced by Jenkinson and Oades (1979). In this technique, microbial
cells are disrupted rapidly with the help of suitable reagents; the ATP thus released is
stabilized by deactivation of degradative and synthetic enzyme processes. Then,
ATP is extracted from the soil matrix and its amount is determined by luciferin-
luciferase system to calculate soil microbial biomass contents. The limitation of this
method is that there is rapid degradation of ATP during extraction and it is absorbed
by soil constituents. The measurement of soil microbial biomass is many a time
uncertain because of low recovery, storage, season of collection, and also, it is
weakly correlated to soil microbial biomass measured with chloroform fumigation
method.
(e) Freeze-dried soil extraction method
The principle of this method is that freeze-dried soils when treated with 0.5 M
NaHCO3 or 0.5 M K2SO4 release cytoplasmic carbon compounds from desiccated
and disrupted microbial cells and these are extracted (Islam et al. 1997). The analysis
of extracted carbon is carried out with rapid colorimetric method (Islam and Weil
1998) to calculate the soil microbial biomass contents. This method is safe, reliable,
and precise; however, it requires sophisticated equipments and trained personnel.
(f) UV-Spectroscopic method
In this method of estimation of microbial biomass, absorption of near UV light

(at 280 nm) by nucleic acids/nucleotides of microbial cells from chloroform
fumigated soils is taken into account. There is a significant correlation between the
absorbance and soil microbial biomass measured by chloroform fumigation method.
These methods are fast and simple for determination of microbial biomass. But, the
results are often undermined because of interferences of soil colloidal and precipita-
tion of electrolyte (Nunan et al. 2002).
(g) Rehydration method
Air-dried soils are rewetted with water or dilute salt solution for determination of
microbial biomass (Blagodatskiy et al. 1987). Because of rehydration, the desiccated
microbial cells are disrupted and released intracellular carbon compounds which are
extracted and analyzed by rapid K2Cr2O7 oxidation method for determination of soil
microbial biomass content.
8.2.2.2 Soil Microbial Eco-Physiological Indices

Eco-physiological indices are calculated by basing physiological performances
(carbon uptake, respiration, growth/death, etc.) on the total microbial biomass per
unit time (Anderson 1994). Soil microbial quotient is mostly estimated by microbial
respiration and assimilation which are largely influenced by soil resources and
environmental conditions (Manzoni et al. 2012). Calculation of microbial ratios
have been given by Moore et al. (2000). Ratio of Cmic:Crg (microbial biomass
carbon:soil organic carbon) is related to the percentage of Cmic (microbial biomass)
in the total Corg (soil organic carbon), which indicates the contribution of microbial
biomass to the soil organic carbon (Anderson and Domsch 1989). Cmic:Corg ratio
can also estimate the availability of soil labile carbon. (Moscatelli et al. 2005). As
Cmic mainly relies on the soil labile carbon as carbon source, a high ratio indicates
accumulation of soil labile carbon and suitable environment for growth of microbes,
whereas low ratio indicates poor quality of organic matter. Similarly, Nmic:Ntot
(microbial biomass nitrogen:total soil nitrogen) ratio corresponds to the percentage
of Nmic in the total soil nitrogen (Ntot). Cmic:Csoil and Nmic:Nsoil are important
indicators of assimilation of carbon by microorganisms (Xu et al. 2014). Similarly,
ratio of Cmic:Nmic usually describes the microbial community structure and its
state. A higher fraction of fungi in the microbial biomass is indicated by high Cmic:
Nmic ratio and dominance of bacteria is indicated by low ratio (Campbell et al.
1991).
8.2.2.3 Microbial Diversity

In the modern eras, advanced methodologies have been developed for characteriza-
tion of structural and functional microbial diversity in soil. With the available
conventional cultivation techniques, 80–99% of microbial species cannot be
cultured (Arias et al. 2005). Hence, for the study of these uncultured microbes,
three types of methodologies have been suggested: (i) differentiation between
communities based on their capacity to utilize different carbon sources in microwell
plates (Gardland and Mills 1991); (ii) profiling of microbial phospholipid fatty acids
(PLFA) (Zelles et al. 1994); and (iii) profiling of nucleic acid of soil microbial
population reflecting the genetic diversity of the microbes (Kennedy and Gewin
1997). The results of the first method provide information regarding functional
diversity of microbial community, whereas the later two groups reflect the structural
diversity.
222 D. Amat et al.
BIOLOGTM
The pattern of utilization of different carbon sources by the microbes can be
measured by the BIOLOGTM assay (Gardland and Mills 1991). Briefly, a soil extract
is incubated in a microtiter plate containing upto 95 different carbon sources, and the
microbial activity is indicated by the use of tetrazolium blue (redox dye). For the
study of microbial communities in the soil, distinct carbon sources have been
chosen. It is a qualitative assay and it shows a metabolic fingerprint of the culturable
microbial community in the soil sample. Then multivariate statistics can be used to
show the difference in the profile. The advantageous part of this technique is the
simplicity and requirement of inexpensive equipment.
Phospholipid Fatty Acid(PLFA) Analysis

It involves extraction of lipids from soils and is followed by hydrolysis to release
their respective fatty acids. PLFAs are extracted by single-phase solvent extractions
and analysis is carried out by: (1) analysis of the phosphate after hydrolysis using
colorimetric assay, (2) esterification of fatty acids and its analysis using colorimetric
analysis or gas chromatography (GC) (3) capillary GC, and (4) GC mass spectrome-
try. Phospholipid classes and their abundance are obtained by mass profile in the
mass spectrometry. The presence of signature PLFAs specific to particular microbe
is deduced by using a pure culture. The signature PLFAs widely accepted are:
(1) branched chain fatty acids (iso, anteiso) for Gram positive bacteria;
(2) cyclopropyl fatty acids for Gram negative bacteria; and (3) 18:2ω6, 9 for fungi,
etc. Though PLFA analysis is a widely accepted technique for study of structural
diversity of microbes, it has many limitations as the extracted lipids are not only
from membranes of living organisms and cellular storage compounds, but also from
plant tissues in different stages of decomposition. So use of fatty acids profile poses
difficulties for drawing conclusions about changes in structure of microbial commu-
nity (Drenovsky et al. 2004). But due to their rapid degradation after death of
microbial cell and taxonomic specificity, they are often used as signature molecules
for determination of microbial abundance and structure of microbial community
under various environmental conditions (Frostegard et al. 2011; Ruess and Cham-
berlain 2010).
Fluorescent In Situ Hybridization (FISH)

Fluorescent in situ hybridization techniques help in identification of specific micro-
organism or taxonomic group directly in their habitat (Amann et al. 1995). This
technique requires the fixation of whole cells, hybridization of their 16S or 23S
rRNA with fluorescently labeled oligonucleotide probes, and then viewing of
labeled cells under scanning confocal laser microscope (SCLM) (Hill et al. 2000).
Specific probes can be designed for kingdoms (Eukarya, Eubacteria, and Archaea),
families, genera, species, or subspecies and labeled differentially and used in
combination to observe the presence of several taxonomic groups simultaneously
in a soil sample (Manz et al. 1992). Though FISH technique is extensively applied,
the limitations are that it is time consuming and expensive and requires trained
personnel.
Molecular Microbial Diversity

Molecular methods give detailed view of structures and diversity of microbial
community (Kennedy and Gewin 1997). Genetic diversity is mainly studied by the
analysis of diversity of gene for 16S rRNA for prokaryotes (bacteria, actinomycetes)
and 18S rRNA for eukaryotes (fungi). These genes are present in all microorganisms
and difference in their nucleotide base compositions varies with species. Analysis of
variation in sequence of 16S (and 18S) rDNA in total DNA extracted from soil
microbial communities is examined using three techniques: denaturing gradient gel
electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and
terminal restriction fragment length polymorphisms (T-RFLP).
(a) Denaturing gradient gel electrophoresis (DGGE)
Denaturing gradient gel electrophoresis based on the differences in the melting

point of 200–700 base pair, small DNA fragments. As small as single base substitu-
tion can be detected by this technique (Muyzer 1999). Heat (a constant temperature
of 60 C) and urea (0–7 M) and formamide (0–40%) in a fixed ratio are used as
denaturants. The position in the gradient gel where a domain of a DNA fragment
melts and thus stops migrating is dependent on the nucleotide sequence of that
region (GC%). It generates a molecular fingerprint of the microbial community for
each soil. Each band of a lane in the gel corresponds to a different bacterial species.
Furthermore, this unique band can be excised and sequenced for species
identification.
(b) Temperature gradient gel electrophoresis (TGGE)
The separation of DNA fragments (Heuer and Smalla 1997) is based on a

precisely defined and controllable temperature gradient. As the melting properties
depend on the sequence of nucleic acid, nucleic acids can be separated and sequence
variation can be detected. TGGE was used to decipher bacterial community diversity
of subtropical, subalpine, and temperate soils of the Western Himalaya (Soni et al.
2010). Both DGGE and TGGE are helpful for qualitative and quantitative study of
spatial and temporal variation of microbial community structure. The limitations
include labor intensive process and low reproducibility for bands pattern and
reduced band intensity after separation by electrophoresis.
(c) Terminal restriction fragment length polymorphism (T-RFLP)
In this technique, microorganisms can be differentiated based on patterns derived

from restriction digestion of their DNA (Liu et al. 1997). Total microbial DNA is
extracted from soil sample followed by its PCR amplification with a primer which is
fluorescently labeled. After purification, using a restriction enzyme, the amplicon
mixture is digested, generating fragments of different sizes which are separated by
capillary or gel electrophoresis. With this technique, a fingerprint of microbial
community based on the polymorphism of certain gene can be obtained. T-RFLP
224 D. Amat et al.
is a reproducible, high-throughput, method that helps in semiquantitative analysis of

the diversity of a particular gene in a community. Diversity of soil bacterial commu-
nity in virgin and cultivated soils were revealed using T-RFLP (Buckley and
Schmidt 2001).
There are many limitations of the study of microbial diversity using molecular
methods. Firstly, extraction DNA or RNA representing the microbial community is
always a cumbersome process (Dick et al. 1996). Secondly, polymerase chain
reaction (PCR) that is used in this technique may not amplify all populations at a
time (Kennedy and Gewin 1997).
8.2.2.4 Microbial Activity
Basal Respiration Rate

During soil respiration, the oxidation of organic matter takes place to produce carbon
dioxide and water. CO2 released by soil respiration includes both from soil microor-
ganism and roots of the plant. The respiration is estimated either as the rate of
evolution of carbon dioxide or consumption of oxygen by using methods like
electrical conductivity, titration, infrared spectroscopy, and gas chromatography
(Alef 1995). Estimation of CO2 evolution rate is most frequently used for soil
respiration, as it requires numerous automated, relatively simple equipments for
continuous measurement of CO2 (Nordgren 1988; Heinemeyer et al. 1989). When
the soil contains carbonate, the O2 consumption rate is recommended (Anderson
1982) as there is a release of abiotic CO2 which may interfere with the results. Under
standardized conditions of water tension and temperature, the preincubation and the
actual measurement should be carried out for reproducible and comparable results.
Organic Matter Decomposition

Any disturbance to the activity of the microorganism will alter the rate of organic
matter (OM) degradation and will affect the availability and cycling of organically
bound nutrients (carbon, nitrogen, sulphur, and phosphorus) within the ecosystem.
Hence, knowledge of organic matter decomposition rate is required for understand-
ing the recycling and the availability of these nutrients. Litter bag techniques
(Hossain et al. 2011) or cotton strips assay (Mendelssohn et al. 1999) are the most
widely used methods for study of organic matter decomposition rate at the field level
and measurement of soil respiration (Anderson 1982) in the laboratory study.
Soil Enzymes
Microorganism, the living entity in the soil such as fauna and plant roots, has great
potential to contribute to diverse groups of enzymes. Soil enzymes play a crucial role
in soil organic matter degradation and nutrient cycling and respond quickly to
changes in soil management practices, and hence, have potential to act as soil quality
indicators. Soil enzymes have been classified as C cycle enzymes which include
cellulase, amylase, lipase, invertase, and glucosidases; Ncycle enzymes comprising
of proteases, amidases, ureases, and deaminases; P cycle enzymes which include
phosphatases; and S cycle enzymes include arylsulphatases. The use of these
enzymes for soil quality assessment is not always reliable as soil enzyme content
may vary depending upon the presence of the substrates in the soil as energy source
for microorganisms (Awale et al. 2017). Furthermore, the extracellular enzyme’s
activity is not necessarily associated with active cells and these enzymes occur in
both living and dead components of the soil (Skujins 1978). As a result, the
interpretation will be unpredictable. However, Visser and Parkinson (1992)
advocated dehydrogenase activity to be an exception to other soil enzymes as it is
truly intracellular enzyme. It reflects total oxidative activities of soil microorganisms
and is related to organic matter degradation (Dick 1997). Nevertheless, enzyme
activities are widely measured for soil ecological assessment (Dick 1997). Beck
(1984) evaluated several enzymes (hydrolases and reductases) together with total
microbial biomass while working with a series of agricultural soils and observed that
all the measured parameters are significantly correlated and suggested that an
“overall index of soil microbial activity” could be derived from them.
Metabolic Quotient (qCO2)

The metabolic quotient (qCO2) is defined as the ratio of basal respiration to micro-
bial biomass carbon, also called specific respiration rate. Anderson and Domsch
(1985) established this eco-physiological characteristic for its specific activity. A
low value of qCO2 is indicative of economic energy utilization and demonstrates a
more stable ecosystem (Anderson 1994). There are some limitations with the use of
qCO2 as a soil health indicator. According to Wardle and Ghani (1995), the results of
metabolic quotient may be complicated because of stresses and disturbance in the
soil. For example, the stress imposed on the soil because of a low pH or deficiency of
nutrients would lead to high qCO2 and lesser microbial biomass production as
compared to the absence of such stress. Similarly, by tillage or manuring, the
qCO2 would increase, but this increase may also lead to an increase in the microbial
biomass.
Measurement of Specific Soil Microbial Processes

(a) Nitrogen mineralization
Nitrogen mineralization is the conversion of organic form of nitrogen to inorganic

form of nitrogen (e.g., ammonium ion) during the decomposition of organic matter.
The mineralization process is mediated by several groups of aerobic and anaerobic
microorganisms. Nonspecific extracellular enzymes play an important role in this
process (Ladd and Jackson 1982) and both mineralization and assimilation of
nitrogen in microbial cells (immobilization) take place simultaneously; hence, the
net mineralization rate is estimated which reflects the potential nitrogen mineraliza-
tion. Waring and Bremner (1964) developed anaerobic incubation methods using
waterlogged soils which are more reliable for testing soil quality as they require
shorter incubation period and maintaining uniformity in moisture condition through-
out the incubation period is not a problem. Also, nitrification process is inhibited
because of anaerobic situation and only ammonical nitrogen has to be assessed. The
emissions of gases due to denitrification can also be avoided. The net mineralization
226 D. Amat et al.
is calculated as the difference in amount of ammonium ion in the soil slurry before
and after incubation period.
(b) Autotrophic nitrification
The autotrophic nitrification is performed mainly by aerobic chemolithotrophic

bacteria. In nitrification, formation of nitrate from the ammonium is a two-step
process. Firstly, ammonium ion is converted to nitrite due to action of ammonia
oxidizers and, in the subsequent step, nitrite is further oxidized to nitrate by nitrite
oxidizers. These are carried out by microbial species of Nitrobacteriaceae family and
are commonly called nitrifying bacteria, and the process of nitrification is sensitive to
disturbances due to management practices. Therefore, it may be considered as
potential soil quality indicator, as there is less influence by the other physiological
groups. Ammonium oxidation assay is measured to estimate nitrification process, in
which soil is incubated with an excess of ammonium under aerated condition. In this
assay, chlorate is also supplemented to prevent the conversion of nitrite to nitrate;
hence, nitrite gets accumulated as the end product which is quantified (Belser and
Mays 1980).
(c) Denitrification
Denitrification is the process of conversation of oxides of nitrogen, mainly NO3

and NO2, to gaseous form like NO, N2O, and N2. Under anaerobic condition, due
to absence of oxygen (O2), nitrogenous oxides serve as the terminal electron
acceptor by the denitrifiers and therefore nitrogen oxides are reduced. Denitrifying
ability is observed in the majority of soil bacteria compared to nitrification (Zumft
1992). Hence, the denitrification process can serve as an important indicator of soil
health. The protocol for estimation of denitrification (Smith and Tiedje 1979) rate is
based on inhibition of N2O reduction by acetylene and chloramphenicol (CAP)
during incubation to prevent synthesis of new denitrifying enzymes. This leads to
a linear accumulation of N2O and the initial production rate is calculated. Limitation
of this method is that use of CAP may lead to decline in the activity of other
metabolic enzymes (Pell et al. 1991).
(d) Nitrogen fixation
Rhizobium is the abundant bacterial genus in soil; they truly exist in a symbiotic
relationship with the roots of the legume. The bacteria mainly live in the nodules, the
prime site for N2 fixation, and supply nitrogen to the plant for better growth and
development. Similarly, the plant in turn provides organic substrates to the bacteria
for optimal growth. The legume-Rhizobium symbiosis is highly host-specific in
nature. Several authors have advocated to use Rhizobium species as soil health
indicator (Brookes 1995) based on sensitiveness of soil organisms to the pesticides
(Domsch et al. 1983) and heavy metals (McGrath et al. 1988; Chaudri et al. 1993;
Chambers et al. 1999). Diversity and abundance of Rhizobium in soil are assessed by
pot test, where different species of legume are grown in the test soil and then nodules
are counted after a specific stage of growth. The enumeration of rhizobia from test
soil may also be carried out on specific culture media and characterized for morpho-
logical and physiological features (Laguerre et al. 1993; Tong and Sadowsky 1994;
Bromfield et al. 1995; Hungria et al. 2001). Many biotechnological methods like
profiling of plasmid and insertion sequence fingerprints (Hartmann et al. 1998),
detection of specific genes using PCR (Tesfaye and Holl 1998), 16S–23S rDNA
spacer sequences (Tan et al. 2001), colony hybridization method (Laguerre et al.
1993), RAPD (Baymiev et al. 1999), and RFLP (Laguerre et al. 1994) can be
employed to estimate the diversity of the rhizobia present in different soils.
Unlike Rhizobium, the cyanobacteria or blue-green algae are nonsymbiotic nitro-
gen fixer. They produce exo-polysaccharide, and thus, help in improving soil
structure. Cyanobacteria have mainly been used as indicators of heavy metal con-
tamination in soil due to application of sewage sludge. Higher concentrations of
heavy metals have detrimental effect on abundance and activity of nitrogenase
enzymes of cyanobacteria (Brookes 1995; Lorenz et al. 1992; Dahlin et al. 1997).
Population can be estimated by most probable number (MPN) method (Rippka et al.
1979) and the activity can be determined by estimating nitrogenase enzyme activity
using acetylene reduction assay (ARA) (Hardy et al. 1968). In ARA, acetylene is
reduced to ethylene due to action of nitrogenase enzyme which can easily be
measured by gas chromatography(GC).
8.3 Conclusion
Among the different soil health indicators, biological indicators are the most sensi-
tive and also rapidly respond to the management practice than the physical and
chemical parameters, and thus can serve as reliable parameters to assess soil health.
Recent advances in technology have improved the precision and reproducibility of
measurement of biological parameters, thus giving more information about the
processes occurring in soil mediated by soil flora and fauna. High-throughput for
genomic and enzyme study is added to understand microbial community structure
and thus for a broader understanding of comparative study across the ecosystem.
This will help in identifying suitable agricultural practices for long-term soil health
maintenance. Each method has its limitation and its use depends upon the purpose of
the end user. As characteristics of soil change because of spatial variation, these
methods need to be optimized for different types of soil for their better practical
utility.
Acknowledgments The authors are grateful to the Director, Indian ICAR-Institute of Soil Science,
Bhopal, Madhya Pradesh, India for his constant support and encouragement.
228 D. Amat et al.
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Rhizosphere Microbes: Driver for Soil
Health Management 9
H. K. Patel, R. V. Vyas, A. Ramesh, and J. P. Solanki
Abstract
The soil is a living entity and its significance and functions are irrefutable.
Organisms present in the soil play a major role in sustainable soil health manage-
ment. Current agrochemical-based nutrient management strategies can fulfil the
demand of food to feed 7.8 billion humans living on earth, however, potential
uses of agriculturally important microbial bioresources have been ignored, which
can lead to compromise towards soil health and agro ecosystem sustainability.
Major soil functions like nutrient and organic matter recycling, fertility, soil
reclamation and ultimately tiny soil creatures called microbes, which comprise
major portion of soil biota, manage soil and ecosystem health management.
ABMs (Agriculturally beneficial microbes) include bacteria, actinobacteria,
fungi and others which live in a mutualistic relationship along with plants
(symbiotic/associative/free living/ endophytic) and make the nutrients available,
play a major role in organic matter decomposition, produce phyto-hormones,
phytoremediation and act as biocontrol agents for plant pests and diseases. These
ABMs are supposed to be present in the soil, but looking to the sustainability and
soil health, to enhance nutrient use efficiency, it is desirable to elevate their
strength through direct application or following different agronomic interventions
that enhance their abundance and activity. This review details the role of
rhizospheric microorganisms and soil organic matter in soil quality management.
H. K. Patel (*) · R. V. Vyas · J. P. Solanki

Department of Agricultural Microbiology, B A College of Agriculture, Anand Agricultural
University, Anand, Gujarat, India
A. Ramesh

23, https://doi.org/10.1007/978-981-15-9154-9_9
236 H. K. Patel et al.
9.1 Introduction
Awareness among people regarding the link between the trillions of microbes that
live within our bodies and human health are increasing day by day. Studies have
found that a healthy population of microbes or a microbiome in a human body can
prevent food allergies and other disorders to improve human health (Kumar and
Chordia 2017). Just as in the human body, these types of tiny creatures also play a
crucial role in soil health management. Growth enhancing microorganisms (MOs)
can be added to plants or soil in a variety of ways—as seed coating, suspended in
water and sprayed on plants or soil or mixed into manure and mulches that are added
to the soil or placed around the plants. Studies of this unseen world have been going
on for so many decades, but now they are gaining momentum and researchers are
looking into ways to improve agriculture in an eco-friendly manner (Wander et al.
2019).
Agricultural management strategies such as intensive/conventional systems
involving excessive inorganic nutrient addition, tillage and other interventions by
modifying soil processes in long-term affect soil environment. The conventional
inorganic fertilization supplies nutrients to crop plants immediately for plant assimi-
lation for improved crop growth and productivity. However, fertilizer application
has a cascading effect on soil quality, soil degradation and groundwater contamina-
tion (Hernández et al. 2016). In conventional agricultural systems, the loss of soil
organic carbon content is a common phenomenon and resorting to improving soil
organic carbon content through proper management practices viz. resource conser-
vation technologies such as reduced tillage/no-till and organic management is the
way forward. These practices tend to increase the concentration of carbon, microbial
proliferation and diversity, improve physical, chemical, microbiological
characteristics of soil and overall environment quality. The world human population
has reached 7.8 billion and is bound to touch approximately nine billion by the year
2050 (https://www.worldometers.info). The biggest challenge is to feed them with-
out compromising the sustainability of agriculture and soil health. Soil is a vital
component of our agro-ecosystem, not only for production of sufficient food and
quality of produce but also for maintaining a sustainable environment for our future
generations (Kumar et al. 2015). Looking into the performance of the existing
technologies, it seems we have reached a plateau and it is no longer sufficient to
fulfil the increasing food production. Therefore, to cope with the food demand for
ever increasing population in an eco-friendly manner, reconsideration, exploration
and exploitation of eco-friendly low-cost technologies or natural resources are
needed to sustain our ecosystem and soil health and prevent further degradation.
Agriculture sustainability cannot be attained without proper management of micro-
bial population directly or indirectly through agronomic interventions in soil
(Timmusk et al. 2017).
The soil ecosystem contains microorganisms as a key player of its living biomass.
The ecosystem services performed microorganisms include improvement of plant
nutrition, management of soil health and quality (Lugtenberg 2015). Overall, effi-
cient management and functions of agro-ecosystem mainly depends on its dynamic
9 Rhizosphere Microbes: Driver for Soil Health Management 237
soil microbiota. Regulation of nutrient cycling in soil through mineralization of

organic matter and solubilization of inorganic nutrients are performed by its efficient
soil microbiota. The symbiotic and saprophytic microbiota residing beneath the soil
improves soil fertility by decomposing organic matter, acting as plant probiotics and
reclaiming soil from pollutants. The efficient plant probiotics must effectively
proliferate in the rhizosphere with native microorganisms, while expressing their
beneficial traits (Martinez-Viveros et al. 2010). The beneficial soil microbes gear up
nutrient transformation and uptake processes viz. fixation of nitrogen, phosphate
solubilization, phytate mineralization, potassium solubilization and micronutrients.
They also inhibit harmful microbes and other insects via different strategies like
production of antimicrobial compounds, through competition for nutrients, creating
infection among harmful insect populations (Barea et al. 2013; Barea and
Richardson 2015). The rhizospheric soil contains an array of microorganisms,
which are categorized into beneficial and deleterious microorganisms based on
their effect on soil quality, crop growth and ultimately productivity (Jat et al.
2015). Without soil microbes, one cannot imagine life on the planet. In-depth
understanding of rhizosphere processes could pave ways for sustainability in agri-
culture and management of soil health without affecting the agro-ecosystems.
Therefore, beneficial rhizosphere microorganisms play a persistent role in improving
soil quality and agriculture sustainability simultaneously.
9.2 Need of Soil Health Management
Soil health management is important for improved productivity of crops, quality of

the produce for humans and animals and sustainability of the environment. Keeping
the current agricultural scenario including the nutritional security and the projected
increase in human population, the soil health management will be of great concern in
the coming years as today’s agriculture practices may not be able to meet the food
requirements for the next generation (Watson 2014).
Soil organic carbon content determines the extent of soil health and encompasses
many soil processes. However, a large amount of SOC that exists in recalcitrant
pools and natural variability in soils under different climatic situations makes it
difficult to gauge the influence of agricultural management practices in short term.
Soil organic matter is not homogenous in nature rather it is composed of materials
with varying chemical composition and recycling/turnover rates. The intricate role of
soil organic carbon stocks in long-term accumulation and carbon sequestration is
pertinent in recent times as it impacts the sustainability of agro-ecosystem function-
ing, mitigation of climate change paradigm, and improves soil nutrient availability
for crop plants, reducing soil erosion, nutrient runoff, and improving crop produc-
tivity. In agro-ecosystems, it is feasible to increase soil organic carbon stocks
through appropriate agricultural management strategies such as appropriate crop
rotations, integrated organic and inorganic nutrient management, reduced tillage and
reducing organic carbon decomposition. The soil organic carbon dynamics in agro-
ecosystem mainly depends on agricultural management practices (Bhattacharyya

and Jha 2012).
Soil organic carbon plays a pivotal role in influencing the soil microbiome as it
provides substrates for the proliferation of soil microorganisms. The activity of soil
microorganisms determines nutrient transformation, plant growth/suppression,
decomposition of organic residues, and physical soil processes (Tamilselvi et al.
2015). The microbial activity greatly determines short-term dynamics and stability
of SOC in the long term. Higher microbial activities results in higher carbon turnover
and carbon release and any management strategy should aim at reducing microbial
access to organic matter decomposition and aid in soil C accumulation. Therefore,
identification of appropriate agricultural management strategies that not only
improve soil organic carbon content but also mitigate the negative impact of CO2
emission into the atmosphere (Tu et al. 2006) is a plausible solution. It has been well-
documented that agricultural management through organic agriculture is a viable
option for sustaining agricultural intensification.
Recent studies have suggested that nurturing an effective soil microbiota can
speed up soil rejuvenation. Soil microorganisms have the capacity to transform
complex compounds like polysaccharides in to simple sugars and the simple sugars
to number of complex compound needed for soil health. Complex compounds
broken by microbes are utilized in part by microbes to fuel up growth of their
population and thereby they indirectly improving soil health. For maximum utiliza-
tion of carbon stored in plant remains, growth of soil microbiome, which can readily
convert plant carbon in to SOM, should be promoted. Healthy soils have a rich
microbiome that can limit disease-causing organisms, can recycle nutrients and
alleviate stress of plant (Bier et al. 2015).
9.3 The Rhizosphere: Junction of Microbial Activities
Lorenz Hiltner in 1904 for the first time used the term ‘rhizosphere’. It is the
immediate thin soil periphery around the plant roots and is governed by the rhizo-
sphere effect. Plant root secretes different types of exudates in varying
concentrations. These exudates act as a messenger for induction of interactions
between plant and soil organisms (Canarini et al. 2019). Therefore, the rhizosphere
determines microbial population structure and diversity as shown in Table 9.1
(Brimecombe et al. 2001; Verma et al. 2017b). The mutual association between
Table 9.1 Abundance of culturable microbes in the rhizospheric and non-rhizospheric soil
Rhizospheric Non-rhizospheric
Microbial group Number g 1 soil R:S ratio
Bacteria 1.3 109 5.4 107 24.0
Fungi 1.2 106 1.0 105 12.0
Actinobacteria 4.6 107 7.0 106 7.0
Algae 5.0 103 2.7 104 0.2
Fig. 9.1 An overview: role of agriculturally beneficial microorganisms (ABMs) in soil and plant
health management
microorganisms and plants results in lots of positive effects on soil health, crop
growth and development, increase in nutrient assimilation along with disease con-
trol. This relationship between plants and microorganisms leads to improvement in
crop yield along with soil and plant health management (Fig. 9.1; Sarkar et al. 2017).
9.4 Effect of Rhizosphere Microorganisms on Soil Quality
The rhizosphere plays a decisive role in affecting crop growth and productivity by
influencing important soil processes. The interactions between plants and microbes
may be classified as mutualistic or antagonistic to the plant, depending on the
growth-promoting or limiting effects of microbes on plants (Bais et al. 2006).
Revealing the dynamic multi-component system of rhizospheric microbes and its
mutual relationship to soil sustainability is going to become very much important
towards maintaining food production with no or minimal use of agrochemicals
(Velazquez et al. 2016).
9.4.1 Microbe-Mediated Rhizosphere Interactions and Its

Implication on Soil Nutrient Availability and Crop
Productivity
Rhizodeposition and facilitation determines the shift in microbial structure as well as

diversity. Plant species have a differential influence on rhizosphere microbial
communities as amount and composition of rhizodeposition vary (Hartmann et al.
2009; Dennis et al. 2010). To exemplify, white lupin excretes high amounts of citric
acid in the rhizobiome that shifts the fungal community structure and secretion of
cis-aconitic, citric, and malic acids is attributed to shift in bacterial community
structure (Marschner et al. 2001). Roots also exude secondary metabolites and
signalling molecules that determine microbial communities and as a consequence
of root-root and root-microbe communication. Rhizosphere microbial communities,
therefore, vary between plant species depending on plant genotype–soil interactions
(Marschner et al. 2006). Rhizodeposition coupled with rhizospheric pH also affects
soil microbial community’s structure and diversity (Hinsinger et al. 2009). Several
studies have shown differences in microbial community structure in the rhizosphere
with intercropping as against sole cropping (Song et al. 2007; Wang et al. 2007; Li
et al. 2010). The intermingling of roots in the intercropping system results in a
common microbial community structure involving both the plant species with
greater diversity as root exudation composition varies (Wang et al. 2007). The
effects pertaining to intercropping system on microbial community structure are
contradictory. It is pertinent to note that microbial community structures as well as
changes in microbial biomass and activity are ought to influence plant growth as they
alter root growth via hormonal production or nutrient availability through minerali-
zation (Richardson et al. 2009a; Fan et al. 2011). However, there are discrepancies in
microbial community composition and activity, as functions are carried out by a
wide range of microorganisms. Functional communities with low levels of redun-
dancy such as nitrifiers, their community structure did not vary by intercropping (Fan
et al. 2011). Nevertheless, indications prompt us to believe that changes in microbial
community composition were better associated with certain microbial activities such
as phosphatase activities. The rhizosphere is a zone where plants coordinate with
other plants, herbivores and microorganisms and help in nutrient mobilization and
disease suppression. The rhizosphere contains innumerable microbes and shapes the
rhizosphere microbiome in order to recruit beneficial and protective bacteria or fungi
(Barea et al. 2005).
Plant growth rhizobacteria (PGPRs), from a wide range of genera, including
Acinetobacter, Alcaligenes, Azospirillum, Bacillus, Pseudomonas, Rhizobium and
others are able to colonize plant roots. These rhizobacteria assume significance due
to their ability to produce plant growth hormones, solubilization/mineralization of
nutrients and capacity to tolerate abiotic and biotic stresses (Yang et al. 2009;
Bhattacharyya and Jha 2012). The PGPRs are classified as biofertilizers,
phytostimulators, and biopesticides (Bhardwaj et al. 2014). The biofertilizers pro-
mote plant growth by increasing the nutrient-supplying capacity to the host. Slew of
biofertilizers have been well-documented (Bhattacharyya and Jha 2012). The
phytostimulators, viz. species belonging to Bacillus, Pseudomonas, Azospirillum,

Enterobacter, Azotobacter, Pantoea, Streptomyces, and Rhizobium, however, pro-
duce phytohormones such as indole acetic acid (IAA), GA3, and cytokinins, which
play a role in altering the root architecture and thereby promote plant growth (Duca
et al. 2014) and help in disease suppression and plant growth (Bhattacharyya and Jha
2012). Besides these there are other PGPRs that are involved in abiotic stress
tolerance. For instance, Paenibacillus polymyxa, Achromobacter piechaudii, and
Rhizobium tropici mitigate drought stress in Arabidopsis, tomato (Solanum
lycopersicum), and common bean (Phaseolus vulgaris), respectively, through
abscisic acid production, reactive oxygen species and 1-aminocyclopropane-1-car-
boxylate (Yang et al. 2009). Achromobacter piechaudii and B. subtilis are also
associated with salinity tolerance in plants (Mayak et al. 2004; Yang et al. 2009).
In the present scenario, the need is to increase crop productivity, in an environment
of climatic changes, minimizing mineral fertilizers and tolerance to abiotic stresses is
of international priority.
Shen et al. (2013) pointed out that to achieve the highest crop productivity, plants
require optimal nutrient input and the ability to be resilient under stress conditions.
This is through maximizing root/rhizosphere efficiency for improved nutrient mobi-
lization and acquisition. Agricultural interventions need PGPRs, and root exudation
enables beneficial root colonization thereby increasing plant growth, nutrient avail-
ability, enhancing seed emergence, and promoting plant health (Lugtenberg and
Kamilova 2009).
Microorganisms being ‘biological engine of the earth’ are considered as a
microbiological soil quality indicator and are used widely as compared to physical
and chemical methods (Kennedy and Smith 1995; Pankhurst et al. 1995). A micro-
bial indicator has been defined as that which represents properties of the environ-
ment or impacts to the environment, which can be interpreted beyond the
information that the measured or observed (indicator) represents itself (Nielsen
and Winding 2002). Selective and specific indicators with high-discriminating
potential for agricultural systems are used as microbiological soil quality indicators.
Soil enzymes activity could be used to evaluate the soil quality changes due to
different agricultural management practices, since it is easy to estimate and relate
well with the microbiome as well (Dick et al. 1996; Dick 1997; Jimenez et al. 2002).
This evaluation provides changes in soil processes due to different agricultural
interventions and is used as a tool to monitor management induced changes on the
long-term basis (Acosta-Martinez et al. 2007). Nannipieri (1994) recommended that
this assay provides us early and sensitive information and these are widely used to
demarcate changes due to organic manure application, crop residue, tillage, herbi-
cide application, green manure application so on and so forth (Aparna et al. 2014;
Tamilselvi et al. 2015).
Dehydrogenase provides information on endogenous soil microbial processes
(Biedrebeck et al. 2005) and is a potential soil quality indicator to demarcate changes
due to agricultural management (Aparna et al. 2014). According to Liang et al.
(2005) and Tejada et al. (2006), the organic amendments do stimulate the dehydro-
genase activity (DHA) due to increased microbial proliferation as a consequence of
readily available food that contains both extra and intracellular enzymes. Moreover,
the microbial population’s proliferation due to rhizodeposition also contributes to
increased DHA in the organic amended soils. This is consistent with the findings of
other researchers, wherein they also observed increases in DHA in organically
managed agro-ecosystems (Aparna et al. 2014; Tamilselvi et al. 2015; Hernández
et al. 2016). A decrease in DHA under conventional agricultural systems can be
ascribed to higher bulk density and compaction which is also observed in the present
study as compared to organic systems as it decreases air filled pore space and macro-
porosity resulting in deficient aeration conditions in soils, this being unfavourable
for microbial activity (Roldan et al. 2005). ß-glucosidase is also a reliable indicator
of microbial activity under the influence of different agricultural management
systems and therefore included as an important component in soil quality
assessments (Stott et al. 2010). This enzyme involves the decomposition of carbo-
hydrate compounds especially cellulose and helps in providing substrates for micro-
bial proliferation due to management-related perturbations. Both acid and alkaline
phosphatases are produced by microorganisms, while plant roots secrete only acid
phosphatases (Tarafdar 1989; Rao and Tarafdar 1992). Metabolized organic
substrates in organic system are critical factors in increasing acid and alkaline
phosphatase activity. Many workers also reported increased phosphatase activity
with organic management (Aparna et al. 2014; Hernández et al. 2016).
Arylsulphatase (ARS) is recognized to mineralize organic sulphur involving the
hydrolysis of ester-S to sulphate sulphur, which is a readily available source for plant
sulphur assimilation. That is why this enzyme was chosen as a functional marker of
S cycling. Soil arylsulphatases are principally microbial in origin. Since carbon
(C) substrates largely control microbial growth in soil, the availability of C is also
a key factor governing S transformations in soil (Knights et al. 2001). Microbial
respiration rate indicates carbon availability to soil microorganisms and subsequent
mineralization. Moreover, substrate induced respiration (SIR) is indicative of meta-
bolic and physiological state of soil microorganisms also increased under organic
management and also at critical stages of crop growth and construed as increased
carbon availability, rhizodeposition with increased microbial activity is in conso-
nance with other researchers (Tamilselvi et al. 2015; Hernández et al. 2016).
Different geochemical processes take place among microorganisms resulting in
nutrient transformation in rhizobiome (Table 9.2). Activities of microorganisms vary
at different levels in rhizospheric soil as against non-rhizospheric one (Marschner
et al. 2003). In field, the target plant has to face competition for all resources with
unwanted plants in its surroundings, e.g. weeds. Among the most essential
resources, soil moisture coupled with temperature and nutrient availability is critical
for crop growth (Akhtar and Siddiqui 2010).
On average, half of SOM is composed of organic carbon, and remaining half is
composed of nitrogen, phosphorus, sulphur and other nutrients and microbes resid-
ing in soil exclusively cycle these nutrients through different geochemical cycles.
Table 9.2 Important nutrient and their transformation processes governed by microorganisms
Nutrient Microbial activity (transformation)
N N2 fixation, immobilization, nitrification, denitrification, urea hydrolysis
P Mineralization, mobilization, immobilization
K K mobilization
S Oxidation, reduction, mineralization, immobilization
Zn Zn solubilization
Fe Siderophore production
Mn Change in oxidation state
Various efficient plant beneficial rhizosphere microorganisms’ viz. symbiotic, asso-

ciative and free-living bacteria facilitate nutrient demand (Table 9.3).
In nature, atmospheric nitrogen is fixed to plant available forms via microbial-
mediated nitrogen fixation, N-fixing microorganisms with the use of nitrogenase
enzyme with concomitant reduction of nitrogen to ammonia (Kim and Rees 1994)
and accounts for two-thirds of the total fixed N. The microbial nitrogen fixation
(MNF) can be classified as symbiotic, associative and free living. Some examples
being Azotobacter, Gluconoacetobacter and Azospirillum spp. are some of the
known free-living nitrogen-fixing bacteria prevailing in nature and fixing nitrogen.
Legume–rhizobium symbiotic system is the most important biological nitrogen
fixation (BNF) system in nature, provides 65% of biosphere’s available nitrogen
(Lodwig et al. 2003) and is environmentally sustainable (Herridge et al. 2008). Other
rhizobacteria present in soil or rhizosphere are also equipped with the nitrogenase
enzyme and contribute in microbial nitrogen fixation for non-legumes, e.g.,
diazotrophs fixing nitrogen asymbiotically (Glick et al. 1998). Inoculation of
legumes with Rhizobium of a specific cross inoculation group can be helpful in
improving the nodulation capability, nitrogen uptake and ultimately the yield of
crops under field conditions (Ahmad et al. 2013; Patel et al. 2016). Other
rhizobacteria present in soil or rhizosphere are also equipped with the nitrogenase
enzyme and contribute in microbial nitrogen fixation for non-legumes,
e.g. diazotrophs fixing nitrogen asymbiotically (Glick et al. 1998). Rhizobium
inoculation in legumes improves the nodulation capability, nitrogen uptake and
crop productivity under field conditions (Ahmad et al. 2013; Patel et al. 2016).
Besides degradation of SOM in nature, microbes also play a critical role in
mineralization/solubilization of nutrients, which are insoluble or fixed in soil affect-
ing its availability to plants. The principle mechanism behind solubilization of these
nutrients is lowering down of pH in root microclimate by acidification by proton
extrusion and organic acid production. Bacteria and fungi have ability to restore
fertility of degraded lands by improving nutrient bioavailability (Rashid et al. 2016).
Phosphorus (P) is one of the essential nutrients, and its deficiency affects crop
growth and production. Many soils contain relatively large amount of total P, albeit
soil solution P concentration is very low (frequently less than 1 μM) for plant uptake.
The low native soil P availability is accompanied by low utilization efficiency of
applied P due to sorption and precipitation reactions. Plants have developed a
Table 9.3 Effect of agriculturally beneficial microorganisms (ABMs) on growth and development
of different crops
Crop ABM Effect on crop Reference
Rice Pseudomonas Improvement in growth, yield, and Chamani
fluorescens, P. Putida yield-contributing parameters et al. (2015)
Maize Klebsiella sp., Pantoea Increased crop growth and indole Rodrigues
sp., Enterobacter sp. acetic acid production in maize. and Forzani
(2016)
Pseudomonas Inoculation enhanced the vegetative Ghorchiani
fluorescens and reproductive traits, nutrient et al. (2018)
assimilation, productivity of maize
Wheat Azotobacter Increase seed and straw yield of wheat Desai et al.
chroococcum, Bacillus (2015)
coagulans
Azospirillum sp. Significant increase of root length, and Singh et al.
root fresh and dry weight (2017)
Azorhizobium Inoculation improved the number and Liu et al.
caulinodans weight of leaves and roots (2017)
Bacillus subtilis (LDR2) Inoculation improved tolerance Barnawal
towards abiotic stress and improved et al. (2017)
growth of seedlings
French Cellulosimicrobium Enhanced seed germination rate, plant Karthik
bean funkei KM032184 vigour and carotenoid contents by et al. (2016)
modulating oxidative damage
Tomato Azotobacter Crop growth improvement and Viscardi
chroococcum overcoming by reducing the abiotic et al. (2016)
stress
Soybean Bradyrhizobium sp. Increased nutrient accumulation and Raja and
yield in soybean Takankhar
(2018)
Groundnut Rhizobium huautlense Inoculation along with FYM Patel et al.
GNR I, Rhizobium enhanced seed and haulm yield and oil (2016)
giardinii GNR II content
Bradyrhizobium sp. Inoculation enhanced the nutrient Argaw
uptake and nodulation in groundnut (2018)
Chilli Azotobacter Increase in number of transplantable Shelat et al.
chroococcum, seedling and seedling vigour (2017)
Azospirillum sp.,
Bacillus coagulans
number of mechanisms that enable P assimilation from soil (Vance et al. 2003).
Plants as well as microorganisms increase P availability by solubilization of inor-
ganic P and mineralization of organic P. These includes changes in root architecture
(Ge et al. 2000), mycorrhizae (Smith and Read 1997), fungi, bacteria,
rhizodeposition and phosphatase production (Yadav and Tarafdar 2001) that
catalyse the hydrolysis of P from organic-P (Ramesh et al. 2004) and to enhance P
availability as most of the soil P (up to 80%) occurs as organic P. Phosphatase
activity in soil is derived by root activity as well as microorganisms (Yadav and
Tarafdar 2001). Enzyme that hydrolyses derivatives of phytate is of paramount

importance as phytate constitutes up to 50% of total soil organic P (Turner et al.
2002) with limited proportion as phosphate esters such as phospholipids (Richardson
et al. 2005). To overcome low P fertility constraints in soils, it is prudent to apply
inorganic P fertilizers, however its application is economically and environmentally
not sustainable because high quantity is required as P fixation is a major impediment
of high P fixing capacity of these soils. Therefore, microorganisms play a key role in
soil–plant continuum where their interactions affect crop growth (Vassilev et al.
2006). Phosphorus solubilization through rhizospheric microorganisms is known to
increase available soil P and help in crop P assimilation. It can solubilize and
mineralize P from inorganic and organic pools of total soil P and therefore are
used as microbial inoculants for improved P assimilation (Richardson 2001; Patel
et al. 2010). It has been found that rhizobacteria used as microbial inoculants should
have the capability to compete with native microbes. Some plant growth-promoting
microorganisms (PGPM) such as Bacillus amyloliquefaciens, B. laevolacticus,
B. subtilis, and Aspergillus niger, Penicillium rubrum are known to be active
phytases producers (Yadav and Tarafdar 2001; Gulati et al. 2007). Studies have
indicated that phytate and phosphate solubilizing bacteria improve P mobilization
and acquisition by plants and is better associated with enzymes pertaining to P
cycling such as soil phosphatases and phytases (Ramesh et al. 2011). Rhizosphere
microbial community influences soil quality through its action such as decomposi-
tion, mineralization and solubilization of native nutrients (Xiao et al. 2017).
Rhizospheric microorganisms also contribute in solubilizing insoluble potassium
to plant available K (Meena et al. 2015a; Etesami et al. 2017). In recent years, several
zinc solubilizing bacteria were reported from different soil and environmental
situations to improve plant zinc availability. Among the bacterial species,
Acinetobacter, Bacillus, Gluconacetobacter, Enterobacter, Sphingomonas and
Pseudomonas have been reported (Ramesh et al. 2014a, b) as zinc solubilizers.
Iron is an essential element required by most biological systems, albeit at low
concentrations. Low-solubility product of Fe minerals and their strong pH depen-
dency make its inorganic forms unavailable to plants and microbes, especially at
neutral to alkaline pH. In a pH range of 7 to 9, the sum of the soluble inorganic forms
of Fe does not exceed 10 10 M (Lindsay 1979), which is far below the minimal
requirement for normal plant growth. Under iron-limiting conditions,
microorganisms produce low-molecular weight molecules and are called
siderophores which have a higher affinity for ferric iron. In the rhizosphere, the
concentration of siderophore around microbes and roots assumes significance in
overcoming iron deficiency parlance in iron-stressed environment.
Many microorganisms also help in solubilizing and mineralizing secondary,
macro- and micronutrients available to agricultural production systems (Eriksen
2009). The arbuscular mycorrhiza (AM) also greatly affects the availability of a
range of nutrients including many secondary, macro and microelements, particularly
P and K to plants (Sharma et al. 2016). For agricultural ecosystem, fungi are as
important as rhizobacteria for performing essential functions of nutrient cycling and
biotic stress management in plant. Fungi including arbuscular mycorrhizal
(AM) fungi play a very important role in agriculture ecosystem (Bianciotto and
Bonfante 2002). The AM fungi play a significant role in nutrient mobilization and
assimilation by crop plants through their extra radical hyphae. In addition to their
contribution in plant nutrition, AM fungi also improve soil physical properties as
well as abiotic stresses through production of several cementing compounds and
metabolites (Raghavendra et al. 2016; Verma et al. 2017a).
9.4.2 Biological Control of Soil-Borne Phytopathogens

and Insect Pests
Rhizospheric microorganisms enhance nutrient availability to plant through the

release of plant growth regulators that help in soil-borne disease suppression or
control. Rhizospheric microbes have emerged as an eco-friendly biological weapon
that triggers the mechanism of disease reduction through systemic acquired resis-
tance (SAR) and induced systemic resistance (ISR). Beneficial microbes or disease
suppressive soil microbes are those that improve crop growth and soil health and
also provide essential nutrients and minerals from soils which are normally not
available to the plant, e.g. Bacillus, Trichoderma, Pseudomonas, Rhizobium.
(Table 9.4) (Antoun and Kloepper 2001). Microbial disease suppression involves
siderophore production, antibiosis and production of secondary metabolites
(Richardson et al. 2009b).
Table 9.4 Bio-control of important plant pathogens by rhizospheric microbes

PGPR Plant disease References
Bacillus spp.
Bacillus strain Blight of squash Zhang et al. (2010)
B. pumilus strain SE34 Blue mould disease in Zang et al. (2002)
tobacco
B. cereus strains B101R Foliar diseases in tomato Silva et al. (2004)
B. subtilis strain GBO3, B. pumilus Downy mildew in pearl Niranjan et al. (2003)
strain INR7 millet
Bacillus sp. Rice blast Naureen et al. (2009)
Bacillus strains BB11 and FH17 Blight of bell pepper Jiang et al. (2006)
Pseudomonas spp. and Burkholderia spp.
Pseudomonas spp. Rice sheath rot Saravanakumar et al.
(2009)
P. fluorescens Sheath blight disease Radjacommare et al.
(2002)
P. fluorescens strain CHA0 Banana bunchy top virus Kavino et al. (2008)
(BBTV)
Burkholderia strains MBf21 and Maize rot Rodriguez et al. (2008)
MBf15
9.4.2.1 Siderophore Production

Soil microorganisms, especially rhizobacteria, produce siderophores under iron-
limiting situation, these bacteria have high chelating affinity for Fe3+ than Fe2+
ions, and Fe3+ ions are transferred for normal growth. Wilson et al. (2006) reported
that iron, being an essential nutrient when becomes limiting, can affect metabolisms
in living organisms. Disease suppression through siderophores has been reported for
Fusarium oxysporum and Pythium spp.
9.4.2.2 Antibiosis and Production of Volatile Compounds

Rhizobacteria suppress diseases through antibiotic production, anti-microbial
peptides, hydrolytic enzymes etc. The Gram-positive bacteria Bacillus spp.
possesses a variety of structures that can produce several antibiotics (Stein 2005;
Maksimov et al. 2011). Pseudomonas fluorescens, a source of pyoluteorin and
pyrrolnitrin, was found to be effective in preventing damping off disease in cotton.
Geldanamycin produced by Streptomyces hygroscopicus var. geldanus decreased
Rhizoctonia root rot in pea. Production of HCN as a volatile compound also helps in
disease suppression (Defago et al. 1990).
9.4.2.3 Induction of Plant Systemic Resistance

Studies related to induced systemic resistance (ISR) reported that PGP bacterium
Pseudomonas spp. was residing into the plant as an endophyte and effectively
controlled plant pathogenic fungus F. oxysporum. Induced resistance is the ability
of plants to develop a defensive mechanism/strategy when appropriately stimulated.
Some pathogenesis-related proteins (PRs) such as 1, 3-glucanases and chitinases are
capable of hydrolysing fungal cell walls and cuticles of insects (Singh et al. 2017).
Pseudomonas and Bacillus spp. are the predominant bacteria that induce ISR. These
include Phytophthora spp., Fusarium spp., Rhizoctonia solani, Pythium spp., etc. In
addition, microbes such as P. fluorescens CHA0 induces signal transduction that is
mediated by salicylic acid (SA) (Siddiqui et al. 2005).
9.4.3 Organic Matter Decomposition and Nutrient Supply
The soil organic matter (SOM) is of paramount importance as it determines nutrient

availability through various rhizospheric processes involving microorganisms for
crop growth. The organic matter in the soil ecosystem is continuously transformed
into various ranges of soil building blocks by different biological and chemical
mechanisms (Marschner and Rengel 2007). This organic matter composed of vari-
ous compounds is a major energy source for the soil microbiota. As shown in
Table 9.5, soil organisms have appropriate bioconversion machineries (Kumar
et al. 2017). The increase in SOC storage favours the formation of soil aggregates
and protects SOC encapsulated inside these stable aggregates from oxidation and by
modifying the edaphic factors such as bulk density, pore size distribution, climate
that dithers SOC biodegradation. Increased carbon sequestration will help in
mitigating carbon emission. Tejada et al. (2006) reported that long-term application
Table 9.5 Soil microbes and their significance in agro waste decomposition
Biodegradation
Microorganisms activity Nature of organic matter References
Pleurotus eryngii Lignocellulose Agricultural wastes Yildirim
degradation, laccase et al. (2015)
activity
Aspergillus Niger Cellulase, xyalanase Decomposition of Singh and
production organic matter, Sharma
sugarcane bagasse (2002)
Aspergillus awamori Cellulases Agro wastes Pleissner
et al. (2013)
Paecilomyces marquandii Keratinase Poultry waste (feather Veselá and
waste) Friedrich
(2009)
Pseudomonas putida Manganese Agro waste Ahmad et al.
peroxidases and (2010)
Laccase
Cellulomonas, Pleurotus, Cellulase Banana stem waste Dabhi et al.
Propionibacter, (2014)
Lactobacillus
Citrobacter freundii Manganese Agro waste, saw dust Ali et al.
peroxidases, lignin (2017)
degradation
B. cereus, B. megaterium Breakdown of Organic substrate Ribeiro et al.
cellulose and (2017)
hemicelluloses
of organic manure significantly increased SOC, increased crop productivity and

quality of produce and was consistent with the findings of other researchers.
Rapid composting is a controlled composting process governed by microbes,
which depends on specific microbiological/biochemical processes to degrade agri-
cultural residues (OM) for effective field application (Singh and Prabha 2017).
9.4.4 Bioremediation of Polluted Soil
Many microorganisms have the ability to detoxify pollutants, and it is a practical and
effective remedy for disposal of hazardous soil contaminants (Murali and Mehar
2014; Kao et al. 2006). The degradation by soil microbes is performed through
several enzymatic processes in the rhizobiome. The phyto-degradation of these
pollutants is also carried out by several plant species and microorganisms. The
ectomycorrhizal fungi also reported to bioremediate heavy metals and abiotic
stresses (Fomina et al. 2005). Bioremediation as a strategy revolves around the
changes in metabolic capacity of microorganisms to detoxify xenobiotic compounds
(Xu and Zhou 2017; Kulshrestha and Husain 2007) as compared to any physical or
chemical methods.
9.4.5 Drought, Salinity and Nutrient Stress Management
Water stress, salinity and various other abiotic stresses are directly associated with
soil-derived limitations and other environmental factors. These stresses include
metabolic, morphological and genetic-level changes in the plant under abiotic
stresses. Although after application, only 30–50% of nitrogenous fertilizers and
only 30% of phosphatic fertilizers are utilized by crop plants for its growth and
development (Adesemoye and Kloepper 2009). Now-a-days due to compromised
soil health and agriculture ecosystem, a trend of curtailing use of agrochemicals in
agriculture is increasing worldwide. Various biotic and abiotic factors and their
interactions govern uptake of soil moisture and nutrients by plant roots that involves
microorganisms thereby increasing the fertilizer use efficiency (Meena et al. 2015b).
During stress condition, plant produces more amounts of ethylene. Ahmad et al.
(2011) reported that increased ethylene concentration adversely affected crop
growth. Rhizosphere microorganisms are known to produce ACC deaminase, a
precursor of ethylene under stress and are known to improve crop growth under
stress through decreased ethylene production (Glick et al. 1998; Glick 2012).
9.5 Management of Rhizosphere System for Sustainable

Production
Rhizodeposition (a complex mixture of various acids, sugars and enzymes) and the
activity of microorganisms in the rhizobiome are of particular significance in nutrient
cycling for plant growth promotion activities. The rhizosphere physico-chemistry
can improve phosphate and potash solubilization, mobilization and acquisition from
soil by plants (Zhang et al. 2010). Recommended agricultural management practices
and integrated soil and nutrient, cropping sequences, reducing tillage shift the
rhizosphere processes favourably and enable sustainable agricultural production
9.5.1 Reduction of Chemical Inputs by IBNM
Indiscriminate use of agrochemicals as fertilizer has resulted in substantial decrease

of rhizospheric microflora and eco-friendly soil biota affecting ecosystem and
environment. The way out is to adopt the Integrated Bio Nutrient Management
(IBNM) strategy, which curtails excessive use of agrochemicals for plant nutrition
(Hakim et al. 2013). Options to manage the fertility of agricultural soils include
supplying nutrients through micro irrigation systems, applying sufficient amount of
organic manures (FYM, VC) along with microbial inoculants or biofortified manures
to increase buffering capacity of the soil (Glaser et al. 2002).
9.5.2 Biofertilizer and Organic Inputs Application
Now-a-days more and more focus has been given on food quality for human and
animal consumption and also on environmental concerns and due to this a new trend
of organic agriculture or natural farming has become popular. Different organic
amendments for supply of balanced nutrition is becoming more and more popular
among farming communities which is directly helping to maintain soil quality and
crop productivity in environmentally sustainable manner. These includes improve-
ment (1) in soil microbial activities, (2) in colonization of ABMs, (3) in physical
properties and (4) nutrient-supplying capacity from recalcitrant pool of organic
nitrogen, phosphorus, potash along with other secondary and micro nutrients in
the soil, reducing their loss by leaching or by fixation in the form of mineral salts
(Singh and Mandal 2000).
9.5.3 Cultural Practices in Crops
Proper combination of rhizosphere bioengineering and root physiology in the

cropping systems will lead to sustainable rhizosphere health management. The
subsurface irrigation systems (water management) and nutrient management (appro-
priate nutrient supply) in the field improves soil physico-chemical properties and
root distribution of the crop plant (Yang et al. 2004). Moreover, use of previous crop
residues as organic mulching can solve major problem of weed infestation and water
loss along with nutrients (Chaudhari et al. 2019). Thus, using different cultural
practices, it is easy to maintain soil biological and chemical properties for better
soil health management.
9.6 Conclusion
Agriculturally important beneficial rhizospheric microorganisms (ABMs) are of

paramount importance in soil health management. Effective microbiota like
AMF/rhizobacteria contributes to improvement in sustainable agricultural produc-
tion. Use of ABMs in agriculture ecosystem is known to improve soil and plant
health under adverse soil conditions. The rhizosphere health management involves
rhizosphere bioengineering, accompanied by efficient crop varieties that have a
capacity to mine nutrients for effective nutrient acquisition by plants instead of
injudicious use of agrochemicals. The use of nutrient inputs in today’s conventional
farming must be balanced to increase crop productivity by exploiting rhizosphere
competence in nutrient acquisition by crops. So, all together, these actions will make
the rhizosphere the liveliest environment in the soil.
Acknowledgements Authors are thankful to the Anand Agricultural University, Anand,

Gujarat and ICAR-Indian Institute of Soybean Research, Indore, Madhya Pradesh for providing
the necessary facilities.
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Ralstonia solanacearum: Biology and its
Management in Solanaceous Vegetable 10
Crops
A. Balamurugan, K. Sakthivel, R. K. Gautam, Sushil K. Sharma, and

A. Kumar
Abstract
Ralstonia solanacearum is a soil-borne bacterium causing bacterial wilt disease

on wider host plants, especially in tropical, subtropical, and some warmer tem-
perate regions of the world. Bacterial wilt disease caused by
Ralstonia solanacearum on solanaceous crops is of serious concern in vegetable
cultivation all over the world, as it could cause deadly effect on host plants with
severe yield losses. Numerous research attempts were made to control this disease
in the field level, but all have limited or no success, mainly due to the complex
nature of pathogen. Hence, understanding of pathogen biology and its behavior is
important before devising any effective disease management strategies. This
review is focused upon taxonomy, symptomatology, host range, pathogen biol-
ogy, diversity, economic impact, mechanisms of infection, pathogenicity
determinants, disease cycle, detection and diagnosis techniques, and also on
A. Balamurugan
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural
University, Coimbatore, Tamil Nadu, India
Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi, India
K. Sakthivel
Crop Protection Section, ICAR—Indian Institute of Oilseed Research, Hyderabad, Telangana, India
R. K. Gautam
Division of Germplasm Evaluation, ICAR—National Bureau of Plant Genetic Resources, New
Delhi, India
S. K. Sharma
ICAR—National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
A. Kumar (*)
e-mail: kumar@iari.res.in

23, https://doi.org/10.1007/978-981-15-9154-9_10
260 A. Balamurugan et al.
available integrated disease management strategies for successful control of

bacterial wilt disease, especially in solanaceous vegetables.
Keywords
Bacterial wilt · Solanaceous vegetables · Diagnosis · Pathobiology · IDM
10.1 Introduction
The world human population is estimated to increase 9.7 billion in 2050 and 11.2
billion by 2100 according to United Nations Population Fund, UNFPA
(Alexandratos and Bruinsma 2012). In the ecosystem, healthy plants play an impor-
tant role and provide foundation for all life. Every year, as much as 40 % of the
world’s potential harvest in agricultural and horticultural crop plants and their
produces are lost due to insect’s damages, plant diseases, and weeds according to
the report of Food and Agriculture Organization of the United Nations (FAO 2015).
Keeping in current view, the United Nations and International Plant Protection
Convention (IPPC) announced the year 2020 as International Year of Plant Health
(IYPH), to recognize the plant health protection on global level. To feed the growing
mankind, the production of adequate amount of food crops and their products will be
the main challenges in the twenty-first century (Rana et al. 2020), and it is first
priority for human life to ensure food security from crop damages (Varshney et al.
2017).
In the growing population, vegetables are contributing important constituents of
agriculture in attaining food and nutritional security. The yield loss due to pest and
diseases is of serious concern in successful vegetable cultivation all over the world,
among which soil-borne pathogens play important role (Delgado-Baquerizo et al.
2020). Cultivation of solanaceous crops is much prone and their yield production
was limited by diverse group of both infectious and noninfectious agents. Several
diseases in solanaceous vegetables crops incited by fungal, bacterial, and viruses are
reported from time to time. Among all other bacterial diseases in solanaceous crops,
the bacterial wilt or sudden wilt caused by Ralstonia solanacearum, formerly called
Pseudomonas solanacearum (Smith), is one of the most important diseases world-
wide (Denny 2007). In this chapter, the bacterial wilt disease in solanaceous
vegetables crops is discussed in detail in terms of pathogen biology, nature of
infection, pathogenicity determinants, disease diagnosis, and integrated disease
management (IDM) approaches.
10.2 Economic Impact of R. solanacearum
Ralstonia solanacearum is considered as the world’s most important bacterial plant

pathogen due to its deadly effects on its host plants, especially the solanaceous
vegetables (Genin 2010). Based on the economic importance, the R. solanacearum
10 Ralstonia solanacearum: Biology and its Management in. . . 261
is listed in second rank among the top ten important plant pathogenic bacteria
worldwide (Mansfield et al. 2012). Elphinstone (2005) has estimated the crop losses
to be up to the cost of US$1.0 billion every year due to the R. solanacearum infection
in potato alone. Further, R. solanacearum was listed out in the selective agent of
agriculture bioterrorism Act in 2002 in USA, which considered the pathogen as a
potential tool for developing microbial bioterrorist weapon (Anon 2005). In India,
the yield losses due to R. solanacearum infection of 2.0–95.0% in tomato (Singh
et al. 2010), 80.0–100.0% in eggplant (Selastin Antony et al. 2015), 20.0–100.0% in
chili (Shekhawat et al. 1978), and 50.0% in potato were recorded (Mukherjee and
Dasgupta 1989).
10.3 Symptomatology
Identification of disease symptom is conceptually an interlinked process and likely to

give first clue for pathogen identification (Denny 2007). Although R. solanacearum
has wider host range over 450 plant families, sudden wilting is the very specific
symptom which is also applicable to solanaceous crops including tomato, eggplant,
and chili. On the plant apical portion, the first noticeable symptom was drooping of
green leaves downwards followed by one-side wilt symptom. The progression of
infection extends and then the whole plant wilts typically with remains showing
green-wilt appearance (Fig 10.1a, b, d, e, g and h). In serious cases, water-soaked
necrotic lesions will be produced on collar region of the basal stem as well as near
branching shoots of the plants which culminated in plant death (Balamurugan et al.
2018a). The transversal section of infected stem may show brown vascular
discolorations in the xylem track which is also evident in bacterial wilt disease
(Fig. 10.2).
In tobacco and potato, early stages of the bacterial wilt disease are notably varied
from tomato, eggplant, and chili as follows: wilt-infected tobacco plants show leaves
yellowing, scorching appearance between the veins and leaf distortion followed by
stunted growth (Stevenson et al. 2001; Denny 2007; Garcia et al. 2019). In case of
potato, R. solanacearum causes two different phases of symptoms, i.e., (i) wilt phase
includes leaf chlorosis and drooping symptoms initiate, later dark-brown streaks
develop on the collar region followed by petiole epinasty accompanying with wilt
symptoms occur in the standing plants; whereas (ii) rotting phase occurs in the field
and storage conditions which includes ring-like brown discoloration can visible on
the cut tubers, and in some cases the tuber exudes the droplets of creamy-white
bacterial ooze from the tuber eyes as well as on cross section of tubers, which is
characteristic sign for R. solanacearum identification especially in potato (Stevenson
et al. 2001; Sagar et al. 2013; Garcia et al. 2019; Charkowski et al. 2020).
Traditionally, R. solanacearum infection could be detected through ooze-out or
bacterial streaming or exudation test from any infected portion of the plants before
performing the isolation and further identification process (Denny 2007). The freshly
cut-end of an infected stem, root, and tuber portion may exude the thread-like milky-
white, slimy ooze from vascular system into clear water which is the confirmative
Fig. 10.1 Field symptoms of bacterial wilt disease in tomato (a); eggplant (d) and chili (g); close-
up view of drooping of green leaves downwards observed on wilt infected tomato (b); eggplant (e)
and chili (h) plants; Milky-white, thread-like bacterial ooze exuding from the cut-end of wilt
infected tomato (c); eggplant (f) and chili (i) stems indicated bacterial etiology of the disease
test for R. solanacearum infection (Fig. 10.1c, f, i). This ooze-out test is a prelimi-
nary diagnostic tool for quick and easy detection of R. solanacearum in the field and
under laboratory conditions (Balamurugan et al. 2018b). In the natural field
conditions, the bacterial wilt disease is somewhat difficult and confused with fungal
wilts in solanaceous vegetables crops, especially of very important soil-borne
pathogens including fusarium wilt (Fusarium oxysporum f. sp. lycopersici), southern
blight/sclerotial wilt (Sclerotium rolfsii), and verticillium wilt (Verticillium dahlia
and V. albo-atrum). However, the specific symptomatology characters of bacterial
Fig. 10.2 Bacterial

wilt affected tomato stem
showing brown color vascular
discoloration from the xylem
track after transversal cross
section
wilt can exclude and be differentiated from fungal wilts as summarized in the
Table 10.1.
10.4 Taxonomy of Ralstonia solanacearum
The pathogen R. solanacearum is classified into numerous genera since it was first
described by E.F. Smith and named as Bacillus solanacearum (Smith 1896). Then
Smith changed its name to Pseudomonas solanacearum Smith (1896) in 1914
(Smith 1914). Further, it was included in the approved lists of bacterial names
(Skerman et al. 1980) and subsequent literature was also published under this
name (Hayward 1991). Later, Yabuuchi et al. (1992) transferred P. solanacearum
and nonfluorescent pseudomonads to a novel genus as Burkholderia solanacearum.
Further, the genomic-based molecular approaches to characterize the genus of
bacterial pathogens have been suggested by many workers. Yabuuchi et al. (1995)
proposed a new genus as Ralstonia having distinct characteristics from
B. solanacearum and B. pickettii based on the phenotypic traits, phylogenetic
analysis using 16S rRNA gene, rRNA-DNA hybridization, fatty acid, and cellular
lipid analysis, respectively. The pathogen is currently been referred as
Ralstonia solanacearum (Smith 1896; Yabuuchi et al. 1995) among scientific
community worldwide. The current taxonomic status of this pathogen is placed
Table 10.1 How bacterial wilt differed from fungal wilt in the solanaceous vegetable crops
Specific Fungal wilt
symptoms and (Fusarium oxysporum f. sp.
other lycopercisi, Sclerotium rolfsii,
identification Bacterial wilt Verticilium dahlia, V. albo-
S. No. characters (Ralstonia solanacearum) atrum)
1. Early stage Drooping of green leaves Yellowing of leaves on one
symptoms on downwards during hottest time side of the plant is the first sign
leaves of the day is the very first sign of the disease. In case of
of the disease Verticillum wilt, premature leaf
chlorosis followed by
leaf necrosis is first evident
2. Late stage In serious cases, whole plant Yellowing of leaves rapidly
symptoms on wilt typically and upper leaves advanced to part or entire plant.
leaves remains show green-wilt In serious cases, these leaves
appearance, whereas lower/ turn to blighted appearance and
older leaves show extensive tend to cause wilt symptoms on
wilting and dropped off part or entire plant. In case of
Verticillum wilt, the infected
leaves become of limp and
flaccid appearance
3. Vascular Brown discoloration in the Brown discoloration in the
discoloration vascular bundles is common in vascular bundles is most
the matured plant, but less commonly associated with
conspicuous on young fungal wilt disease; In case of
seedlings Verticillium wilt, light-tan
color discoloration could be
observed
4. Surface infected Water-soaked lesions will Due to disfunctioning of
stem develop on the basal as well as vascular systems, the infected
aerial branching stem that leads stem becomes blightening, and
to darken and collapsed stem, drying leads to death of plant.
fall-off to soil, and plant death The fungal mycelium, spores,
occurred and resting structures remain
present in the vascular systems
and return to soil environment
along with dead plant debris
5. Signature When performing the ooze-out None produce such ooze. On
identification or streaming test, the collar region, stem girdling,
test in the field characteristic thread-like, mycelia growth, and
milky-white, bacterial ooze presence of mustard-like
could exude from cut-end of an sclerotia are signs of sclerotial
infected portion of the plant wilt; V. dahlia produces
confirming bacterial etiology of minute, black color
the disease microsclerotia; V. albo-atrum
produces dark thick-walled
mycelium on the infected plant
debris. Fusarium wilt infection
does not fall under any of two
these categories
6. Mechanism of Due to accumulation of Due to secretion of toxin and
wilting bacterial cells, slimy cell wall degrading enzyme
(continued)

Specific Fungal wilt
symptoms and (Fusarium oxysporum f. sp.
other lycopercisi, Sclerotium rolfsii,
identification Bacterial wilt Verticilium dahlia, V. albo-
S. No. characters (Ralstonia solanacearum) atrum)
extrapolysachharides, and from fungus, the xylem vessels
virulence-related functions in are blocked and movement of
the xylem, the functional sap-flow environment stopped
vessels are blocked and tending to cause wilt disease
movement of sap-flow
environment is stopped tending
to wilt disease
7. Dry fish odor Emit a dry fish odor from Do not emit any odor from
severely infected field infected field
8. Favorable Tropical, subtropical (race 1, 2, F. o. f. sp. lycopersici &
climate 4, 5) and S. rolfsii occur in tropical and
warm temperate regions (race subtropical regions;
3) Verticillium spp. occurs in all
region but more severely in
temperate regions
9. Optimum 29 to 35 C (race 1, 2, 4, 5) and F. o. f. sp. lycopersici — 28 C;
temperature 27 C (race 3) S. rolfsii — 30 to 35 C;
V. dahlia — 20 to 25 C;
V. albo-atrum — 25 to 28 C
under Domain: Bacteria; Phylum: Proteobacteria; Class: Beta-Proteobacteria; Order:

Burkholderiales; Family: Burkholderiaceae; Genus: Ralstonia; and Species:
R. solanacearum, R. pseudosolanacearum, and R. syzygii (Garcia et al. 2019).
10.5 Diversity of R. solanacearum
Bacterial wilt pathogen R. solanacearum is distributed in almost all the continents

including many islands between the Tropics of Cancer and Capricorn (Denny 2007).
R. solanacearum is generally described as a ‘species—complex’ or ‘heterogeneous
species’ (Safni et al. 2014). The term ‘species—complex’ was first applied to
R. solanacearum by Gillings (1994), in light of its wide host range, geographical dis-
tribution and genomic diversity, variations in its physiological characteristics, cross-
transmissible ability, pathogenic-specialization, as well as variations in its phylogeny
from whole-genome sequences (Palleroni and Doudoroff 1971; Hayward 2000;
Kumar et al. 2012; Safni et al. 2014). It was distributed widely in tropical, subtropi-
cal, and certain warm temperate countries of the world (Hayward and Hartman 1994)
and prevalent as endemic form in much of United States, Indonesia, Brazil,
Colombia, Israel, South Africa, and many other vegetable growing countries
(Floyd 2008). To date, R. solanacearum has been reported to infect 50 different
families over 200 plant species including Solanaceae family (tomato, eggplant, chili,
potato, tobacco) and other important crops from monocots (banana, ginger), legumi-
nous plants (peanut, French bean), ornamentals (Pelargonium, Anthurium and
Rose), shrubs and trees (mulberry, olive, cassava, eucalyptus), weed hosts (Sola-
num nigrum and S. dulcamara) as well as model plant Arabidopsis thaliana (Genin
and Denny 2012; Mansfield et al. 2012; Garcia et al. 2019). Further, Moraes (1947)
reported that R. solanacearum is capable to infect the potato even in the temperature
of 24 C in the temperate zones (cold race).
10.6 Pathogen Classification
Classification of R. solanacearum is based on the phenotypic, host range, and

geographical origins, as well as variation in their core sequence alignments from
whole-genome assembly. Historically, R. solanacearum strains were classified into
five races (Table 10.2) based on the host specificity (Buddenhagen and Kelman
1964; Lozano and Sequeira 1970). The race 1 is having many host range in
Solanaceae family (tomato, eggplant, chili, and tobacco), causing bacterial wilt
disease in Southern USA, Asia, Africa, and South America with an optimum
required temperature of 29 to 35 C (warm with lower elevations), and as equally
required for other races like race 2, 4, and 5 (Buddenhagen and Kelman 1964; Denny
2007; Sakthivel et al. 2016; Singh 2017; Garcia et al. 2019). Race 2 infects
Musaceae family and attacks principally on triploid bananas and Heliconia spp.
inciting of moko wilt or bacterial wilt found mainly from Southeast Asia and Central
America (Denny 2007; Garcia et al. 2019). Race 3 infects predominantly potato and
Table 10.2 Current classification of Ralstonia solanacearum species—complex

Phylotype/
Ralstonia species Origin Race Biovar Host plants
R. pseudosolanacearum I (Asia) 1, 3, 4, 5 BW of Solanaceae, ginger, and
4, 5 mulberry
R. solanacearum IIA (America) 1, 2-T, Musa spp. and BW of
2, 3 1, 2 Solanaceae
R. solanacearum IIB (America) 1, 2-T, Moko wilt; highly virulent to
2, 3 1, 2 Anthurium, Heliconia,
cucurbits (but nonpathogenic
to banana); potato brown rot;
BW in tomato and geranium
R. pseudosolanacearum III (Africa) 1 2-T, 1 BW of Solanaceae
R. syzygii IV (Indonesia, 1 2-T, Sumatra disease in clove;
Australia, 1, 2 blood disease in banana; BW
Philippines, of Solanaceae
and Japan)
BW, Bacterial wilt; 2-T refer to ‘tropical’, i.e., extended carbohydrate panel of biovar 2 found in
lowland origin (Denny 2007)
Adopted from Safni et al. (2014), Garcia et al. (2019)
Table 10.3 Biovars of Ralstonia solanacearum species—complex (Denny 2007)

Biovar
S. No. Carbohydrate media 1 2 2-T 3 4 5
1. Dextrose (+) (+) (+) (+) (+) (+)
2. Mannitol ( ) ( ) ( ) (+) (+) (+)
3. Sorbitol ( ) ( ) ( ) (+) (+) ( )
4. Dulcitol ( ) ( ) ( ) (+) (+) ( )
5. Trehalose (+) ( ) (+) (+) (+) (+)
6. Lactose ( ) (+) (+) (+) ( ) (+)
7. Maltose ( ) (+) (+) (+) ( ) (+)
8. D (+) Cellobiose ( ) (+) (+) (+) ( ) (+)
9. myo-Inositol (+) (+) (+) (+) (+) (+)
10. Nitrite from nitrate (+) (+) (+) (+) (+) (+)
11. Gas from nitrate ( ) ( ) ( ) (+) (+) (+)
(+), Positive reaction; ( ), Negative reaction
it is special from other four races as it required optimum temperature of 27 C (cool
with higher elevation) found mainly in the temperate climate (Buddenhagen and
Kelman 1964; Sagar et al. 2013; Singh 2017). This race is also called ‘temperate
race or potato race or cold race’ primarily isolated and associated with potato brown
rot/bangle blight disease. Race 4 specifically infects Zingiberaceae family and
especially causes bacterial wilt disease in ginger and small cardamom found in
Asia and Hawaii (Persley 1986; Kumar and Sarma 2004; Kumar et al. 2004, 2012,
2014a, 2017; Kumar 2006; Kumar and Abraham 2008). Race 5 particularly causes
wilt disease on mulberry (Moraceae family), so far reported from China (He et al.
1983; Denny 2007; Garcia et al. 2019).
R. solanacearum is also traditionally classified into six biovars including biovar1,
2, 2-T, 3, 4, and 5 (Table 10.2), based on the utilization or acidification of the basal
media containing different carbon sources like disaccharides and sugar alcohols as
given in the Table 10.3 (Hayward 1964). Few strains infecting potato isolated from
Amazon basin differ with biovar 2; hence, they utilize the extended carbohydrates
panel and this new group is known as biovar 2-T (T refers, tropical) in the recogni-
tion of its lowland origin (Garcia et al. 2019). The biovar 3, 4, 1, 2 are associated
with race 1 (Solanaceae family); biovar 2-T, 1, 2 associated with race 2 (Musa spp.);
biovar 2, 1, 2-T associated with race 3 (particularly potato); biovar 3, 4 associated
with race 4 (ginger, small cardamom); and biovar 5 mostly associated with race
5 (mulberry). Exact race assignment for biovar 2-T strains is problematic because
they are identified to have a wider host range than archetypal biovar 2, known as race
3. Biovar assignations are somewhat confusing because reports demonstrated that
due to single frame-shift mutation in the plants or in storage, and horizontal gene
transfer could change phenotypic reaction of the strain. However, these subgrouping
or classification systems for race and biovars identification in R. solanacearum are
neither biological means of predictions nor genetically meaningful (Lowe-Power
et al. 2018) and are still to be verified at strains level (Garcia et al. 2019), or using
new alternative methods. Although there is no specific correlation between races and
biovars, strains belong to biovar 2 almost race 3, and strains belong to biovar
5 usually race 5 (Denny 2007). Additionally, the phylogenetic studies indicated
that strains belonging to biovar 3, 4, and 5 shared separate genetic-lineage and were
formed distinct from other biovars (Hayward 1994).
Further, using multiplex-PCR assay R. solanacearum is classified into four
phylotypes (Table 10.2) based on geographical origins are as follows: the
Phylotype—I: belong to Asia origin (specific amplicon size is 144 bp);
Phylotype—II: belong to America origin (specific to 372 bp); Phylotype—III:
belong to Africa origin (specific to 91 bp); and Phylotype—IV: belongs to Indonesia
and Australia (specific to 213 bp), which was originally suggested by Fegan and
Prior (2005) according to their sequence variation of the internal transcribed spacer
region. The ‘phylotype’ is a cluster of monophyletic strains identified by the analysis
of phylogenetic relationship from their sequences, and it can be used to identify the
subspecies level to even a separate species (Fegan and Prior 2005). The proposal of
Safni et al. (2014) to the International Journal of Systematic and Evolutionary
Microbiology revealed that high intraspecific variations in genomic profile of
R. solanacearum species—complex are classified into three species or genospecies
such as (i) Ralstonia pseudosolanacearum (corresponding to Phylotypes—I and III
strains); (ii) Ralstonia solanacearum (corresponding to Phylotype—II strains); and
(iii) Ralstonia syzygii (corresponding to Phylotype—IV strains) based on whole-
genome sequences. Each phylotype can be further subdivided into sequevars
(sequence variants); it is a group of strains having the highly conserved partial
sequence regions of endoglucanase (egl) gene (Fegan and Prior 2005), and currently,
around 20 sequevars have been identified for all the four known phylotypes (Garcia
et al. 2019). Among the five races, six biovars, and four phylotypes, the race 1, biovar
3, and phylotype—I is genetically and geographically well-distributed in nature and
causes bacterial wilt outbreak in major solanaceous crops worldwide (Denny 2007;
Mansfield et al. 2012). In India, prevalence and distribution of race 1, 3, and 4;
biovar 1, 3, 4; and phylotype—I, IIB were previously reported by several workers,
both from solanaceous crops and also from ginger and small cardamom (Shekhawat
et al. 1978; Kumar and Hayward 2005; Kumar et al. 2014a, b; Ramesh et al. 2014;
Sagar et al. 2014; Sakthivel et al. 2016; Singh 2017; Balamurugan et al. 2018a).
Among all, race 1, biovar 3, and phylotypes—I was considered as much important as
a consequent of bacterial wilt disease in solanaceous vegetable crops and its genetic
diversity also wider spread across the mainland as well as Andaman Nicobar islands
regions.
10.7 Diagnosis and Detection
Bacterial pathogen, R. solanacearum, is difficult to identify on nutrient medium until

otherwise when selective media were used. Kelman (1954) developed the nonselec-
tive medium called casamino-acid peptone glucose (CPG) agar amended with 1%
triphenyl-tetrazolium chloride. It is the most common and routinely used growth
Fig. 10.3 Colony characters of R. solanacearum; irregularly round, white-fluidal, opaque single
colonies along with pinkish center on CPG agar amended with 2, 3, 5 triphenyl-tetrazolium chloride
is the characteristic to R. solanacearum (a); Virulent colony of R. solanacearum appears as highly
white-fluidal with less pink center (b); Avirulent colony appears to flatten, non-fluidal, butyrous/dry
and narrow streak (c); Absence of exopolysaccharides (EPS) makes difference between these two
colonies (b, c)
medium for selective isolation of R. solanacearum from infected specimens and

from frozen stocks. Usually, R. solanacearum colonies are visible after 36 to 48 hrs
of incubation at 28–30 C, but some strains prefer 27 C as lower optimal growth
temperature (Prasannakumar et al. 2011). In the process of isolation, by streaking of
bacterial ooze on to 1% triphenyl-tetrazolium chloride amended CPG medium, there
are two types of R. solanacearum colonies differentiated such as (i) virulent/wild
type and (ii) avirulent/mutant type. Due to secretion of exopolysaccharide (EPS) by
the bacteria, the virulent type colonies appeared to be white or cream-colored, highly
fluidal, opaque, irregularly round centered with pinkish pigmentation, whereas
avirulent colonies appeared non-fluidal or butyrous, narrow streak, dark red, and
evidence in absence of EPS compositions (Fig. 10.3a, b and c). This spontaneous
mutation in R. solanacearum referred as ‘phenotypic conversion’ due to the muta-
tion in the phcA gene usually occurs during the process of preservation, repeated
subculturing, or under oxygen stress (Poussier et al. 2003). Virulent colonies directly
measure and are highly pathogenic to host plants, whereas avirulent colonies are
nonpathogenic or weakly pathogenic (Poussier et al. 2003; Lowe-Power et al. 2018)
and/or antagonistic against even virulent isolates of R.solanacearum (Zheng et al.
2019). Reversal of the phenotypic conversion is recently reported by Sahu et al.
(2020) under certain physiochemical pressure that generates virulent type colonies
from avirulent ones. In this study, a protocol was developed using nutrient
deprivation and Phyllanthus emblica fruit extract for the reversal of EPS production
in avirulent type colonies under laboratory experiment.
The pathogen R. solanacearum is a soil-inhabitant, gram negative, aerobic,
rod-shaped, motile, and non-spore forming bacterium. Individual cell size varies in
length from approximately 1.5 to 2.0 μm to 0.5 to 0.7 μm width (Champoiseau
2009). Transmission electron microscopy study using negatively stained
R. solanacearum cells revealed to have lophotrichous type of flagella (two or three
flagella at one polar end), which ensure their motility in the aqueous environments
(Clough et al. 1997). Various biochemical methods to characterize the
R. solanacearum demonstrated that R. solanacearum is positive for potassium
hydroxide (3 % KOH) test (Suslow et al. 1982), appears pink-red color rod-shaped
cells on gram-staining reaction (She et al. 2017), positive for catalase test (He et al.
1983), and negative for fluorescent-pigments production on King’s B medium (Clara
1934) and indole production on Kovac’s reagent (Prior and Steva 1990). The 1%
NaCl amended liquid nutrient broth supports the R. solanacearum growth, whereas
beyond the 1% NaCl concentration no growth or poor growth was observed (Kumar
and Sarma 2004; She et al. 2017). R. solanacearum isolates also highly sensitivity to
1% bleaching powder amend nutrient medium where complete growth inhibition
was also recorded (Singh et al. 2012; Balamurugan et al. 2018b).
Phenotypically, R. solanacearum can be identified through Biolog (Biolog Inc.,
Hayward, CA) based approach. Biolog is a phenotypic metabolic finger printing
method and applies by single panel system using microtiter plate which contains
71 carbon sources and 23 chemical sensitivity preselected assay solely for bacterial
substrate utilization. It is easy to handle, reliable, can detect the identity of the
organisms within 24 hrs of incubation, and several attempts were reported on
R. solanacearum (Tawfik et al. 2008; Albuquerque et al. 2016).
Serological-based diagnostic kit is also available for the detection of
R. solanacearum both from field and laboratory condition. Nitro-cellulose-mem-
brane-based ELISA originally developed by International Potato Center, Lima, Peru,
for the detection of R. solanacearum in the seed tubers of potatoes was also adopted
for indexing ginger rhizomes (Priou et al. 1999; Kumar et al. 2002). Agdia® (Agdia,
Elkhart, IN) is the commercially available, farmer-friendly, field diagnostic
R. solanacearum-specific immunoassay strip and can detect the result within
10 minutes of use from freshly infected plant samples (Selastin Antony et al.
2015; Ireland et al. 2016). The Central Science Laboratory, UK, developed the
lateral flow device kit to detect the R. solanacearum in a single step process within
3 minutes (Singh 2017; Thind 2019).
Methods described above are generally reliable and easy to perform, but each
technique has certain limitations. False-positive results in the serological-based
approach are one of the drawbacks and the results proportionally increase with
number of dead cells and contaminants in the samples (Garcia et al. 2019).
DNA-based approaches using PCR techniques are very reliable, accurate, and
more sensitive method to identify the R. solanacearum (Weller et al. 2000) than
other methods. With the advancement of molecular biology, many genomic
techniques are available to verify and investigate the phylogeny of
R. solanacearum (Denny 2007). The universal bacterial primers, 27F and 1492R, for
the target of 16S ribosomal RNA (16S rRNA) gene become unique for the identifi-
cation of bacteria at the genus level and several workers reported for identification
(Taghavi et al. 1996; Kumar 2006; Balamurugan et al. 2018a). Ireland et al. (2016)
used the fD1 and rP2 primer probe for identification of 16S rRNA region and
confirmed the sequence result as R. pseudosolanacearum which incited the wilt
disease in tomato. For specific identification of R. solanacearum through PCR-based
assay, Opina et al. (1997) have developed the species-specific primers using 759F
and 760R to attain the 281 base pairs, which are specific to R. solanacearum. Till
date, these primers are widely adopted and used for the species level identification of
R. solanacearum (Kumar and Abraham 2008; Sakthivel et al. 2016; Balamurugan
et al. 2018a; Mollae et al. 2020). The primer probes of Y2F and OLI1R also were
reported for species-specific identification of R. solanacearum (Singh et al. 2010;
Sagar et al. 2013). Additionally, recombination (recN) gene-based makers for
intraspecific classification of R. solanacearum are also reported (Kumar et al.
2013). Other molecular tools that include total genomic fingerprinting by restriction
fragment length polymorphisms (Van Der Wolf et al. 1998), amplified fragment
length polymorphism (Poussier et al. 2000; Mollae et al. 2020), repetitive element-
PCR (Horita and Tsuchiya 2001), qPCR (Chen et al. 2010), microarray (Cellier et al.
2017), real-time loop-mediated isothermal amplification for field detection of
R. solanacearum (Prameela et al. 2017), multilocus sequence typing (Kumar et al.
2014a; Sakthivel et al. 2016), draft genome (Zou et al. 2016; Kotorashvili et al.
2017; Kumar et al. 2017), and whole-genome sequencing (Salanoubat et al. 2002)
are also investigated to describe the phylogeny of R. solanacearum.
Although PCR techniques are available to characterize the R. solanacearum
through 16S rRNA gene and species-specific primers, none of these primer probes
allow them for identification of the phylotypes which identify pathogen based on
broad geographical origin. To address these limits, Fegan and Prior (2005) have
developed multiplex-PCR primers which provide the genomic gateway for identifi-
cation of the geographical origin of R. solanacearum and the genetic variations in the
pathogen have been successfully studied by several workers (Safni et al. 2014;
Lowe-Power et al. 2018; Mollae et al. 2020).
For the past several years, MLST/MLSA (Multilocus Sequence Typing/Analy-
sis)-based genotyping systems become popular to investigate microbial populations
of bacterial pathogens (Almeida et al. 2010; Kumar et al. 2020). The MLST-based
identification of R. solanacearum was developed by Castillo and Greenberg (2007)
to exploit the variation in housekeeping genes in a diverse group of the strains which
consisted of five housekeeping genes, including gapA (glyceraldehyde 3-phosphate
dehydrogenase oxidoreductase), gdhA (Glutamate dehydrogenase Oxidoreductase),
ppsA (phosphoenolpyruvate synthase), adk (adenylate kinase), and gyrB (DNA
gyrase), and three megaplasmid-borne virulence-related genes such as egl
(endoglucanase precursor), fliC (encoding flagellin protein), and hrpB (regulatory
transcription regulator), respectively, based on its specific conserved regions present
in each R. solanacearum strain. MLST-based genetic characterization of
R. solanacearum is used to not only identify the bacterial diversity, but also enable
to understand the pathogen migration across the regions which reflect the horizontal
spread of the bacterium through movement of any planting materials (Kumar et al.
2014a; Sakthivel et al. 2016; Balamurugan et al. 2018a), also reported on other
baterial pathogen (Kumar et al. 2020).
10.8 Mechanisms of Bacterial Wilt Disease Development

in Solanaceous Vegetables
R. solanacearum is a typical soil-inhabitant pathogen and survives in soil for many

years even in the absence of main hosts. In the tropical and subtropical regions, the
pathogen is favored by many factors including contaminated soil, susceptible host,
virulent strains, temperature between 29 to 35 C (27 C for potato race in the
temperate climate), and prolonged soil wetness with warmer condition which are
highly conducive for R. solanacearum infection (Prasannakumar et al. 2011).
During the process of infection, the cells of R. solanacearum enter the root systems
typically through root wounds, root tips, natural opening, emerging lateral roots, as
well as nematode (Meloidogyne incognita) injury and facilitate the easier entry of the
bacterium (Lowe-Power et al. 2018; Furusawa et al. 2019). Bacterial cells locate the
host roots by polar flagellar fashion motility is triggered through chemotaxis signals
from root exudates (Tans-Kersten et al. 2001). Initial contact and attachment with
host surfaces is achieved through secretion of EPS, adhesion protein, and
appendages like pili present in the bacterial cell-surface (Hoffman et al. 2015).
Once attached, it then turns to form micro-colonies at the favorable entry sites,
lateral emerging roots as well as root elongation zone (Vasse et al. 1995; Hawes et al.
2016). Firstly, the bacterium moves towards the host roots where it attaches to the
epidermis layer and infects the root cortex. Upon its entry into the root cortex, the
bacterium then systemically spreads to the xylem network, where it rapidly
multiplies and produces high densities of extra-polysaccharides, and functioning
of cell wall degrading enzymes accounted for its pathogenicity and virulence
determinants. This leads to blockage of the xylem vessels due to sap flow stops
and which further leads to dysfunctioning of xylem cylinder which is accompanied
by wilt symptoms followed by plant death (Lowe-Power et al. 2018). Then the
bacterium is released from the dead host debris, returns to the soil environments
(Alvarez et al. 2008), and continues for its survival or saprophytic life state on weed
hosts, soil, water, tubers, etc., ensuring as a potential reservoir for next disease cycle
(Fig. 10.4).
10.9 Pathogenicity Determinants of R. solanacearum
During pathogenesis, the most important virulence functions involved in

R. solanacearum infection are discussed below. The exopolysaccharide (Orgambide
et al. 1991) and lipopolysaccharide (Kocharova et al. 1993) play an important role to
block the xylem vessels and thereby develop sudden wilting symptom. A syringe-
Fig. 10.4 Schematic representation of R. solanacearum disease cycle in solanaceous vegetable

crops. R. solanacearum can survive in soil, infected plant materials, seeds, and weed hosts over the
periods (a); Bacterium contact with the healthy plant in the soil (b); The bacterium recognizes and
attaches the host roots, then forms micro-colonies. Once enough population is attained on the root
surface, then the bacterium enters root tissues through natural wounds, root hairs, root tips, and
nematode injury and invade the vascular system, colonize the xylem vessels, thereby developing
wilt symptoms (c); Under favorable conditions, the wilt symptoms associated with multiplication of
the bacterium and secretion of copious amount of exopolysaccharides in the xylem track lead to
cause typical wilt symtpoms and plant death was occur (d); R. solanacearum cells return to the soil
environments from dead or diseased plant tissues and possibly carried through irrigation water,
root-to-root contact, displacement of contaminated soil and farm equipments, and nematode injury
(e); R. solanacearum spreads to entire fields, leading to wilt disease outbreak (f); During the
off-season, the bacterium survives as saprophytic life stage on the soil and weed hosts and starts
new infection cycle in the next season (g)
like projection called type-III secretion system (T3SS) present on the bacterial cell
and used to inject the ‘effector proteins’ to plant cell cytosol also contribute to the
virulence (Coll and Valls 2013). In addition to these biological weapons, others
factors also contribute to the virulence functioning such as protein appendages called
flagella and pili used for motility, attachment, and biofilm formation on the surface-
roots (Tans-Kersten et al. 2001), type 4 pili used to move on the solid surface by
twitching motility fashion (Strom and Lory 1993), hrp is the pathogenicity gene
control wilt induction and hypersensitive reaction (Boucher et al. 2001), as well as
type-II (T2SS) secreted enzymes used to collapse the plant cell wall (Kang et al.
2002) were reported. However, other molecular and physiological functions also
contributes to the R. solanacearum virulence and were discussed (Alvarez et al.
2008; Genin and Denny 2012; Peeters et al. 2013; Lowe-Power et al. 2018).
10.10 Transmission, Spread, Survival, and Inoculum Source
Transmission of R. solanacearum can also be carried over long distances through the
movement of latently infected planting materials and seed tubers which are studied
well (Brito et al. 2002; Sagar et al. 2013; Charkowski et al. 2020). Potato bacterial
wilt is a well-known fact for its local and international spread through latently
infected seed potatoes (Tiaden et al. 2010) and geranium cuttings produced in Africa
and Central America (Williamson et al. 2002). As a consequence of seed potato
movement, the potato strain grown in planta in the lower altitudes (27 C) often
expresses latent infection in the higher altitudes (29 to 35 C) from tropical and
subtropical potato growing areas, interestingly this same strain can cause wilt disease
on tomato (Charkowski et al. 2020). Potato tubers may carry the pathogen on surface
of seed potatoes, lenticels, and inside the vascular tissue (Genin and Denny 2012;
Charkowski et al. 2020). True seed transmission of R. solanacearum has long been
suspected and even confirmed by its seed-transmitted nature by several workers
(Devi and Menon 1980; Shakya 1993; Sanchez Perez et al. 2008; Genin and Denny
2012). However, the possibility of latently infected true seed in case of tomato,
eggplant, chili, and tuber potatoes needs to be investigated with more study using
sensitive techniques using DNA-based probes and monoclonal antibodies, etc.
(Genin and Denny 2012).
R. solanacearum spreads over long distance and nearby field through displace-
ment of contaminated soil, movement of surface irrigation water from infected field
to disease-free field, dissemination by farm equipments, and improper cultural
practices that can translocate the bacterium (Kelman 1965; Marchetti et al. 2010;
Remigi et al. 2011; Genin and Denny 2012). Root wounding by root-knot nematode
(M. incognita) infection seems to play an important role in increasing bacterial wilt
incidence in tomato (Deberdt et al. 1999; Furusawa et al. 2019) and eggplant (Zakir
and Bora 2009).
During overwintering, the cells of R. solanacearum stay as saprophytic life stage
in association with reservoir plants, infected plant stubbles, and soil (Hayward 1991;
Elphinstone 1996). Several weed hosts including Solanum dulcamara (bittersweet
nightshade), Solanum nigrum (black nightshade), and Urtica dioica (stinging nettle)
are frequently reported as potential host for R. solanacearum survival (Olsson 1976;
Wenneker et al. 1999; Pradhanang et al. 2000; Charkowski et al. 2020).
The R. solanacearum cells exude and are released from infected plant materials
and enter inside the soil by a matrix of protective extra-polysaccharide substance
(Shekhawat and Perombelon 1991). van Elsas et al. (2000) reported that
R. solanacearum survived in agricultural soil up to one year; up to two years after
removal of crops (Shamsuddin et al. 1979); and holds the wilt causing capacity even
four year intercropping period with nonhost plants (Graham et al. 1979). Pathogen
has the ability to survive in contaminated water and multiply in pure water in the
absence of nutrients. Potentially, irrigated field with contaminated water responsible
for bacterial wilt outbreak on several crops and water-channel are considered as
major routes for R. solanacearum dissemination (Elphinstone 1996; Janse 1996; van
Elsas et al. 2001; Caruso et al. 2005; Alvarez et al. 2008). Not surprisingly, by
keeping bacterial wilt infected or dead plant debris and seed potatoes within the
growing field itself provides an excellent source of bacterial inoculums in the next
season, and the R. solanacearum population will build-up much higher than the
previous season.
10.11 Integrated Disease Management
Bacterial wilt disease is very difficult to control due to its wider host range and
geographical distribution, species complexity, antibiotic resistance, soil-inhabitant
nature, ability to survive in the soil for many years, and association with weed host
(King et al. 2008; Yuan et al. 2014). Till date, numerous control strategies are well-
practiced and recommended regarding usage of chemical fumigants, tillage
practices, use of organic amendments, external nutrient supplements, usage of
resistant cultivars and other beneficial microbes, etc. Chemical usage through soil
disinfection has only temporary effect, but it may also pose serious threat to
nontargeted beneficial microbes and can cause environment hazards to food chain
and human health (Gamliel et al. 2000; Yi et al. 2007). Soil solarization, a steam
therapy method is another strategy for controlling soil-borne pathogens, but it is not
economically feasible under larger field conditions (Tamietti and Valentino 2006).
In recent days, control of R. solanacearum by using potential beneficial microbes
has been suggested for suppression of bacterial wilt under field conditions either
alone or in consortia with different formulations (Ji et al. 2008; Zhang et al. 2008;
Xue et al. 2009; Ramesh et al. 2009; Ling et al. 2010; Liu et al. 2012). However,
these potential biocontrol activities persist and perform well only under in vitro trails
than the field grown experiment (Kamilova et al. 2005; Liu et al. 2012). The usage of
transgenic crops, avirulent strains of R. solanacearum, and bacteriophages has also
been suggested for managing the bacterial wilt disease (Dong et al. 1999). But the
usage of genetically edited plant varieties may raise the environmental issues
(Lemessa and Zeller 2007) on diverse organisms.
10.11.1 Host Plant Resistance
Selection of breeding program for development of bacterial wilt resistance to most

solanaceous crops on a regional level is the best option (Champoiseau et al. 2010).
International potato center has developed highly resistant potato cultivar against
bacterial wilt disease that were selected from two wild Andean potato species
(Solanum commersonii S. tuberosum), which reveals the colonization of

R. solanacearum was detected in roots, but not on stems (CIP 2004; Carputo et al.
2009). Tomato cultivar Hawaii 7996 developed through breeding program has
become popular for its high levels of resistance to bacterial wilt disease, but it
produces only small fruit size (Kwak et al. 2018). The commercial cultivars of
FL7514 and BHN 466 become more popular with large-sized fruit and provide
moderate level resistance to bacterial wilt disease (Champoiseau et al. 2010). In
India, four cultivars of tomato, Arka Rakshak (Sadashiva 2013), Sakthi, Anagha,
and Manulakshmi (Nandakumar 2014); three cultivars of eggplant, Swetha, Surya,
and Haritha; two cultivars in chili, Ujjwala and Anugraha (Nandakumar 2014); and
five cultivars in potato, CIP382381.13, CIP381381.20, CIP382193.9, CIP378699.2,
and CIP387792.5 (Nagesh et al. 1997; Patil et al. 2012) were shown to be resistant to
bacterial wilt disease and widely adopted among Indian growers.
Another option is imparting host plant resistance through grafting of susceptible
cultivars onto resistance root-stock of Solanum torvum, which proves effective
control of bacterial wilt incidence (Saddler 2005; Singh et al. 2015). Tomato plants
grafted onto ‘Dai Honmei’ and ‘RST-04-105-T’ root stocks conferred high and
moderate levels of resistance against bacterial wilt disease (Rivard et al. 2012).
Upon soil inoculation, the highly virulent strains of R. solanacearum isolated from
tomato, eggplant, chili, and potato (race 1, biovar 3, phylotype—I) were found to be
highly resistant against wild species of S. torvum (unpublished data), but these
strains were highly pathogenic to all these crops by respective host as well as
cross-inoculation (Sakthivel et al. 2016; Balamurugan et al. 2018a).
10.11.2 Biological Control
Root-associated bacteria are an important functional group of beneficial bacteria

widely used for control of soil-borne pathogens worldwide. Several potential
rhizobacteria proved their antifungal, antibacterial activity as well as plant growth
promotion against R. solanacearum, and it will facilitate the frontline defenses of
roots to invading soil-borne pathogens (Gamalero et al. 2009; Kurabachew and
Wydra 2013; Guo et al. 2014; Tahir et al. 2017). Large members of biocontrol
agents were reported to manage the R. solanacearum in solanaceous vegetables as
furnished in the Table 10.4. Among all, Bacillus species were most studied against
R. solanacearum due to their five special advantages from other genus as follows:
Bacillus species have (i) ability to form heat- and desiccation-resistant spore called
‘endospore’ which can be used to formulate dry white powder with longer shelf life;
(ii) Bacillus species are playing the important role for its plant growth promot-
ing function; (iii) Bacillus species produce array of antimicrobial lipopeptides, viz.,
fengycin, bacillomycin, iturins, and surfactin; (iv) Bacillus species counterattack the
invading phytopathogens by outcompeting (root surfaces) for colonization in the
rhizosphere zone; (v) Bacillus species are able to survive as epiphytes, endophytes,
phyllosphere, spermosphere, and natural inhabitants of the crops microflora, etc.
(Tahir et al. 2017; Sakthivel et al. 2019).
Table 10.4 Potential antagonistic rhizhosphere bacteria against R. solanacearum for disease
management
S. No. Antagonistic bioagents Host used References
1. Bacillus species: Tomato, Saddler (2005), Zhou et al.
Bacillus amyloliquefaciens Eggplant, (2008), Ramesh et al. (2009),
B. velezensis Chili, Potato, Ramesh and Phadke (2012),
B. cereus and Tobacco Hyakumachi et al. (2013),
B. subtilis Kurabachew and Wydra
B. artrophaeus (2013), Raza et al. (2016a),
B. sacchari Tahir et al. (2017), Cao et al.
B. tericola (2018), Balamurugan et al.
B. pyrrocinia (2018b), Sakthivel et al.
B. thuringiensis (2019), Alamer et al. (2020)
Bacillus spp.
2. Pseudomonas species: Eggplant, Saddler (2005), Ramesh
Pseudomonas fluorescens Tomato, and (2006), Ramesh et al. (2009),
P. brassicacearum Chili, Ramesh and Phadke (2012),
P. aeruginosa Vanitha et al. (2009), Zhou
P. mallei et al. (2012), Raza et al.
P. cepacia (2016b), Alamer et al. (2020)
P. putida
Pseudomonas sp.
3. Burkholderia species: Tomato, Nion and Toyota (2008),
Burkholderia nodosa Eggplant, and Ramesh et al. (2009)
B. sacchari Spinach
B. tericola
B. pyrrocinia
B. cepacia
4. Other bacterial agents: Tomato, Hoa et al. (2004), Saddler
Serratia marcescens Eggplant, (2005), Momma et al. (2007),
Serratia sp. Chili, Ramesh et al. (2009), Xue et al.
Enterobacter cloacae Tobacco, and (2009, 2013), Tan et al. (2011),
Chryseobacterium daecheongense Potato Yang et al. (2012), Huang et al.
C. indologenes (2013), Wei et al. (2013), Li
Clostridium sp. and Dong (2013), Kurabachew
Flavobacterium johnsoniae and Wydra (2013), Yuliar et al.
Myroides odoratimimus (2015)
Paenibacillus marcerans
Ralstonia pickettii
Sphingomonas paucimobilis
Staphylococcus auricularis
Streptomyces rochei
S. virginiae
Escherichia sp.
Actinomycetes
5. Avirulent strain of R. Tomato and Arwiyanto et al. (1994), Khan
solanacearum shown potential Eggplant et al. (1997), Grimault and
antagonistic to virulent strains of Prior (2008), Zheng et al.
R. solanacearum: Avirulent strain (2019)
FJAT-1458 and hrp-mutants
6. Virulent bacteriophage against Tomato and Toyoda et al. (1991),
R. solanacearum: Phage φP4282, Eggplant Sambrook and Russell (2001),
(continued)

S. No. Antagonistic bioagents Host used References
φPK101, φP4282, φRSA1, Ozawa et al. (2001), Kawasaki
φRSL1, φPK-101, φRSS1, and et al. (2007), Fujiwara et al.
φRSM1 (2008), Yamada et al. (2010)
Apart from rhizosphere microbes, the mutant/avirulent strains of

R. solanacearum become important and also identified as a good antagonistic effect
against virulent strains of R. solanacearum both in in vitro and in vivo studies
(Arwiyanto et al. 1994; Grimault and Prior 2008; Zheng et al. 2019). Several studies
reported that the severity of bacterial wilt disease can be checked by avirulent strains
of R. solanacearum (Kempe and Sequeira 1983; Spadaro and Gullino 2005). The
avirulent strain FJAT-1458 of R. solanacearum was isolated from healthy tomato
plant and proved to have high antagonistic efficiency against R. solanacearum under
pot culture and greenhouse condition (Zheng et al. 2016, 2017). Recently, the
combined use of microbial restoration substrate using avirulent strain (FJAT-1458)
of R. solanacearum has proved their antagonistic potential against bacterial wilt in
tomato (Zheng et al. 2019). On the other hand, the stability of avirulent strains of
R. solanacearum needs to be addressed upon its field application; since the reversal
of virulent colony types from avirulent ones is reported recently (Sahu et al. 2020), it
may aggravate the disease.
Phage therapy is one of other methods in biological control also studied against
R. solanacearum. In recent decades, the phage therapy has been explored for control
of phytopathogens (Fujiwara et al. 2011). In case of R. solanacearum, two
bacteriophages called P4282 and PK101 have been isolated, characterized, and
infected only few range of R. solanacearum (Sambrook and Russell 2001; Ozawa
et al. 2001). Phage ɸRSA1 is a Mycoviridae, isolated and shown susceptibility to
race 1, 2, 3, and 4 strains of R. solanacearum (Fujiwara et al. 2008). The phage
ɸRSL1 is another Mycoviridae approximately 231 kb in size which is reported to
lyse the 15 strains of R. solanacearum (Yamada et al. 2010). Phage ɸRSB1 belongs
to Podoviridae having a T7-like appearance and infects the 14 to 15 strains belong-
ing to race 1, 2, 3, and 4 (Kawasaki et al. 2009). The increased antibacterial efficacy
of phage therapy-based methods in biocontrol seemed to be useful for successful
eradication of bacterial wilt pathogen in the field condition. However, better under-
standing of phage-ecology and phage-host interaction is needed to be studied in
more details (Fujiwara et al. 2011).
10.11.3 Chemical Control
Complete control of bacterial wilt disease in the field conditions is very difficult
because bacterium localizes inside the xylem vessels and is able to survive at depth
in soil (Mariano et al. 1998). Fumigating the soil using methyl-bromide, vapam,
chloroicrin, or antibiotics is done in some countries, but has only limited efficacy
and/or no more usage. Also, production of methyl-bromide gas was phased out
during 2005 since it causes depletion of the stratospheric ozone layer according to
Montreal Protocol and Clean Air Act (Fujiwara et al. 2011). Bacteriostatic nature of
1.0% NaCl and 0.25 to 1.0% bleaching powder amended nutrient media able to
inhibit the growth of R. solanacearum was reported earlier only under laboratory
condition (Kumar et al. 2004; Singh et al. 2012; Balamurugan et al. 2018b). Actigard
(Syngenta) is a plant resistance inducer and enhances the resistance against
R. solanacearum, only when used on moderately resistance cultivar if available
(Champoiseau 2009). However, there is no specific chemical identified for success-
ful control of R. solanacearum under field conditions (Singh 2017).
10.11.4 Cultural Control
Several agronomic/cultural practices can reduce the bacterial wilt disease such as
crop rotation with nonhost plant species, but only partial or less control of the wilt
disease can be achieved (Akiew and Trevorrow 1994; Mariano et al. 1998).
R. solanacearum is a ubiquitous soil-borne pathogen; hence, implementations of
phytosanitary measures are important where the disease is of an endemic form and/or
recently introduced (Saddler 2005). Use of disease-free planting, seed (seed
potatoes), and other vegetative propagated materials and avoidance of weed hosts
through herbicides application also have been considered. Intercropping with use of
cereals crops, such as maize and wheat, and other plant species such as onion,
cabbage, bean, and pea proved variable efficiency to control bacterial wilt disease
(Pradhanang and Elphinstone 1996; Terblanche 2002; Katafiire et al. 2005).
10.12 Conclusion
Due to its extreme diversity in host range and strainal variability, this bacterial wilt
pathogen, R. solanacearum, is now called R. solanacearum species complex
(RSCC). The successful management of this disease in field conditions is of serious
concern. The available host plant resistance is limited, and hence, resistant cultivars
are not much reported. Also, no specific chemical has been reported for successful
management unlike other diseases. Nowadays, biological control using native
antagonistic microbes seems to be a promising approach towards disease manage-
ment under field conditions. Bacterial wilt disease is continually reported in new
locations and hosts, and hence, imposing strict quarantine measures even in intra-
regional transportation on seed and seed potatoes or other vegetative planting
material would be helpful to avoid the rapid spread of this disease in newer locations.
At present, pathogen biology and mechanisms of infections of R. solanacearum are
well-understood using recent molecular approaches, and hence, the future research
should focus on resistance breeding programs for effective disease management
against wider strains of bacterial wilt pathogen with the available knowledge.
Acknowledgments Dr. A. Kumar is thankful to Consortium Research Project on Genomics

[ICAR-CRP, (Genomics)] for financial assistance to carry out whole-genome sequencing and
genotyping of Ralstonia solanacearum isolates. Facilities provided by Directors ICAR-IARI,
New Delhi, and ICAR-IIOR, Hyderabad, & Head, Department of Plant Pathology, TNAU,
Coimbatore is gratefully acknowledged.
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Genome Announc 4(2):e00241–e00216
Seed Endophytes: The Benevolent
Existence in the Plant System 11
Shrey Bodhankar and Minakshi Grover
Abstract
Endophytic microbial communities of crop plants interact intimately with hosts

and are associated with almost every plant. Endophytic microorganisms exhibit
an array of plant beneficial traits similar to those reported for plant growth–
promoting rhizobacteria (PGPR). Their localization inside the plant gives them an
advantage over rhizobacteria in terms of availability of nutrients and shield
against external unfavorable conditions. However, many microbial and host traits
as well as environmental factors influence endophytic colonization. In the last
decade, several reports have been published on the isolation of endophytic
microorganisms from different plant tissues and their role in promoting plant
growth under various environmental conditions. Seed endophytes are of great
interest because of their capability to colonize the developing seeds, stay latent in
the dormant seed, and transmit to the next generation after germination and plant
development. Endophytic bacteria isolated from seeds have been found to pro-
duce antimicrobial compounds, phytohormones, enzymes, secondary
metabolites, etc. that make them industrially as well as agriculturally very
important. Application of seed endophytes has been reported to improve plant
growth and physiological status under biotic and abiotic stress conditions. Lim-
ited studies have also reported their influence on plant gene expression resulting
in altered physiological response. However, focused research is needed to study
the vast diversity of endophytes in different crops and also to unravel their
interaction with host plants, for their potential application in agriculture. The
present book chapter aims to focus on the significance of seed endophytes, their
S. Bodhankar
ICAR-Central Research Institute for Dryland Agriculture, Hyderabad, Telangana, India
ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India
M. Grover (*)
ICAR-Indian Agriculture Research Institute, New Delhi, India

23, https://doi.org/10.1007/978-981-15-9154-9_11
292 S. Bodhankar and M. Grover
entry, establishment, diversity, and interactions with host plants along with the
applications in agriculture under changing climatic conditions. A better under-
standing of plant-endophyte interaction can help in the optimization of plant
microbiome and microbe assisted breeding in the near future.
Keywords
Abiotic stress · Climate change · Gene expression · Transmission
11.1 About Endophytes
Anton de Bary first introduced the terms “epiphytes,” for the fungi living on the host
plant’s surface and “endophytes” for those present in the plant tissues (De Bary
1886). The term “endophyte” means “in the plant” (endon Gr. ¼ within, phy-
ton ¼ plant); however, with the inclusion of added information its elucidation has
changed over time (Petrini 1986). Petrini (1991), expanded the definition further as
“all organisms inhabiting plant organs that, at some time in their life cycle, can
colonize internal plant tissues without causing apparent harm to their host.” Thus,
the microorganisms like fungi and bacteria, including actinobacteria, can be called
endophytes if they, during their life cycle, colonize internal tissues of a healthy plant,
without causing any apparent disease (Dutta et al. 2014). Besides microorganisms
inside the plants (Kobayashi and Palumbo 2000; Stone et al. 2000), plants and
insects inside the plants (Marler et al. 1999; Feller 1995), and algae inside algae
(Peters 1991) can also be termed endophytes as per the literal meaning of the term.
On the other hand, plant pathogens also colonize the internal plant tissues, but there
are critical levels of disagreement over considering them as endophytes, since the
endophytes need to be asymptomatic and in harmony with the host plant (Freeman
and Rodriguez 1993; Sinclair and Cerkauskas 1996). The endophytes have also been
defined based on the purpose for which they are isolated. Bacon and White (2000)
proposed an inclusive and widely accepted definition of endophytes as “Microbes
that colonize living, internal tissues of plants without causing any immediate, overt
negative effects.” The endophytic microorganisms can be isolated from surface-
sterilized plant tissues, but to establish their “true” endophytic nature, additional
validation by inoculating the tagged microorganisms and then tracing them inside
plant tissues by microscopic and/or molecular techniques is required. A true endo-
phyte should be able to infect and establish in the internal tissues of the disinfected
plants from which it was initially isolated.
11.2 Significance of Seed Endophytes
The seed carries forward the progeny and can escape the unfavorable conditions by
remaining dormant for years and germinates into a new plant, when the conditions
become congenial (Nelson 2004). The soil that immediately surrounds the seed
11 Seed Endophytes: The Benevolent Existence in the Plant System 293
provides an intricate interface for interactions between soil, microbes, and

germinating seed is called spermosphere. Despite the transient nature of the
spermosphere, the microbial communities present in this small zone which are
influenced by the host plant’s root exudates can have significant and long-lasting
impacts on the developing host plant (Schiltz et al. 2015). Generally, some of the
rhizospheric and phyllospheric bacteria eventually infect the plant and become
endophytic, however, some of them may be transmitted vertically from the parent
plant through the seed. Colonization by endophytes in the internal tissues has been
observed in every plant studied, exhibiting varied relationships including symbiosis,
mutualism, commensalism, trophobiosis, etc. The role of endophytic microbes in
plant growth promotion and disease control has been reported by many workers.
Besides, their role in soil reclamation through enhancing phytoremediation and soil
fertility through phosphate solubilization and nitrogen fixation has been documented
(Ryan et al. 2008). Although the plant-associated beneficial microorganisms have
gained due attention in recent decades, the role of seed-associated microorganisms
largely remains unnoticed. Germinating seed and the developing plant benefit from
seed-associated microorganisms as they are already present in the seed at the early
stages of development (Truyens et al. 2015). According to Mastretta et al. (2009)
endophytic bacteria isolated from seeds of Nicotiana tabacum grown in the field
with high soil cadmium levels induced higher plant growth and also reduced metal
toxicity in the plant during the early stages of growth. Seed-borne strains of
Pseudomonas, Acinetobacter, and Bacillus sp. enhanced plant growth of cardon
cactus by increasing the availability of solubilized rock minerals (Puente et al. 2009).
Seed endophytic bacteria belonging to genera Paenibacillus and Pantoea exhibited
plant growth–promoting traits and could improve chlorophyll content in barley
seedlings (Herrera et al. 2016). Moreover, it has been suggested that colonization
with safe bacteria in the plants can help in out-competing the human pathogenic
bacteria. Cooley et al. (2003) demonstrated that colonization by
Enterobacter absuriae could out-compete and eliminate human pathogens Salmo-
nella enterica and the enterohemorrhagic Escherichia coli from Arabidopsis
thaliana.
Soil type, climatic conditions, geographical factors, and plant genotype and age
have been identified as major factors influencing seed microbiota. Besides, domesti-
cation of crops and agricultural management practices for intensive cultivation have
also influenced the seed microbiota, leading to loss of microbial diversity, which has
been showing consequences for human health. The microbial diversity can be
restored by optimizing and designing the seed microbiome by taking clues from
the rich microbial diversity of seeds of wild ancestors or other native plants. The
reconstructed seed microbiome can be harnessed for sustainable agriculture through
improving growth and productivity and improving the resilience of important crops
to various stress factors (Wassermann et al. 2019).
11.3 Diversity of Seed Endophytes in Different Crops
Recent studies indicate that almost all plants harbor endophytes. In recent years,
attempts to study diversity of seed endophytes in various crop plants, to understand
their role and to harness their beneficial effects in agriculture have increased
exponentially. Both cultivation-dependent and independent techniques are being
used to study endophytic diversity in crop plants. Liu et al. (2012) compared the
seed endophytic bacterial communities of four hybrid maize offspring and respective
parents by preparing 16S rDNA libraries and showed significant differences in
number and species of seed endophytic bacteria among the offspring hybrids and
their parents, indicating the vertical transmission of microorganisms from both
parents to offspring plant. Vega et al. (2005) isolated different fungi from the seed
of coffee including Acremonium, Aspergillus, Eurotium, Fusarium, Gibberella,
Penicillium, Pseudozyma, and an undescribed Clavicipitaceous species. Among
the bacterial genera Bacillus and Pseudomonas are commonly reported as seed
endophytes in different plant species. In addition, Paenibacillus, Micrococcus,
Staphylococcus, Pantoea, and Acinetobacter have also been found frequently as
seed endophytes (Truyens et al. 2015). Okunishi et al. (2005) observed that majority
of the rice seed endophytes were motile that has enabled their migration into the
seeds. Kaga et al. (2009) isolated and identified bacterial endophytes from
germinating rice seedlings as Bacillus fusiformis, B. firmus, B. pumilus, Caulobacter
crescentus, Micrococcus luteus, Kocuria palustris, Methylobacterium
radiotolerans, M. fujisawaense, and Pantoea ananatis. The study indicated that
bacterial endophytes colonizing the rice seed infect the subsequent generation via
seeds and establish as the dominant endophytic species in the developed plant.
Fluorescence in situ hybridization (FISH) helped in detecting the colonization by
Bacillus spp. inside the grapevine seeds, as indicated by their presence along the cell
walls of some cells (Compant et al. 2011). Gagne-Bourgue et al. (2013)
demonstrated the presence of the same Bacillus spp. and Microbacterium spp. in
the harvested switch grass seeds and the plants grown from these seeds indicating
transmission of endophytes from the seeds to the developing plant. Cankar et al.
(2005) collected fresh seeds from four differently located Norway spruce trees
(Picea abies) in the Slovene subalpine region and attempted to detect the presence
of endophytes by cultivation method and by 16S rDNA gene amplification. Both
approaches detected the presence of Pseudomonas and Rahnella in the surface-
sterilized spruce seeds. Endophytic colonization studies of germinating wheat seed
by gfp tagged Streptomyces sp. strain EN27 revealed successful colonization of
inoculated strain in the embryo, endosperm, and emerging radicle indicating that
colonization of wheat seedling started at very early stage of plant development
(Coombs and Franco 2003). In a study by Ferreira et al. (2008), endophytic bacteria
belonging to Bacillus, Enterococcus, Paenibacillus, and Methylobacterium could be
isolated from seeds and seedlings of ten species and two hybrids of eucalyptus,
indicating their vertical transfer from seeds to seedlings. In addition, seed-inoculated
gfp tagged endophytic bacteria Pantoea agglomerans could be re-isolated from the
grown up seedlings indicating its vertical transmission (Ferreira et al. 2008).
Bodhankar et al. (2017) isolated eighty maize seed endophytic bacteria (MSEB)
from 30 maize genotypes, and characterized them by 16S rRNA sequence analysis.
Among the identified MSEB isolates, Bacillus sp. was found as the most dominant
genus under Phylum Firmicutes, few isolates belonging to genus Staphylococcus
and one isolate was identified as Corynebacterium sp. under Phylum Actinobacteria
(Bodhankar et al. 2017). In a study by Johnston-Monje and Raizada (2011) compo-
sition of seed endophytic microbial community varied with host plant phylogeny and
a core microbiota of endophytes was conserved in maize seeds across boundaries of
evolution, ethnography, and ecology. Three genera (Paenibacillus, Acidovorax, and
Pantoea) were seed specific in rice and as the seed matured, the flora of culturable
endophytic bacteria changed in a different manner from that of culturable surface
bacteria (Mano et al. 2006). López-López et al. (2010) isolated a novel rhizobial
species Rhizobium endophyticum from Phaseolus vulgaris seed and it was observed
that endophytic rhizobia were not capable of forming nodules, indicating a different
functional profile as endophyte. Shen et al. (2014) isolated 350 strains of fungi from
the seeds of uncommon moso bamboo (Phyllostachysedulis) and reported four new
genera including Leptosphaerulina, Simplicillium, Sebacina and an unknown genus
under Basidiomycetes.
Venkatesagowda et al. (2012) attempted isolation of endophytic fungi from seeds
of oil producing crops (castor, coconut, neem, peanut, pongamia, rubber, and
sesame). They could morphologically identify 40 isolates as belonging to 19 taxa
(Alternaria, Aspergillus, Chalaropsis, Cladosporium, Colletotrichum, Curvularia,
Drechslera, Fusarium, Lasiodiplodia, Mucor, Penicillium, Pestalotiopsis, Phoma,
Phomopsis, Phyllosticta, Rhizopus, Sclerotinia, Stachybotrys, and Trichoderma).
Brownbridge et al. (2012) used two methods: (1) seed coating and (2) root dipping
to study endophytic colonization of entomopathogenic fungus Beauveria bassiana
in the pine seedlings. Interestingly, B. bassiana could successfully colonize pine
seedlings by both methods. Parsa et al. (2016) attempted isolation of endophytic
fungi from seeds of 11 Colombian cultivars of common bean (Phaseolus vulgaris).
Identification of isolated 394 endophytic fungi by ITS region sequence analysis
revealed representation by 42 taxa. Aureobasidium pullulans was the most dominant
endophyte (isolated from 46.7% of the samples) followed by Fusarium oxysporum,
Xylaria sp., and Cladosporium cladosporioides detected in 13.4, 11.7, and 7.6% of
seedlings, respectively. Ottesen et al. (2013) observed that different organs of tomato
plant were colonized distinct groups of microbial communities. Further, a gradient of
compositional similarity of microbial communities was observed in different plant
parts depending on their distance from the soil. The fruits and flowers of tomato
plants harbored unique bacterial phylotypes including Microvirga, Pseudomonas,
Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas, and
Microbacterium. Among the bacterial taxa, Pseudomonas and Xanthomonas were
observed as most frequent taxa across aerial plant parts. Among the identified fungal
taxa, Hypocrea, Aureobasidium, and Cryptococcus were found to be abundant. A
study by Rosenblueth et al. (2012) revealed difference in bacterial population and
diversity among the individual seeds harvested from the same cob of maize (Zea
mays L.) and among the bean (Phaseolus vulgaris L.) seeds harvested from different
pods as well as the same pod. The study indicated role of fertilization step in creating
seed microbial diversity. Thus, various studies indicate toward the diverse groups of
microorganisms that could colonize seeds as endophytes. The microbial variability
observed within seeds of a plant may be useful in the adaptation of plant species to
diverse environmental conditions and, thus, can be explored for enhancing agricul-
tural productivity.
11.4 Population of Seed Endophytes in Different Crops
Population of endophytes include dominant types that are isolated on a frequent

basis in large numbers having rare types that are difficult to isolate. Mano et al.
(2006) reported endophytic bacteria in the range of 102 to 104 CFU g1 in rice seeds.
Khalaf and Raizada (2016) isolated 169 unique bacteria associated with cucurbit
seeds. Taxonomic studies revealed that cucurbit seed–associated bacteria belonged
to two classes, Bacilli (under phylum Firmicutes) and γ-proteobacteria (under
phylum Proteobacteria). The dominance of Bacillus (50% of all strains) was
observed in all tested cucurbit species. A larger diversity of wheat endophytic
actinobacteria could be revealed by molecular approach than that by culturable
approach (Conn and Franco 2004). Misganaw et al. (2019) reported a high popula-
tion of endophytic bacteria in finger millet growing regions of Ethiopia. Endophytic
bacterial population in dry seeds of finger millet collected from six locations ranged
between 8.7 104 and 1.6 106 CFU g1 dry seeds. Rosenblueth et al. (2012)
could detect 1 to 55 CFU g1 seeds of bacterial endophytes in different bean seeds
taken from different pods or from within the same pod. Similarly from maize
kernels, bacterial endophytes in the range of 101–2 CFU g1at day two, to 105–8
CFU g1 at seven days after germination could be detected. Chen et al. (2014)
reported the highest bacterial population at the seedling stage in ginger plant. The
endophytic populations of the wild strawberries were also well represented through-
out the plant and seed, but with a much lower diversity (Kukkurainen et al. 2005).
Martinez-Rodriguez et al. (2019) attempted isolation of endophytic bacteria from
different Agave species and reported a population of endophytic bacteria in the range
of Log10 3.36 to 6.54 CFU g1 seed. Highest population of endophytes was reported
from the seeds of Agave angustifolia, and the lowest from those of A. tequilana
indicating species specific influence on endophytic populations. The various reports
indicate that seeds of the same plant located differently show variation in endophytic
population indicating influence of many factors, namely, plant source, plant age,
tissue type, time of sampling, and environment, further growth stage of growing seed
also influence endophytic population (Hardoim et al. 2008; Rosenblueth et al. 2012).
11.5 Entry and Colonization of Endophytes
The term “endophytic colonization” refers to the way in which endophyte

populations enter, multiply, and establish inside the host tissues. Endophytes living
inside the plant are acquired horizontally from the surrounding environment and/or
can be vertically transmitted from one generation to subsequent generation via seed
(Fig. 11.1). Endophytes may enter the host plant via rhizosphere (branching sites,
cracks, wounds) and/or phyllosphere including stems (cracks, wounds), leaves
(stomata), flowers (pollen tubes), and cotyledons (Zinniel et al. 2002; Santoya
et al. 2016; Okungbowa et al. 2019). In the rhizosphere, some of the rhizospheric
and soil bacteria eventually enter into plant tissues through cracks at the site of root
hair formation and root branches. This is considered to be the most common mode
for the entry of microorganisms that establish inside the host tissues as endophytes.
Root exudates play a key role in attracting and recruiting soil microbial communities
in the root zone, facilitating host-microbe cross talk and endophytic colonization
(Kawasaki et al. 2016). Several factors affect this endophytic recruitment process
including microbial quorum sensing, soil and environmental conditions, host geno-
type, plant age, etc. (Zúñiga et al. 2013). The penetration of endophytes into the host
plant can be mediated by a passive or active process. The passive penetration occurs
through the cracks present in the areas of lateral root emergence, root tips and the
wounds caused by injury or plant pests, whereas active penetration requires
Fig. 11.1 Entry and establishment of endophytes

attachment by exopolysaccharide, structural components, quorum sensing for move-

ment, and multiplication of endophytes inside the plant tissues (Okungbowa et al.
2019). Other sites of entry include stomata, particularly on leaves and young stems
(Santoya et al. 2016). Most fungal spores that land on a leaf surface require water to
germinate and then penetration through leaf surface, while others enter the leaf via
stomata for horizontal transfer from one plant to the other (Hardoim et al. 2015).
Many bacteria produce lytic enzymes that help them to lyse the plant tissues to gain
entry and establish as plant endophyte (Hardoim et al. 2008; Truyens et al. 2015).
Amylase activity has been frequently observed among seed endophytic bacteria
which can help them proliferate by utilizing stored seed starch at the onset of seed
germination (Mano et al. 2006). In addition, lytic enzymes play important role in the
control of phytopathogens and protect the plant at a sensitive stage of seed germina-
tion. Bacteria enter into the plant as endophytes through rhizosphere or aerial parts
and then they spread in the root cortex and vascular tissues of the host, thus making
way toward seed colonization. Bacterial taxa identified as seed endophytes have
been generally found to be similar to common soil bacteria indicating that soil
bacteria get entry into the host plant and find their route toward seeds and establish
as seed endophytes. The colonization studies of rifampicin-resistant mutants of
Bacillus sp. isolated from surface sterilized maize seeds revealed that seed inoculated
Bacillus sp. strains could gain entry into the host plant and aggressively colonize the
roots, shoots, and leaves of maize plants (Bodhankar et al. 2017). Lamb et al. (1996)
demonstrated the capillary transport of Pseudomonas aureofaciens from the root
surface to the seeds above the hydroponic solution in the hydroponically grown corn
inoculated with the bacteria.
It is clear that the majority of bacteria that form endophytic relationship with the
host plant come from the soil. These bacteria migrate toward the rhizosphere and
subsequently to the rhizoplane under the influence of root exudates of the host plant
and exhibit plant beneficial effects. Some of these bacteria may get the chance to
penetrate plant roots, and some of these penetrated strains may move to aerial plant
parts and some finally end up in seeds. In general, there is a decreasing bacterial
density from the soil populations that colonize the rhizosphere and subsequently the
plant aerial part. Microscopic visualization techniques are generally used to study the
endophytic colonization processes. Besides, generation of mutants with dysfunc-
tional colonization genes can also help in studying the colonization behavior of an
endophytic strain. Analysis of endophytic bacteria in flowers by fluorescence in situ
hybridization (FISH) revealed Gamma-proteobacteria, Pseudomonas spp.,
Firmicutes, and Bacillus spp. inside the flowers, showing their colonization in
ovary, xylem, and ovules (Compant et al. 2011). Microbial colonization of plant
roots through soil is the most important and well-studied transmission route. How-
ever, more research focus is needed on the vertical transmission to aerial parts and
stomatal colonization by microorganisms (Frank et al. 2017). Deckert et al. (2019)
propounded the transmission of microbial propagules into a pine seed through
various routes including megastrobilus, male sporogenous tissue, pollens, etc. The
endophytic colonization may be tissue-/organ-specific depending on the ability of
endophytes to adapt and adjust with the different physiological processes inside the
plants (Rai and Agarkar 2014; Rodrigues and Samuels 1999).
Populations of endophytes increase after seed germination and colonize different
tissues including roots and endorhizosphere and may also reach exorhizosphere
(Herrera et al. 2016). Mano et al. (2006) observed that although seed endophytes
in the germinating rice seed mainly colonized shoots, some strains could reach the
rhizosphere and also spread to the soil. López-López et al. (2010) could recover
almost all bacterial genera from bean seeds and also from the emerging roots of
germinating seed. Thus, introduced microorganisms need to compete and/or share
their ecological niche with endophytic communities that spread in the rhizosphere
during germination (Herrera et al. 2016). The plant’s immune system can influence
the colonization of endophytes. However, endophytic bacteria produce their
microbe-associated molecular patterns (MAMPs), which do not trigger plant
immune responses and, thus, can escape elimination by the plant system
(Vandenkoornhuyse et al. 2015; Liu et al. 2017). Pitzschke (2018) studied molecular
dynamics in the endophyte colonized germinating seeds of quinoa. Quinoa seeds and
seedlings exhibit remarkably complex and dynamic mitogen-activated protein
kinase (MAPK) activity profiles. Variances have been reported in MAPK patterns
and probably also in endophyte assemblages, depending on the seed origin. The
ability to degrade mucilage enabled the quinoa endophytes to colonize Salvia
hispanica (chia) seeds, a non-host species. The motile quinoa endophytes caused
expansion of the cells, were able to move across the cell wall, and elicited damage-
associated molecular patterns and MAPKs in the host plant. The study indicated that
the bacteria may hasten the germination process and confer a primed state to the
germinating seeds. There is a possibility of transfer of these endophytes into
non-native crops for desirable results. Mitter et al. (2017) described an innovative
approach to modulate seed microbiomes of elite crop seed embryos and concomi-
tantly design the traits to be mediated by seed microbiomes. They found that the
introduction of the endophyte Paraburkholderia phytofirmans PsJN to the flowers of
parent plants can add it to the microbiome of the progeny seeds, thus facilitating its
vertical transmission to the next generation. They demonstrated the introduction of
PsJN to seeds of monocot and dicot plant species and the resulting changes in the
composition of seed microbiome and effect on plant growth parameters. The study
illustrated the potential role of modified seed microbiomes in influencing plant traits.
Thus, methods to optimize the seed microbiomes of major crops can have
far-reaching implications for plant breeding and crop improvement to enhance
agricultural productivity.
11.6 Seed Endophytes in Climate Resilient Agriculture
Climate change has become the most important issue in the twenty-first century,
affecting agricultural productivity worldwide. It is a real challenge to supply suffi-
cient food for the ever-increasing population, while maintaining the environment
sustainability. Water availability is one of the major constraints limiting crop
production, and, thus, a real challenge for global food security. The emerging
climatic variabilities are exerting pressure on agriculture. Drought affected areas
have been increasing under all major crops including wheat, maize, rice, sorghum,
soybean, etc., as expressed in terms of the Palmer Drought Severity Index (PDSI)
(Palmer 1965). In addition, rising atmospheric CO2 and increasing average temper-
ature are bound to impact agricultural productivity directly by changing crop
physiology and indirectly through influencing pests and disease dynamics. Simi-
larly, unpredictable rainfall events causing frequent floods can affect wider areas,
and lead to soil anaerobic conditions, water logging, and sometime wiping out the
entire crops causing huge losses (Gornall et al. 2010). Seed persistence is a complex
phenomenon that allows its survival after death of the parent plant, thus preserving
the genotype through time. It is a complex expression of seed and species
characteristics that resist germination during pre-dispersal and post-dispersal envi-
ronmental conditions determining the fate of the seed toward germination or death.
Thus, environment has a strong influence on seed persistence even before the
dispersal of seeds (Donohue 2009; Kochanek et al. 2011). The environment of
parent and even grandparent plant at different stages of life cycle can influence
seed traits like size, germination efficiency, seed coat properties, dispersal, dor-
mancy, longevity, and fecundity, thus influencing the complexities of seed persis-
tence (Gallagher and Fuerst 2006; Long et al. 2015). Seed endophytes may play
important role in the germination, establishment and growth of the miniature plant
coming out of the seed. Microorganisms have the capability to actively respond to
various biotic and abiotic stress factors. Many reports have demonstrated the role of
endophytes in conferring tolerance to abiotic stress in the host plants. Abiotic
stresses like salinity and drought cause disruption of homeostasis and ion distribu-
tion in fescue plant; however, infection with Epichloë coenophialum, a seed-
transmissible systemic fungal endophyte, confers stress tolerance to the fecuse plants
(Singh et al. 2011). Moreover, production of epoxy-janthitrem alkaloids by Epichloë
festucae var. lolii protects the plants from herbivory. The alkaloid content depends
on genetic predisposition of the endophyte and host plant, and is also influenced by
seasonal variations and plant age. This study indicated that global warming may
increase incidences of endophyte mediated intoxications of livestock feeding on
European grasslands (Fuchs et al. 2017). Absence of fungal endophytes has been
found to impair the tolerance of host plants to habitat imposed abiotic and biotic
stresses. For example, colonization of Dichanthelium lanuginosum plants found in
geothermal soils of Yellowstone National Park by fungal endophyte Curvularia
protuberate enables the plants to tolerate high temperatures (60 C) of the geother-
mal habitat and neither the fungus nor the plant can survive in separation from one
another at temperatures above 38 C (Redman et al. 2002). Higher amount of
ethylene is generally secreted by plants due to pathogen attack or other stresses,
which cause plant growth inhibition and cell death in the infected part. This
phenomenon of triggered cell death can be avoided by reducing the level of ethylene
(White and Verma 2019). The bacterial enzyme ACC (1-aminocyclopropane-1-
carboxylate) deaminase catalyzes the conversion of ACC, the immediate precursor
of ethylene synthesis in plants into ammonia and α-ketobutyrate. The sequestration
of ACC by bacterial ACC deaminase reduces ethylene levels and thus helps in
ameliorating plant stress under various abiotic and biotic stress conditions (Glick
2015). Many studies have demonstrated the promotion of plant growth by some
ACC deaminase producing bacteria under various abiotic stresses like drought,
salinity, and flooding (Glick et al. 1999). Isolation of ACC deaminase-producing
bacteria from seeds of wild plants and their application in crop plants can help in
better growth even under stressed environmental conditions (White and Verma
2019). Treatment of tomato seeds with the ACC deaminase-producing endophytic
bacteria (Pseudomonas fluorescens YsS6 and P. migulae 8R6) resulted in priming of
the emerging plant to tolerate imposed salt stress more effectively due to
pre-colonization of developing plant with the endophytes. Even under non-stressed
conditions, treatment with endophytic bacteria resulted in substantial increase in the
growth of tomato plants as compared to uninoculated treatment (Ali et al. 2014). A
non-nodulating legume, marama bean (Tylosema esculentum), indigenous to the arid
regions of Southern Africa, thrives in stressful and harsh environmental conditions
in soils with nutritional and moisture deficiencies (Council 2006; Chimwamurombe
et al. 2016). Investigation revealed the existence of seed-associated bacterial com-
munity in surface-sterilized seeds and gnotobiotically grown marama bean seedlings
with Firmicutes and Actinobacteria as dominant groups. Firmicutes are endospore
formers that can help host plant in dealing with harsh and unfavorable environmental
conditions. Actinobacteria are known to be well adapted to environmental
fluctuations such as moisture deficit conditions. Members of genera Curtobacterium
and Microbacterium present as seed endophytes in marama beans are also desicca-
tion tolerant and can persist through dry periods (Goodfellow and Williams 1983)
and can enhance tolerance of host plants to drought (Ali et al. 2014; Lucas et al.
2014). Walitang et al. (2017) isolated Flavobacterium sp., Microbacterium sp., and
Xanthomonas sp. type strains from the salt-sensitive and salt-tolerant rice cultivars.
Flavobacterium sp. strains tolerant to salinity and osmotic stress also exhibited IAA
production and phosphate solubilization. Certain groups of bacteria, including
Flavobacterium, Pantoea, Curtobacterium, Microbacterium, Kosakonia, and
Enterobacter, were more abundant under stress conditions. The study indicated
their potential role in enhancing tolerance of rice plant to stress (Walitang et al.
2018). Conservation and transmission of these dominant bacterial groups to the
subsequent generations of rice plant via seeds even after crossbreeding and
recultivation indicated an evolutionary co-existence between the rice plant and its
endophytic bacteria (Walitang et al. 2019). Gond et al. (2015) isolated Bacillus
sp. from modern varieties of corn and reported isolation of only Pantoea
agglomerans and Agrobacterium sp. from teosinte. Treatment with
P. agglomerans enhanced tolerance of the tropical corn seedlings to hypersaline
conditions. Endophytic bacteria isolated from seeds of Nicotiana tabacum grown
under metal stress induced plant growth promotion as well as reduced cadmium
phytotoxicity in the newly grown tobacco plants. Endophytic strains of
actinobacteria Streptomyces coelicolor DE07, S. olivaceus DE10 and
S. geysiriensis DE27 isolated from wheat exhibited plant growth–promoting traits
such as IAA production and intrinsic osmotic stress tolerance (0.05 to
0.73 MPa). Culture and cell-free extracts of these endophytic actinobacteria when
applied to wheat seeds under moisture deficit conditions enhanced plant growth and
yield of wheat as compared to untreated control plants (Yandigeri et al. 2012).
Treatments with direct seed coating of cultures showed superior performance than
seed coatings with cell-free extract and, further, co-coating of seeds with culture and
cell-free extract exhibited better performance than those with only culture coatings.
Bacteria isolated from stressed habitats exhibited a high level of stress tolerance
along with the plant growth–promoting traits and, hence, could be potential
candidates for seed inoculants (Tiwari et al. 2011).
11.7 Plant Beneficial Mechanisms of Seed Endophytes
The exact role of seed endophytes in plant growth is not understood yet. It has been
demonstrated that seed endophytes benefit the host plant by using similar
mechanisms as used by rhizobacteria. However, seed endophytes get advantage
over the rhizosphere microorganisms in terms of time and space, which means that
when the seed undergoes germination, endophytes are the closest partners that can
be triggered for timely action. Some of the seed endophytes have been reported to
enhance plant growth through phytohormones production, helping in nutrient acqui-
sition, specially N and P, control of phytopathogens, etc. (Gagne-Bourgue et al.
2013; Johnston-Monje and Raizada 2011; Xu et al. 2014; Herrera et al. 2016). Many
studies have reported the positive effect of IAA producing endophyte strains on seed
germination and establishment and growth of plant (Prasad and Dagar 2014).
Burkholderia sp. strain KJ006, an endophytic bacterium associated with rice root
bacterial strain benefits its host plant by providing plant growth substances and a
broad range of antifungal activities (Cho et al. 2007). Whole genome sequence
revealed that the strain KJ006 harborsvital genes related to plant growth promotion,
antimicrobial activities, and degradation of several kinds of aromatic pollutants (Cho
et al. 2007; Pérez-Pantoja et al. 2012). Expression of these genes including accD
gene, encoding 1-aminocyclopropane-1-carboxylate deaminase; the pqq operon for
biosynthesis of pyrroloquinoline quinone, and the nif gene cluster contributes to
plant growth. A similar whole genome sequences of some endophytic Bacillus
species revealed the genes that enabled them to act as biocontrol agent, stimulate
plant growth, produce phytohormones (like GAs and IAA), and ameliorate drought
stress in host plant (Forchetti et al. 2007). Bacterial endophyte Bacillus
amyloliquefaciens RWL-1 isolated from rice (Oryza sativa L. “Jinsomi”) seeds
could produce gibberellins (GAs), and influence host-plant physiology. Rice plants
treated with GAs producing RWL-1 exhibited significant improvement in growth
parameters and endogenous levels of salicylic acid as compared to the control plants
treated with water or exogenous GA3, indicating their potential role in plant growth
and hormonal modulation (Shahzad et al. 2016). Seed entophytes of finger millet
(Eleusine coracana) exhibited multiple plant growth–promoting traits like produc-
tion of indole-3-acetic acid (IAA), biologically nitrogen fixation, phosphate solubi-
lization, and hydrogen cyanide production (Misganaw et al. 2019). The multiple
plant growth–promoting traits can help in better growth and nutrient status of host
plant finger millet under limited moisture and nutrient conditions. Besides growth
promotion, endophytic microorganisms can also protect the host from biotic stresses.
Several seed endophytic bacteria (such as Bacillus and Pseudomonas) have been
reported to exhibit antagonism against plant fungal pathogens (Sundaramoorthy and
Balabaskar 2013; Bodhankar et al. 2017). Production of antifungal volatiles and
lipopeptide metabolites like surfactin, iturin, and mycobacillin have been reported in
many of Bacillus strains frequently mentioned as seed endophytes (Gagne-Bourgue
et al. 2013). Members of genus Bacillus are known to promote plant growth under
adverse conditions, besides helping in disease control (Bent and Chanway 1998).
Bacillus-based commercial biofertilizers and biopesticides are popularly used in
agricultural crops (Borriss 2011). Seed plant growth–promoting (PGP) endophytic
Bacillus sp. isolated from different maize genotypes exhibited multiple PGP traits
including antagonism against fungal phytopathogens (Bodhankar et al. 2017). The
biocontrol trait can be highly advantageous for the host at the seed germination stage
which is particularly prone to fungal attack. The trait could probably be one selection
criteria for recruiting endophytic population by the host plant (Bodhankar et al.
2017). Moreover, endophytic microorganisms have been found to produce toxins
effective against insects that results in weight loss and increased death rates of pests
(Azevedo et al. 2000). Beauveria bassiana, the entomopathogenic fungus known to
causes disease in various insects, including bark beetle pests of plantation forest
trees, has been found to exist as an endophyte in plants, including the commercial
Pinus radiata plants in New Zealand (Brownbridge et al. 2012).
Endophytic microorganisms have been reported to alleviate abiotic stress in host
plants. Adaptation to various environmental stresses is governed by cascades of
molecular networks that result in series of metabolic, physiological, and morpholog-
ical changes in plant (Table 11.1). In an important response to drought stress,
osmotic level in the cytosol of the plant cell and the vacuoles is modulated by
accumulating higher levels of osmolytes, which can counteract the loss of turgor
caused due to desiccation. These osmolytes help to maintain cellular osmotic
balance without interfering with normal biochemical reactions of the cell. Osmolytes
include an array of compounds like amino acids such as proline and quaternary
ammonium compounds (glycine, betaine, etc.), carbohydrates (sucrose, trehalose,
fructan, etc.), polyols (pinitol, mannitol, etc.) and polyamines, and hydrophilic
proteins (late embryogenesis abundant proteins) (Chaves et al. 2003; Liu et al.
2004). Accumulation of osmolytes like proline and glycine betaine under drought
and salinity stress has been demonstrated in different plants under the influence of
endophytic bacteria (Delauney and Verma 1993; Rhodes and Hanson 1993).
Barnawal et al. (2016) isolated an ACC deaminase-producing salt-tolerant endo-
phyte Brachybacterium paraconglomeratum SMR20 from the roots of salt stress–
sensitive medicinal plant Chlorophytum borivilianum. Inoculation with SMR20
successfully protected C. borivilianum from negative effects of salt stress by poten-
tial deamination of ACC leading to reduced levels of stress ethylene, thus delaying
chlorosis and senescence. Apart from reducing stress ethylene, inoculation with
endophyte enhanced levels of indole-3-acetic acid and abscisic acid in plants, altered
Table 11.1 Alleviation of abiotic stress by endophytes in host plants

Stress
Endophyte Plant Alleviated Observed effects Reference
Burkholderia Grapevine Chilling Significant increase in Barka et al.
phytofirmans strain (Vitis levels of starch, proline, (2006)
PsJN vinifera) and phenolic
compounds. Better root
growth and plant
biomass
Arthrobacter sp., Sweet Osmotic Improved plant growth Sziderics
Bacillus sp. pepper and proline level, et al. (2007)
(Capsicum reduced expression of
annuum) the stress-inducible
genes CaACCO and
CaLTP
Piriformospora indica Barley Salinity Improved plant growth, Baltruschat
(Hordeum reduction in NaCl- et al. (2008)
vulgare) induced lipid
peroxidation, metabolic
heat efflux and fatty
acid desaturation,
increased antioxidant
enzyme status
Trichoderma hamatum Cacao Drought Enhanced plant growth, Bae et al.
219b (Theobroma altered gene (2009)
cacao) expression. Delay in
the onset of the drought
response in leaves
Piriformospora indica Chinese Drought Increased antioxidant Sun et al.
Cabbage enzymes activity in the (2010)
(Brassica leaves. Upregulation of
rapa) drought-related genes
DREB2A, CBL1,
ANAC072 and RD29A
and increased
expression CAS protein
Fusarium culmorum Rice (Oryza Salinity 20–30% reduction in Redman
(FcRed1), Curvularia sativa) and water requirement and et al. (2011)
protuberata drought higher growth rate,
(Cp4666D), reproductive yield, and
Curvularia biomass of plants
protuberata
(CpYNP5C)
Penicillium Soybean Salinity Improved plant growth, Khan et al.
minioluteum LHL09 (Glycine chlorophyll content, (2011)
max) leaf area and nitrogen
assimilation; increased
daidzein and genistein
contents
(continued)

Stress
Endophyte Plant Alleviated Observed effects Reference
Streptomyces Wheat Drought Improvement in plant Yandigeri
coelicolor DE07, (Triticum growth parameters and et al. (2012)
Streptomyces olivaceus aestivum) yield
DE10, Streptomyces
geysiriensis DE27
Burkholderia Maize (Zea Drought Improved biomass, leaf Naveed
phytofirmans strain mays) area, number of leaves et al. (2014)
PsJN, Enterobacter per plant, and yield.
sp. FD17 Higher efficiency of
photosystem II (PSII)
Pantoea agglomerans Maize Salinity Improved plant growth Gond et al.
and biomass and 2015
up-regulation of
aquaporin genes
Pantoea alhagi Wheat Drought Improved growth, Chen et al.
increased accumulation (2017)
of soluble sugars,
decreased
accumulation of proline
and malondialdehyde
(MDA), and decreased
degradation of
chlorophyll in leaves
Azospirillum Maize Drought Higher carbon, Curá and
brasilense, nitrogen, and Franz
Herbaspirillum chlorophyll levels, (2017)
seropedicae relative water content
and better
osmoregulation under
drought conditions
Bacillus licheniformis Maize Drought Improved chlorophyll Bodhankar
MSEB17, content, plant biomass, et al. (2017,
B. subtilis leaf proline content, 2019)
MSEB78, B. subtilis total sugars, relative
MSEB72 water content and soil
moisture content
Bacillus tequilensis Agave Cold Induction of secondary Martinez-
(Agave metabolites (solanidine Rodriguez
tequilana) and plakinamine B) et al. (2019)
the amount of total leaf pigments and biochemical compounds like proline and
malondialdehyde, and increased foliar nutrients. Maize seed endophytic bacteria
possessing PGP traits exhibited tolerance in vitro to abiotic stresses and could also
alleviate the effect of moisture deficit stress in maize plants, thus indicating their
potential application under drought conditions (Bodhankar et al. 2019). Endophytic
fungus Curvularia protuberata has been associated with the survival of the grass
Dichanthelium lanuginosum at high soil temperatures in geothermal habitats

(Márquez et al. 2007). Thus, the mechanisms reported for seed endophyte mediated
plant growth are similar to those reported for rhizobacteria. However, the localiza-
tion of endophytes in the seeds gives them edge over rhizospheric microorganisms to
respond quickly to the needs of germinating seed.
11.8 Endophytic Microorganisms Influence Plant Gene

Expression
Many reports have confirmed the influence of endophytes on plant physiological

parameters which could be the result of altered gene expression under the influence
of endophytes. Although very limited studies have been carried on effect of
endophytes on plant gene regulation, it is assumed that endophyte can influence
the expression of certain plant genes responsible for growth, yield, stress tolerance,
and modulation of secondary metabolite biosynthesis (Ray et al. 2019). Studies on
expressed genes may provide clues about effects of endophytes in plants.
Gene expression analysis by Gond et al. (2015) revealed that inoculation with
P. agglomerans caused upregulation of the aquaporin genes, particularly plasma
membrane integral protein type 2 (PIP2–1) genes in maize plants under salt stress
conditions. Inoculation with endophytic Trichoderma harzianum mitigated abiotic
stress in rice by upregulation of aquaporin, dehydrin, and malonialdehyde genes
(Pandey et al. 2016a, b, c). Colonization of Zea mays with root-associated dark
septate endophyte (DSE), Exophiala pisciphila, enhanced plant tolerance to high
concentration of cadmium by decreasing phytotoxicity and repartitioning subcellular
Cd into the cell wall and also triggered plant antioxidant system, resulting in
significant improvement in growth. Gene expression analysis of maize roots and
leaves revealed influence of DSE on the three key genes (pathways) involved in the
uptake, detoxification, and transport (ZIP, PCS, and MTP) of Cd, causing down
regulation of ZIP, and upregulation of PCS and MTP under high soil Cd
concentrations (Wang et al. 2016). Song et al. (2017) demonstrated that endophytic
fungi from the calabash-shaped root of Tetrastigma hemsleyanum could regulate
plant growth, expression of expansin gene and flavonoid content of the host plant
under high temperature. Maize seed endophytic bacteria when inoculated could
enhance tolerance of maize plant to drought stress and could influence the expression
of drought responsive genes in leaves (Bodhankar et al. 2020, communicated for
publication). Phytohormones are known to play an important role in plant tolerance
to abiotic stresses (Wani et al. 2016). The abscisic acid (ABA)-mediated stomatal
closure and modulation of plant growth contributes toward abiotic stress tolerance in
the plant (Waqas et al. 2012). The endophytic microorganisms may modulate
ABA-mediated signaling pathways, enhancing plant tolerance to stress conditions.
Halo-tolerant endosymbiont Dietzia natronolimnaea could induce salinity tolerance
in wheat plants by modulating an ABA-signaling cascade as revealed by the
upregulation of TaABARE (ABA-responsive gene) and TaOPR1 genes
(12-oxophytodienoate reductase 1) upon inoculation (Bharti et al. 2016;
Ilangumaran and Smith 2017). Sheibani-Tezerji et al. (2015) observed that potato
endophyte Burkholderia phytofirmans PsJN could modulate expression of genes for
a cell surface signaling element, which helped bacteria to sense and respond to
changing environmental conditions. The strain has been found to enhance stress
tolerance of host plants. The inoculation with PsJN enhanced cold tolerance of grape
plants earlier and faster, and resulted in higher levels of stress-related gene
transcripts and metabolite levels than in un-inoculated counterparts (Theocharis
et al. 2012). Endophytic bacteria Enterobacter sp. SA187 previously isolated from
a desert plant could colonize inner tissues of Arabidopsis roots and shoots. Produc-
tion of 2-keto-4-methylthiobutyric acid (KMBA) by this bacteria could modulate the
plant ET signaling pathway, resulting in enhanced salt tolerance and higher yield of
alfalfa crops under salt stress conditions (de Zélicourt et al. 2018). Shittu et al. (2009)
observed an upregulation of the genes for R-carotene hydroxylase, Rubisco and
glutamyl tRNA synthase and increase in photosynthetic activity in Craigella
tomatoes (Lycopercicum esculentum Mill.) plants having Verticillium dahlia
Dvd-e6 as endosymbiont, which is likely to result in an increased vigor and a
stronger resistance response against pathogen. A study by Singh and Gaur (2017)
revealed the role of endophytic Streptomyces sp. in eliciting systemic resistance in
plant and mitigating oxidative stress in chickpea plants infected with fungal patho-
gen Sclerotium rolfsii. Seed treatment with Streptomyces griseus caused significant
increase in levels of defense enzymes such as phenylalanine ammonia lyase (PAL)
and polyphenol oxidase (PPO), and total phenolics and flavonoids in pathogen
compromised chickpea plants. The inoculation also caused a significant increase in
levels of antioxidant enzymes viz superoxide dismutase (SOD), peroxidase (PO),
ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) and reduced lipid
peroxidation in chickpea under biotic stress. The findings were further supported
by real-time polymerase chain reaction analysis of genes encoding SOD, PO, PAL,
APX, catalase, chitinase, and β-glucanase that showed significant change in expres-
sion. The results indicated that the chickpea defense pathway is triggered after
perception of endophytes to synthesize various enzymes, leading to an enhanced
resistance against pathogen. Ray et al. (2019) used a consortium of two
endophytes—Acinetobacter sp. SM1B and Marmoricola sp. SM3B—to improve
the in planta morphine yield of poppy (Papaver somniferum cv. Sujata). The study
revealed that Acinetobacter sp. SM1B upregulated most of the genes of morphine
biosynthesis except T6ODM and CODM, which were upregulated by Marmoricola
sp. SM3B in alkaloidless poppy plants. Dual inoculation was superior in improving
the photosynthetic efficiency of the plants, resulting in higher biomass and yields as
compared to single bacterial inoculation. The increase in morphine content was
attributed to the bacterial consortium mediated upregulation of related biosynthetic
genes that altered the metabolic-flow of key intermediates and enhanced expression
of COR, key gene for morphine biosynthesis. The study emphasized the potential of
microbial based strategies for upregulation of metabolic pathways for
overexpression of genes similar to transgenics. Recent findings on micro-RNAs
(miRNAs) and plant gene expression revealed endophytic microorganisms mediated
response of genes and pathways in host plant. Khare et al. (2018) observed the
effects of microbial inoculation on signaling pathways of ethylene (ET)/jasmonic

acid (JA)/salicylic acid (SA). Thus, various studies indicate the influence of micro-
bial partners on host plant’s gene expression and plant biosynthetic pathways under
various environmental conditions. However, the picture is still incomplete and there
is need for more research in this direction by studying natural plant-endophyte
associations from various habitats (extreme conditions) and validating these
interactions under controlled conditions. The validated systems can be used to
generate the complete map of plant responses mediated through various metabolic
pathways and the expression of respective genes.
11.9 Conclusion and Future Prospects
Endophytic microorganisms act as promising resource for production of various

metabolites and have plant beneficial traits. Endophytes typically associated with
seeds appear to have co-evolved with the host plant. These seed endophytes that
colonize the seeds, remain in the dormant seed until germination and become active
in the germinating seeds to be transmitted to the next generation. They must have
been selected by the host plant to perform some special functions. Many reports have
demonstrated their role in influencing plant growth, physiology responses, and gene
expressions. However, a lot more research is needed to understand the specificity
and diversity of seed endophytes in different crops for optimizing the seed
microbiome of major crops. Further, seed endophytic diversity of major crops
needs to be mapped in the domesticated varieties, as well as their wild ancestors
and landraces which can have implications in the breeding programs. In-depth
knowledge of plant-seed-endophyte interactions could have major implications in
developing strategies for microbial assisted breeding and crop improvement for
agricultural sustainability.
Acknowledgements Authors of this manuscript are thankful to ICAR-Central Research Institute

for Dryland Agriculture, Hyderabad, and ICAR-Indian Agricultural Research Institute, New Delhi,
and ICAR-AMAAS (Application of Microorganisms in Agriculture and Allied Sectors) for
providing the financial support and necessary facilities.
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Exploitation of Plant Tissue Invading
Rhizospheric Microbes as Bio-Fertilizers 12
Himani Chaturvedi and Anil Prakash
Abstract
Maintaining the fertility of the soil has been the major concern of the farmers as
this factor directly rules the productivity of plants. With the continuously increas-
ing application of chemical fertilizers for a span of the last few decades, the
fertility of soil has deteriorated due to acidification and mineral depletion. Plant-
microbe interactions have been providing an excellent alternative for these
chemicals hazardous to plants, animals, humans, and notably soil health. This
makes these interactions the main motif for sustainable agriculture. Rhizospheric
microbes have been the main focus being considered responsible for plant growth
promotion, prominently known as plant growth-promoting rhizomicrobes
(PGPRs).
A few of these rhizospheric microbes can enter the plant and reside there
without showing any deleterious effects. Such microbes are known as
endophytes. The site of wound formations due to lateral root formation and the
differentiation zones are the major entry points of endophytes into the roots of
host plant. Thereafter, these microbes produce various hydrolysing enzymes like
cellulase, pectinase, etc. and migrate towards the shoot tissues. Endophytes
confer more advantages to the plant as compared to the rhizospheric population
because of the decreased concentration inside the host plant. Endophytes promote
plant growth by various direct as well as collateral mechanisms that involve
providing nutrients to the plants. Endophytes are known to metabolically accli-
matize plants by forming mutualistic relationships. This makes their contribution
significant as biological fertilizers. Carefully selected in a knowledge-driven
manner, endophytic microbes must be combined and formulated in accordance
with the environmental conditions for the development of efficient endophytic
biofertilizers. A deeper insight into various gene-level mechanisms and
H. Chaturvedi · A. Prakash (*)

Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India

23, https://doi.org/10.1007/978-981-15-9154-9_12
316 H. Chaturvedi and A. Prakash
conditions that limit the colonizing efficiency of these endophytes is necessary for
the successful exploitation of plant-microbe interactions for agricultural
sustainability.
Keywords
Rhizospheric microbes · Endophytes · Endophytic colonization · PGPR ·

Bio-fertilizers
12.1 Introduction
Plants cannot be considered solo entities. They are the holobiomes where multiple
plant-microbe interaction complexes exist. Plant-microbe interactions have been the
focus of researchers these days. The microbes residing in the vicinity of roots in the
rhizosphere are known to secrete regulatory chemical substances, which intensify
the plant growth. Such microbes responsible for promoting plant health are known as
plant growth-promoting rhizomicrobes (PGPRs), which include both fungi and
bacteria (Kloepper et al. 1980). There are various direct as well as indirect
mechanisms incorporated by PGPRs to augment plant growth, improve nutrient
uptake and antagonism against pathogens.
Some of these rhizospheric microorganisms are opportunistic to inhabiting plant
tissues and become symbionts. These tissue-invading microbes colonize plants
without exerting any subversive effects on the host and are known as endophytes
(Hallmann et al. 1997; Azevedo et al. 2000). They can be obligate or facultative and
are usually harmless to the host plants. They form a mutualistic association with
plants usually but can also behave antagonistically at times. Plants, in response to
their colonization, limit the growth of these endophytes. The successful endophytes
then develop and use mechanisms to gradually revamp to the restricted environmen-
tal conditions (Gehring and Whitham 1994; Gehring et al. 1997; Dudeja et al. 2012).
The interaction between plant and microbes leads to many physiological changes
that help both the partners in gaining maximum benefits.
The studies conducted by different authors have shown that the potential of plant
growth promotion is much higher in endophytes as compared to the exophytic
microbes surviving in the rhizosphere and rhizoplane areas (Lledó et al. 2016; Van
Overbeek and Saikkonen 2016). Endophytes confer more advantages to the plant as
compared to the rhizospheric population because of the decreased contention inside
the host. In order to establish a symbiotic association between plant and microbes
exhibiting endophytism, different signalling molecules and expression of specific
genes are required (Compant et al. 2010; Chaturvedi et al. 2016). An insight into
these symbiotic interactions, the signalling molecules involved and the expression of
different genes can facilitate the development of new schemes for the promotion of
plant-endophyte interaction to be applied for agricultural sustainability (Coutinho
et al. 2015).
12 Exploitation of Plant Tissue Invading Rhizospheric Microbes as Bio-Fertilizers 317
12.2 Entry and Colonization of Plant Tissues by Endophytes
The root-colonizing endophytic microbes are crucial in promoting growth in host

plant by producing a plethora of bioactive compounds responsible for bioremedia-
tion, plant growth enhancement and resistance against diseases (Compant et al.
2010; Ijaz et al. 2015, 2016). There are many environmental factors that are
responsible for controlling the colonization events of endophytes. These factors
cannot be ignored when the reliability of endophytes as plant growth-enhancing
microbes is to be improved (Zhu et al. 2014; Gupta et al. 2015). In order to
accomplish as a potential plant growth promoter, the endophytes need to attain
sufficient numbers inside the plant and, for that, they need to recognize the exudates
secreted by the host roots (Glick 2012). Synchronized expression of multiple genes
and traits is required for successful colonization of plants by endophytes. The
endophytic colonization is a multistep process that begins with chemotaxis. The
microbes migrate chemotactically in response to signals produced by plants followed
by attachment on the root surface, entry, and distribution inside root and growth and
survival of the microbes inside the plant (Gupta et al. 2012).
12.2.1 Chemotactic Migration Towards Roots
The root exudates produced by the plant roots stimulate chemotaxis by microbes
towards root surfaces. The root exudates are a mixture of multiple compounds like
enzymes, ions, amino acids, organic acid, vitamins, etc. For the movement as well as
colonization of interior roots, chemotaxis has the most important role to play. In a
study carried out by de Weert et al. (2002), the absence of gene cheA in a mutant
Pseudomonas fluorescens strain, reduced efficiency for chemotaxis was observed
towards tomato rhizosphere, which further resulted in reduced root colonization
(Gupta et al. 2019). A two-way interaction is required for upregulating genes coding
for chemotactic proteins, motility proteins and the ones required for adhesion. Mark
et al. (2005), in his study, observed that the down-regulated expression of genes
cheA and pctA in the presence of exudates produced by sugar beet cultivar, while it
remained unaltered by another cultivar. This differential response attractive or
repulsive, to different root exudates, affects the gene expression.
The crosstalk between the host and the microbes begins on arrival of microbes in
the rhizosphere and this results in preferential colonization of some microbes over
other because of the secretion of selective signalling molecules (Compant et al.
2010). The root-driven chemical elicitors are responsible for this preferential
behaviour. Jha et al. (2015), in his study, mentioned about this selective mechanism
as rhizo-engineering. This mechanism is known to have a major involvement when
microbes compete with each other for limiting nutrients in the rhizosphere (Bais
et al. 2006). Different competing bacterial strains show varied chemotactic efficiency
in response to root exudates, which is crucial for colonization (Kloepper 1993).
12.2.2 Attachment on Root Surface
The invading endophytic microbes attach to the root surface after being attracted by
the chemotactic molecules. This attachment process is carried out due to interaction
between the surface molecules of the host as well as microbes. Both attachment and
motility are contributory to the process of endophytic colonization. A number of
surface proteins are found to be responsible for early recognition of host for
attachment. Burdman et al. (2000) reported a bacterial major outer membrane protein
(MOMP) identified in Azospirillum brasilense. These MOMPs are reported to show
a greater inclination towards the chemicals secreted by cereals and a reduced
attraction to the ones secreted by legumes and tomatoes. The study shows that
MOMPs play a very crucial role at all the three steps as adhesion to host roots,
adsorption on root and cell aggregation.
The mechanism of fungal adhesion to the plants has been studied for years, but
the compounds responsible for the mediation of this adhesion have not been
characterized fully till date and very less information is available for molecular
basis of this interaction. It is difficult to explain the fungal attachment to the host
because adhesion is found to occur at multiple stages of fungal morphogenesis
(Epstein and Nicholson 1997).
12.2.3 Entry and Distribution Inside Roots
A number of different mechanisms are used by to enter the plant tissues specifically
roots. Endophytes employ distinct mechanisms to invade into the plant tissues,
particularly in roots. Some of the microbes are already present inside the seeds.
Rest of the endophytes gain access to the tissues via root cracks both primary as well
as lateral, and also via tissue wounds occurring in the process of plant growth. These
sites attract microbes because these wounds and cracks lead to secretion of various
plant metabolites. The other openings of the plant, including stomata, lenticels, etc.,
are also the sites of ingress. (Sprent and de Faria 1988; Hardoim et al. 2015).
Chi et al. (2005) illustrated that rhizoplane is where colonization begins at the site
of lateral root emergence and then is followed by entry into stem base, stem, and
lastly leaves. The whole study was carried out with the help of gfp-tagged rhizobia.
The amount of time required to colonize the roots varies from 7–10 days, which is
later extended into aerial parts of the plant with the help of two degradative enzymes
responsible for dissolving the cell walls of plant tissues. These two enzymes are
cellulases and pectinases, which are produced by majority of the endophytes as a part
of their strategy to colonize plants (Dong et al. 2003; Compant et al. 2008).
12.3 Plant Responses upon Colonization by Endophytes
Scientists are still trying to understand how plants distinguish between beneficial and
harmful microbes. Studies suggest that the host immune system has a major role to
play in this (Fesel and Zuccaro 2016). Research findings have suggested that the
endophytic microbes regulate the responses of genes, which is evident by studies
carried out on plant gene expression and micro-RNAs (miRNAs). However, signal-
ling pathways of ethylene (ET)/jasmonic acid (JA)/salicylic acid (SA) remain
affected by the presence or absence of endophytic microbes. In a study conducted
by Kusajima et al. (2018), it was observed that bacterial endophyte Azospirillum
sp. B510 induces systemic disease resistance in rice
There are many reports that suggest that mutualistic partners of plants like
rhizobia or arbuscular mycorrhizal fungi (AMF) down-regulate plant defence
pathways during colonization. (Fouad et al. 2014; Benhiba et al. 2015; Sarkar
et al. 2016). For example, when symbiosis is to be established between the host
and microbes, almost all the pathways targeted by miRNAs for plant defence system
are switched off that would lead to obstruction of endophytes (Plett and Martin
2018). Also, in a study carried out by Plett and Martin (2018), it was suggested that
there is a late induction of defence pathways, which prevents the colonizing
microbes from ‘overstepping’ and ‘overpowering’ the plant. It suggests that different
group of genes might get activated in response to colonization by different set of
micro-organisms.
12.4 Endophytes as Bio-fertilizers
Plants, when existing in association with endophytes together, form holobiont,

which is assembly of varied species, which work together as a single ecological
unit. The term holobiont was originally proposed by Margulis (1990) to describe
different microbes combine asexually to form new hereditary units, but now this
term has been expanded more generally to hosts as well as their microbial flora. All
plants and animals, including humans, host these microbes and have been found to
be extremely beneficial in both cases (Rogers 2016; O’Malley 2017).
These endophytic microbes confer multiple advantages as compared with con-
ventional plants like better and deeper root systems, increased yields, efficient
uptake of nutrients, better resistance against pests and pathogens, and better photo-
synthetic efficiency. There are various mechanisms used by these microbes to
promote plant growth.
12.4.1 Nutrient Uptake
Agricultural soils normally lack one or more nutrients essential for the optimum
growth of plants like nitrogen, iron, and phosphorus. Endophytes directly enhance
plant growth by providing these nutrients.
12.4.1.1 Nitrogen Fixation

A lot of studies have confirmed that many bacterial species like Rhizobia spp.,
Azospirillum spp., have the ability to fix nitrogen for plants (Bashan and Levanony
1990). A cluster of various structural genes called nitrogenase (nif) genes are
responsible for responsible for coding the enzyme nitrogenase, which carries out
nitrogen fixation. These structural genes are responsible for iron activation, synthesis
of iron-molybdenum cofactor, and electron donation. In diazotrophic (nitrogen-
fixing) bacteria, the size of nif genes is around 20–24 kb and has seven operons,
which encode for 20 different proteins.
Oxygen is the major inhibitor of nitrogenase enzyme and a negative regulator of
nif genes. The haemoglobin produced by the nitrogen-fixing bacteria eliminates
oxygen, therefore carrying out nitrogen fixation successfully. In a study conducted
on bean plant, transformed strain of Rhizobium etli was used. It was transformed
using haemoglobin gene from Vitreoscilla sp. (a gram-negative bacterium). It was
observed that the transformed rhizobial cells had a two- to threefold higher respira-
tory rate than the non-transformed strain even at low levels of dissolved oxygen.
Further, it was observed that in a greenhouse experiment, bean plants inoculated
with haemoglobin-containing R. etli had 68% more nitrogenase activity than plants
inoculated with wild-type R. etli. The resultant seeds also had a 16% increase in the
nitrogen content due to this difference of around 25–30% in leaf nitrogen content
(Ramírez et al. 1999; Kamfwa et al. 2019).
12.4.1.2 Phosphate Solubilization

‘Water, water everywhere not a drop to drink’- Samuel Taylor Coleridge. This
saying partially holds true in case of availability of phosphorus to plants. The soil is
generally found to have a very high amount of phosphorus, around 400 mg/kg of soil
but is in insoluble form and not available for the plants to absorb. This insoluble
phosphate is solubilized by some bacteria and fungi known as phosphate-
solubilizing microbes as reported by Pikovskaya (1948). A number of bacteria and
fungi endophytic in nature have been proclaimed to efficiently solubilize phosphate
(Sharma et al. 2017). The most common bacterial genus known for phosphate
solubilization include Pseudomonas and Bacillus (Mehta et al. 2015) and Aspergil-
lus and Penicillium are the fungi most predominant (Wakelin et al. 2004). The
microbes solubilize phosphate by producing organic acids having low-molecular-
weight such as gluconic acid and citric acid (Bnayahu 1991; Rodríguez and Fraga
1999; Richardson 2001; Rodriguez et al. 2004). The availability of phosphorus in
limited quantities and the inefficiency of the plant to uptake phosphorus result in
reduced plant development (Feng et al. 2004). By increasing the availability of
phosphorus, these endophytes promote plant growth (Fig. 12.1).
12.4.1.3 Sequestering Iron

The amount of iron, specifically in its ferric form, is required by plants for various
reactions but is not readily assimilated by them and its availability is also extremely
low (Ma 2005). There is a high competition in microbes for obtaining iron as it is an
essential element (Loper and Buyer 1991; Guerinot and Ying 1994). The microbes
Fig. 12.1 Some rhizospheric microbes colonize the internal tissues of plants and confer benefits to
the host by various direct as well as indirect mechanisms like nitrogen fixation, solubilization of
minerals like phosphorus and zinc, siderophore production, HCN production and by priming the
immune system of the plant by induced systemic resistance
have developed a mechanism to cope up with this stress. They synthesize a com-
pound known as siderophore, which has extremely high affinity for Fe+3. They also
have membrane receptors able to bind the Fe-siderophore complex, thereby
facilitating iron uptake (Neilands 1981; Hider and Kong 2010). In various studies,
it has been reported that siderophore production also helps to alleviate stress
imposed on plants due to heavy metal contamination of soil (Diels et al. 2002;
Belimov et al. 2005; Braud et al. 2006).
12.4.1.4 Zinc Solubilization

Zinc is an element required in minute quantities by the plant but is a key element for
its growth and development. In plants, it is crucial for carbohydrate metabolism,
plays a major role in auxin metabolism and is also an important antioxidant (Hafeez
et al. 2013; Alloway 2004, 2008). Zinc has a major role to play in sexual reproduc-
tion and associated aids like floral tissues, flowering, fertilization and also fruiting
(Epstein and Bloom 2005). The deficiency of zinc has been found to be linked with
adverse effect on shoot growth, chlorosis, leaf size (Alloway 2004). The unavail-
ability of zinc in sufficient amount may also lead to susceptibility to heat, light and
fungal infections, grain yield, pollen formation, root development, water uptake and
transport (Tavallali et al. 2010).
The form of zinc, which can be easily up taken by plants, is in the form of divalent
cations, but very less amount of zinc is available in this form. (Kabata-Pendias and
Pendias 2001). The remaining amount of zinc is present in insoluble form (Alloway
2008). The methods incorporated by zinc-solubilizing microorganisms to solubilize
zinc mainly include acidification. The pH of the soil is balanced by the organic acid
produced by the microbes, thereby making it suitable for zinc solubilization (Alex-
ander 1997).
12.4.2 Modulating Phytohormone Levels
The surrounding environment of the plants determines the growth and development
of plants and the plant hormones are the regulatory factors, which control the
responses of the plant (Davies 2004). Plants are subjected to many non-lethal
stresses, which directly or indirectly affect the plant growth. In order to overcome
such conditions, plants try to modulate the levels of phytohormones just as it is the
case with humans and animals. Some microbes are known to produce these
phytohormones, which add on to this strategy adopted by plants to overcome
negative effects of environmental stress (Salamone et al. 2005; Glick et al. 2007).
12.4.2.1 Cytokinins and Gibberellins

Diverse plant hormones have different functions in the process of plant growth and
mechanisms responsible for the overall development of plants. Cytokinins and
gibberellins being the most crucial ones, several studies have shown that many
soil microbes specifically bacteria can produce either of them or even both (Williams
and de Mallorca 1982; Salamone et al. 2001). A list of different hormones produced
by different microbes and their function has been mentioned in Table 12.1.
12.4.2.2 Indole Acetic Acid (IAA)

Multiple studies have shown that IAA has a very important role to play in the plant
physiology. It is responsible for cell elongation and growth coordination. Varying
concentration of IAA is known to have varying effects and may differ from one plant
to another. The concentration of IAA may differ depending upon the stage of
development of plant as well. The amount of IAA can be altered by microbes
which produce IAA. The IAA synthesized by the plant can determine whether
bacterial IAA stimulates or suppresses plant growth. In plant roots, endogenous
IAA may be suboptimal or optimal for growth (Pilet and Saugy 1987), and
additional IAA produced by bacteria could alter the IAA level to optimal or supra-
optimal, resulting in plant growth promotion or inhibition, respectively (Badenoch-
Jones et al. 1984; Mathesius et al. 1998).
Table 12.1 Different endophytic bacteria are known to produce different plant hormones which
play a major role in plant growth promotion
Plant
hormone Function Bacterial strains References
Indoleacetic plant cell division, Pseudomonas putida GR12-2, Patten and Glick
acid extension, and Rhizobium, Bradyrhizobium (2002), Tsavkelova
differentiation; elkanii, et al. (2006),
affects Methylobacterium extorquens, Fukuhara et al.
photosynthesis, Methylobacterium zatmanii (1994), Pattnaik
pigment formation, et al. (2017)
biosynthesis of
various metabolites,
and resistance to
stressful conditions
Cytokinins Cytokinins promote Azotobacter spp., Rhizobium Atzorn et al. (1988),
and cytokinesis and spp., Pantoea agglomerans Joo et al. (2005),
Gibberellins gibberellins Rhodospirillum Kang et al. (2009)
stimulate shoot rubrum, Pseudomonas
elongation, seed fluorescens,
germination, and Bacillus
fruit and flower subtilis,
maturation Paenibacillus polymyxa.
12.4.2.3 Ethylene
Another plant hormone ethylene, which is known to be one of the simplest
molecules, is found to affect plant growth and development in multiple ways,
which include regulation of root development and elongation, fruit development
and maturation, initiating seed germination, etc. Ethylene is also synthesized when
plant is under stress both biotic and abiotic, also known as ‘stress ethylene’ (Abeles
et al. 1992; Morgan and Drew 1997). Miscellaneous environmental stresses may
lead to an intense increase in production of ethylene, which can exacerbate some of
the symptoms of the stress. It might also result in an increased resistance to adverse
conditions.
12.4.3 Induced Systemic Resistance
The endophytes upon colonization are known to improve overall plant health by
activating the immune system of the plant the mechanism known as induced
systemic resistance. Using this mechanism, the plant growth-promoting microbes
prime the whole plant body for enhanced defence against a wide range of pathogens
and insect herbivores. A wide variety of microbes, including Pseudomonas, Bacil-
lus, Trichoderma, and mycorrhizae species, strengthen the plant immune system for
enhanced defence (Bardoel et al. 2011).
Boller and Felix (2009), in their study, found that the plant immune system
contains pattern recognition receptors (PRRs) specific for recognizing common
microbial compounds like bacterial flagellin or fungal chitin just like that of humans,
called pathogen or microbe-associated molecular patterns (PAMPs or MAMPs).

These PRRs are considered the first line of defence known as called PAMP-triggered
immunity (PTI), which keeps most potential invaders in check (Zipfel 2009; Dodds
and Rathjen 2010).
12.5 Conclusion
Probiotics, also known as ‘good’ or helpful bacteria, are beneficial for human beings.
They help in modification of microbiota by replacing the harmful microbes with
helpful microbes. These friendly bacteria are known to improve the immune system
and are also found to be effective in treatment of various diseases. Plants are also
known to associate with some good microbes, which can be considered plant
probiotics.
Plants in nature have been known to form beneficial associations with microbes,
which are essential for their survival and growth also affecting their biodiversity and
ecosystem functioning. The area surrounding the plant roots called the rhizosphere
contains an extensive range of bacteria as well as fungi, which promote plant growth
orchestrated by their ability to interact with plants using complex chemical
mediators. Some of these rhizospheric microbes have the capability to enter the
plant and reside there without showing any deleterious effects. Such microbes are
known as endophytes.
Endophytes confer more advantages to the plant as compared to rhizospheric
population because of the decreased contention inside the host plant. Endophytes
promote plant growth by various direct as well as indirect mechanisms, which
involve providing nutrients to the plants, which are not available in absorbable
forms, an example of which is phosphate. Endophytes help in solubilization and
uptake of phosphate from soil, which is otherwise available in insoluble forms of
phosphorus. Other mechanisms include modulation of phytohormone levels like
auxins and gibberellins. Endophytes also help in suppression of plant diseases either
directly by antagonizing the plant pathogens or indirectly by eliciting plant immune
responses. The mechanism involved is analogous to the one responsible for devel-
oping the human immune system. The process by which the pathogen activates host
plant’s immune system shares similarities as well as differences with beneficial
effects. It is similar to the way a vaccine works in human body. Introduction of a
weakened pathogen or another similar non-pathogenic strain provokes an immune
response, which helps the body in developing resistance against the pathogen. An
excellent example is Pseudomonas bacteria; the cell-surface components of patho-
genic as well as beneficial Pseudomonas strains are potent inducers of similar
immune responses, the former causing disease and the latter conferring resistance
to the particular disease. It is not the stress that kills but the reaction to it. Plant
pathogens, when interacting with plants, induce stress, which leads to production of
ethylene. The aftereffects of heightened ethylene production may lead to a signifi-
cant inhibition of plant growth and survival. Endophytes help in reduction of stress
by synthesizing indole acetic acid (IAA), which is also a type of plant hormone. This
IAA, produced by endophytic microbes together with endogenous plant IAA, can
stimulate plant growth. In other cases, they may also induce the synthesis of the plant
enzymes, which convert immediate precursor of ethylene to ammonia and
α-ketobutyrate, compounds that are readily assimilated. This enzymatic activity
reduces the amount of ethylene produced by the plant.
Agriculture, being the most important sector of Indian economy, has been the
target area of enormous research and development. Sustainable agriculture is the
need of the hour as the environment has been ignored by us so much so that we are at
the verge of breaching the critical threshold. Microorganisms provide a promising
alternative to the extensive use of chemical fertilizers. However, the feasibility of
these strains in open field trials still remains a challenge as there are many factors,
which affect the interactions as compared to in vitro conditions where all factors
under control.
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Contribution of Microbe-Mediated
Processes in Nitrogen Cycle to Attain 13
Environmental Equilibrium
Humera Quadriya, Mohammed Imran Mir, K. Surekha,

S. Gopalkrishnan, M. Yahya Khan, Sushil K. Sharma, and
Hameeda Bee
Abstract
Nitrogen (N), the most important element, is required by all living organisms for
the synthesis of complex organic molecules like amino acids, proteins, lipids etc.
Nitrogen cycle is considered to be the most complex yet arguably important cycle
next to carbon cycle. Nitrogen cycle includes oxic and anoxic reactions like
organic N mineralization, ammonia assimilation, nitrification denitrification,
anaerobic ammonium oxidation (anammox), dissimilatory nitrate reduction to
ammonium (DNRA), comammox, codenitrification etc. Nitrogen cycling is one
of the most crucial processes required for the recycling of essential chemical
requirements on the planet. Soil microorganisms not only improve N-cycle
balance but also pave the way for sustainable agricultural practices, leading to
improved soil properties and crop productivity as most plants are opportunistic in
the uptake of soluble or available forms of N from soil. Microbial N
transformations are influenced by plants to improve their nutrition and vice
versa. Diverse microorganisms, versatile metabolic activities, and varied biotic
H. Quadriya · H. Bee (*)

Department of Microbiology, Osmania University, Hyderabad, Telangana, India
M. I. Mir
Department of Botany, Osmania University, Hyderabad, Telangana, India
K. Surekha
ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, Telangana, India
S. Gopalkrishnan
ICRISAT-International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, Telangana,
India
M. Yahya Khan
Kalam Biotech Private Limited, Hyderabad, Telangana, India
S. K. Sharma
ICAR-National Institute of Biotic Stress Management (ICAR-NIBSM), Raipur, Chhattisgarh, India

23, https://doi.org/10.1007/978-981-15-9154-9_13
332 H. Quadriya et al.
and abiotic conditions may result in the shift in the equilibrium state of different
N-cycling processes. This chapter is an overview of the mechanisms and genes
involved in the diverse microorganisms associated in the operation of nitrogen
cycle and the roles of such microorganisms in different agroecosystems.
Keywords
Nitrogen cycle · N-linked microbial processes · Environmental factors ·

Agroecosystems
13.1 Introduction
Global nitrogen cycle deals with the movement of various forms of N, either reactive
or unavailable across various reservoirs and ecosystems. Nitrogen availability can be
predicted by the presence of diverse and metabolically versatile microorganisms.
Reactive nitrogen (Nr) species include NO3, NO2, NH3, NH4+, NOx, N2O, urea,
amides, etc. These nitrogen transformation result into production of available forms
of nitrogen leading to shift in nitrogen cycle equilibrium. The associated reactions
either reduce or intensify global environmental change and produce/consume green-
house gases. Microorganisms play a vital role in maintaining geochemical N cycle
across the globe. These microbial processes namely nitrogen fixation, nitrification,
ammonification, denitrification, etc. are mediated by Cyanobacteria, Azotobacter,
Nitrosomonas, Nitrobacter, Nitrococcus, Rhizobium, etc. Assimilation of nitrates
and ammonium ions are mediated through conversion into complex molecules, like
amino acids, proteins, etc. in living beings. While some plants provide amino acids
and/or nutrients to bacteroids, these bacteria in turn fix nitrogen and provide amino
acids back to plants through symbiotic relationship. Further, conversion of nitrate to
ammonium is mediated by microorganisms through the process of dissimilatory
nitrate reduction. Anammox is anaerobic ammonium oxidation, wherein nitrite and
ammonium combine to form nitrogen. The key genes steering microbial N cycle
include nif (nifHDK); amoA; narG; nirS/K; and nosZ genes (David et al. 2014).
Agroecosystem is considered to be the major ecosystem in India, with highest
quantum of N fertilizer utilization and loss as well. This excess nitrogen fertilization
is a threat to human, animal, and environment. According to Rockstrom et al. (2009),
three of the nine interlinked planetary boundaries (rate of biodiversity loss (terres-
trial, marine), interference with the N cycle, and climate change) have been crossed
as a result of industrialized form of agriculture and anthropogenic activities, which if
not stopped or rectified can lead to disastrous consequences or irreversible environ-
mental changes. Interference with N cycle is due to excess fertilizer application to
enhance cereal production, followed by energy, industrial, and wastewater manage-
ment (Abrol et al. 2017). According to Pathak (2015), greenhouse gas (GHG)
emissions increased 75% from 1970 to 2010, of which N2O emissions doubled,
and N2O was the second largest emitted GHG after CH4. In addition to this, Nr
(reactive nitrogen) emissions are due to puddled rice; horticultural production
13 Contribution of Microbe-Mediated Processes in Nitrogen Cycle to Attain. . . 333
systems; burning of crop residues, livestock and poultry etc. which lead to nitrate
leaching and groundwater pollution (Abdullah et al. 2017). Due to this, we are
witnessing nitrogen-deficient food and food products (Abrol et al. 2017). In this
chapter, we will discuss the role of microorganisms, the genes involved in N cycle,
microbial diversity in different ecosystems, and the role of microbes in maintaining
N equilibrium.
13.2 Nitrogen Cycle: The Role of Microorganisms
Nitrogen is important for muscle movement and other body functions, an essential
component of protein, and an integral part of protein synthesis, photosynthesis, and
other critical biological processes. It is predicted that N fixed by human activities
would be greater than N fixed by microbial processes (Fowler et al. 2013; David
et al. 2014; Silvia and Brendan 2016; Ramiro and Silvia 2018).
N cycle is the most important cycle after carbon cycle as both nitrogen and carbon
(C) are required mainly for the synthesis of essential biomolecules. Nitrogen cycle
includes organic N mineralization, ammonia assimilation, bacterial and archaeal
nitrification (autotrophic and heterotrophic), comammox, anammox, dissimilatory
nitrate reduction, and denitrification and codenitrification. The major genes
associated with the biogeochemical cycle of nitrogen are as follows: (1) nif for
nitrogen fixation; (2) amoA and amoB for archaeal and bacterial ammonification/
ammonia oxidation; (3) chi A for mineralization; (4) nar and nap for nitrate
reduction; (5) nirS and nirK for nitrite reduction; (6) norB for nitric oxide reduction;
(7) nosZ for nitrous oxide reduction; (8) nrfA for DNRA (dissimilatory nitrate
reduction to ammonium); (9) hzo, hh and hzs for anammox; (10) pmoA/amoA,
norB and hao for comammox and (11) nap, nor, nir and nos together in different
steps of denitrification (Fig. 13.1). Microorganisms respond differently to various
biotic and abiotic factors and result in a shift in the equilibrium state of N cycle
(Shoun et al. 2012; Lisa and Martin 2016; Olivia et al. 2017; Florence et al. 2018).
Almost 80% of Earth’s atmosphere contains nitrogen gas, but the availability of
nitrogen is still limited. Nitrogen fixation becomes accelerated/limited based on the
presence/absence of factors like iron (Fe), molybdenum (Mo), phosphorus (P), and
cobalt (Co) (Dilworth et al. 1978; Howarth et al. 1988; Vitousek 1999). Net
biologically available nitrogen is determined by balancing between denitrification
and nitrogen fixation. Nitrogen status in the environment is controlled as a result of
interactions among N2 fixation and other biogeochemical processes of cycle. The
process of getting biologically available forms from dinitrogen is called nitrogen
fixation. Dinitrogen is chemically inert due to triple bond and requires a large
amount of energy, i.e., eight electrons and 16 adenosine triphosphate (ATP)
molecules, to convert a dinitrogen molecule into ammonia. On the other side,
334
Extracellular
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Fig. 13.1 Processes and associated genes of microorganisms involved in operation of N cycle. [Sources: David et al. (2014), Holger et al. (2015), Inoue et al.
H. Quadriya et al.
(2015), Lisa and Martin (2016), Olivia et al. (2017), Florence et al. (2018), Marcel et al. (2018), Ramiro and Silvia (2018), Black et al. (2019) and Yan et al.
(2020)]
environmental lighting also adds to N fixation naturally. Industrial nitrogen fixation

(Haber-Bosch method) is also carried out, but it is not that much economic.
Nitrogen fixers may be symbiotic and asymbiotc (free-living). Symbiotic fixers
are divided into (1) aerobic and anaerobic and (2) phototrophic and chemotrophic,
based on their oxygen requirement and light or chemical requirement for fixation,
respectively. In order to maintain symbiosis, specific mechanisms and enzyme
complexes are required. Though prokaryotes have diverse physiological processes
among N fixers, they contain similar enzyme complex. One of the enzymes is
nitrogenase, which is oxygen sensitive and reduces dinitrogen to ammonia. With
time, N fixers have gradually developed ways to protect nitrogenase from oxygen,
like heterocysts in Cyanobacteria, while some others carry out nitrogen fixation
during the night, when oxygen is not synthesized due to its dormant photosystem
(David et al. 2014; Danovaro et al. 2017; Dilfuza 2018).
N2 þ 8Hþ þ 8e ! 2NH3 þ H2 " and 16ATP

! 16ADP þ 16Pi ðwith Mo nitrogenaseÞ
N2 þ 14Hþ þ 12e ! 2NH4 þ þ 3H2 " and 40ATP

! 40ADP þ 40Pi ðwith V nitrogenaseÞ
Nitrogenase enzyme complex is comprised of two multisubunits metalloprotein.

In conventional nitrogenases, Fe-S subunits are bridged by Mo, i.e., Mo-nitrogenase
(Fe-Mo-S), whereas alternative nitrogenases contain either V or Fe, i.e.,
V-nitrogenase (Fe-V-S) or Fe-only nitrogenase (Fe-Fe-S). All the three types of
enzymes are similar in sequences and structural and functional properties but varied
in metal cofactor. Azotobacter vinelandii contains all the three types of enzymes,
while Trichodesmium spp. have Mo-nitrogenase and V-nitrogenase, which is rarely
present. The gene clusters for nitrogenase enzymes based on the above three metal
requirements are, respectively, nif and non-nif; vnf or anf, comprising major clusters
of genes nifHDK and cys, mod; and vnfHDGK or anfHDGK, along with minor
clusters (QBXWO). As depicted in the above reactions, it is clear that
Mo-nitrogenase requires comparatively less electron flux, less energy/ATP, and
less nitrogen pressure, thereby resulting in 1:1 N-fixed:H2 evolved ratio, while the
other two have increased values up to 7:1 to 9:1 N-fixed:H2 evolved ratio (Florence
et al. 2018; Marcel et al. 2018).
N fixation is a fairly expensive process metabolically, where approximately 20 kb
DNA (genes) encodes proteins required for N fixation (Chen et al. 1998; Kim et al.
1999; Hoover 2000). Factors regulating N fixation can be checked by transcribing
nifHDK operon, which is not constitutively expressed. A strong reductant, iron
protein, “Ferredoxin,” carries electron and reduces catalytic components during
nitrogen fixation. This reduction and electron transfer need iron and catalytic protein
to dissociate and reassociate (Marcel et al. 2018).
According to Graham et al. (1988), rhizobia behave as free-living organism when
present in soil and as a symbiont in root-nodule. Various nutrients affect their growth
in the root like calcium (Ca) for multiplication, cobalt (Co) for nodule initiation, and
boron (B) and iron (Fe) for nodule development. Cobalt accelerates nitrogen fixa-
tion, participates in “cobamide” coenzyme catalyst formation, and is the center of
cyanocobalamin. There are enzymes like methylmalonyl-coenzyme A mutase and
methionine synthase that are dependent on cobalamin, especially in rhizobia, which
would affect the concentration of leghemoglobin. Cobalt (Co) acts as a cofactor for
cobalamin during N fixation and nodule growth. It has a role in the activity and
population of nitrogen fixers like Azotobacter and Cyanobacteria.
The presence of Co only or Mo when applied along with Co has shown an
increase in nodule formation, nutrient content, and yield and growth in legumes, and
studies also revealed lesser use of fertilizer dose than recommended dose. There is
antagonistic relation between Fe and Co (Dilworth et al. 1978; Graham et al. 1988;
Nadia 2012; Weria et al. 2013; Campo et al. 2000).
13.2.2 Nitrification
Nitrification is an oxidation process that converts ammonium into nitrate via nitrite.
It involves two pathways: autotrophic nitrification and heterotrophic nitrification.
Factors contributing to both include the nature and availability of substrates in soils.
In addition to this, acidic soils inhibit the activity of autotrophic nitrifiers while
favoring heterotrophic nitrifiers. In heterotrophic nitrification, oxidation of ammonia
is not coupled to energy conservation and the enzymes regulating both pathways are
different.
13.2.2.1 Autotrophic Nitrification
Aerobic Nitrification
Nitrification is a process wherein ammonia in converted in nitrate via nitrite. A major
percentage of nitrification is carried out by prokaryotes under aerobic conditions.
Hydroxylamine is the intermediate form in the first step, where aerobic ammonia
oxidizers convert ammonium to nitrite, generating a little amount of energy that
utilizes two different enzymes—ammonia monooxygenase (AMO) and hydroxyl-
amine oxidoreductase (HAO). It is carried out not only by a few bacteria but majorly
by archaea (David et al. 2014). In the next step, oxidation of nitrite to nitrate is
carried out by nitrite-oxidizing bacteria; some genera involved are Nitrospira,
Nitrobacter, and Nitrospina. These ammonium and nitrite oxidizers are autotrophic,
slow growers that synthesize organic carbon but utilize ammonia as their energy
source instead of light. Nitrospira moscoviensis respires nitrate while utilizing
hydrogen in aerobic and organic acids in an anaerobic environment. Ammonia and
nitrite oxidizers help maintain environmental balance by the removal of potentially
toxic ammonia level.
Aerobic nitrite oxidation conserves energy, while anaerobic process does not.
Mostly, aerobic and anaerobic nitrite oxidation is carried out by NXR (nitrate
oxidoreductase), with few exceptions like Thiocapsa sp. KS1 and
Rhodopseudomonas sp. LQ17 (Marcel et al. 2018).
1Þ NH3 þ O2 þ 2e ! NH2 OH þ H2 O ! NO2 þ 5Hþ þ 4e

Ammonia Oxygen Hydroxyamine Nitrite
2Þ 2 NO2 þ O2 ! 2 NO3

Nitrite Oxygen Nitrate
Comammox
Comammox can be defined as complete microbial oxidation of ammonium/ammo-
nia to nitrate in one step. Chemolithotrophic microorganisms in second cohort utilize
nitrite as reductant and are confined to Cohort II of lineage II of Nitrospira genus of
Proteobacteria class in phylum Nitrospirae and third cohorts utilize ammonia as
reductant for cellular growth and Cohort III have high affinity for ammonia, lower
growth rate, and higher growth yield than typical ammonia oxidizers. A few
examples include Candidatus Nitrospira nitrosa, Candidatus Nitrospira nitrificans,
and Nitrospira inopinata with new gene camoA, which is similar to amoA; addition-
ally, new enzymes AMO and hydroxylamine oxidoreductase (HAO) are similar to
typical enzymes of ammonia-oxidizing bacteria (AOB). These organisms lack
enzymes for assimilatory nitrite reduction and instead are able to take up
ammonia/urea-based ammonia via urease-encoding genes (Holger et al. 2015;
Kessel et al. 2015; Lisa and Martin 2016; Hu and He 2017).
NH4þ =NH3 ! NH2 OH ! NO2 ! NO3

Ammonium=Ammonia Hydroxylamine Nitrite Nitrate
OR
NH4þ ! NO2 ! NO3
Ammonium Nitrite Nitrate
Anammox
In contrast to nitrification, anammox (anaerobic ammonium oxidation) takes place in
an oxygen-limited environment like the rhizosphere of rice and oceans. Strous et al.
(1999) discovered that ammonia oxidation is even carried out in anoxic conditions
by prokaryotes. Candidatus (Ca) Brocadia anammoxidans is the first anammox-
performing bacterium reported, and it uses nitrite as electron acceptor to oxidize
ammonia and synthesize nitrogen. Haem4 is the catalytic center of octaheme
hydroxylamine oxidoreductase (HAO) in anaerobic ammonium-oxidizing bacteria,
Kuenenia stuttgartiensis (Marcel et al. 2018). Anaerobic ammonium oxidation or
anammox take up both ammonium and nitrite to form dinitrogen from nitric oxide
and/or hydrazine as intermediate form/s; hence, it is also referred to as nitrification-
denitrification process catalyzed by hydrazine synthase (HZS). This process is
carried out in the “anammoxosome,” a specific membrane-bound organelle
containing hydrazine dehydrogenase (HDH), HZS, NIR, hydroxylamine oxidase
(HOX), and NOR enzymes in Kuenenia and members of Brocadiaceae of
Planctomycetales. The gene hzsA (hydrazine synthase) is considered as genetic
markers for anammox (Harhangi et al. 2012). It is a major nitrogen removal process,
may be nitrite or ammonium without the production of N2O, and hence ecologically
beneficial and applied industrially for wastewater treatment (Lisa and Martin 2016;
Marcel et al. 2018).
NH4þ =NH3 ! NH2 OH ! NO2 ! NO3

Ammonium=Ammonia Hydroxylamine Nitrite Nitrate
OR
NH4þ ! NO2 ! NO3
Ammonium Nitrite Nitrate
NH4þ =NO2 ! NO‐ ! N2 H4 ! N2

Ammonium=Nitrite Nitric Oxide Hydrazine Dinitrogen
OR
NH4þ =NO2 ! N2 H4 ! N2
Ammonium=Nitrite Hydrazine Dinitrogen
OR
NH4þ þ NO2 ! N2" þ 2 H2 O
Ammonium Nitrite
According to David et al. (2014), bacterial and archaeal nitrifiers are significantly
different. Crenarchaeota oxidizes ammonia by nitroxyl (HNO) as it lacks HAO.
Archaea here requires less oxygen and is useful in anoxic zones of soil. Bacterial
ammonia oxidation is restricted to β- and γ- Proteobacteria. Methanotrophs are
capable of utilizing ammonia in scarcity of methane to produce hydroxylamine by
methane monooxygenase (MMO). HAO is an octahaem protein that involves end-
ergonic reaction for the oxidation of hydroxylamine to nitric oxide, and then it is
converted to nitrite (Marcel et al. 2018).
Autotrophic nitrification contributes to proportional relatedness with increased
soil pH and raised N2O level and is inversely related to denitrification, soil pH, and
N2O emission (Zhang et al. 2015). Low pH and low ammonia are assumed to hinder
ammonia oxidation by chemolithoautotrophs, but this was proved reverse by the
isolation of Candidatus Nitrosotalea devanaterra. Soils with sandy and silty clay
texture witness abundant Thaumarchaeota than bacteria. Ammonia is produced
from the hydrolysis of organic N compounds like urea and cyanate by Nitrosospira
sp., Nitrososphaera viennensis and Nitrososphaera gargensis with ureases and
cyanase, respectively (Marcel et al. 2018).
Soils fertilized by manure or compost which receive ammonical substrate
represents increase in the ratio of gross nitrification rate to nitrification potential
(GNR:NP). Agricultural soils are acidified due to higher rates of nitrification and
leaching of nitrates. Population abundance and ecotype of ammonia and nitrite
oxidizers more or less regulate nitrification rate in the presence of higher substrate
concentration. There are examples, such as one from Utah agriculture soils, where
ammonia-oxidizing archaea (AOA) represented lower Vmax at lower substrate
concentration. During the first few weeks of fertilization, ammonia-oxidizing
bacteria (AOB) mediated net and gross nitrification rates as ammonium got
depleted and AOA overcame AOB. Another example can be of Oregon soils,
where lower ammonium concentration saturates nitrification via AOA. Zhu et al.
(2018) revealed that bacteriovorous nematodes significantly reduced AOB while
increasing AOA abundance (Norton and Yang, 2019). In a few plants, root
exudates synthesize inhibitors that hinder nitrification activities (Subbarao et al.
2013, 2015).
13.2.2.2 Urea Hydrolysis

Nitrification include ammonia-oxidizing microbes (AOM) and nitrite-oxidizing
bacteria (NOB) of genera Nitrospira and Nitrospina those convert ammonia to
nitrite followed by nitrate. It has been recently reported that the same AOM also
converts urea into ammonia and bicarbonate using own produced urease enzyme.
Urea is first hydrolyzed to ammonia and carbamate, which is quickly converted
into second ammonia molecule and a bicarbonate by urease-positive AOM. This
ammonia is then converted to nitrite by urease-negative AOM or NOB, thereby
reducing the competition for free nitrite availability. The genus Nitrospira contains
genomically and functionally variable organisms, i.e., AOM (bacteria and archaea)
and NOB like urease-positive Nitrospira moscoviensis and urease-negative
N. defluvii and N. europaea coexisting in natural soils and activated sludge. Based
on ammonia availability, AOM and NOB outcompete each other or sometimes
behave mutually symbiotic. Therefore, soils should be fertilized in an efficient
way that would not alter much the community or composition of the microorganisms
present (Kocha et al. 2015).
NH2 CoNH2 ! NH3 þ COONH2 ! 2NH3 þ HCO3

Urea Ammonia Carbanate Ammonia Bicarbonate
13.2.2.3 Heterotrophic Nitrification

Heterotrophic nitrification is predominantly seen in acidic soils utilizing either
organic or inorganic nitrogen compounds other than ammonia as substrates. It is
assumed that acid-tolerant and acid-sensitive ammonium-oxidizing bacteria majorly
contribute to the synthesis of nitrate in acid forest soils saturated with nitrogen. It is
not coupled with energy conservation and has different enzymes than autotrophic
nitrification. Organic nitrogen’s heterotrophic nitrification contributes significantly
to N2O emissions from soils having near neutral pH, acidic pH, and high organic
matter (grasslands). Heterotrophic nitrification is proportional to C:N ratio in the
soil; i.e., higher C/N contributes to greater heterotrophic nitrification rate. It is more
in coniferous acidic forests than broad leaf acidic forest soils. Conversely, NO2
emission would be in higher concentration in pasture soils and arable soils than in
forest soils due to the quantity of microbial community present and/or the organic N,
C content available (Zhang et al. 2015). Bacterial and fungal genera like Absidia and
Alcaligenes, Arthrobacter, Aspergillus, Paenibacillus, Paracoccus, Pseudomonas,
Thiosphaera, and Verticillium are involved in heterotrophic nitrification (Siddarame
Gowda et al. 1977; Moir et al. 1996; Brierley and Wood 2001; Behrendt et al. 2010;
Dilfuza 2018).
Organic N=Inorganic N ! NH2 OH ! NO3‐

Organic=Inorganic N‐ containing Substrate Hydroxylamine Nitrate
13.2.3 Denitrification
It is an anaerobic reduction step where bioavailable nitrogen, i.e., nitrate, is reduced

to bioinert form, i.e., dinitrogen gas. This reduction step utilizes energy, pro-
duce intermediates such as nitrous oxide, nitric oxide, etc being greenhouse gases
that are players in air pollution and react with ozone. It is carried out not only by
chemoorganotrophic prokaryotes, like few species of Aerobacter, Bacillus, Pseudo-
monas, Klebsiella, Escherichia coli, Paracoccus, Thiobacillus, Thiomargarita, and
Thioploca (T. araucae and T. ingrica species), but also by some fungal members of
Ascomycota and Basidiomycota (Risgaard-Petersen et al. 2006; David et al. 2014;
Danovaro et al. 2017). Denitrifiers contain enzyme nitrate reductase, which can be
membrane-bound nitrate reductase (NAR) and/or periplasmic nitrate reductase
(NAP) responsible for catalyzing dissimilatory nitrate reduction to nitrite.
Microorganisms like Beggiatoa reduce nitrate to ammonium via nitrite. NAR can
be considered a transmembrane protein as it has cytoplasmic catalytic dimer
encoding narG,H and a membrane-bound protein anchoring between cytoplasm
and periplasm encoding narI, which transfers protons to periplasm, and hence proton
motive force is seen. In agriculture, denitrification is detrimental as most of the
nitrate in fertilizer is lost, while it is beneficial in wastewater treatment.
NO3 ! NO2 ! NO ! N2 O ! N2
Nitrate Nitrite Nitric oxide Nitrous oxide Dinitrogen
10e þ 12Hþ ! 6H2 O

It can also be understood as the anaerobic respiration of nitrite, nitric oxide, and
nitrous oxide to dinitrogen. Microorganisms carrying out all the three reactions are
termed as classical or canonical denitrifiers. Nitrous oxide reductases (NOS) present
in the periplasm of diverse bacterial species have diverse NOS variants. Inhibition of
NOS resulted in nitrous oxide emission during reduction of nitrate to nitrogen by
denitrifiers. Nitric oxide reduction is catalyzed by heme-copper oxidases containing
cytochrome c-dependent nitric oxide reductase (cNOR), quinol-dependent qNOR,
and copper-containing CuANOR. There exists an unusual qNOR, viz., nitric oxide
dismutase (NO-D), that catalyzes dismutation of nitric oxide to dinitrogen and
oxygen gas, a lesser known process in organisms like Candidatus Methylomirabilis
oxyfera.
Certain bacteria and archaea do not complete the pathway, and this results in the
release of greenhouse nitrogenous gases to the environment, like ammonia-oxidizing
chemolithotrophic bacteria that reduce nitrite to nitrous oxide, while eukaryotes such
as fungi and foraminifers reduce nitrite/nitrate to nitrous oxide/dinitrogen, respec-
tively. A denitrifying intra-oxygenic methanotroph, Candidatus Methylomirabilis
oxyfera found in anoxic environment, reduces nitrous oxide to dinitrogen without
producing an intermediate by utilizing methane as a source of energy, reductant and
carbon (Lisa and Martin 2016; Dilfuza 2018; Marcel et al. 2018).
13.2.4 Ammonification
Various fungi and prokaryotes decompose organic nitrogen from dead and decaying
organisms, excreta etc. to inorganic bioavailable nitrogen form, ammonia. This
process of converting complex organic nitrogenous compounds such as proteins,
vitamins, nucleic acids and urea to simple available nitrogenous forms like ammonia
and ammonium ion is known as ammonification. Nitrogen is returned to the ecosys-
tem by enzymes such as proteases, nucleases and ureases of different heterotrophic
fungi and bacteria like Bacillus, Microbacterium, Streptomyces, Clostridium, Pro-
teus, Pseudomonas, Zygorhynchus, Penicillium, Rhizopus etc. (Jeanette and Joshua
2011; Dilfuza 2018).
Complex Organic0 N0 → NH3 =NH4þ

Ex: Proteins=Urea ! Peptide, Amine, Amides=Ammonia
8e‐ þ 8 Hþ ! 3 H2 O
Ammonia fungi are the chemoecological group of fungi that exclusively develop
their reproductive structure or fruiting bodies when soils are rich in ammonia, urea,
and other nitrogenous compounds. Examples include (1) early-phase fungi—ana-
morphic and cup fungi in Ascomycota when ammonium content in soil was
decreased to pH-7—and (2) late stage of early-phase fungi—with the oxidation of
ammonia and decrease in pH from 7 to 6, Basidiomycota were in larger number and
still decrease in pH witnessed in late-phase fungal species with larger fruiting bodies.
Previous studies have shown that early-phase species Amblyosporium botrytis and
Ascobolus denudatus had deadlock competition/inhibition with nonammonium
fungi Aspergillus niger and Penicillium citrinum on substrate-containing urea
while medium and weak competition in ammonium-, nitrite-, and nitrate-containing
media (Barua et al. 2012).
13.3 Dissimilatory and Assimilatory Nitrate Reduction

to Ammonia (DNRA and ANRA)
DNRA can be defined as reduction of nitrite to ammonia or ammonium ions at

cellular level or between organisms in a community utilizing dehydrogenases, which
evolved from respiratory homologous multiheme cytochrome c reductases with nrfA
gene product. It is stimulated by negative redox potential and removes or reduces the
loss of dinitrogen from soil by conserving energy and generating proton motive force
for cellular growth. DNRA to ammonium is catalyzed by periplasmic cytochrome c
nitrite reductase (ccNIR) encoded by nrfAH, octahaem nitrite reductase (ONR), or
octahaem tetrathionate reductase (OTR). ANRA is similar to DNRA except that the
enzymes participating would be nitrite reductases that are evolutionarily unrelated
from bacteria and/or fungi. Microbes carrying ANRA utilize assimilatory cytoplas-
mic ferredoxin or NADH-dependent nitrite reductases with a single siroheme and an
iron-sulphur [4Fe-4S] center containing nirA of Cyanobacteria, ε Proteobacteria, or
nirB/nasB gene product of β- and γ- Proteobacteria, respectively (Zhang et al. 2013;
Lisa and Martin 2016; Marcel et al. 2018).
NO3 ! NO2 ! NH4þ

Nitrate Nitrite Ammonium
Recent metagenomic and microscopic studies have shown that the volcanic
eruption consists of different genera of prokaryotes and eukaryotes that can be
involved in different metabolic pathways for the uptake of sulfur and nitrogen
compounds. Of the different microorganisms reported in this study, it was observed
that Thiolava veneris has genes for both dissimilatory and assimilatory nitrate
reduction (Philippot and Germon 2005; Danovaro et al. 2017).
13.4 Codenitrification
The microbially mediated cosynthesis of N2O and N2 via N-nitrozation reaction is

known as codenitrification, where fungi play a significant role over bacteria. Fusar-
ium oxysporum converts N compounds like azide, salicylhydroxamic acid and
ammonium ions to either N2O or N2, which will then combine with another N
atom from nitrite to form a hybrid N2O species. It is considered to be a potentially
significant process in all the three domains, viz., Archaea, Bacteria, and Fungi in
Eukarya. Most of the codenitrifying bacterial and fungal species, such as Pseudo-
monas spp., Fusarium solani, Cylindrocarpon tonkinense, Fusarium spp., Strepto-
myces etc. are already known as typical denitrifiers. It is a process where nitrogen gas
is formed by a variety of possible biotic nitrozation reactions, i.e., N-N or N-C
nitrozation reactions. Bionitrozation is another nitrozation reaction as a result of
which release of nitrogen gas or immobilization of nitrogen is seen, independently of
participating microbial species or environmental conditions (Tanimoto et al. 1992;
Masahito et al. 2007; Oliver et al. 2011).
NO ! □
2 =NO
N
N2
N2 O Nitrate=Nitric oxide, Nitrous oxide ! N Nitrogen □ Dinitrogen
13.5 Microbial Diversity: N Cycle
Both culture-dependent and independent techniques can be used to understand the

microbial diversity involved in N cycle. Usually, traditional pure culture techniques
include (1) modified N-free solid/semi-solid media for the isolation of N fixers,
followed by acetylene reduction assay; (2) ammonia-oxidizing bacteria (AOB) and
archaea on AOB media (John and Robert 1936); (3) ammonia producers on routine
growth medium (RGM) or hyperammonia producers on selective/enrichment
(SE) medium; and (4) identification of nitrate or nitrite producers using nitrosamine
strips in peptone broth (Terence and Michael 2003). Nitrogen fixation can also be
assessed by crown- and lateral-nodulation count in nodulating plants, measuring
acetylene reduction after 4 h of sunrise, measuring cobalamin (Raven and Barkhem
1974), calculating GHG flux by static gas chambers, calculating Co by AAS,
measuring leghemoglobin (Coventry and Dilworth 1976), and calculating available
N by Kjeldahl method (Dilworth et al. 1978).
The presence of nase genes in N fixers has been carried out at varied habitats like
soils, plants, lakes, rivers, oceans, invertebrate guts, bioreactors, etc. (Zehr et al.
2003). Short-term changes, for example, in nitrifying activities, can be known via
specific proteomic approach by extracting, purifying and assaying significant
enzymes like AMO and NXR. If the enzymes are membrane bound, then indirect
quantifying activity of the enzyme or activity-based protein profile can be carried out
(Norton and Yang 2019). Relevant biochemical and physiological characteristics of
an environment can be determined by using pure cultures. Other culture-independent
techniques include utilization of N15 nitrogen isotope, known as N fixing probes for
rRNA phylotypes or gene sequences for nitrogenase. The relation and contribution
of microbial process to N cycle can be estimated via stable isotopes, specific assays,
and selective inhibitor techniques on the basis of the metabolism and enzyme
properties of microbes responsible for N transformation. Metal homeostasis studies
can be carried out to know the role or significance of metal cofactors such as V, Mo,
or alternate metals for biological nitrogen fixation (BNF) in nitrogenase. Enzyme
activity can be demonstrated via isotopic fractionation technique like isotopic
acetylene reduction assay (ISARA) (Romain et al. 2017). For the analysis of specific
metabolites, methods like isotope pool dilution (IPD) and hydrolytic activity assay
for specific amino sugars and amino acid enantiomers at varied temperatures in N15
labeled media to analyze free amino groups, soil-free amino acids, total amino sugar,
and amino acid content can be used. PLFA (phospholipid fatty acid analysis) profile
followed by gas chromatography (GC) and ISQ single quadrupole mass spectrome-
ter can be used to analyze microbial communities that are involved in N cycle
(Yuntao et al. 2018).
Biochemical and gene-based coupling studies can also be used to determine the
presence of nitrogen fixers by nif and of AOB by amo primers. Ammonia synthesis
or evolution can be analyzed by peptone broth and quantitatively by indophenol
spectrophotometry. Nitrite production can be confirmed by Griess reagent, and
nitrate/nitrite concentration can be known by ion exchange chromatography. Primers
are designed using degenerate oligonucleotides or modified nucleotides that enable
us to estimate and determine nitrogen-cycle-related microbes quantitatively (Shinn

1941; Poly et al. 2001; Baldani and Baldani 2005; Hirotsugu et al. 2015).
Not all the microbes are culturable, even after the development of few new
methods by Zengler and coworkers which combined culture-based and molecular
techniques to clearly understand the actual roles of microbes in N cycle. Conversely,
molecular techniques to study microorganisms (both culturable and nonculturable)
involved in N cycle include DNA isolation, PCR amplification, Sanger sequencing
(of pure cultured isolates, which should be carried out to know further details about
the organisms), DGGE (denaturing gradient gel electrophoresis), cloning, DNA
arrays, FISH (fluorescent in situ hybridization), NGS (next-generation sequencing),
or high throughput sequencing. For qPCR analysis, RT-PCR provides information
about the distribution of microorganisms involved in N cycle across varied habitats,
while phylogenetic analysis requires bioinformatics parallelly, along with results
obtained from NGS, DGGE, restriction fragment length polymorphism (RFLP),
DNA microarrays, library preparation by cloning, and sequencing. The 16S rRNA
gene is involved in molecular analysis via targeted metagenomics by sequence-
based and function-based approaches involving—sample preparation, purification,
separation, sequencing, data analysis, and interpretation. NGS/HTS is carried out via
Roche 454 Genome Sequencer, HiSeq 2000, and AB SOLiDTM system. DNA-based
stable isotope probing (DNA-SIP) approach was used to study six grass species. It
revealed a variation of N uptake in conservative and exploited species, which was
also related to root exudation levels. Furthermore, metatranscriptomics approach is
being used to evaluate functional abundance and diversity changes related to N
cycle. Additionally, metaproteomics provides protein fingerprints via mass spec-
trometry. Meta-analysis still deals with challenges with sampling and data procure-
ment (Wang et al. 2017; Nicholas and Babalola 2018).
13.6 Different Ecosystems: Microbial Diversity (Gene Based)

and N Cycle
13.6.1 Forest Ecosystem
Ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group affiliated to

groups 1.1a and 1.1b are significant in the oxidation of ammonia in forest soils.
Forest soils contain greater nifH abundance than agricultural soils. Tropical forests
contain leguminous trees, which constitute symbiotic nitrogen fixers, while conifer-
ous forests dominate free-living nitrogen fixers, except Frankia-Alder symbiotic
diazotrophy. Acid forest soils have less nifH abundance. Rosch et al. (2002)
observed that acid forest soils contained nifH from highly conserved symbiotic
and nonsymbiotic diazotrophs. It is known that the abundance of free-living
diazotrophs results in greater soil nitrogen than fertilization. Studies carried out by
Wang et al. (2013) suggested the presence of distinct communities of nifH from
different ecosystems like subtropical wet forests, dry forests, grasslands, boreal
forests, etc., usually driven by pH, annual precipitation, annual temperature, sunlight
exposure and soil organic matter, i.e., C:N ratio (David et al. 2014).
Nitrification is carried out by chemolithotrophic ammonia oxidizers—bacteria
and archaea—and nitrite oxidizers. Nitrosospira sp. clusters 1, 2, 3, and 4 predomi-
nate AOB amoA in forest soils. AOA and Crenarchaeota affiliated to groups 1.1a
and 1.1b are significant in the oxidation of ammonia in forest soils. The amoA of
AOA copies are 16 times higher in sandy grasslands with a pH of 7 than in arable
grasslands with pH 5.5. In silty clay, AOA is one to ten times higher in abundance
than AOB in clay agri-undic soil, which is similar to that in semi-arid soil. Archaeal
nitrifiers are directly correlated with N2O flux in the forest ecosystem (David et al.
2014).
N2O emission and fungal:bacterial ratio is higher in low pH soils, either in forest
soil or drained peatland soil. Rosch et al. (2002) suggested an effective link between
biological nitrogen fixation and denitrification by nirS and nosZ; nirS and nirK gene
diversity is low in oak and spruce, i.e., acid forests. Positive correlation of napA gene
is observed with exchangeable manganese (Mn) and soil C; narG is positively
correlated with soil C. Various factors influence the quantity or concentration of
nirS and nirK, like soil moisture; soil temperature; total N; available P, ammonium,
nitrate, soil organic matter, dissolved organic carbon; and soil pH. Forest soils have
abundant nosZ, while agricultural soils are home to nirS. Nitrogen addition via
ammonical sulfates reduces soil pH, leading to lowered narG, nirK, nirS, and
nosZ, while the addition of organic fertilizers increases soil pH to near neutral,
thereby increasing narG, nirK, and nosZ abundance (David et al. 2014).
13.6.2 Agroecosystems
The effects of the application and management of nitrogen can be apparently

observed in the farming, in wild plants, in the health of all living beings, and in
economic and social domains (Robertson and Vitousek 2009; Foley et al. 2011).
Reduced soil organic carbon, crop production and greater erosion are result of poor
land management practices, which lead to a reduction in biological diversity.
Agroecosystems are managed by efficient or substitution approaches that focus on
alternative practices in order to reduce or replace external, synthetic and nonrenew-
able inputs, and increase ecofunctional intensification. Other practices include effi-
cient N utilization by biological nitrogen fixers that help maintain soil biota, time-
based fertilizer application and decreased soilborne pathogens by bioactive
amendments in soil-like manure, compost etc. Integrated crop management practices
include the use of a push-pull cropping system in order to distract pests from primary
crops, conservation agriculture practices such as crop rotation, mixing of legumes,
perennial/annual cropping, implementation of a zero-till system, reducing soil dis-
turbance by direct seeding, maintained grazing of animals, and reducing pathogen
effect by integrated pest management through utilization of genetically less suscep-
tible/resistant varieties (Lampkin et al. 2015). However, greater quantities of
nutrients, energy, and water are required for mechanical- and chemical-intensive
agriculture, i.e., ecological intensification (Abdullah et al. 2017). Response to

changes in the environment can be explored via an integrative approach toward
functional traits, soil conditions, microbial diversity and community dynamics
(Espenberg et al. 2018). Several model fungal nitrate assimilations and regulations
are well known, whereas the contribution of fungi in total nitrogen cycling in
agricultural soils is under study. It is a well-known fact that fungal-dominated
agricultural systems are less prone to leaching and denitrification. Available carbon
in addition to fungal assimilatory nitrate reductase plays a crucial role where a
horizontal gene transfer event is commonly observed (Markus et al. 2014).
13.6.2.1 Agroecosystem: Legume-Cultivated Soils

Microbes inhabit or colonize in leguminous plants, forming plant-microbe
complexes known as holobiomes. They increase yield; promote growth; enhance
nutrient uptake; enable fixation by nodules; improve CO2 sequestration from air;
impart resistance toward biotic stresses like pests, diseases, and abiotic stress;
improve plants’ photosynthetic capacity etc. These are referred to as EPHs
(enhanced plant holobiomes) when the association is with a purpose. Legume-
related microbial EPHs are usually seen in (a) bacteria from Rhizobiaceae,
(b) plant obligate arbuscular mycorrhizal fungi (AMF), (c) selected endophytic
strains of Trichoderma, and (d) fungi from order Sebicales, especially
Piriformospora indica (Gary and Norman 2019). Each of these organisms produces
chemical-signaling molecules, viz., SAMPs and MAMPs (symbiont-associated and
microbial-associated molecular patterns). EPH-induced plant defense systems
include (1) improved upregulated defense gene expression—mpk3, mpk6, wrky22,
wrky29, and pdf1.2—via quorum sensing and increased release of antifungal phenol
compounds and H2O2.; (2) induction of systemic resistance; and (3) hormonal
signaling, leading to acquired systemic resistance (Abraham and Babalola 2019).
Genes like nosZ, nifH, amoA, nirK and nirS were abundant in bulk soil, while
nifH, amoA, hao, narG, nirS/K, norB, and nosZ were abundant in maize rhizospheric
soils. Abundance of nosZ and bacterial amoA genes was observed in organically
amended soils. Soils with higher organic carbon content showed increased nrfA and
nosZ genes (Jennifer et al. 2019). N-fertilized soils have abundant AOA and AOB,
while nxrB abundance was not affected by N fertilization. Repeated ammonium-
sulfate-based fertilized soils have higher AOB as a result of nitrification than AOA.
Among the treatments, the difference was insignificant. Plants take either nitrate or
ammonium, which in turn affects the community and diversity of nitrifiers. Nitrogen
transformations were more significant in rhizospheric soils as against bulk soils.
Certain plants secrete nitrification inhibitors in the rhizosphere, thereby reducing
nitrification (Norton and Yang 2019). Nitrification is carried out by nifHDK and/or
pnfA, which is a similar sigma factor; comparatively, Agrobacterium species
colonizes Sesbania and rice plants having sym plasmid that comprises nifH and
nodA genes (Nicholas and Babalola 2018). NirK was less abundant in legume-based
soils, whereas amoA, narG, nirS, nosZ and nifH were in greater abundance. Mean-
while, amoA, narG, and nifH diversity was significantly affected. It is known that
these soils have higher organic C, nitrate, available potassium (K), and ammonium
ion concentration, and lower available P. The more significantly found bacteria in
such soils include Tumebacillus, Bacillus drentensis, Bradyrhizobium and Strepto-
myces (Zhou et al. 2019). Bacterial amoA, nirK and nirS genes abundance were
observed in early vegetative stage, irrespective of fertilizer application, which
decreased gradually with the passing of the developmental stages of plant and was
lowest at flowering stage. Meanwhile, AOA was abundant in all stages, organic
fertilizer application increased its abundance, nifH gene was abundant during the
flowering stage when manure-based fertilizer was applied, and nirK and nirS genes
were abundant in legume-cultivated soils (Hai et al. 2009).
13.6.2.2 Agroecosystem: Paddy-Cultivated Soil

As paddy is a staple crop cultivated by Asians, changes in microbial population in
paddy soils bring about crucial variance globally. Most of the soil bacterial
communities decreased with deeper layers of paddy soils; however, ammonia-
oxidizing archaea (AOA) communities are enhanced. Genes belonging to AOA
are in abundance than AOB genes in soils cultivated with paddy. In top-layer
(0–20 cm) soil, nirS are abundant, while in deep layers, nirK is abundant. It is
observed that variance in N-cycling genes is a result of changing environmental
conditions. It is known that N-cycling gene abundance is related to the fertilizer
applied, the type of soil used in cultivation, soil depth, and soil properties. Deeper
soils generally witnessed low abundance of AOA and AOB genes, but up to a depth
of 0–30 cm, and the reverse is observed in ammonium-rich soils (Leininger et al.
2006; Hofferle et al. 2010). Abundance in nifH gradually decreases with depth. In
deeper layers of soil, AOA amoA dominates AOB amoA copy number, shooting
peak at 20–40 cm based on region and way of interaction. Increased abundance of
nirK, AOA, and 16S rRNA gene is directly proportional to the total C, N, C/N ratio
and inversely proportional to annual precipitation and frost-free period. Altitude and
abundance of 18S rRNA gene are negatively related; a remarkable positive correla-
tion is found with C/N ratio and nifH, as well as changes in the abundance of 18S
rRNA and AOA. Soil physical structure is positively correlated with most microbial
and N-cycling functional genes, whereas negative correlation is observed with
dissolved organic carbon (DOC) concentration and changes in nirS gene abundance.
Abundance in AOA gene is positively correlated with nitrate concentration, soil
water content, soil pH, sand content and DOC in correlation with nirK gene. In
rhizospheric and alkaline soils, nirK gene is abundant, while nirS is abundant in bulk
and acidic soils (Henry et al. 2004; Barta et al. 2010; Wang et al. 2017).
13.6.3 Terrestrial Antarctic Soils
Studies in Antarctic soils revealed that the addition of nitrogen and temperature had a
significant effect on microbial community diversity. Ammonia-oxidizing bacteria
are quantitatively more than archaea in Antarctic soils. Fungi and archaea are mostly
found in colder temperatures. Warm temperature reduces ammonia-oxidizing
archaea (amoA) and nitric oxide reductase (norB) genes and is even detrimental to
ammonia-oxidizing archaea. Warm temperature increases nifH and nirK gene abun-
dance, while nirS is less sensitive to it. Nitrogen addition increased nifH and nirK
genes. Greater numbers of denitrifiers (nirS and nirK) are observed upon nitrogen
addition, whereas archaeal communities remain unaffected.
Nitrous oxide emissions usually depict a truncated denitrification pathway. Bac-
terial norB and nosZ abundance is lesser than bacterial narG, nirS and nirK abun-
dance due to the reduction of nitric or nitrous oxides, hence resulting in incomplete
nitrogen pathway. The nirS gene is predominantly abundant than nirK; however, a
significant increase in NirK abundance was observed if the temperature of soil was
maintained at 10 C. Temperature plays a crucial role in denitrifying community size
and composition. There are certain temperature-sensitive amoA communities, and a
few of them can be made adaptive or less detrimental by favorable environmental
factors like nutrients. Soils fertilized with nitrogen witnessed amoA at 4 C and narG
at 10 C (Jung et al. 2011).
13.6.4 Tropical Peatland: Drained and Natural Soils
Tropical peat can be defined as Histosol, i.e., with high organic C and low bulk
density of approx. 0.2 mg/m3, which mostly contains carbon-water storage in
tropical latitude covering Southeast Asia, Africa, and Central and South America.
Peatland ecosystems are very much affected by climate changes, anthropogenic
activities like logging and building of drainage, and land conversions to agriculture.
Denitrification or conversion of nitrous oxide to dinitrogen in natural tropical
peatlands is usually controlled by nosZ clade I genes and in drained peatland soils
by clade II genes mainly. Archaeal abundance is observed in drained peatlands,
while bacteria are dominant in natural peatland soils. Genera Mycobacterium,
Conexibacter, Burkholderia, Rhodoplanes, Pseudomonas and Paenibacillus of bac-
terial phyla Proteobacteria, Actinobacteria, Acidobacteria and Firmicutes are domi-
nant in both natural and drained peatland soils. NirS, nirK, nosZ I, nosZII, nifH, and
archaeal amoA genes are abundantly found in both soils, while nrfA is seen in natural
peatland soils only. Bacterial amoA is not seen in peatland soils. Drained peatland
soils are comprised mostly of archaeal community, while natural peatland soils
comprise both bacterial and archaeal communities equally. Warm temperature
favored AOA than AOB, just like acidic tea orchards and forest soils (Lu et al.
2012; Wu et al. 2013). This AOA abundance can be related to chemical properties of
soil like temperature, pH, and available C, O, and N content. Anammox has a very
minor role here (Espenberg et al. 2018).
13.7 Strategies to Maintain N Equilibrium
Principal N management practices include the application of the 4R approach—right

source, right rate, right time, and right place (Clarke and Beegle 2014). Excess
fertilizer-based reactive nitrogen (Nr) contributes to wash off or runoff, leading to
(1) accumulation as nitrate form in drainage or waste and atmospheric deposition of
denitrified dinitrogen and nitrous oxide (GHG); (2) emission of trace amounts as
ammonia and nitrogen oxides in the atmosphere, which paves the way for secondary
pollutants like ammonical nitrates and sulfates, aerosols, ozone, and photochemical
oxidants; and (3) sedimentation in water bodies as a result of leaching. These forms
are available for a few weeks in the atmosphere and a few decades in terrestrial
ecosystems, and the longest are in oceans, which are difficult to be controlled
(Fowler et al. 2013 and Huhe Borjigin et al. 2016). Therefore, alternate strategies
for nitrogen use efficiency (NUE), along with biotic and abiotic factors, need to be
worked out in order to maintain N equilibrium.
Blanket usage of N chemical fertilizers; variations of climatic conditions and the
conditions of soil and crops and their cultivars; and conventional management
practices lead to excess N application, which has an impact on NUE and thereby
adds more Nr to the environment. Therefore, proper agronomical practices and
methods that are less prone to leaching and volatilization and retain available N
for the plants can lead to sustainable agriculture and N equilibria in the environment.
Understanding management strategies based on the ability of soils to emit or mitigate
N2O (GHG) is also important. Another approach that can be suggested, depending
on the environmental conditions of a particular site, is an alternate legume-based
cropping system which will automatically replenish nitrogen-based microbial com-
munity in the soils. Alternatively, organically coated urea/fertilizers can also be used
which will serve as slow/controlled release urea; application of nitrification
inhibitors to soils or modified fertilizer practices like Karanj oil coating, N-(n-butyl)
thiophosphoric triamide (NBPT) coating, allicin coating, use of dicyandiamide
(DCD), 3,4-dimethylpyrazole phosphate (DMPP), or nitrapyrin; etc. have inhibitory
effect on nitrification and soil urease (Ranitha et al. 2017; Shilpha et al. 2017; Wang
et al. 2018; Yaying et al. 2018).
13.8 Conclusion
Nitrogen occupies more than two-third part of the atmosphere and is in an unavail-
able form. Besides, its presence in every cell of living beings in different stable
forms, combined with various elements, demonstrates its importance. Nitrogen
circulates globally in three major ways: (1) nitrogen fixation, (2) nitrification, and
(3) denitrification, apart from other lesser known processes. Anthropogenic activities
such as return of animal waste to soil, wastewater treatment, application of chemical-

based nitrogen in the form of fertilizers and fossil fuel combustion have led to the
disturbance of N cycle. Percolation of nitrogen into the deeper layer and water
streams, especially the biosphere region, led to a shift to isotopically light nitrogen
in lake sediments and heavy nitrogen in temperate regions. There is a possibility of
an “open” N cycle instead of a “closed” N cycle due to excessive fertilization;
leakage of natural N (denitrification); inhibition of natural N deposition/fixation,
which would disturb the food web or energy pyramid; global temperature increase
leading to changes in gaseous composition in the atmosphere, which in turn may
have negative effects on vegetation and positive effects for certain vegetation. In
addition, N2O accounts for 0.03% of the total GHG emission. It has 300-fold greater
potential for causing global warming due to its radiative forcing capacity compared
with other GHGs. Hence, in order to maintain an environmental equilibria status of
N cycle, understanding the diversity of microorganisms involved in N cycling is
fundamental for sustainable environmental management. Recent studies and molec-
ular methodologies have led to a better understanding of the role of the different
microorganisms in N cycle.
13.9 Future Perspective
In order to understand microbial diversity and its impact on environmental N

equilibria in the midst of a fluctuating environment, it is necessary to understand
and link deeper physiological processes that involve different microbial
subpopulations. It is important to study a broader range of samples (i.e., more than
one site) and conduct quantitative in situ studies on the primary reactions of N cycle
and lesser known processes like anammox, comammox, and codenitrification, along
with the function of microbial extracellular enzymes. Interactive studies between N
fixers, nitrifiers, ammonifiers, denitrifiers, and anammox communities in varied
environmental conditions will decrease the knowledge gap and facilitate our under-
standing of the natural distribution pattern of microorganisms. Different experimen-
tal set up can be planned by varying different concentrations of nutrients (N, P, K
and microelements); their interactive effect on culturable and non-culturable diver-
sity of microorganisms and their correlation with global N cycle and other geochem-
ical cycles can be implicated (Table 13.1).
Table 13.1 Genes involved in N cycle

Name of genes Details of genes
nifD,K,H, vnfDGK and Nitrification—Mo-nitrogenase, vanadium nitrogenase, and Fe-only
anfHDK nitrogenase major cluster genes
nfH, vnfH, and nifH Nitrogenase reductase or Fe protein—Fe-containing electron transfer
protein, used as a genetic marker for nitrogen fixers
ifD,K; vnfDGK; and Encodes catalytic component of nitrogenase with Fe, V, and Mo in
anfDK active center
nifQXWE-XQVY Mo-nitrogenase minor cluster genes—for synthesis and insertion of
FeMo cofactor into FeMo protein
ifENB Helps in nitrogenase cofactor synthesis
odA Nodulation gene
pnfA Nitrogenase—electron transfer/O2 protection, thereby controlling
nitrogenase activity
hesB Fe-S cluster assembly protein
cysE Serine acetyltransferase
hh Hydrazine hydrolase
hzo Hydrazine oxidoreductase
amoA, B, C Ammonia monooxygenase—subunits A, B, C
pmoA, B, C Methane monooxygenase—subunits A, B, C
camoA Nitrification by comammox
norB, C Nitric oxide reductase—subunit B, subunit C
hao Hydroxylamine dehydrogenase
nir-nirA Nitrite assimilation—ferredoxin nitrite reductase
nirB, D, K/S Nitrate reductase—(NADH) large subunit, small subunit,
(NO) forming/nitrite assimilation—(NO) forming hydroxylamine
reductase
nor Nitric oxide reductase
nosZ Nitrous-oxide reductase
napA, napB Periplasmic nitrate reductase, cytochrome c-type protein
narG, narH, narL, Nitrate reductase—α subunit, β subunit, γ subunit, δ subunit
narJ
narG, H Nitrate reductase catalytic dimer, narG to detect denitrifiers
narI Membrane-bound nitrate reductase—codes membrane anchor
nxr Nitrite: nitrate oxidoreductase
nas-nasA, nasB Assimilatory nitrate reductase—catalytic subunit, electron transfer
subunit (NADP(H))
hzsA, B and C Hydrazine synthase—hzsA and B to detect anaerobic ammonia
oxidizers
nar Assimilatory nitrite reductase
glnA Glutamine synthetase
glnP,H Glutamine transporter gene
glu2 Glutamate synthase ferredoxin
gltB Glutamate synthase large chain
gltD Glutamate synthase small chain
amt Ammonium transporter
(continued)

Name of genes Details of genes
gdhA Glutamate dehydrogenase
gudB Glutamate dehydrogenase
nrt Extracellular nitrate transporter
narB Ferredoxin-nitrate reductase
nrfA Dissimilatory nitrate reduction to ammonia (DNRA)
nap Denitrification
chiA Mineralization
Sources: David et al. (2014), Holger et al. (2015), Inoue et al. (2015), Lisa and Martin (2016), Olivia
et al. (2017), Florence et al. (2018), Marcel et al. (2018), Ramiro and Silvia (2018), Black et al.
(2019) and Yan et al. (2020)
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Contribution of Zinc-Solubilizing
and -Mobilizing Microorganisms (ZSMM) 14
to Enhance Zinc Bioavailability for Better
Soil, Plant, and Human Health
Ramesh Chandra Yadav, Sushil K. Sharma, Aketi Ramesh,

Kusum Sharma, Pawan K. Sharma, and Ajit Varma
Abstract
The impetus on agricultural productivity and food security to feed the burgeoning
population is a major challenge faced by researchers and policy makers. Thus,
there is a need for enhancing not only productivity but also the quality of produce
for human and animal consumption. Micronutrient malnutrition is due to low
concentration of micronutrients in farm produce. The micronutrient content is
appreciably low, especially in cereals, and is below the dietary requirements. Zinc
(Zn) is one of the integral components of many metabolic processes involving
crop growth, human and animal development. Zinc deficiency is a universal
problem among crops, humans, and animals. Globally, 33% of the human
R. C. Yadav
ICAR-National Bureau of Agriculturally Important Microorganisms , Maunath Bhanjan, Uttar
Pradesh, India
S. K. Sharma (*)
Pradesh, India
ICAR-National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
e-mail: sushil.sharma1@icar.gov.in
A. Ramesh
K. Sharma
ICAR-Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India
P. K. Sharma
Pradesh, India
A. Varma

23, https://doi.org/10.1007/978-981-15-9154-9_14
358 R. C. Yadav et al.
population, especially in developing countries, is prone to Zn deficiency as a

consequence of overdependence on cereal diets to fulfill dietary intake. Defi-
ciency in zinc is the major cause of morbidity and death in humans. Therefore,
agricultural intervention is the need of the hour in order to minimize Zn defi-
ciency in humans and thereby overcome zinc malnutrition. One strategy for
agricultural intervention is the application of zinc-enriched fertilizers to soil for
biofortification of grain/edible portions of crops, but this strategy is not environ-
mentally friendly. In recent years, zinc-solubilizing and -mobilizing
microorganisms (ZSMM) alone or with organic materials have been advocated
to improve soil zinc mobilization from the native and applied pool for increasing
crop assimilation. However, several bacterial genera (Bacillus, Burkholderia,
Enterobacter, Exiguobacterium, Gluconacetobacter, Pseudomonas,
Sphingomonas, Xanthomonas, Pseudomonas and Serratia), fungal strains
(Aspergillus, Penicillium, and arbuscular mycorrhizal fungi (AMF), and other
microbes have been recognized to solubilize zinc. Nevertheless, these
microorganisms have potential to be harnessed as bioinoculants for zinc
biofertilization and biofortification in crops for combating Zn malnutrition in
human population. The chapter highlights the roles of ZSMM on enhancing zinc
content through solubilization of insoluble soil zinc compounds for better soil,
plant and human health.
Keywords
Biofortification · Food security · Zinc · Zinc solubilization · Zinc deficiency and

ZSMM
14.1 Introduction
Micronutrient deficiency in soils is the most recognized abiotic stress that affects
crop productivity globally. With regard to micronutrient deficiencies, zinc deficiency
is often prevalent in most soils in almost all countries, including India (Cakmak et al.
1999; Alloway 2009). Zinc is associated with metabolic activity in plants, animals,
human beings, and microorganisms (Broadley et al. 2007) and is a constituent of
many proteins required for structural and functional integrity (Andreini et al. 2009).
Zinc deficiency not only affects agricultural productivity but also inflicts human and
animal well-being and is prevalent in a majority of countries (Hotz and Brown 2004;
Myers et al. 2015). The deficiency depends on many factors such as high pH, organic
matter, bicarbonate content, soil phosphorus, and iron concentration (Wissuwa et al.
2006; Alloway 2009) in soils. Other reasons for deficiency in micronutrients include
intensive cropping system, dispensing with organic matter application, recycling of
crop residues, and high-analysis inorganic fertilization that is devoid of
micronutrients (Hafeez et al. 2013). Soluble zinc sulfate applied to agriculture fields
is either sorbed or precipitated, affecting its availability to crop plants (Sarathambal
et al. 2010), and hence efficiency of zinc fertilizer use is very low (Ma and Uren
14 Contribution of Zinc-Solubilizing and -Mobilizing Microorganisms (ZSMM) to. . . 359
Table 14.1 List of zinc-solubilizing and -mobilizing microorganisms (ZSMM)

Bacteria Acinetobacter sp. Arthrobacter (Arthrobacter humicola), Azospirillum
lipoferum, Azotobacter, Bacillus (Bacillus megaterium, B. alcalophilus,
B. barbaricus, B. subtilis, B. circulans, B. licheniformis, B. atrophaeus,
B. amyloliquefaciens, B. firmus, B. pumilus, B. thuringiensis, B. cereus),
Brevibacillus parabrevis, Burkholderia cepacia, Curtobacterium,
Corynebacterium callunae, Delftia sp., Duganella violaceusniger,
Enterobacter (E. cancerogenus, E. cloacae), Exiguobacterium acetylicum,
Gluconacetobacter diazotrophicus, Kocuria sp., Lysinibacillus xylanilyticus,
Microbacterium saperdae, Paenibacillus (P. dendritiformis, P. tundra),
Pantoea (P. ananatis, P. dispersa, P. agglomerans), Plantibacter, Providencia
rettgeri, Pseudomonas (Pseudomonas sp., P. monteilii, P. thivervalensis,
P. stutzeri, P. fuscovaginae, P. lini, P. fragi, P. fluorescens, P. aeruginosa),
Psychrobacter fozii, Ralstonia picketti, Rhizobium, Serratia, Sphingomonas,
Stenotrophomonas, Variovorax, and Xanthomonas
Fungi Aspergillus niger, Oidiodendron maius, and Penicillium simplicissimum
Actinomycetes Leifsonia poea and Streptomyces (S. fradiae, S. cinnamonensis, S. canus,
S. netropis, S. scabiei, S. violarus, and S. griseus)
Cyanobacteria Cyanobacteria
AMF Glomus etunicatum and other genera
1997; Zhang et al. 2017). Moreover, the high cost of inorganic zinc fertilizers makes
them further inaccessible to resource-poor farmers (Cakmak 2008; Ramesh et al.
2014a). The total soil Zn content does not translate to increased zinc availability, and
much of the zinc in soil is in discreet chemical forms, and the availability depends on
their solubility (Sillanpää and Vlek 1985). Apparently, the bioavailability of zinc to
crops and the quality of produce for human and animal dietary intake have declined
(Cakmak and Hoffland 2012). Genetic biofortification takes a longer time, and its
performance differs in different soil environments and acts as an impediment for
successful implementation (Cakmak 2008). Agricultural interventions involving the
application of fertilizer zinc through soil and foliar application, crop rotation
(Dimpka and Bindraban 2016; White and Broadley 2009), organic manure applica-
tion (Aghili et al. 2014; Helfenstein et al. 2016), and bioinoculation (Gandhi and
Muralidharan 2016; Ramesh et al. 2014b; Wang et al. 2014; Kaur et al. 2020;
Kushwaha et al. 2020) are gaining prominence (Table 14.1). In recent years, efforts
are being made to isolate zinc-solubilizing and -mobilizing microorganisms with
multiple plant-growth-promoting traits, including innate ability to solubilize insolu-
ble Zn to bioavailable Zn for crop assimilation. Many ZSMM have been reported to
play a decisive role in crop zinc assimilation. Still, there is a need at a global level to
explore potential microorganisms that solubilize and mobilize native soil Zn pool to
improve Zn content in edible portions of crops to improve the health of all. This
chapter focuses on the status and contribution of ZSMM for improving bioavail-
able zinc in soil, plant, and human growth and development.
14.2 Contributions of Zinc in Different Life Forms
14.2.1 Microorganisms
Zinc is known for its immense role in both eukaryotic and prokaryotic microbial
metabolisms (Hughes and Poole 1989). In bacteria and fungi, reduced zinc avail-
ability affects pigment formation (Chernavina 1970). Some fungi are known to
tolerate zinc even at high concentrations. Aspergillus niger has been reported to
survive both at higher concentration (1000 mg Zn) and at low zinc concentration
(2 mg kg 1 available zinc) (Bullen and Kemila 1997). This is also the case with
lichens and poisonous mushrooms (Vinogradov 1965). Some bacteria such as
Thiobacillus thiooxidans, T. ferrooxidans, and facultative thermophilic iron
oxidizers are reported to solubilize zinc from sulfide ore (sphalerite) present in soil
(Hutchins et al. 1986).
14.2.2 Plants
Micronutrients are essential at different stages of plant growth (Uchida 2000),

thereby augmenting crop growth and productivity. Zinc is involved in plant metab-
olism, such as in respiration, photosynthesis, carbohydrate metabolism, protein
synthesis, phytohormone synthesis, growth regulation (Hussain et al. 2015), assimi-
lation of major nutrients, and enzymatic and redox reactions. It plays a role in
biological systems by its involvement in umpteen metabolic processes (Thenua
et al. 2014; Yu et al. 2017). Furthermore, zinc is involved in gene expression,
which confers tolerance to environmental stresses (Cakmak 2000). Zinc regulates
carbonic anhydrase, superoxide dismutase, and catalases (Marschner 1995) and
protects plants from oxidative stresses. Moreover, zinc is an integral part of all six
classes of enzymes, which enables a catalytic role in various biochemical reactions
in plants. Zinc inhibits highly toxic hydroxyl radicals in Haber–Weiss reactions in
the thylakoid lamellae with its high affinity with cysteine and histidine (Brennan
2005; Disante et al. 2010; Tsonev and Lidon 2012). Zinc is involved in the formation
of complexes with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
(Påhlsson 1989; Coleman 1992). Tryptophan biosynthesis (precursor for indole
acetic acid production) involves zinc, which induces signal transduction (Brown
et al. 1993; Alloway 2004a, b; Hansch and Mendel 2009). With the combination of
phospholipids and sulfhydryl, groups of membrane proteins result in the regulation
of membranes. Therefore, zinc deficiency affects crop growth, productivity, and
nutritional quality.
14.2.3 Human
Zinc plays a catalytic, cocatalytic, or structural role in human beings, in whom many
enzymatic activities are maintained (Tapiero and Tew 2003). Similar to other
organisms, zinc is required for the activity of six classes of enzymes. Skeletal
muscles possess 86% zinc, followed by other body parts like hippocampus, pan-
creas, prostate, and kidney cortex (Vallee and Falchuk 1981). Furthermore, meta-
bolic homeostasis is dependent on zinc content. Evidence shows that the number of
zinc-protein complexes govern gene expression (Welch 2001). Therefore, a defi-
ciency in micronutrients affects brain development and wound healing and has a part
in causing pneumonia, diarrhea, and malaria (Prasad 2013). Zinc deficiency causes
many health-related complications in human beings and need to be looked into
(Graham 2008; Benton 2008). Moreover, infertility and congenital diseases like
acrodermatitis enteropathica occur due to zinc deficiency in men (Zimmermann and
Hilty 2011; Kumar et al. 2016).
14.3 Zinc Deficiency and Malnutrition
Zinc deficiency in soil and plant is directly related to zinc deficiency/malnutrition in

human beings at a global level (Fig. 14.1). Approximately, 33% of the world’s
population suffers from Zn deficiency (Hotz and Brown 2004; Stein 2010; Zhang
et al. 2011). Zinc is essential, and deficiency in zinc is recognized as the 11th major
risk factor and accounts for a number of human fatalities yearly (WHO 2002). In
China, zinc deficiency has been recognized to be fatal in peoples living in rural areas
(Zhang et al. 2011; Ma et al. 2008). Additionally, children below five years are prone
to this deficiency in Pakistan (Harvest Plus 2012). In vitro trials revealed that food
fortification decreases the severity of common infections in babies (Harvest Plus
2012). WHO recommended that supplementation of zinc can deter diarrhea infection
due to severe zinc malnutrition (WHO 2004). In China, zinc-fortified flour has been
found to overcome zinc deficiency in pregnant women (Brown et al. 2009). The
major cause of Zn malnutrition with concomitant insufficient digestion is zinc
nutrient deficiency, accompanied by antinutritional factors, especially phytate, in
humans and animals (Welch and Graham 2004; Cakmak et al. 2010). Most of the
micronutrients are essential in low quantity in edible portion of farm produce as
Fig. 14.1 Geographical distribution of zinc deficiency in soils, plants, and humans at global level
(Sources: Alloway 2008a; Wessells and Brown 2012; Cakmak et al. 2017)
against animal-derived foods. This has led to billions of humans around the globe
suffering from micronutrient malnutrition (Cakmak 2008; White and Broadley 2009;
Rehman et al. 2018a). Alloway (2004a) reported lower content of Zn in the grains of
most of the wheat crops. Moreover, the adoption of high-yielding crop varieties has
further aggravated this problem (Zhao and McGrath 2009; Cakmak et al. 2010).
Furthermore, wheat grain processing lowers not only Zn concentration, but also the
concentration of other essential nutrients (Zhang et al. 2010; Kutman et al. 2011).
This can be overcome by adopting agronomic (fertilizer, microbial inoculation),
biotechnological, and plant-breeding strategies that improve zinc bioavailability in
crops.
14.4 Status and Fate of Zinc in Soil
The wide-spread zinc deficiency is well recognized around the world (Katyal and
Rattan 1993). Low solubility of native and applied zinc is the major cause of low
zinc bioavailability in soil. Generally, to overcome zinc deficiency, zinc sulfate is
commonly applied to increase the growth and productivity of crops; however, the
efficiency of zinc use is very low as the applied zinc is sorbed on colloidal surfaces or
precipitated. Zinc is known to exist in different chemical forms in soil, such as
exchangeable, organic-bound, calcium-carbonate-bound, and Mn-oxide-bound crys-
talline and amorphous sesquioxides and clay lattices. Zinc sorption is associated
with cation exchange in acidic soils, whereas zinc fixation occurs in the sorption and
precipitation of calcium and in complexation in high pH (Alloway 2008a). The
bioavailability of zinc determines zinc supply for proper crop growth and is a
determinant of soil characteristics (White and Zasoski 1999). Zinc deficiency is
widespread in Australia (Sillanpaa 1990) China (Liu 1991) and India (Takkar 1996;
Singh 2008; Behera et al. 2009a) and other countries. Zinc deficiency is prevalent in
calcareous soils with high pH (Liu et al. 1983; Katyal and Vlek 1985). In acidic soils,
its deficiency is due to low organic matter content and lack of plant available
nutrients (Rautaray et al. 2003). The acidic soils in India cover about 49 million ha
of area, whereas more than 800 million ha of acidic soils are found worldwide
(Sharma and Singh 2002). Therefore, soil acidity is also a huge problem affecting
food production across Asia, Africa, and Latin America and is imposing heavy costs
on farmers in Europe and North America. Excessive accumulation of phosphorus in
the soil has also been found to interfere in zinc uptake by plants, and thus, it has been
found to cause zinc-induced deficiency in plants (Salimpour et al. 2010). Differential
uptake of zinc by crops among different soil zinc fractions gives credence to the fact
that zinc gets redistributed between different pools due to cropping. Behera et al.
(2009b) reported a decline in organic matter and carbonate-bound Zn in an inceptisol
as a result of intensive cropping with maize and wheat for more than three decades.
High organic matter (Lindsay 1972; Moody et al. 1997) increased exchangeable and
organic fractions of zinc and decreased the oxide fractions of zinc in soil because of
reducing conditions that enhance zinc availability for uptake by the plants. To
evaluate the bioavailability of zinc in soils, several extractants are being used,
which include mineral acids, chelating agents, buffered salts, and neutral salts.
Diethylenetriaminepentaacetic acid (DTPA) is the most widely used extractant for
the extraction of plant-available Zn in different soil types.
Ethylenediaminetetraacetic acid (EDTA), hydrochloric acid, ammonium
bicarbonate-DTPA (ABDTPA), Mehlich 1, and Mehlich 3 are also being used
(Alloway 2008b). The non-availability of zinc fertilizers at proper time, poor quality,
exorbitant cost, and the lack of awareness among farmers of its importance in crop
productivity and human health are major impediments and have to be recognized
(Das and Green 2013).
14.5 Zinc-Solubilizing and -Mobilizing Microorganisms (ZSMM)
ZSMM with multiple plant-growth-promoting traits have the ability to improve crop
quality through their intense shift in rhizobiome, leading to increased mobilization
and assimilation by plants (Ahmed et al. 2011; Bhatt and Maheshwari 2020). There
are many instances of microbial inoculation, and their role in improving crop
productivity and the quality of produce are recognized (Rawat et al. 2020; Sharma
et al. 2020). Plant-growth-promoting microorganisms (PGPM) present in the
rhizobiome promote plant growth by different mechanisms, like biological nitrogen
fixation; mobilization of nutrients; solubilization of phosphorus; synthesis of
siderophores, which can solubilize insoluble iron from the soil; production of
organic acids and production of phytohormones such as auxins, cytokinins, and
gibberellins; altering the native level of phytohormones; and improving plant stress
tolerance to salinity, toxicity, drought, metal, and pesticide load, and also act as
biocontrol for the plant (Bänziger and Long 2000; Glick 2005; Reeves and Chaney
2008; Senadheera and Cvitkovitch 2008; Khalid et al. 2009; Yoshihara et al. 2010;
Ahmad et al. 2014).
14.5.1 Solubilization by Bacteria
Rhizobacteria and endophytic bacteria solubilize sorbed or precipitated zinc on soil

colloids for plant assimilation and contribute to improving crop productivity and
human and animal health. In recent years, there has been increased usage of these
microbes to counteract the indiscriminate use of inorganic fertilizers and
agrochemicals, which have inherent negative effects on soil quality. Microbial
inoculation has been proposed as one of the strategies for overcoming malnutrition.
The exploration of promising rhizospheric soil bacteria with the innate ability to
solubilize inorganic zinc, along with multifaceted plant-growth-promoting traits, has
been considered to overcome nutrient deficiencies, increase nutrient assimilation,
and improve crop productivity. This is true in the case of zinc, wherein the microbial
transformation of native zinc to plant-available zinc is a viable option to achieve the
aim of overcoming zinc deficiency and malnutrition problem in humans (He et al.
2010; Mader et al. 2010). In recent years, many microbial isolates belonging to the
genera Enterobacter, Sphingomonas, Gluconacetobacter, Pseudomonas,

Acinetobacter, and Bacillus have been reported to solubilize zinc from different
environments and thereby increase zinc availability to crops (Di Simine et al. 1998;
Fasim et al. 2002; Hafeez et al. 2013; Kayalvizhi and Kathiresan 2019; Ramesh et al.
2014a, b; Saravanan et al. 2007a; Sunithakumari et al. 2016; Wang et al. 2014).
Strains belonging to the genus Bacillus as zinc-solubilizing bacteria (ZSB) are of the
utmost importance and are ubiquitous in soil environments; endospore formation
entails them to overcome extreme climatic conditions associated with crops
(Ramirez and Kloepper 2010; Perez Garcia et al. 2011). Inoculation of
Gluconacetobacter diazotrophicus has been reported to increase zinc availability
and zinc assimilation in sugarcane (Sarvanan et al. 2007a), Bacillus sp. in soybeans
and wheat (Sharma et al. 2012; Ramesh et al. 2014a), Sphingomonas sp. in rice
(Wang et al. 2014), Enterobacter cloacae subsp. dissolvens in soybeans and wheat
(Ramesh et al. 2014b), Providencia sp. in wheat (Rana et al. 2012), Serratia sp.,
Pseudomonas sp., and Bacillus sp. in wheat (Abaid-Ullah et al. 2015).
The innate ability to solubilize insoluble zinc compounds under in vitro
conditions has been reported in many bacterial species. Moreover, it has been
recognized that zinc solubilization ability in liquid medium varies from strains to
strains and bacterial genera to genera (Table 14.2). For instance, strains belonging to
Gluconacetobacter, Bacillus, Ralstonia and Pseudomonas solubilized maximum
zinc from zinc oxide and/or zinc carbonate (Fasim et al. 2002; Sarvanan et al.
2007a; Sunithakumari et al. 2016; Gontia-Mishra et al. 2017, Mumtaz et al. 2019),
while maximum solubilization by Bacillus aryabhattai strains MDSR7 and
MDSR14 in medium containing zinc phosphate, as compared to ZnO and ZnCO3,
has been reported by Ramesh et al. (2014a). On the other hand, a study by Abaid-
Ullah et al. (2015) revealed that Serratia liquefaciens, S. marcescens, and Bacillus
thuringiensis solubilized insoluble zinc carbonate and zinc oxide to an appreciable
extent, and these strains did not solubilize zinc sulfide ore.
14.5.2 Solubilization by Fungi
Fungi being a significant component of the microbiota in the terrestrial environment

have a role to play in nutrient transformation (White et al. 1997; Burford et al. 2003).
The solubilization of minerals through fungal processes was shown to increase metal
concentration in such environments (Burford et al. 2003). Solubilization of metals by
fungi is the result of many mechanisms such as (i) acidolysis or protonolysis,
(ii) chelation or complexolysis and siderophore production, (iii) redox reactions,
and (iv) mycelium functioning as a “sink” (Burgstaller and Schinner 1993). The first
two mechanisms are called “heterotrophic leaching” and results in the efflux of
protons from hyphae, siderophore, and organic acid production (Gadd and Sayer
2000). Generally, acidification is the result of four main processes: excretion of
protons (proton-translocating plasma membrane ATPase), proton influx, organic
acid exudation, and acidification through carbon dioxide production following
respiratory activity (Burgstaller and Schinner 1993).
Table 14.2 Zinc-solubilizing and -mobilizing microorganisms and their capacity to produce soluble zinc from different insoluble zinc sources
14
Soluble zinc (μg/ml) produced from insoluble zinc

Name of compounds
microorganisms Zinc oxide Zinc phosphate Zinc carbonate Mode of action (organic acid/chelation/others) References
Pseudomonas 14 mM 5 mM – Production of gluconic acid and 2-ketogluconic Fasim et al.
aeruginosa CMG 823 acid (2002)
Gluconacetobacter 420.0 7.20 300.0 8.49 157.0 5.66 Production of 5-ketogluconic acid Saravanan et al.
diazotrophicus (2007a& 2007b)
Serratia liquefaciens 18 – 135 Proton extrusion and production of organic acid, Abaid-Ullah et al.
FA-2 i.e., gluconic acid (2015)
B. thuringiensis FA-3 126 – 152
Serratia marcescens 93 – 164
FA-4
Pseudomonas and ZnO – ZnCO3 Production of gluconic acid and 2-ketogluconic Saravanan et al.
Bacillus spp. acid (2004)
Mycorrhizal fungi ZnO Zn3 (PO4)2 – Production of organic acid fumarate, citrate, and Martino et al.
malate (2003)
ZnO – – Production of 6 and 7 different organic acids such Kushwaha et al.
as gluconic, malonic, oxalic, lactic, succinic acids, (2020)
etc.
Aspergillus niger ZnO Zn3 (PO4)2 – Production of organic acids such as oxalic acid Sayer et al.
Penicillium ZnO Zn3 (PO4)2 – (1995)
simplicissimum
Penicillium ZnO – – Production of organic acids such as citric acid, Artmann et al.
ochrochloron gluconic acid, lactic acid, succinic acid, and (2020)
itaconic acid
Aspergillus niger – Zn3 (PO4)2 – Production of organic acids Bakri (2019)
Contribution of Zinc-Solubilizing and -Mobilizing Microorganisms (ZSMM) to. . .
Pseudomonas 28–625 247–753 Production of gluconic acid and 2-ketogluconic Azadeh Bapiri
fluorescens acids et al. (2012)
(continued)
365
366

Name of compounds
Stenotrophomonas 15.71 0.77 13.87 0.34 9.78 0.31 Production of gluconic acid Sunithakumari
maltophilia ZSB-1 et al. (2016)
Mycobacterium 11.58 0.31 12.17 0.65 9.70 0.12
brisbanense ZSB-10
Enterobacter 30.60 2.28 32.33 4.83 18.07 0.82
aerogenes ZSB-13
Pseudomonas 51.33 6.88 31.30 5.47 19.87 0.51
aeruginosa ZSB-22
Xanthomonas 46.39 + 6.57 30.34 6.56 17.85 1.36
retroflexus ZSB-23
Bacillus megaterium ZnO – ZnCO3 Production of organic acids and proton extrusion Bhatt and
CDK25 Maheshwari
(2020)
Bacillus AZ5 and ZnO – – Production of citric, malic, lactic and succinic acids Ahmad et al.
AZ17 (2019)
Bacillus megaterium – 4.09 3.21 Production of organic acid Sharma et al.
KHBD-2-2A (2012)
B. subtilis BDKH-3 – 4.08 2.86
B. circulans BD-3-1B – 3.45 2.95
B. licheniformis – 4.22 3.26
BDSD-2-2C
Bacillus sp. BDN-5 – 3.58 3.10
B. atrophaeus KHTH- – 4.17 3.05
4-1
B. amyloliquefaciens – 4.65 3.13
KHBAR-1
R. C. Yadav et al.
14
B. firmus KHBD-6 – 4.87 3.65

B. pumilus KDMR-1-1 – 3.98 3.11
B. cereus – 4.53 3.11
Bacillus KT221633 ZnO – – Production of organic acids Hussain et al.
Bacillus sp. AZ6 (2020)
B. aryabhattai 40 1 148 3 30 2 Production of organic acid Ramesh et al.
MDSR7 (2014a)
B. aryabhattai 25 2 137 4 28 2
MDSR11
B. aryabhattai 39 1 154 2 32 1
MDSR14
Bacillus sp. ZM20 26.29 – – Production of organic acid Mumtaz et al.
B. aryabhattai ZM31 25.94 – – (2017)
B. subtilis ZM63 27.15 – –
B. aryabhattai S10 27.66 – –
Azospirillum sp. As20 11.1 1.03 – 10.2 0.947 Production of gluconic acid Desai et al.
Azospirillum sp. As22 8.49 0.78 – 10.3 0.949 (2012)
Bacillus sp. B40 8.54 0.79 – 5.82 0.53
Bacillus sp. B61 5.66 0.52 – 12.7 1.17
Bacillus sp. B113 5.57 0.51 – 9.96 0.91
Bacillus sp. B114 9.68 0.89 – 14.7 1.35
Bacillus sp. B116 13.12 1.21 – 11.6 1.07
Bacillus sp. B118 11.80 1.09 – 16.3 1.50
Pseudomonas sp. P17 10.00 0.92 – 10.9 1.01
Pseudomonas sp. P21 10.530 0.97 – 13.9 1.28
Pseudomonas sp. P24 10.32 0.95 – 9.58 0.883

Pseudomonas sp. P29 9.46 0.87 – 9.62 0.887
Pseudomonas sp. P33 10.47 0.97 – 10.34 0.95
367
(continued)
368

Name of compounds
Pseudomonas sp. PIII 11.30 1.04 – 10.16 0.93
105
Bacillus subtilis KKI-4 183.13 21.21 183.84 11.05 45.79 1.00 Production of orμganic acids such as citric acid, Khande et al.
B. thuringiensis KKI-6 296.13 19.54 171.25 8.69 64.81 2.45 oxalic acid, gluconic acid, pantoic acid and (2017)
B. cereus KKI-8 272.00 15.64 181.16 11.29 48.66 2.70 2-ketogluconic acid
B. cereus KMR-5 333.50 18.45 223.00 13.04 61.69 3.82
B. cereus KKS-2 434.38 21.08 185.94 15.96 46.13 2.74
B. cereus KKH-1 251.38 17.57 190.69 8.81 60.76 4.31
B. cereus KBY-1 288.00 8.99 161.34 18.70 52.91 9.30
B. cereus KBY-5 247.25 8.45 142.94 10.20 61.29 2.63
B.s cereus BHKD-6 254.38 9.43 98.13 11.51 37.84 1.55
B. thuringiensis 246.25 15.23 94.94 7.82 63.44 0.85
JUKD-2
B. tequilensis JUKD-5 89.00 5.76 191.97 7.95 59.87 1.94
B. anthracis MUKD-1 499.50 40.59 152.53 7.85 60.59 2.04
B. cereus MUKD-2 421.50 23.99 196.61 18.39 49.74 1.65
B. cereus MUKD-4 388.13 14.46 190.28 12.44 44.34 3.17
B. anthracis KHKD-1 451.38 9.91 205.44 20.08 59.64 3.07
B. cereus KHKD-4 455.75 18.28 84.22 11.21 55.93 2.46
B. anthracis DBU-1 377.75 14.10 198.56 16.60 63.05 2.82
B. cereus DBU-5 507.38 31.72 204.22 17.73 59.65 3.11
B. cereus BBU-5 547.38 31.65 230.94 10.81 61.58 2.10
B. anthracis THB-2 519.63 25.54 167.72 12.62 47.94 2.92
R. C. Yadav et al.
14
Bacillus sp. AZ6 13.55 – – Production of organic acids like cinnamic acid, Hussain et al.
ferulic acid, caffeic acid, chlorogenic acid, syringic (2020)
acid and gallic acid in a liquid medium
Bacillus ZM20, ZnO – – Production of organic acid such as lactic and acetic Mumtaz et al.
Bacillus cereus acid (2019)
Bacillus KT221633 ZnO – – Production of organic acids Hussain et al.
Bacillus sp. AZ6 (2020)
Curtobacterium strains ZnO – – Production of glutamic acid Kushwaha et al.
(2020)
Chelation
Pseudomonas – – – Chelation is the dominant phenomena to improve Whiting et al.
monteilii Zn bioavailability and uptake by plant roots (2001)
Microbacterium – – – through microorganisms
saperdae
Enterobacter – – –
cancerogenesis
Pseudomonas – – – Producing chelating agent Tariq et al. (2007)
sp. 96–51, ethylenediaminetetraacetic acid
Azospirillum lipoferum – – –
JCM-1270, ER-20,
Agrobacterium – – –
sp. (Ca-18)
Penicillium bilaji – – – Chelation Kucey (1987)
Changes in root architecture
Arbuscular – – – Obtain Zn from a distance of 40 mm from the root Bürkert and
mycorrhizae surface Robson (1994)
(continued)
369
370

Name of compounds
Mycorrhizal fungus – – – Increased Zn concentration in the grain and Subramanian
increased root length as compared to plants with no et al. (2009a)
fungal inoculation
Zinc-solubilizing – – – Increased root weight, length, and volume and zinc Tariq et al. (2007)
bacteria biofortification of rice with inoculation
R. C. Yadav et al.
Heterotrophic zinc solubilization in fungi (White et al. 1997) has been widely
studied. Many free-living saprophytic fungi, ericoid, and mycorrhizal fungi have
been studied for their ability to solubilize metal-bearing minerals (Sayer et al. 1999;
Sayer and Gadd 2001; Martino et al. 2003; Artmann et al. 2020). Sayer and Gadd
(2001) reported that Aspergillus niger produces citric acid and plays a significant
role in zinc phosphate solubilization. Burgstaller et al. (1994) investigated the role of
gluconic, citric, and oxalic acids excreted by the fungus Penicillium simplicissimum
in zinc oxide solubilization. Beauveria caledonica, an entomopathogenic fungus,
dissolves zinc phosphate in medium by the production of acetic, malic, and citric
acids (Fomina et al. 2005). Acid production, complexation, and accumulation of
metals help in zinc phosphate solubilization by ericoid; ectomycorrhizal plant
symbionts like Hymenoscyphus ericae, Oidiodendron maius, Laccaria laccata,
Paxillus involutus, Suillus bovinus, S. luteus, and Thelephora terrestris; and the
entomopathogenic fungus Beauveria caledonica (Martino et al. 2003; Fomina et al.
2004; Fomina et al. 2006). For example, zinc phosphate solubilization is carried out
by the production of gluconic acid by H. ericae DGC3(UZ) and S. bovinus MG1;
oxalic and citric acids by B. caledonica; gluconic and succinic acids by L. laccata;
and succinic acid by T. terrestris; malic, succinic, and fumaric acids by S. luteus. The
application of zinc phosphate significantly increased acetic acid synthesis by
H. ericae DGC3(UZ). As acidolysis is a predominant mechanism of zinc solubiliza-
tion by most of the fungi, Trichoderma harzianum T-22 follows different
mechanisms of zinc solubilization, such as by chelation and reduction but not at
all by acid production (Altomare et al. 1999). Several species of fungi are known to
convert immobile metal compounds to available forms through proton extrusions
(plasma membrane ATPase); production of enzymes, especially phosphatases; and
production of organic acids (Franz et al. 1991; Sigler and Höfer 1991; Burgstaller
and Schinner 1993; Gadd 1993; Winkelmann and Winge 1994; Artmann et al. 2020;
Bakri 2019). Ericoid mycorrhizal and ectomycorrhizal fungi solubilize zinc from
insoluble forms by the excretion of organic acid with high complexing ability
(Martino et al. 2003; Fomina et al. 2005). AMF are ubiquitous (Schüβler et al.
2001) and are integral to the root components of crop plants (Smith and Smith 2011).
The presence of hyphae, arbuscules in AMF, provides an alternative plant nutrient
assimilation pathway (Parniske 2008; Smith and Read 2008). This association is
known to increase the assimilation of immobile nutrients, particularly phosphorus,
zinc, and iron (Cavagnaro 2008; Subramanian et al. 2008; Subramanian et al. 2013;
Lehmann et al. 2014; Balakrishnan and Subramanian 2016), from soil components.
14.6 Mechanisms of Zinc Solubilization and Mobilization by

Microorganisms
The presence of zinc in insoluble forms affects crop assimilation (Barber 1995).
Zinc-mobilizing microorganisms assume significance for zinc solubilization and
assimilation by plants. There are different types of mechanisms for the solubilization
of insoluble zinc by zinc-solubilizing and -mobilizing microorganisms.
14.6.1 Organic Acid Production
The exact mechanism of zinc solubilization by microbes is still not clear. Some of
the mechanisms reported are production of organic acid, sequestration or active
efflux, cell wall modification, and bioprecipitation (Sunithakumari et al. 2016).
Organic acid production, extrusion of protons, and secretion of metabolites play an
important role in metal solubilization (Upadhyay and Srivastava 2014; Bakri 2019;
Mumtaz et al. 2019). Many organic acids have been identified that have chelating
properties and are reported to solubilize metals (Panhwar et al. 2013). Various
mechanisms have been studied to understand the mechanism of zinc solubilization
and zinc tolerance by microbes.
14.6.2 Zinc Chelation
It is well known that the bioavailability of zinc in soil is low and depends on soil
characteristics and climatic conditions. Zinc-chelating agents have a role to play in
zinc solubilization and are derived through root exudation and also by proliferating
microorganisms (Obrador et al. 2003; Tarkalson et al. 1998) that chelate zinc and
increase its availability. It is one of the dominant processes by which the bioavail-
ability of zinc become more for plant assimilation. Pseudomonas monteilii,
Microbacterium saperdae, and Enterobacter produce metallophores that chelate
zinc, thereby enhancing zinc bioavailability (Whiting et al. 2001). Tariq et al.
(2007) reported that Pseudomonas sp. 96–51, Azospirillum lipoferum
ER-20 (JCM-1270) and Agrobacterium sp. Ca-18 had the ability to produce chelat-
ing agents like ethylenediaminetetraacetic acid.
14.6.3 Changes in Root Architecture for Zinc Uptake
Zinc is an immobile nutrient, and its uptake is diffusion controlled (Havlin et al.
2005). Native zinc bioavailability being low in soils, zinc bioavailability can be
increased either by an external application of zinc fertilizer or changing the root
architecture, enabling the roots to traverse long distance. In this context, the role of
mycorrhiza on root architecture is well documented (Subramanian et al. 2008, 2013).
Mycorrhizal fungi are known to increase root surface areas and help mobilize zinc
for plant uptake from long distances, even up to 40 mm (Bürkert and Robson 1994).
Subramanian et al. (2009a) reported that mycorrhiza fungi increase zinc content in
grains with concomitant increase in root parameters as compared to those without
inoculation. Similarly, Tariq et al. (2007) also recorded an increase in root-
associated parameters that influence increased assimilation and yield.
14.6.4 Zinc Solubilization and Mobilization by

Exopolysaccharide-Producing Microorganisms
The significance of exopolysaccharides in zinc solubilization was assessed by

Kamran et al. (2017). They found that microbial exopolysaccharides (EPSs) pro-
duced by Pseudomonas fragi EPS 1, Pantoea dispersa EPS 6, Pantoea agglomerans
EPS 13, Enterobacter cloacae PBS2, Enterobacter cloacae PBS1 and Rhizobium
sp. LHRW1, Pantoea sp. LS1-b and Pseudomonas fragi EPS 15 solubilize zinc.
Many exopolysaccharide (EPS)-producing ZSMM also exhibit multiple plant-
growth-promoting (PGP) traits such as 1-aminocycloproparane-1-carboxylic acid
(ACC) utilization and phosphate and potassium solubilization (Gontia-Mishra et al.
2016). EPS production by ZSMM is also useful to mitigate desiccation and disease
incidence.
14.7 Soil Zinc Mobilization by Zinc-Solubilizing and -Mobilizing

Microorganisms (ZSMM) for Crop Zinc Assimilation
and Productivity Enhancement
The application of rhizobacteria and endophytic bacteria is sustainable as compared

to fertilizer and pesticide application and provides soil nutrients for crop assimilation
and improved crop performance (He et al. 2010; El-Sayed et al. 2014; Chen et al.
2014; Ramesh et al. 2014a; Wang et al. 2014; Majeed et al. 2015; Shakeel et al.
2015; Shen et al. 2015; Dhiman et al. 2020). Many soil microorganisms have been
reported to improve zinc accumulation and the growth of many plant species (Fasim
et al. 2002; Saravanan et al. 2007b; Hassan and Aarts 2011; Ma et al. 2011; Sharma
et al. 2011; Rana et al. 2012; Ramesh et al. 2014a, b; Abaid-Ullah et al. 2015;
Shakeel et al. 2015). Zinc-solubilizing Bacillus aryabhattai MDSR14 (Ramesh et al.
2014a) and Enterobacter cloacae subsp. dissolvens MDSR 9 (Ramesh et al. 2014b)
improved zinc solubilization and the uptake of zinc by soybean and wheat crops.
They reported redistribution among the different pools of zinc, thereby culminating
in increased growth promotion and zinc accumulation in seeds of soybean and
wheat. Arbuscular mycorrhizal (AM) fungi facilitate nutrient diffusion of phospho-
rus, zinc, iron, and copper and are also involved in rhizospheric biochemical changes
such as phosphatases, dehydrogenase, and shift in microbial biomass carbon (Kim
et al. 1997; Kandeler et al. 2002; Wamberg et al. 2003; Ryan and Angus 2003; Wang
et al. 2006; Subramanian et al. 2009b). This intense rhizospheric processes
accompanied by glomalin production (Wright and Upadhyaya 1996) by AM fungi
results in improved nutrient mobilization and assimilation by crops (Subramanian
et al. 2009b). The inoculation of AM fungi resulted in the increase of organically
bound zinc and decreased crystalline and residual zinc fraction, resulting in
increased Zn availability to maize crops and, thereby, increased productivity. Fur-
thermore, isolates AGM3 and AGM9 of Acinetobacter sp. were found to increase
exchangeable oxides of iron (Fe)-, aluminum (Al)-, and manganese (Mn)-bound zinc
and might have resulted in increased zinc mobilization (Gandhi and Muralidharan
2016).
Zinc bioavailability and zinc concentration increased by 5.6 times and 113% in
rice, respectively, through bacterial inoculation (biopower) (Tariq et al. 2007). The
inoculation of maize (Zea mays) with Azospirillum and Azotobacter increased grain
zinc content (Biari et al. 2008). Abaid-Ullah et al. (2015) reported that the
co-inoculation of zinc-solubilizing Bacillus thuringiensis and Serratia
sp. increased wheat yield by 9%. Similarly, Mishra et al. (2012) reported that a
consortium of Rhizobium leguminosarum-pr-1 and Pseudomonas sp. increased zinc
content in shoots. The inoculation of endophytic bacterium Sphingomonas SaMR12
increased shoot biomass in rice by 40 to 64% and also zinc accumulation. Strain
SaMR12 also increased root branching and root surface area and increased root
exudation of oxalic acid (Chen et al. 2014). Changes in root architecture and root
exudation by both endophytic bacteria and rhizobacteria improved crop performance
(Meharg and Killham 1995; Hinsinger et al. 2003; Taghavi et al. 2009), thereby
effecting redistribution of different forms of zinc and increasing zinc bioavailability.
Similarly, Wang et al. (2014) reported that endophytic bacteria Sphingomonas
sp. SaMR12 and Enterobacter sp. SaCS20 modulated root morphology and
increased zinc accumulation in shoots and roots and also increased grain yields as
well as zinc concentration in grains in brown and polished rice. This was also
accompanied by a considerable increase in DTPA-extractable zinc in soil. The
inoculation of ZSB has been shown to increase the root and shoot parameters of
many crops (Sharma et al. 2012; Goteti et al. 2013; Ramesh et al. 2014a; Vaid et al.
2014). The increase in biomass could be attributed to their zinc-solubilizing and
-mobilizing characteristics as well as to other PGP traits (ACC deaminase, ammonia
production, siderophores, indole-3- acetic acid (IAA) production, P solubilization, K
solubilization, etc.). Zinc application in combination with strain Pseudomonas
spMN12 through seed priming increased the grain yield of wheat against control.
Soil and foliar application of zinc with and without Pseudomonas sp. MN12
increased protein content and zinc concentration in wheat (Rehman et al. 2018b).
Singh et al. (2017a, b) reported that inoculation with endophytic Bacillus subtilis
DS-178 and Arthrobacter sp. DS-179 increased zinc concentration in wheat gains
as compared to control and attributed this to higher IAA synthesis and improved
root architecture. Zinc-solubilizing and -mobilizing rhizobacterial isolates of Bacil-
lus sp.ZM20, Bacillus aryabhattai ZM31 and S10, and Bacillus subtilis ZM63 with
multifaceted plant-growth-promoting traits resulted in improved crop growth and
productivity and can be the potential the biofertilization and biofortification
strategies for overcoming zinc deficiency in maize (Mumtaz et al. 2017; Hussain
et al. 2020). Transformation of native soil nutrients by microorganisms to plant-
available forms is one strategy to increase the assimilation of nutrients (microbial
biofortification) in staple food crops to meet the recommended dietary intake in
human beings to overcome zinc deficiency malice in human populations (Mader
et al. 2010). Any biofortification strategy should be commensurate with the
decrease in phytic acid content and increase in zinc concentration in seeds. Phytic
acid/zinc molar is a good indicator of zinc bioavailability (Weaver and Kannan
2002; Ramesh et al. 2014a). Combined application of Pseudomonas and Zn (soil

and foliar) decreased phytic acid and phytate:Zn molar ratio and subsequently
increased the bioavailable Zn in wheat grains (Rehman et al. 2018b). Inoculation
with AM fungi registered increased grain zinc concentration and decreased phytic
acid concentrations. A negative correlation between grain zinc and phytic acid
content was also reported (Kaya et al. 2009). Phytase activity in maize grains also
increased in AMF+ as against AMF-maize plants.
It was reported that AMF inoculation caused redistribution among the different
zinc fractions, resulting in increased zinc assimilation (Subramanian et al. 2008;
Balakrishnan and Subramanian 2016). Co-inoculation of AMF and plant-growth-
promoting rhizobacteria (PGPR) improved plant nutrient assimilation even under
adverse soil conditions (Al-karaki et al. 2004; Artursson et al. 2006; Smith and Read
2008). Inoculation with AMF and PGPR (Pseudomonas jessenii R62; Pseudomonas
synxantha, R81) increased the grain yield of wheat by 41% and improved soil
quality parameters (Mader et al. 2010). The role of AMF in zinc, growth, yield,
and uptake was explicitly reviewed by Imran et al. (2014). Other fungal
bioinoculants of the genera Penicillium and Aspergillus increased plant zinc con-
centration and can be used to increase zinc availability to crops (Kucey 1988;
Burgstaller and Schinner 1993; Sayer et al. 1995; Imran et al. 2014).
14.8 Genetic Engineering of Zinc-Solubilizing and -Mobilizing

Microorganisms (ZSMM)
Information on genetic pertaining to zinc-solubilizing and -mobilizing

microorganisms being scanty, efforts at molecular level are being made to unravel
how precisely these microorganisms bring out the solubilization of insoluble zinc
compounds. However, some genes involved in solubilization have been isolated and
are being characterized. Preliminary studies on the manipulation of these genes
through genetic engineering and subsequent gene expression in selected
rhizobacterial strains open an impressive perspective in evaluating ZSMM strains
for the enhancement of their zinc solubilizing capacity for their use as bioinoculants.
The introduction or overexpression of genes for solubilization (both organic and
inorganic) in naturally occurring rhizospheric bacteria has been considered as a
plausible approach for improving the capacity of microorganisms as microbial
inoculants. The insertion of zinc-solubilizing genes into microorganisms with subtle
solubilizing capability may overcome the present need for microbial consortia
(nitrogen fixers and phosphate solubilizers). There are many advantages to develop-
ing genetically modified ZSMM over transgenic plants for better crop performance:
(1) it is easier to modify a bacterium than complex higher organisms, (2) there are
multifaceted plant-growth-promoting traits in a single organism, and (3) single-
engineered microbial inoculants can be used for a number of crops. Because of
this, significant progress in genetically engineered microorganisms for agricultural
use has been made (Armarger 2002). Overall, further studies on this aspect of
ZSMM will provide crucial information in the future for better use of these ZSMM in
varied environmental conditions.
14.9 Conclusion
Zinc deficiency in soils and other environments has serious impact on all forms of
life, including human beings. It is observed that globally most of the soils are zinc
deficient, which is considered to be a major constraint hindering zinc enrichment in
crops and resulting in decreased consumption of grains/edible portions of crops,
particularly cereals. This does not fulfill the dietary requirements of human popula-
tion, especially in developing countries. As such, it has led to several health
problems in human population. Strategies like agronomic and genetic interventions
for increasing zinc density in seeds seem to have immense potential; however, these
interventions have their own set of issues such as environmental pollution, exuberant
cost, socioeconomic issues and policy issues. The use of zinc-solubilizing and
-mobilizing microorganisms (ZSMM) opens up a new horizon for overcoming
micronutrient deficiency (zinc) in soil and plants and thereby improving human
health (Fig.14.2). These strategies are sustainable in nature with the aim of increas-
ing zinc concentration and fulfilling human dietary requirements. ZSMM can be
used as biofertilizers, which may proliferate in the rhizobiome and influence crop
growth, soil fertility, and yield and zinc content in crops, thereby obviating the
indiscriminate use of inorganic fertilizers. The use of effective microbial
technologies in agriculture in recent years is progressing at a rapid pace with the
reorganization of novel bacterial strains and due to their role in plant growth
promotion. The exploitation of multifunction PGPR will be a good approach for
biofertilizer formulations. The introduction and popularization of biofertilization and
biofortification could be a promising and apt strategy for avoiding micronutrient
deficiencies in an era of sustainable agriculture. To sustain ZSMM technology,
proper availability and distribution of good quality microbial inoculants with longer
shelf life are of paramount importance. Therefore, consistent efforts in identifying
ZSMM and extensive field validation to allow identification with greater efficiency
are important. Moreover, microbial inoculants should be selected not for a single
trait but for multiple plant-growth-promoting traits so that they may perform in
different agroecological zones. Understanding contributions of ZSMM is required
for sustainable agriculture with a generic consideration of a green revolution and an
evergreen revolution, which needs to be accomplished for societal progression.
14.10 Future Line of Work
Although significant work on ZSMM has been carried out over the last few decades,
the effects of these microorganisms on the zinc content of crops are still scanty. The
future challenges in the area of ZSMM and their potential use for the health of soils,
plants, and humans need to be explored:
14
Fig. 14.2 The role of zinc-solubilizing and -mobilizing microorganisms (ZSMM) in zinc biofertilization and biofortification in crops and the impact of zinc on
plant and human health
377
1. Development of an efficient region-specific microbial strain that can work under

different soil types and climatic conditions, which may prove a boon in the
agricultural sector
2. Selection of ZSMM that have potential to trigger specific translocation genes in
crop plants to allow more zinc uptake in plants
3. Developing crops with biased/engineered rhizosphere that recruits ZSMM for
better zinc nutrition in crops
4. Smart formulation of ZSMM for the biofortification of crop plants and bioreme-
diation in contaminated areas
5. Developing probiotic zinc-solubilizing and -mobilizing bacteria (ZSMB) for
direct consumption by human beings to meet the zinc requirements of their
daily diets
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Fungal Siderophore: Biosynthesis,
Transport, Regulation, and Potential 15
Applications
Keshawanand Tripathi, Narendra Kumar, Meenakshi Singh, and

Ravi Kant Singh
Abstract
Iron (Fe) is an essential nutrient for life and the fourth most abundant element in
the earth. The availability of ferric iron (Fe III) is less in soil solution due to the
low solubility of ferric hydroxides, oxides, and oxyhydroxides. Therefore, the Fe
availability to microbes and plants is limited, albeit its abundance in the environ-
ment. Therefore, the availability of Fe to microbes and plants has evolved
strategies based on acidification through proton extrusion and organic acid
production, chelation, ligands like siderophore and phytosiderophore production,
and enzymatic reduction involving reductase enzymes. This review attempts to
explain the fungal siderophore, its biosynthesis, transport, and practical applica-
tion. Thus, siderophore is an iron-binding molecule synthesized by fungi, bacte-
ria, cyanobacteria, and plants. The common types of siderophore are
hydroxamates, catecholates, carboxylates, but hydroxamate type is dominant in
fungi. L-ornithine is a biosynthetic precursor of siderophore and synthesized
through multimodular large enzymes complex nonribosomal peptide synthetase
(NRPSs) dependent/independent. Siderophore-Fe chelators protein (FIT1, FIT2,
and FIT3) helps in the retention of siderophore. Saccharomyces cerevisiae
expresses two genetically separate systems (reductive and a non-reductive sys-
tem) at the plasma membrane, which converts Fe III into Fe II by ferrous-specific
K. Tripathi (*)
Centre for Conservation and Utilization of Blue-Green Algae, ICAR-Indian Agricultural Research
Institute, New Delhi, India
N. Kumar
Depaqrtment of Biotechnology, IMS Engineering College, Ghaziabad, Uttar Pradesh, India
M. Singh
Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India
R. K. Singh
Amity University Chattisgarh, Raipur, Chhattisgarh, India

23, https://doi.org/10.1007/978-981-15-9154-9_15
388 K. Tripathi et al.
metallo-reductases enzyme complex, FRE reductases. Regulation of the

siderophore gene expression on the promoter region by Fur Box protein depends
on the availability of Fe in the external medium. Biotechnologically, it is more
important due to its wide range of applications that include medical, remediation
of heavy metal, biocontrol of plant pathogens, and enzyme inhibitions.
Keywords
Siderophore · Iron · Fungi · Hydroxamate · Biocontrol
15.1 Introduction
Bioweathering of the minerals in the soil ecosystems plays an important role to

mitigate the iron (Fe) limitation in the surrounding environments (Mustoe 2018). In
the earth’s crust, iron is the fourth most abundant element after oxygen, aluminum,
and silicon, and the availability of iron to living organisms is limited in oxygenic
environments. Fe is one of the essential elements for the growth of living organisms.
In natural environments, the solubility of the Fe III is 10 17 M (pH 7) but the plant
requires (10 5 to 10 7 M) of Fe II for growth and developments (Martinez et al.
1990). In aerobic conditions, Fe is converted into insoluble oxyhydroxide polymers
at biological pH (oxidized form) (Paul and Dubey 2015). During the evolution
period, increasing O2 concentration in the natural environments and subsequent
oxidation of Fe convert soluble Fe II into insoluble Fe III forms. Microorganisms
like fungi, bacteria, and cyanobacteria sense quickly to the decreasing iron availabil-
ity. In this situation, microbes overcome iron deficiency by the synthesis of
siderophore (Khan et al. 2018). Siderophore is low molecular weight
(200–2000 Da), Fe-chelating, high-affinity natural compounds, mostly water-
soluble, chemically diverse and synthesized by various microorganisms like fungi,
bacteria, cyanobacteria, and some plants during iron starvation (Schwyn and
Neilands 1987; Crowley 2006) Approximately, 500 different siderophores have
been reported by fungi and bacteria (Boukhalfa and Crumbliss 2002). In the
human serum, transferrin is an iron transport protein that maintains Fe III concentra-
tion (10 24 M) and makes it inaccessible for the human pathogens (Raymond et al.
2003).
Fe is essential for various vital functions such as respiration, photosynthesis,
enzyme cofactor, electron transfer, and synthesis of DNA, RNA, and protein. Fe
acquisition from the extracellular environment by fungi and other microorganisms
has adopted various strategies including (1) metal ion transporters (Howard 1999),
(2) acquisition from heme and heme-containing proteins (Lawen and Lane 2013),
(3) acquisition from transferrin, lactoferrin, and ferritin (Crawford and Wilson 2015;
Mech et al. 2014), reductive systems of iron uptake, (5) siderophore-mediated iron
transport (Khan et al. 2018).
Siderophore-mediated Fe-scavenging is important and improves the iron bio-
availability in the soil by the dissolution of bioweathering of the minerals. The
15 Fungal Siderophore: Biosynthesis, Transport, Regulation, and Potential. . . 389
siderophores are mainly scavenging iron and formation of complexes with the other
metals in addition to iron like molybdenum, cobalt (Bellenger et al. 2008). This
compound enhances plant growth, biocontrol (Verma et al. 2011) and bioremedia-
tion agents (Ishimaru et al. 2012). In addition to microbes, mammalian siderophore
is also reported (Devireddy et al. 2010). Characterization of siderophores by spec-
trophotometric titration, electrophoretic mobility, mass spectrometry, acid hydroly-
sis, and biological activity is frequently used (Pluhacek et al. 2016).
15.2 Iron in the Environment
Iron is a ubiquitous element predominant in the terrestrial ecosystem and one of the
essential micronutrients for both microorganisms as well as plants albeit at low
concentrations. Paradoxically, though the total Fe content is high but its availability
is controlled by stable oxides and hydroxides and never correlates with its availabil-
ity to plants and microbes. The factors that determine its availability is solubilization
and dissolution rates of oxides of Fe. The solubility and dissolution rates are pH
dependant, and its minima exist at neutral and alkaline range especially in calcareous
soils under aerobic environment (Lindsay 1979; Cornell and Schwertmann 2003). In
a pH range of 7–9, the sum of the soluble inorganic forms of Fe does not exceed
10 10 M (Lindsay 1979), which is far below the minimum requirement for normal
plant growth. This indicates the Fe acquisition under oxic and carbonate-rich
environment to be strongly sensitive for plant and microbial Fe acquisition. More-
over, redox potential and complexing agents also play a significant role in regulating
Fe availability through the rhizosphere and microbial effect. The low availability of
Fe in calcareous soils can be ascribed to (1) an extremely low solubility of soil Fe,
which is essentially in ferric oxide form (Miller et al. 1984; Mengel 1994), and
(2) reduced Fe uptake from the apoplast into the symplast, which can be related to
the pH of the former (Yu et al. 2000), influenced by a high bicarbonate concentration
in calcareous soils (Mengel 1994; Lucena 2000). This leads to the widespread
occurrence of lime-induced Fe chlorosis (Wiersma 2005; De Santiago et al. 2008).
Therefore, to acquire Fe(III) has become a major challenge crop plant as well as
microorganisms as they are essential for growth and metabolism. To overcome this,
plants and microorganisms have evolved strategies that include (1) acidification of
soil solution through excretion of organic acids or protons, (2) chelation of Fe(III) by
ligands with very high affinity for Fe(III), and (3) reduction of Fe (III) to Fe(II) by
reductases and reducing compounds (Lemanceau et al. 2009).
Acidification of the rhizobiome is one process to overcome Fe limitation in
calcareous soils. Organic acid exudation from plant cells, microbes, and organic
matter decomposition is one strategy that enables us to overcome Fe limitations.
Dicots and non-graminaceous plants exhibit this strategy and are known as strategy
I. Moreover, organic ligands with high affinity for Fe like phytosiderophores are
exuded in response to Fe limitation. They are generally exuded by graminaceous
plants by ligand-controlled dissolution processes (strategy II). Enzymatic dissolution
also plays a significant role in Fe availability and mostly in circumneutral pH with

high redox potential especially in the oxic environment (Lemanceau et al. 2009).
The low solubility of Fe bearing mineral phases has prompted microorganisms
and plants to devise strategies to increase Fe bioavailability. One such strategy is the
production of siderophores which are low molecular weight chelating/binding agents
and is predominant in the circumneutral oxic environment. Siderophore is conspicu-
ous in having high thermodynamic affinity to bind Fe oxides which on chelation
helps in dissolution and solubility of Fe oxides. The other function of siderophore is
to control redox properties for Fe uptake. The response of siderophore production by
aerobic microorganisms is prevalent under Fe stress. Moreover, the production and
assimilation of siderophore with higher affinity enables Fe availability under Fe
limitation conditions is an important role played by microbes.
15.3 Microbial Siderophores
The term “siderophores” is a Greek word for “iron carriers” (Ishimaru 1993) as these
molecules are produced by microorganisms and were found to have an extremely
high affinity for ferric iron (Lankford 1973). Siderophores bind Fe (III) ion and
transport it into the bacterial cell. They are low molecular weight (350–1500
Daltons) organic molecules, which can compete for Fe (III) in ferric hydroxide
complexes (Postle 1990). There are over 500 described siderophores (Corneils and
Matthijs 2002; Wandersman and Delepelaire 2004) that are classified based on their
chelating group specific for Fe (III). Microbial siderophores are classified as
catecholates (phenolates), hydroxamates, α-carboxylates, and “mixed” depending
on the chemical nature of their coordination sites with Fe (Winkelmann 1991, 2002;
Renshaw et al. 2002). Catechol-type siderophores bind Fe (III) with adjacent
hydroxyls of catechol rings and are almost always derived from
2,3-dihydroxybenzoic acid (DHBA) (Crosa and Walsh 2002). The best-studied
example of a catechol-type siderophore is enterobactin, which is produced by
E. coli (O’Brien and Gibson 1970). Hydroxamate-type siderophores contain a
carboxyl group attached to the adjacent nitrogen, which chelates Fe (III). An
example of this type is ferrichrome, a fungal siderophore produced by Ustilago
sphaerogena (Emery 1971). Hydroxamates are generally more complex structurally
and are also considered more hydrophilic nature. Hydroxamate types are produced
by fungi and bacteria, whereas catecholates are produced exclusively by bacteria and
comprise catechol and hydroxy groups as ligands. In addition to these classes, a
miscellaneous class called “mixed-type” of siderophores does occur. Heterobactins
and pyoverdins, produced by Rhodococcus erythropolis (Carran et al. 2001) and
Pseudomonas species (Meyer and Hornsperger 1978; Meyer and Stintzi 1998),
respectively, contains both hydroxamate and catecholate functional groups. A third
mixed molecule called “anguibactin” is produced by Vibrio anguilarum and contains
combinations of all three siderophore types hydroxamate, catecholate, and carbox-
ylate (Crosa and Walsh 2002).
The process of Fe transport begins with the binding of siderophore with Fe (III) in
the external environment. The Fe (III) siderophore complex is then recognized by the
corresponding outer membrane receptor protein. Binding of the Fe (III) siderophore
complex induces considerable conformational changes, perhaps signaling to initiate
TonB interaction. Using energy presumably provided by the TonB complex (proton
motive force), the Fe (III) siderophore complex is actively transported into the
periplasm. Once in the periplasm, the Fe siderophore complex is bound to a
periplasmic binding protein that transports the complex to the ABC-type transporter
in the cytoplasmic membrane, which transports the complex into the cytoplasm
utilizing energy from the hydrolysis of ATP. Fe is released from the siderophore by
either reduction via ferric reductases or by chemical modification or breakdown of
Fe (III) siderophore complexes by acetylation and esterases, respectively (Schwyn
and Neilands 1987).
The most common detection method for siderophore production is the universal
assay of Schwyn and Neilands et al. (1987). This assay is independent of the
structure of siderophore and thus is a very useful method for screening
siderophore-producing bacteria. The assay is based on a competition for iron
between the ferric complex of the dye chrome azurol S (CAS) and removal of iron
by siderophore is indicated by a change from blue dye color to orange or yellow or
purple. The CAS assay of liquid supernatants of cultures has been stated to be
quantitative; however, on the solid medium, it is not possible to quantify the CAS
reaction (Schwyn and Neilands 1987; Raaska et al. 1993). Schwyn and Neilands
et al. (1987) observed that detergent hexadecyl trimethyl-ammonium bromide
(HDTMA) used in the preparation of the CAS medium may be toxic to some
microorganisms. To overcome the problem of HDTMA inhibition, a modification
was made in the CAS-agar plate assay in which half of each plate was filled with the
most appropriate culture medium for each organism and the other half with
CAS-blue agar. This modification allowed all fungi and Gram-positive bacteria to
grow properly without any inhibition and also reacted to CAS effectively (Milagres
et al. 1999; Machuca and Milagres 2003). In 2007, Perez-Miranda and coworkers
suggested further modification in which they allowed all microorganisms to grow in
a nontoxic medium followed by overlaying growing microorganisms with CAS
medium containing agarose as a gelling agent and then incubated for change in
color from blue to the orange of the overlaid medium exclusively surrounding
siderophore microorganisms.
To detect the nature of siderophores, a variety of assays have been developed
based on chemical properties. The catecholate nature of the siderophore can be
detected by Neilands spectrophotometric assay (Neilands 1981). Formation of
wine colored complex with FeCl3 that absorbs maximal at 495 nm indicated the
catecholate nature of siderophores. This can also be confirmed with the Arnow test
(Arnow 1937) using nitrite molybdate reagent. The hydroxamate-type siderophore
can be detected by Neilands spectrophotometric assay (1981) or tetrazolium salt test
(Snow 1954). This type of siderophore can also be detected by Gibson and Magrath
(1969), Casky’s methods, and Atkin’s method (Atkin et al. 1970). The carboxylate
nature of siderophore can be detected by the Vogels chemical test. It can also be
confirmed by spectrophotometric assay (Shenker et al. 1992).
15.4 Fungal Siderophores and Classification
Fungi are non-photosynthetic and multicellular eukaryotic organisms (Hagen 2012).

These are mostly heterotrophic and broadly classified into slime molds, aquatic and
terrestrial groups. These are saprophytes or decomposers which break down and feed
on decaying organic matter. They show two major responses, siderophore synthesis
under iron stress and high-affinity ferric iron reductase (Philpott 2006). Siderophore
can also be classified based on coordinating groups and binding to Fe. The common
type of siderophore (Table 15.1) is hydroxamates, catecholates, and carboxylates.
Certain microbes synthesized mixed-type siderophore that binds salicylic acid,
thiazoline nitrogen, or oxazoline, and pyoverdines is classified in the place of Fe
III binding group. Phytopathogenic fungi synthesized unique compounds that che-
late iron but function as phytotoxins. Fungi mainly synthesize hydroxamate-type
siderophores (derived from non-proteinogenic ornithine amino acid) and are further
categorized into three groups (1) fusarinines, (2) coprogens, (3) ferrichromes, and
(4) Rhodotorulic acid. With certain exceptions, the rhizoferrin (carboxylate-type
siderophore) is produced by certain zygomycetes members. Siderophore synthesis
depends on the nutrient medium and culture conditions of the fungi. These are
derived from the common structural unit Nδ-hydroxyornithine. The hydrophilic
siderophore consists of hydroxylated and alkylated ornithine amino acid, while in
bacteria these are acylated and hydroxylated alkylamines (Baakza et al. 2004). It
consists of N6-acyl-N6-Hydroxylysine or N5-acyl-N5-Hydroxyornithine reported by
Winkelmann (2002). All hydroxamate siderophores consist of peptide linkage
(Oberegger et al. 2001), except that fusarinine C which is synthesized by Aspergillus
Table 15.1 List of some fungal species and their siderophore biosynthesis
S. No Fungal species Siderophore References
1. U. maydis Hydroxamate (Ferrichrome) Allen et al. (1989)
2. A. nidulans Hydroxamate (Fusigen, Haas et al. (2003)
Ferrichrome)
3. A. fumigates Hydroxamate (Fusarinine) Haas (2014)
4. C. Hydroxamate (Ferrichrome, Fatima et al. (2017)
heterostrophus Coprogen)
5. F. graminearum Hydroxamate (Ferrichrome) Tobiasen et al. (2007)
6. A. brassicicola Hydroxamate (Coprogen) Chen et al. (2013)
7. N. crassa Hydroxamate (Coprogen) Toth et al. (2009)
8. E. festucae Hydroxamate (Fusarinine) Fatima et al. (2017)
9. A. flavus Hydroxamate (Ferrichrome, Holinsworth and Martin
Fusarinine) (2009)
10. F. oxysporum Hydroxamate (Ferrichrome Ahmed and Holmström
(2014)
nidulans and consists of ester bonds. Two O2 molecules of these groups bind with Fe
known as a bi-dentate ligand. Hydroxamate siderophore is capable to bind
hexadentate octahedral complex with Fe (III) (Saha et al. 2016).
Fungi synthesized more than one type of siderophore and categorized into a
particular structural family, whereas in some cases synthesized different structural
families. For example, Trichoderma pseudokoningii and T. longibrachiatum
synthesizes all the three structural families of siderophore (Anke et al. 1991).
Fusarinine is a siderophore synthesized from young cultures of Fusarium roseum,
but at the older stage of the culture, fusarinines are replaced by malonichrome.
Charlang et al. (1981) suggested that the identity of siderophore can be exploited as a
useful trait for fungal taxonomy.
15.5 Siderophore Biosynthesis
Siderophores’ biosynthetic pathway can be categorized into two major pathways:

(1) nonribosomal peptide synthetase (NRPs) dependent and (2) NRPs independent
(Challis 2005). Nonribosomal peptide synthetases (NRPSs) are multimodular large
enzymes complex that consists of adenylation domain (A), thiolation domain (T),
condensation domain (C), and thioesterase domain (TE). Each module of the NRPS
enzymes is responsible for the addition of amino acid (AA) and the formation of the
peptide bond. The sequence and number of AA of the peptide chain are determined
by NRPS enzymes (Crosa and Walsh 2002). NRPS recognizes and activates AA by
the activation of the A-domain and its acylation of adenylate by the reaction with
ATP. The next step is the thiolation of (T) domain in which activated ester is
covalently linked. The next step is responsible for condensation (C) domain, direct
transfer of another acyl amino acid to form a peptide bond (Ravel and Cornelis
2003). Thioesterase domain (TE) is present in the final unit. Finally, assemble or
release the chain from the NRPs by hydrolysis or cyclization. Cleavage of the acyl
thioester which binds with T domain and reaction is NADH-dependent (Nadia and
Challis 2009). Biosynthetic pathways of fungal siderophores hydroxamate are
similar in their basic unit hydroxyornithine (Hider 1984; Mei and Leong 1994).
Figure 15.1 represents a general schematic biosynthetic pathway (Philpott 2006).
The first step is the conversion of L-ornithine into N-hydroxy-L-ornithine by the
process of hydroxylation catalyzed by enzymes L-ornithine N-oxygenase (De Luca
and Wood 2001). The second step is the acylation of N-hydroxy L-ornithine to N-
acyl-N-hydroxy-L-ornithine in the presence of enzyme transacetylase (Plattner and
Diekmann 1994). The acyl-CoA derivative is an acyl donor, and the reaction is
catalyzed by acyl-CoA: N-hydroxy-L-ornithine N-acyl transferase. This step is
reported in Ustilago sphaerogena (Ong and Emery 1972; Neilands et al. 1987),
and N-acetyltransferase activity in siderophore biosynthetic pathways of
R. pilmanae. N-acetyltransferase has been reported in other fungi such as Fusarium
cubense (Anke and Diekmann 1974), R. glutinis, and Aspergillus quadricinctus
(Plattner and Diekmann 1994). Next steps, condensation of several N-acyl-N-
hydroxy-L-ornithines combined in two to three units and formed dipeptides and
Fig. 15.1 L-Ornithine is a biosynthetic precursor of siderophore in fungi. Intermediate steps

include hydroxylation, acylation, and acetylation and the final product synthesized rhodotorulic
acid, fusarinine C, and coprogen (figure redrawn with slight modification, from source, Philpott
2006)
triesters such as fusarinine C, rhodotorulic acid, and coprogen B (Plattner and

Diekmann 1994). Condensation of amino acid to form the cyclic peptide ferrichrome
was reported in Aspergillus quadricinctus (Hummel and Diekmann 1981). Peptide
biosynthesis in siderophore production and respective genes (sid2) has been reported
in U. maydis (Yuan et al. 2001) and Trichoderma virens (Psy1). Disruption of these
genes in U. maydis and T. virens generates inability to synthesize ferrichrome.
(Leong and Winkelmann 1998; Wilhite et al. 2001).
15.6 Siderophore-Mediated Iron Transport by Fungi
The fungal cell wall is a highly dynamic structure and biochemically is the polymer
of glucan (β-1,3-glucan, and β-1,6-glucan) and chitin (Caroline 2006). The outer
layers are composed of mannoprotein (Orlean et al. 1997). The cell permeability and
mannoprotein composition change dramatically and influenced by growth
conditions. They provide the passage of nutrients across the cell wall to the periplas-
mic space and plasma membrane (Lesuisse and Crichton 1987). Saccharomyces
cerevisiae retains a significant amount of Fe-chelating molecules in their cell wall
and periplasmic space termed as siderophores. Fe-deficient condition, expression of
very high levels of three mannoproteins, termed as a facilitator of Fe transport (Fit1p,
Fit2p, and Fit3p) (Protchenko et al. 2001). Siderophore-Fe chelators help to the
retention of the proteins in the cell wall. Deletion of these genes FIT1, FIT2, and
FIT3 reduces 50% uptake of ferrichrome and ferrioxamine. Fe- transport of
S. cerevisiae expresses in two genetically separate systems known as reductive
and a non-reductive system (Philpott 2006). The reductive system operates in two
steps at the plasma membrane in which reduction of Fe III (oxidized) into Fe II
(reduced) by a ferrous-specific complex that is high-affinity transporter (Kosman
2003).
15.7 FRE Family of Reductases
FRE reductases are Metallo-reductases encoded by FRE 1 and FRE 2 genes

expressed on the plasma membrane. It can reduce or oxidize iron and copper
(Hassett et al. 1995). These reductases are integral membrane proteins (multiple)
possessing binding sites for Fe, coenzymes (FAD, NADPH), and cytochrome
(b-types) (Finegold et al. 1996). S.cerevisiae exhibited reductases and present in
different forms such as FRE1, 2, 3, 4, 5, and 6. These reductases expressed under
iron-deficient condition, whereas FRE1 and 7 expressed copper depleted conditions.
Fre3p expressed on the plasma membrane and showed siderophore reductase activ-
ity, whereas Fre6p located on the vacuolar membrane and involved in the reductive
export of iron into the cytosol. Initially, ferric citrate reductases (Fre1 and Fre2p) of
yeast characterized as broad substrate specificity and able to uptake in a large
number of ferric sources. Siderophores bind ferric iron with high affinity and
conversion of the ferric ion into a ferrous ion and transported by the specific ferrous
ion transporter.
Fre1p and Fre2p catalyzed the reduction of a variety of iron-siderophore chelates
such as ferrichrome and ferrioxamine B (Yun et al. 2000). Fre3p encodes plasma
membrane reductase that catalyzed the reductive uptake of an iron bind with
hydroxamate siderophores and Fre4p catalyzed di-hydroxamate rhodotorulic acid
(Yun et al. 2001). The standard reduction potentials of physiological reductants such
as NADPH are lower than ferric siderophores complex. Ferric-siderophore
complexes are kinetically unfavorable on the cell surface. However, coupling
reduction of ferric-siderophore into ferrous iron-specific at lower pH and in hydro-
phobic environments changes the reduction potential in the level of physiologic
reductants (Boukhalfa and Crumbliss 2002). Therefore, in vivo ferric siderophores
reduced into ferrous ion at reduced pH in the lipid-rich environment. Similarly,
transferrin (high-affinity iron-binding protein and low affinity for affinity ferrous
ion) reduces ferric ion into ferrous ion and removal of the reduced form of
ferrous iron.
15.8 The Fet3p/Ftr1p Oxidase/Permease Complex
Multicopper permease (Ftr1p) is a high-affinity transport complex, responsible for

the transport of reduced form of iron (Askwith et al. 1994). Apparent Km of high-
affinity enzyme complex is 0.2μM (Dancis et al. 1992) and allows to transport at low
concentration of iron. The ferrous iron is oxidized by Fet3p and required molecular
oxygen. The ferric iron is transported into the cytosol via the Ftr1p permease. The
oxidase reaction is copper-dependent, and four copper ions are inserted into Fet3p
during post-translationally in the secretory pathway on the post-Golgi compartment
(Yuan et al. 1997). The copper chaperone Atx1p is responsible for the binding of
copper and transported it to Ccc2p (Lin et al. 1997). Copper transporter pumps
transported copper into the lumen of the post-Golgi vesicle (Yuan et al. 1997). Both
of the proteins are required for the maintenance of adequate cellular copper levels
and functioning secretory pathways (Dancis et al. 1994). These copper-binding
proteins Atx1p and Ccc2p are synthesized during the iron deprivation and not a
copper deficiency, indicating that Fet3-dependent iron uptake. Assembled
complexes (Fet3p and Ftr1p) of protein are retained quality control systems in the
endoplasmic reticulum if they are expressed in the absence of their protein partner
(Felice et al. 2005). Fet3p/Ftr1p complex is rapidly degraded by the process of
ubiquitin-mediated endocytosis in the presence of high levels of iron (Felice et al.
2005).
15.9 Non-reductive Uptake Systems: The Siderophore-Iron

Transporters
Most of the fungi synthesized and secrete siderophores, and are small organic
compounds bind with iron molecules with high affinity with specificity (Neilands
1995). Fungal siderophores bind iron with dissociation constants (10 29 M), which
have greater affinity than any iron-binding ligand in the biological systems. Hsiang
and Baillie (2005) reported that all fungi express a non-reductive uptake system that
is specific for siderophore-iron chelates. Most of the fungi express transporters
specific for siderophores and secreted by other species of fungi. When siderophore
is abundant, the reductive system of transport can catalyze the uptake of siderophore-
bound iron. Although, more than 50% of genes are transcriptionally activated under
iron deprive condition and involved in the uptake of iron chelators. The evolution of
these uptake systems helps fungi to other microorganisms to effectively compete in
limited availability of iron.
15.10 Regulation
Transport of iron via siderophores is investments of biosynthetic energy. The

function of siderophores is mainly iron sequestration from the external medium.
During excretion, siderophore gets lost and few of them are return into loaded by
iron molecule and supported for growth of the siderophore-producing
microorganisms (Ahmed and Holmström 2014). Therefore, the excretion of
siderophore is responded to the availability of iron in the external medium. The
Fur is a ferric uptake regulatory protein responsible for most of the Gram-negative
and Gram-positive bacteria (Ernst et al. 1978). Fur+ gene is repressed along with a
long number of genes involved in iron uptake as compared with control (Escolar
et al. 1999). The proposed regression model requires the internal binding of ferrous
Fig. 15.2 Mechanisms of ferric ion (Fe III) transport in a Yeast cell, and subsequent transforma-
tion into ferrous ion (Fe II) (redrawn with slight modification from source, Philpott 2006)
iron to the Fur protein and then binds to the Fur Box present on the target DNA. The
inhibition of RNA polymerase is responsible for the search of the promoter region of
the iron-regulatory region. Under limited iron conditions, transport systems and
siderophore biosynthesis are activated and Fur protein separated from the Fur box
on the DNA (Tanui et al. 2017) (Figs. 15.2 and 15.3).
Gram-positive bacteria (Streptomyces, Mycobacterium, and Corynebacterium)
contain a high percent of G + C sequences. Diphtheria toxin regulator (DtxR) is
preventing similar types to Fur protein. In fungi, similar transcriptional repressor
proteins are known as GATA factor proteins (Scazzocchio 2000; Kim et al. 2012).
These proteins contain GATA types of zinc fingers that bind to the genes of
siderophore biosynthesis (Gauthier et al. 2010) (Table 15.2).
On the other hand, Aft1 is a transcription factor of C. albicans and S. cerevisiae
which binds to the promoters region of siderophore biosynthetic genes and posi-
tively activates the expression in the genes in the iron-deficient condition
(Yamaguchi-Iwai et al. 1995, 1996). Therefore, external ion concentration is a
regulatory factor for intracellular biosynthesis of siderophores and transport proteins
microorganisms. Thus, we can say the regulation of siderophore production and
transporter proteins is the most important siderophore ecology. Iron sensing in the
natural environment helps for the survival of the competing microorganisms which
are unable to adapt in iron metabolism (Stubbendieck and Straight 2016). Therefore,
Fig. 15.3 Regulatory model for the biosynthesis of siderophore and Fur Box activation or repres-
sion: At the higher concentration of iron the formation of Fur box protein and iron complex binds at
promoter region of the DNA and RNA polymerases unable to binds at promoter to express the gene
while at low concentration of iron Fur proteins released and RNA polymerases binds and lead to the
expression of genes (proposed model redrawn with slight modification from Tanui et al. 2017)
Table 15.2 Fungal regulatory proteins are negatively regulated in the biosynthesis and transport of
siderophores
Regulatory protein similar to
S. No GATE factor Organisms References
1. URBS1 U. maydis Voisard et al. (1993);
2. SRE N. crassa Zhou et al. (1998)
3. SREP P. chrysogenum Haas et al. (1997)
4. SREA A. nidulans Haas et al. (1999); Oberegger
et al. (2001)
5. GAF2p S. pombe Hoe et al. (1996); Pelletier et al.
(2003)
those microorganisms which are highly sensitive to iron regulation are superior to
other colonizing species of microbes. It means that in ecological environments, those
having low iron content will be colonized rapidly by the aerobic microorganisms and
upregulation of siderophore biosynthetic genes and transport proteins (Tshikantwa
et al. 2018). Now, the important question is where is the low iron content in natural
habitats? The marine region especially in the open oceans, calcareous soils, and
freshwater lakes contains iron concentration in surface water in nano-molar
(0.2–1 nM) range (Butler 2005) and inhibits the growth of planktonic, bacterial,
and plant. Free iron is absent in humans but present in protein-bound forms like
transferrin or lactoferrin and ferritin. Under low iron concentration, pathogens can
multiply by sequestering iron from the host protein by the use of ferric binding
protein and transferrin binding protein found in Neisseria. These transport proteins
are transported from periplasm-to-cytosol (Cornelissen and Sparling 2004). Pseudo-
monas aeruginosa cell lysis and degradation of proteins by the proteases and
subsequent iron scavenging by the excretion of siderophores are another method
of iron acquisition. There are different routes by which pathogen can utilize iron
from the host cells, but the most suitable system is siderophore mediate transport
(Butt and Thomas 2017). The further important aspect is siderophore ecology which
is energy saving. The siderophore biosynthesis requires energy which is obtained
from ATP and carbon sources. Siderophore production is just started after the
germination of conidiospores in fungi starts (Wang et al. 2016). The conidiospores
contain a certain amount of siderophores which is packed into the wall of the spore
and secreted at the time of germination (Matzanke et al. 1988). If the siderophore
genes were knocked out from the fungi then the process of sporulation cannot
possible in such fungal strains (Eisendle et al. 2003).
15.11 Biotechnological Applications
15.11.1 Medical
For the treatments of thalassemia, siderophores can use to iron overload conditions
(Faa and Crisponi 1999; Day and Ackrill 1993). This problem is associated with the
use of the compound desferrioxamine (DFO) (Dexter et al. 1999; Kontoghiorghes
1995). DFO is a natural and synthetic analogs of siderophore and few side effects
was associated with its medical use (Peterson et al. 1976). Therefore, it is used as an
alternative to iron and aluminum overload. Rhodotorulic acid is a fungal siderophore
that has been investigated and found that it is not active orally. The mechanism of
rhodotorulic acid is similar to iron excretion of DFO but increased the excretion of
zinc and found toxic at the site of administration (Grady et al. 1979). DFO and other
hydroxamate siderophores have been used for the treatment of cancer, malaria,
actinide contamination, and other infectious diseases (Stradling 1998; Weinberg
1999). Treatment of acute lymphoblastic leukemia has been carried out by using
DFO (Yang et al. 2018). Vergne et al. (2000) reported that several other siderophores
exhibit anticancerous and antitumor activity. For example, triornicin fungal
siderophore, produced by Epicoccum purpurascens, has an inhibitory effect on
tumors in mice (Frederick et al. 1981). For the treatment of infections, siderophore
transport/uptake research can be exploited for the transport of the drugs into the
microbial cell conjugated with siderophore-drug (Lu and Miller 1999; Vergne et al.
2000). The actinides are radioactive elements and capable to create cancer known as
are potent carcinogens. Siderophores and its analogs have enhanced the excretion of
actinides. It might be possible to decorporation of actinides (Durbin et al. 1997).
Although the research on application of siderophores in the medical field is still
under progress and most of the research has been focused on bacterial siderophores
mainly desferrioxamine B (DFO) and siderophore analogs hydroxypyridinones.
15.11.2 Remediation
Sources of metal pollution are a motor manufacturing industry, sewage sludge,

vehicle emissions, and mining (Ledin and Pedersen 1996; Gray 1998). Neptunium
and plutonium are man-made actinides, present in the environment as a pollutant
during the weapon production in nuclear reprocessing plants (Gopalan et al. 1992).
Siderophores are effective for sequestering actinides (Brainard et al. 1992) and form
tetravalent actinides that are very stable complexes. They also play a significant role
in the mobilization of other metals including zinc, copper, lead, and cadmium
(Neubauer et al. 2000). Siderophore-producing microorganisms are most abundant
in the soils (Crowley et al. 1991) and affect the bioavailability, existence of the metal
in long-term and radionuclides present in the environment (Ruggiero et al. 1999).
Thus, siderophores could be used for metal recovery or remediation of waste sites,
including radioactive waste due to its complexing ability (Awad and Romheld
2000).
15.11.3 Growth-Promoting Activity and Biocontrol of Plants

Pathogen
Microbial siderophore provides Fe-nutrition when bioavailability of iron is limited to

enhance the growth of the plant (Crowley 2006). The mechanism of siderophore-
mediated Fe-nutrition is still unknown. Two possible mechanisms were suggested
by which plants can operate to obtain Fe from fungal siderophores: (1) High redox
potential of fungal siderophores can be reduced by the donation donate Fe (II) to the
transport system of the plant. In this mechanism, fungal Fe (III)–siderophores are
transported to the plant root apoplast, where siderophore reduction takes place
(Mengel 1995). As a result, Fe (II) is trapped in the root apoplast and increased
high Fe concentrations in the root (Kosegarten et al. 1999). (2) Siderophores of
fungal origin can chelate Fe from soils and perform ligand exchange with
phytosiderophores (Masalha et al. 2000). These mechanisms depend on several
parameters such as concentrations of phytosiderophores, microbial source, pH, and
redox potential of the root environment (Crowley 2006). Schenk et al. (2012)
suggested that siderophores be an environmentally friendly pesticide. Thus, mycor-
rhizal fungi can also be used as biofertilizers for the enhancement of plant growth
and developments. Caris et al. (1998) reported that root-associated mycorrhizal
sorghum plants take up higher concentrations of Fe than non-mycorrhizal plants.
Further, Van Scholl et al. (2008) suggested that the ectomycorrhizal fungi forms an
associations with plant for nutrition through fungal the siderophores. Plant growth-
promoting activities of the fungi were investigated recently. Yadav et al. (2011)
found that fungal strains such as Trichoderma harzianum, Penicillium citrinum, and
Aspergillus niger produce siderophore and enhance root and shoot length of the
chickpeas. Kloepper et al. (1980) demonstrated that siderophores play a significant
role in the biological control mechanism. This mechanism is based on the
siderophores as competitors for Fe wherein biocontrol agent reduce Fe availability
for the pathogens of plants and thereby jeopardizes survival pathogens due to iron
deficiency (Beneduzi et al. 2012). Wilt diseases of potato caused by Fusarium
oxysporum can be controlled by pyoverdine type siderophores produced by Pseudo-
monas sp. (Schippers et al. 1987). Apart from fungi, the bacterial strain mainly
Pseudomonas species is extensively studied for the improvement in plant growth or
protection from plant pathogen by siderophore production them from plant
pathogens (Kloepper et al. 1980; Gamalero and Glick 2011).
15.11.4 Siderophores as Enzyme Inhibitors
As we know, siderophores are iron chelators and they are capable to inhibit the iron-
dependent activity of enzymes by withdrawing iron. Many reports have been proved
that ribonucleotide reductase activity is reduced by synthetic siderophores (Kurth
et al. 2016), as a result of inhibition of biosynthesis of DNA. In the proliferating
neoplastic cells, the iron delivering transferrin receptors are frequently occurred on
the cell surface and enhanced iron requirement of the cell. Inhibition of iron supply
by the siderophore reduces the growth of neoplastic cells. Therefore, siderophores
can be used as inhibitors of cell proliferation and help drug designing with
anticancerous agents.
15.12 Conclusions
Iron is an essential need for all living organisms. Fungi acquire iron by secreting
siderophores, small low molecular weight, and iron-binding molecules, in extracel-
lular environments. Siderophore plays a significant role in iron homeostasis of the
fungi which shows similarity to bacteria and plants for the mobilization of extracel-
lular iron. However, much more remains to be explored, concerning the biosynthetic
pathway, iron assimilation, and regulation. In this regard, genome sequence analysis
of the fungus and siderophore-mediated iron acquisition in a wide range of fungal
species is still unexplored. Apart from the fungal siderophore, several unexplored
aspects will be the elucidation, e.g., extracellular excretion mechanism, details of the
siderophore biosynthetic pathway, intracellular iron release, iron metabolism, and
storage of iron. The iron requirement of fungi opens up a new area of research for the
development of novel antifungal treatment such as iron chelation therapy. However,
functional studies of siderophore will reveal novel nonribosomal peptide synthetases
are another area that opens the way for the development of new compounds that are
important for pharmaceutical value. In fungal species, iron uptake through
siderophore-mediated is essential not only to the survival of free-living organisms
but also in establishing commensal or pathogenic.
Acknowledgments Review paper supported by the Department of Science and Technology, India,
as the Young Scientist Project (SB/FT/378/2012).
Conflict of Interest None.
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Status of Silicon in Ecosystem, Silicon
Solubilization by Rhizospheric 16
Microorganisms and Their Impact on Crop
Productivity
Prakash B. Nagabovanalli, Sabyasachi Majumdar, and

Sandhya Kollalu
Abstract
Silicon (Si), a beneficial element, plays an overwhelming role in not only

improving the crop productivity but also provides resistance against various
biotic (insects and pathogens) and abiotic (salt stress, heavy metal toxicity,
nutrient toxicity, ultraviolet radiation, drought condition) stresses. It may be
considered as the only element which has multifaceted role in boosting the crop
productivity in future against various environmental stresses. In spite of the
abundance of Si in soil, it is not available for plant uptake. As a result, present
scenario of agriculture demands to improve plant available Si for improving crop
productivity across the world. One way is to apply silicon fertilizers which
sometimes become very costly for the farmers to afford. However, an alternative
is to utilize the potential of soil microorganisms in solubilizing silicates from
minerals. Numerous studies have indicated that soil microorganisms, viz., bacte-
ria and fungi, have the capacity to dissolve silicon from different primary
minerals. Hence, in this chapter, we made an attempt to explore how silicon
accumulates in different microorganisms, sources and sinks of terrestrial silicon
cycle, role of microbes in silicate weathering, importance of rhizosphere microbes
in solubilizing silicates, and the impact of silicate solubilizing bacteria on crop
productivity.
P. B. Nagabovanalli (*) · S. Kollalu

Department of Soil Science and Agricultural Chemistry, University of Agricultural Sciences,
Gandhi Krishi Vignana Kendra, Bangalore, Karnataka, India
S. Majumdar
Department of Soil Science and Agricultural Chemistry, University of Agricultural Sciences,
Gandhi Krishi Vignana Kendra, Bangalore, Karnataka, India
College of Agriculture, Central Agricultural University—I, Kyrdemkulai, Meghalaya, India

23, https://doi.org/10.1007/978-981-15-9154-9_16
410 P. B. Nagabovanalli et al.
16.1 Introduction
Silicon (Si), the second most ubiquitous element in the earth’s surface (Epstein 1994;
Wedepohl 1995), has been considered as beneficial substance by the Association of
American Plant Food Control Officials (AAPFCO) and International Plant Nutrition
Institute (IPNI) in 2014 and 2015, respectively (AAPFCO 2014; IPNI 2015). The
concentration of Si, present as silicic acid, in the soil solution usually varies from 0.1
to 0.6 mM (μg per gram) (Epstein 1994). In spite of the numerous evidences
indicating the benefits of Si in providing resistance against various environmental
stresses in several crop species (Farooq and Dietz 2015; Guntzer et al. 2012; Meena
et al. 2014; Liang et al. 2015; Prakash et al. 2018), the essentiality of Si for growth of
higher plants is still under dilemma (Epstein 1994) as it fails to meet the criteria of
essential nutrient proposed by Arnon and Stout (1939). According to the modified
criteria for essentiality of nutrients advocated by Epstein and Bloom (2005), Si can
be optimistically recognized as an essential element from the point of view of
nutrition of higher plants (Liang et al. 2015). It has also been documented in the
existing literature that sodium (Na), rubidium (Rb) and potassium (K), selenium
(Se) and sulfur (S), cobalt (Co) and nickel (Ni), Si and carbon (C) often substitute
each other in certain nonspecific metabolic processes. Hence, it can be stated that the
element substituting the essential element may be more effective than any other
element (Kaur et al. 2016).
Although Si is omnipresent, majority of Si in soil exist as crystalline
aluminosilicates that are unavailable for plant uptake (Richmond and Sussman
2003). The concentration of Si in soil solution is chiefly affected by solubility of
minerals in soil (Sommer et al. 2006). Soil is considered as a nutrient bin for the
plants, on a broader scale it is a heterogeneous complex ecosystem comprising of
varieties of microbes including bacteria, fungi, protists, and other small animals
(Muller et al. 2016). Microorganisms usually boost plant performance by enhancing
the availability of nutrients present in minerals. Their involvement is crucial in
various biological processes including the conversion of insoluble soil nutrients to
soluble ones (Babalola and Glick 2012). In this regard, Drever (1994) opined that
plants and their related microbiota play a pivotal role in promoting silicate
weathering by production of chelating ligands, modification of pH through produc-
tion of CO2 or organic acids, and alteration of soil physical properties. Henderson
and Duff (1965) reported that numerous bacteria and fungi are capable of
transforming insoluble silicates to soluble silicates through production of mineral
and organic acids and chelating agents.
Although soil contains plethora of microbiota, such as diatoms, radiolarian,
microorganisms, and protists, the role of microbiota in the transformation of Si has
been poorly understood. Das et al. (1992) and Chakrabarty et al. (1988) reported that
Microbacterium and Nocardia species can develop without carbon in the presence of
Si compounds. They further claimed that bacteria can grow automatically under such
environment by fixing CO2 by using energy received from Si metabolism which is
considered as a form of Si autotrophy. Likewise, few fungi were noticed to grow in
ultrapure water in the presence of Si compounds and on nutrient-free silica gel (Tribe
16 Status of Silicon in Ecosystem, Silicon Solubilization by Rhizospheric. . . 411
and Madadaje 1972; Parkinson et al. 1989). Regardless of the plenty of Si in earth’s
surface, only few organisms, viz., diatoms, chrysophytes, silicoflagellates, and some
xanthophytes, absorb and accumulate Si (Heinen 1962). Basak and Biswas (2009)
reported that Bacillus mucilaginosus is a model microorganism in research on
silicate mineral weathering. Bacterial like genera of Proteobacteria, Aminobacter,
Burkholderia, Collimonas, Janthinobacterium, Dyella, and Frateuria, have been
reported to solubilize the biotite, which contains substantial quantity of silicate
minerals (Uroz et al. 2009). Further, Burkholderia cenocepacia KTG, Aeromonas
punctata RJM 3020, and Burkholderia vietnamiensis ZEO3 have been observed to
speed up the solubilization of silicon dioxide (SiO2 originated from quartz (Santi and
Goenadi (2017)). Various studies have also exposed about different isolates of
silicate solubilizing bacterium from root rhizosphere and mineral surfaces (Sheng
et al. 2008; Sulizah et al. 2018; Chandrakala et al. 2019; Vijayapriya et al. 2019).
However, silicate dissolution by bacteria has gained increasing attention due to the
potential of bacteria in the release of potassium (K), calcium (Ca), and magnesium
(Mg) from the silicates (Vasanthi et al. 2018). Thus, in this chapter, we outlined
status of Si accumulation in different microorganisms, sources and sinks of terres-
trial Si cycle, role of microbes in silicate weathering, importance of rhizosphere
microbes in solubilizing silicates, and the impact of silicate solubilizing bacteria on
crop productivity.
16.2 Accumulation of Silicon in Different Microbiota

and Sponges
16.2.1 Diatoms
Diatoms are unicellular algae that contribute for nearly 40% of the total marine
primary production (Falkowski et al. 1998). These algae either live in water or soils
and few diatoms live in the plankton of ponds, lakes, and oceans (Mann 1999).
Although majority of the diatoms are photosynthetic, however, only some are
obligate heterotrophs and can survive in the nonexistence of light but in the presence
of organic source (Armstrong et al. 2000; Lewin and Lewin 1967). They uptake the
dissolved silica from fresh water and sea water and later precipitate in their cell walls
as biogenic silica (Brunner et al. 2009). However, marine diatoms have the ability to
persist at low Si concentrations compared to freshwater diatoms (Olsen and Paasche
1986). This eukaryotic organism serves as the best example from the view of silica
biomineralization and has been widely studied (Lewin 1965; Sumper and Brunner
2006, 2008; Hildebrand 2008; Gordon et al. 2009). It has also been reported that Si
plays a substantial role for DNA synthesis in diatoms (Volcani 1983).
The diatom cell is composed of two halves, each made up of a flat plate, or valve
and marginal connecting, or girdle band. The pore on the valves mainly helps in gas
and nutrient exchange (de Vrind-de Jong and de Vrind 1997). These algae have two
distinguishing shapes: centric diatoms are radially symmetric, while pennate diatoms
are usually bilaterally symmetric (Mishra et al. 2017). Cell wall made of hydrated
silicon dioxide, called a frustule (Lewin 1965), is a distinctive trait of diatom

anatomy. These frustules have structural coloration which is mainly credited to
their photonic nanostructure and as a result, diatoms are habitually named as “jewels
of the sea” or “living opals” (Parker and Townley 2007). However, the biological
role of this structural coloration is still a puzzle, but it may be linked to communica-
tion, camouflage, thermal exchange, and/or UV protection (Gordon et al. 2009).
Rogall (1939) reported that the cell walls of marine diatoms may contain more than
95% SiO2, but only little quantity of Al2O3 or Fe2O3 and water.
Although diatoms absorb Si as monosilicic acid (H4SiO4) (Lewin 1955; Del Amo
and Brzezinski 1999) via special silicon transport proteins (SIT proteins, Hildebrand
et al. 1997), these photosynthetic organisms do not uptake highly polymerized forms
of silicate unless it is depolymerized as noticed in few bacteria (Lauwers and Heinen
1974). Silicon uptake rates in diatoms were detected highest during cell wall
formation. The polymerization of silicic acid monomers leads to composing the
biogenic silica in the cell wall of the diatoms (Mishra et al. 2017). Subsequently,
research conducted on various diatom species to study the Si uptake kinetics showed
that the SIT proteins regulate the uptake of Si particularly at low Si(OH)4
concentrations, whereas at high Si(OH)4 concentration, its uptake is controlled by
diffusion (Thamatrakoln and Hildebrand 2008).
16.2.2 Radiolarians
Single-celled protists, dwelling in open-ocean locations, are called as silica secreting

radiolarians (Anderson 2019). There are single-celled protozoan zooplankton which
vary in magnitude from <100 μm to very large species with diameters of 1–2 mm
(Anderson 2001). They usually present in near surface to great depths of the water
column. They are diverse in low latitudes and most copious in near-surface waters
(Lazarus et al. 2009). They require dissolved silica to construct their opaline shell
(Anderson 1983; De Wever et al. 2001). The radiolarians are the second most
important source of biogenic opal deposited in the ocean sediments (Anderson
2001) and play an overwhelming role only in peripolar and equatorial belts, and
the western sides of continents (Lisitzin 1985; Casey 1993).
The framework of radiolaria is built of opal (SiO2nH2O) (Afanasieva and
Vishnevskaya 1992). It is because of well-established presence of fossil record,
the radiolarians have been comprehensively studied by the paleontologists. The
sturdy crystal structure of opaline silica of the radiolarian helped in well preservation
of the fossil record (Anderson 1983). From one subclass to another that varies
slightly, the skeletons are normally prearranged around spicules, or spines, which
are sharp, dense outcroppings from the main skeletal mass (Casey 1993). The
presence of Si in the ultrastructure of the primary opal provides the basis for the
development of the primary four-rayed spicule of radiolarians (Afanasieva and
Amon 2003). Besides, incline of temperature, silica, and other micronutrient content
may affect the latitudinal profusion patterns of living radiolarians (Abelmann and
Gowing 1996). Although few radiolarian species are skilled enough to compete for
silica in 1.0 mM seawater concentration, changes in seawater silicate may not have
an intense influence on longevity, weight, or skeletal size of the radiolarians
(Sugiyama and Anderson 1997). However, due to the presence of ornate skeletons,
radiolarians have been extensively used in biostratigraphy (De Wever et al. 2001).
16.2.3 Silicoflagellates (Dictyochophyceae)
Silicoflagellates have a star shape skeleton made of silica and chloroplast. They are
also known as silica secreting marine microplankton. Their origin is anticipated to
the early Cretaceous sediments and extends to the present, but was more abundant
during the late Cretaceous (McCartney 1993). The original Silicoflagellates were
first reported by Ehrenberg in 1837. The cell size of Silicoflagellates ranges from
20 to 80 μm (Taylor et al. 2009). Silicoflagellates have two flagella and thin
pseudopodia. Silicoflagellates are considered golden algae or chrysophytes on the
basis of pigmentation, mitochondria, chloroplasts, and siliceous skeleton. The ele-
mentary configuration for the skeleton consists of a basal ring and an apical ring
either slightly curved or dome shaped (McCartney and Loper 1989). The joints of the
various types of bars generate hollows known as portals or windows (Adamonis
et al. 2008). Silicoflagellates account for 1 and 2% of the siliceous components of
marine sediments and are more copious in waters of the equatorial upwelling zone.
Moreover, silicoflagellates are erratic and strongly coupled with ambient near-
surface changes (Boltovskoy et al. 1993).
16.2.4 Siliceous Sponge
Sponges, the members of the phylum Porifera, are the simplest form of multicellular
animals. The phylum Porifera consisting of classes Demospongiae and
Hexactinellida represents a major group for these siliceous sponges. They are
characterized by spicules made of SiO2, unlike calcareous sponges. Siliceous
sponges are usually found in the marine ecosystem but occasionally in fresh water.
In areas with low sediment accumulation rate (<5 103 cm yr1), the siliceous
sponge spicules can build up a significant fraction of the near-interface sediments
(DeMaster 2001). However, spicule begins with the formation of an organic filament
to which silica is secreted (Imsiecke et al. 1995).
Although siliceous sponges are involved in the overall balance of Si (Harriss
1966), few researchers opined that the contribution of spongesis is ignored in global
marine Si cycle (Greenwood et al. 2001; Gallinari et al. 2002; Rickert et al. 2002).
Few studies have highlighted that the Si deposited by sponges is hydrated, amor-
phous, and noncrystalline which is similar to opal (Mihin 1910; Vinogradov 1953;
Jones 1979). Numerous studies described that siliceous sponges may function as Si
sinks (Maldonado et al. 2005, 2010, 2011, 2012; Chu et al. 2011). Moreover,
sponges may influence dynamics of Si cycling and Si availability under
Si-deficient conditions (Maldonado et al. 2005). Recently, Maldonado et al. (2019)
revealed that sponges help to substantial Si burial through their siliceous skeletons
which are resistant to dissolution. As the life span of sponges can range from decades
to millennia (Maldonado et al. 2005, 2010, 2012; Jochum et al. 2012, 2017), it will
be interesting to study annual accumulation of biogenic Si through sponges.
16.3 Biogeochemical Cycle of Silicon
Nutrients move through the ecosystem in biogeochemical cycles. The biogeochemi-

cal cycle of Si can be defined as a systematic pathway by which Si mingles through
the biotic (living) and the abiotic (nonliving) factors of an ecosystem. Consequently,
while moving through different factors, such as weathering of soil and plant uptake
of Si, of an ecosystem, Si is not lost but recycled in sinks where they can be held for a
longer period of time. Through this biogeochemical cycle, Si is translocated from
one organism to another and from one part of the biosphere to another. Hence, the
global Si cycle is considered as a multifaceted interaction of biological, chemical,
and geological processes (Struyf et al. 2009). Moreover, the biogeochemical combi-
nation of Si with the global carbon cycle makes understanding of Si cycle predomi-
nantly significant (Tréguer et al. 1995; Ragueneau et al. 2000; Treguer and
Pondaven 2000; Yool and Tyrell 2003; Struyf and Conley 2009).
16.4 Terrestrial Silicon Cycle
Si is a beneficial element which is utilized by plants, grasses, and microbes in the

terrestrial biosphere. Terrestrial Si cycle consists of mineralogical and biological
pools (Struyf et al. 2009). The mineralogical pool of Si consists of primary minerals,
secondary minerals, and secondary poorly to noncrystalline (amorphous) minerals
(Sommer et al. 2006). The flux of Si in soils and terrestrial biogeosystems is mainly
mediated through water. Plants take up Si mainly as monomeric orthosilicic acid
known as dissolved silicon (DSi) (Drees et al. 1989). A part of DSi in the soil
solution is adsorbed onto clay particles and Fe and Al hydroxides (Hansen et al.
1994; Dietzel 2002). In terrestrial soils, substantial amount of soluble Si is present as
amorphous Si compared to mineral Si (Clymans et al. 2011). The amorphous Si
(ASi) comprises mostly of biogenic silica (BSi), known as phytoliths (Alexandre
et al. 1997), but also as different pedogenic Si forms (Sauer et al. 2006). Phytoliths
are an easily soluble, prospective Si source for plants (Berthelsen et al. 2001; Sauer
et al. 2006; Cornelis et al. 2011). In spite of their higher solubility compared to
silicate minerals (Fraysse et al. 2009, 2010), few phytoliths have the ability to persist
for more than thousand years in soils (Wilding 1967; Parr and Sullivan 2005).
Besides phytoliths, sponges and diatoms can make up a significant ASi pools in
wetland condition (Clarke 2003; Struyf and Conley 2009). Further, Aoki et al.
(2007) reported that testate amoebae could have been well thought-out as a vital
terrestrial bioreactor of Si.
16.4.1 Sources
The most important source of DSi in soil is the weathering of rock forming silicate
minerals. Throughout silicate weathering, dissolved soil CO2 helps in releasing
monosilicic acid (H4SiO4) or DSi from the structural lattice of silicate minerals
(Lindsay 1979) and the DSi is transported through soil and exported to rivers and
ultimately to ocean (Meybeck 1994). For example, during the weathering of albite to
kaolinite, produced DSi is shown in Eq. (16.1):
2NaAlSi3 O8 þ 9H2 O þ 2Hþ ! Al2 Si2 O5 ðOHÞ4 þ 2Naþ þ 4H4 SiO4 . . . : ð16:1Þ
In general, weathering potentially provides 5.6 1012 moles yr1 of DSi to the
ocean via river discharge along with an additional 0.5 1012 moles yr1 contributed
via atmospheric deposition (Tréguer et al. 1995).
16.4.2 Sinks
The chief sink of the terrestrial Si cycle is transported to the ocean by rivers. The rate
of this transport is assumed to be about 6 Teramole (Tmol; 1 Tmol ¼ 1012 mole) Si
yr1 (Conley 2002; Tréguer et al. 2013). This is the major sink and largest source of
the terrestrial silica cycle and marine silica cycle, respectively (Tréguer et al. 2013).
A minor sink for terrestrial silica, deposited in terrestrial sediments, is eventually
exported to the earth’s crust.
16.5 Marine Silicon Cycle
Siliceous organisms such as diatoms and radiolarians are the most important sink of
dissolved silicic acid into opal silica (Yool and Tyrell 2003). Once in the ocean, DSi
molecules are biologically recycled approximately 25 times before export and
permanent deposition in marine sediments on the seafloor (Conley 2002). This
rapid recycling is dependent on the dissolution of silica in organic matter in the
water column, followed by biological uptake in the photic zone. The estimated
residence time of the silica biological reservoir is about 400 years (Conley 2002).
Opal silica is predominately under saturated condition in the world’s oceans. This
undersaturation promotes rapid dissolution as a result of constant recycling and long
residence times. The estimated turnover time of Si is 1.5 104 years (Tréguer et al.
2013). The total net inputs and outputs of silica in the ocean are 9.4 4.7 and
9.9 7.3 Tmol Si yr1, respectively (Tréguer et al. 2013).
Biogenic silica production in the photic zone (surface layer of the ocean that
receives sunlight) is estimated to be 240 40 Tmol Si yr1 (Tréguer et al. 2013).
Dissolution in the surface removes roughly 135 Tmol Si yr1, while the remaining Si
is exported to the deep ocean within sinking particles (Conley 2002). In the deep
ocean, another 26.2 Tmol Si yr1 is dissolved before being deposited to the
sediments as opal rain (Conley 2002). Over 90% of the silica here is dissolved,
recycled, and eventually upwelled for use again in the euphotic zone (layer closer to
the surface which receives sufficient light for photosynthesis to occur) (Conley
2002).
16.5.1 Sources
The major sources of marine silica include rivers, groundwater flux, seafloor
weathering inputs, hydrothermal vents, and atmospheric deposition (aeolian flux)
(Gaillardet et al. 1999a). Rivers are by far the largest source of silica to the marine
environment, accounting for up to 90% of all the silica delivered to the ocean
(Gaillardet et al. 1999a; Tréguer et al. 2013; Huebner 1982). A source of silica to
the marine biological silica cycle is silica that has been recycled by upwelling from
the deep ocean and seafloor.
16.5.2 Sinks
Deep seafloor deposition is the largest long-term sink of the marine silica cycle
(6.3 3.6 Tmol Si year1), and is roughly balanced by the sources of silica to the
ocean (Gaillardet et al. 1999a). The silica deposited in the deep ocean is primarily in
the form of siliceous ooze, which is eventually subducted (a geologic process in
which one edge of one crustal plate is forced below the edge of another) under the
crust and metamorphosed in the upper mantle (Gaillardet et al. 1999b). Under the
mantle, silicate minerals are formed in oozes and eventually uplifted to the surface.
At the surface, silica can enter the cycle again through weathering which can take
tens of millions of years (Gaillardet et al. 1999b). The only other major sink of silica
in the ocean is burial along continental margins (3.6 3.7 Tmol Si year1), primar-
ily in the form of siliceous sponges (Gaillardet et al. 1999a). Due to the high degrees
of uncertainty in source and sink estimations, it is difficult to conclude if the marine
silica cycle is in equilibrium. The residence time of silica in the oceans is estimated
to be about 10,000 years (Gaillardet et al. 1999a). Silica can also be removed from
the cycle by becoming chert (quartz without clay) and being permanently buried.
16.6 Role of Microbes in Silicate Weathering
The complex interaction among precipitation, runoff, lithology, temperature, topog-

raphy, vegetation, and soil microbes results into varying rates of weathering (Drever
1994). Land plants and soil microbiota impact the weathering process of silicate
minerals in different ways through: production of organic chelating ligands, alter-
ation of pH, production of organic acids and CO2, and alteration of physical
properties of the soil (e.g., physical weathering of rocks, increase in the surface
area, longer residence time of water) (Hinsinger et al. 2001; Kelly et al. 1998).
Fig. 16.1 Various substances produced/mechanism operated by silicate solubilizing microbiota

for production of bioavailable Si
Dissolution of mineral silicates may be directly or indirectly controlled by activity

of microbiota (Fig. 16.1). Different microbiota like bacteria, fungi, algae, and lichens
are responsible to a certain extent for soil and sediment formation and release
dissolve silica to surface waters (Canfield et al. 2005). Moreover, microbiota
attached to the surface of weathered minerals may perhaps act as nucleating agents
in mineral neoformation (Macaskie et al. 1992; Schultze-Lam et al. 1995). In
positive situations, attached microbiota are capable of exerting noteworthy mechan-
ical forces which induce physical distortion of the mineral lattice structure that
facilitates chemical weathering (Bonneville et al. 2009). Solubilization of silicates
by microbes is done through production by ligand of cations, organic or inorganic
acids, alkali or extracellular polysaccharides (Canfield et al. 2005). The most
frequently occurring mechanism by which silicate minerals are weathered is by
acidolysis (Jongmans et al. 1997).
16.6.1 Solubilization of Silicates by Ligands
Microbes are capable of weathering silicate minerals by production of ligands of

divalent cations. For example, Webley et al. (1960) reported that production of
2-ketogluconic acid from glucose by Pseudomonas has the potential to dissolve
synthetic silicates of calcium, zinc, magnesium and the minerals such as wollaston-
ite, olivine, apophyllite. Dissolution of silicates resulted from the complexation of
the cationic components of the silicates by 2-ketogluconate has been reported. At the
same time, gluconic acid produced from glucose by different bacteria has been
reported to solubilize bytownite, albite, kaolinite, and quartz at near neutral pH
(Vandevivere et al. 1994). The action of microbes nearby pH 7 indicates that
mechanism of action of gluconate includes chelation. Santi and Goenadi (2017)
reported that Burkholderia cenocepacia KTG, Aeromonas punctata RJM3020, and
Burkholderia vietnamiensis ZEO3 isolates were capable of producing citric, acetic,

and oxalic acid in various optimum incubation times and accelerating the solubiliza-
tion of SiO2 originated from quartz.
16.6.2 Solubilization of Silicates by Organic and Inorganic Acids
Bennett et al. (1988) revealed that the organic acids such as citric, oxalic, pyruvic,
and humic acids produced by fungi or bacteria can dissolve the quartz in slower
rates; however, acetate, fumarate, and tartrate were ineffective in dissolving silica as
a complex. Numerous investigations also indicated that almost dilute solutions of
oxalic, citric, and acetic acid can enhance silicate dissolution by up to one order of
magnitude (Canfield et al. 2005). Nevertheless, the rates of dissolution in solutions
containing organic acids may be up to 10 times higher compared to the rates in
solutions containing inorganic acids at similar pH level (Welch and Ullman 1993).
An experiment conducted by Welch and Ullman (1993) on weathering of three
different Ca-Na feldspar by acids produced by different microorganisms revealed
that oxalate, citrate, succinate, pyruvate, and 2-ketoglutrate were the most effective,
whereas acetate and propionate were less effective. They further indicated that all
organic acids were more effective than the inorganic acids like hydrochloric acid and
nitric acid. Moreover, solubilization of silica from amphibolite, biotite, and ortho-
clase by citric and oxalic acids produced by numerous fungi and yeasts at the
expense of glucose has been revealed by Eckhardt (1980) and Barker et al. (1997).
Few studies have also reported the release of Si from plagioclase and nepheline by
acid produced by Penicillium notatum and Pseudomonas sp. (Aristovskaya and
Kutuzova 1968; Kutuzova 1969).
16.6.3 Solubilization of Silicates by Alkali
Alkaline environments are very comfortable zone for mobilizing silica from
silicates, aluminosilicates or even quartz (Karavaiko et al. 1984). This is ascribed
to the substantial lability of the Al-O and Si-O-Si bonds under alkaline conditions
due to their susceptibility to nucleophilic attack (Karavaiko et al. 1984). Maurice
et al. (2001) reported that Pseudomonas mendocina was able to enhance mobiliza-
tion of Al, Si, and Fe impurities from kaolinite in a succinate-mineral salt medium
where the pH rose from 7.7 to 9.2 in 4 days of growth under aerobic condition.
16.6.4 Solubilization of Silicates by Extracellular Polysaccharide

and Polymers
A high-molecular-weight polymers that are composed of sugar residues which are

secreted by microorganisms into the surrounding environment are termed as extra-
cellular polysaccharide (Staudt et al. 2004). By production of high-molecular-weight
polymers, microbes can also influence weathering of silicates (Canfield et al. 2005).
These polysaccharides are capable enough to react with Si-O-Si (siloxanes) bonds to
form organic siloxanes. It may either derive from bacteria (Avakyan et al. 1986) or
fungi (Holzapfel and Engel 1954). Due to the presence of water in the mucoid
polymers, they increase the contact time between water and the mineral surface
(Canfield et al. 2005). High-molecular-weight polysaccharides formed by microbes
may increase the extent of silica dissolution by complexing with ions in soil solution
and thus lower the solution saturation state (Canfield et al. 2005).
16.7 Rhizosphere Microbes and Silicon Availability
The availability of literature on the potential role of microbial processes in making Si

available to plants is almost nonexistent (Prakash et al. 2018). Despite the abundance
of microorganisms in soil, only a few are capable of solubilizing silicates. During the
degradation of silicate by bacteria, the release of Si has been reported (Hutchens
et al. 2003), and silicate solubilizing bacteria (SSB) have recently attracted great
interest, since solubilization of silicate promotes the uptake of both Si and K, thus
reducing the need for potash fertilizer (Sheng 2005). Soomro (2000) reported that
(a) the bacteria solubilize rock potash which results in releasing free Si into the
medium, (b) the growth of a Penicillium sp. in vitro increased the solubilization of
sodium silicate, but concentrations of free Si decreased when the fungus was grown
in the presence of silicic acid, presumably due to Si-immobilization by the fungus,
(c) addition of silicic acid generally decreased bacterial numbers in all soils at least
over the first few days of the incubation period, and (d) silicic acid had no influence
on nitrification, while the addition of sodium silicate stimulated nitrate production.
Raj (2004) reported that bacteria are abundant in soil and few of them have the
potential to solubilize silicate minerals releasing silica. They also revealed that
potassium (K) is also released simultaneously when K-bearing silicate minerals are
attacked by microorganisms. The organic acid and polysaccharide produced by
microbes are supposed to create favorable condition for silica solubilization. Few
researchers reported that SSB can solubilize the insoluble silicates by production of
carbon dioxide (CO2) and/or exopolysaccharide (Sadzawka and Aomine 1977;
Farshad 2011). However, gluconate promoted dissolution of silicates like albite,
quartz, and kaolinite by subsurface bacteria has been reported earlier (Werner and
Roit 1983). Murali Gopal et al. (2005) observed the presence of substantially higher
population of SSB in the rhizosphere of field-tolerant palms at 0–25 cm depth
compared to root-wilt diseased palms. The numbers of these function specific
bacteria were on par at 25–50 cm depth in both types of palms (Table 16.1).
This study revealed that majority of the silicate solubilizers were Bacillus spp.
and Pseudomonas spp., though at times Aspergillus spp. and Penicillium spp. were
also identified. Large number of SSB in the root zones of field-tolerant palms noticed
in this investigation could improve the available pool of Si to be absorbed by these
palms, which is reflected in their firm and erect leaves arranged to receive maximum
sunlight.
Table 16.1 Population estimation of function specific microbial communities in rhizosphere of

root (wilt)-diseased and field-tolerant coconut palms var. WCT (average of five replicates of each
sample)
N-fixers P solubilizers Cellulose Silicate
Soil sample (102) (102) degraders (102) solubilizers (102)
Root (wilt)-diseased palms (RWD)
0–25 cm 21.68 2.81 2.75 0.08
25–50 cm 14.95 8.41 1.43 1.04
Field-tolerant palms (FTP)
0–25 cm 73.40 4.23 3.23 5.03
25–50 cm 49.24 10.20 1.32 1.96
Mean RWD 18.31 5.61 2.09 0
Mean FTP 61.32 7.20 2.27 3
CD (P ¼ 0.05) RWD vs 1.31 4.10 NS 0
FTP
CD (P ¼ 0.05) 0–25 vs 1.31 4.10 1.62 0
25–50 cm
CD (P ¼ 0.05) 1.85 NS 2.29 0
interaction
16.8 Influence of Silicate Solubilizing Bacteria on Crop

Productivity
Several studies demonstrated the effect of silicate solubilizing bacteria (SSB) on the
nutrient uptake from the soil, their positive influence on photosynthesis, and the
growth of some crops (Han and Lee 2005; Han et al. 2006; Tripti et al. 2017). Few
studies also indicated that SSB can be used as a biofertilizer and found to enhance
the growth, suppress pest and diseases, and increase the yield (Kannan 1996;
Kannan and Raj 1998). Ciobanu (1961) reported that Azotobacterin and
Silicabacterin when applied simultaneously increased the yields of raw cotton by
34–50%. The beneficial effects of SSB on lucerne and maize were also shown by
Vinticova (1964). Inoculation of SSB to sterile and unsterile soil solubilized silica in
water reflected the improvement in the available silica in soils (Tables 16.2 and 16.3)
(Kannan 1996).
Moreover, field trials conducted with SSB showed that this bacterium enhanced
the number of grains per panicle, thousand grain weight, fully filled grains, biomass
and grain yield in rice (Table 16.4) (Kannan 1996). Peera et al. (2016) studied the
effect of silicon (Si) on yield and uptake of rice (var. BPT 5204) based on graded
levels of Fly Ash (FA), SSB and Farm Yard Manure (FYM) at fixed fertilizer
schedule. The highest mean grain yield was recorded by the addition of SSB + FYM
(3622 kg ha1) followed by FYM (3530 kg ha1) and SSB (3310 kg ha1). Further,
Peera et al. (2016) reported that combined application of SSB + FYM recorded the
lowest leaf openness in rice followed by FYM and SSB. Brindavathy et al. (2012)
revealed that the application of silicon mobilizing bacteria (Bacillus edaphyticus)
Table 16.2 Available silica (mg L1) in water from different soils inoculated with silicate
solubilizing bacteria
Red soil Clay soil Sand
Sl No Treatment 0 4 0 4 0 4
1 Sterile soil 203 210 119 215 173 176
2 Sterile soil + SSB 302 321 278 326 258 330
3 Unsterile soil 287 291 152 225 230 258
4 Unsterile soil + SSB 327 363 184 328 248 306
0: Initial; 4: after 4 weeks; SSB—silicate solubilizing bacteria
Table 16.3 Release of available silica (mg kg1) in different soils inoculated with silicate
solubilizing bacteria
Red soil Clay soil Sand
Sl No Treatment 0 4 0 4 0 4
1 Sterile soil 105 106 73 75 430 430
2 Sterile soil + SSB 105 135 73 105 430 494
3 Unsterile soil 105 110 73 105 430 453
4 Unsterile soil + SSB 105 145 73 129 430 496
0: Initial; 4: after 4 weeks; SSB—silicate solubilizing bacteria
Table 16.4 Influence of silicate solubilizing bacteria inoculation on the yield parameters in rice
No of grains 1000 grain Fully filled Biomass Grain yield
Treatment per panicle weight (g) grains (%) (Mg ha1) (kg ha1)
Control 54 22.14 76.7 10.24 3400
SSB 62 22.84 78.1 11.27 3800
SSB—silicate solubilizing bacteria
significantly produced higher cane yields in fresh plantings of sugarcane as well as in

ratoon crop.
The combined application of 25 ton ha1 FA with SSB + FYM has recorded the
highest grain yield of 3710 kg ha1 which was 16.3% more over control. The results
further revealed that 25 ton ha1 FA and SSB + FYM have been proven to be
superior treatments for best management of Si in coastal loamy sand soils under
irrigated rice ecosystem. Maleva et al. (2017) revealed that addition of SSB-enriched
biofertilizer to clay substrate significantly improved the content of total nitrogen,
phosphorus, and potassium in the leaves of Indian mustard (Brassica juncea). The
thickness of mesophyllic layer and the number of mesophyll cells were increased on
an average by 24%. It was correlated with a sharp increase of photosynthetic
pigment content and CO2 uptake (1.5–2.0 times). This study also indicated that
there was a substantial improvement in chlorophyll-a content compared to
chlorophyll-b. Markantonis et al. (2017) revealed that in simplified hydroponic
systems, presence of arsenic (As) oxidizing silicate and phosphate solubilizing
bacteria (SPSB)-inoculums reduced uptake of As. It was possibly due to enhance-
ment of competition between the two ions on aquaporin transporters in the rice roots.
Kang et al. (2017) reported that the isolate Burkholderia eburnea CS4-2 has the
capacity to produce indole-3-acetic acid (IAA) under alkaline conditions, solubilize
silicate, and encourage crop growth in rice. Lee et al. (2019) reported the inherent
ability of the strain Enterobacter ludwigii GAK2 to solubilize silicate that produces
organic acids, IAA, and gibberellic acid (GA). This study further revealed that
isolate GAK2 increased root length, shoot length, fresh biomass, and chlorophyll
content of rice plants and consequently might be considered as an efficient
inoculants for plants as a Si biofertilizer. Chandrakala et al. (2019) characterized a
silicate solubilizing bacterial isolate IIRR-1 from rhizosphere soil of rice and
revealed that the isolate successfully colonized rice seedling roots and improved
seedling vigor by 29.18% as compared to uninoculated control.
16.9 Conclusion
This review explored the potential of microbes in solubilizing silicates from

minerals. Research is needed to quantify the quantum of silicon solubilized by
different microbes under different vegetation cover. Further, it will be interesting
to investigate the quantum of biogenic silicon accumulation through different
microbes.
16.10 Future Line of Work
Living organisms might have impacted Si in earth much longer than was previously
thought, and hence there is necessity to understand the length of time involved. The
researchers need to analyze fossilized spicules in order to know how much Si was
dissolved in the oceans and other water bodies at different times. The need of the
hour is to elucidate mechanism for Si solubilization through organic acid profiling
and its correlation with other enzymes in soil. There is necessity to isolate and
identify group of soil microorganisms which has the potential to be employed as a
plant beneficial for accelerating the weathering process and increasing silicon
availability in the rhizosphere.
Acknowledgements The first author would like to profusely thank Dr. Sushil K. Sharma, Princi-
pal Scientist (Agricultural Microbiology), ICAR-National Bureau of Agriculturally Important
Microorganisms, Mau Nath Bhanjan, Uttar Pradesh, India for the invitation to contribute this
chapter.
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Diversity and Function of Microbes
Associated with Rhizosphere of Finger 17
Millet (Eleusine coracana)
Renu Choudhary, Geeta Rawat, Vijay Kumar, and Vivek Kumar
Abstract
The plant and microbes symbiotic association is known to be an important and

beneficial activity for sustainable agriculture since many decades. Rhizospheric
zone is full of varied microbial population. Different microbes not only interact
with each other, but also influence the host. Finger millet is an important,
nutritionally rich crop of semiarid and tropical regions, and its rhizospheric
zone harbors a wide diversity of beneficial microbes. The microbial community
of finger millet crop consists of rhizobacteria, phosphate solubilizing and
nitrogen-fixing bacteria, arbuscular mycorrhizal fungi, and plant growth-
promoting fungi along with other micro- and macroorganisms. The association
of these rhizospheric microbes with finger millet plant confers protection from
several biotic and abiotic stresses. Association of beneficial or efficient microbes
with millet plant helps it to adapt to hostile environments and also promote its
productivity through various mechanisms either directly or indirectly. These
beneficial means are diverse direct and indirect benefits on the host plant. The
literature on rhizospheric microbes associated with finger millet is scanty. In this
chapter, an attempt has been made to discuss the beneficial aspects of different
types of rhizospheric microflora on finger millet.
Keywords
Finger millet · Microbes · Plant growth · Rhizosphere
R. Choudhary · G. Rawat · V. Kumar · V. Kumar (*)

Himalayan School of Biosciences, Swami Rama Himalayan University, Dehradun, Uttarakhand,
India

23, https://doi.org/10.1007/978-981-15-9154-9_17
432 R. Choudhary et al.
17.1 Introduction
The genus “Eleusine” is derived from the Greek goddess of cereals, “Eleusine,”
whereas the universal name finger millet designates “fingerlike” panicle branching.
Intrinsically, it might be one of the earliest aboriginal cultivated African tropical
cereals. It is very productive crop that can flourish under various severe environ-
mental conditions. Marginal farmers in underdeveloped and developing countries
grow finger millet without any chemical input; therefore, this crop in such conditions
is also organic by default. This crop can be cultivated under low or poor fertility soils
and therefore is not reliant on application of chemical fertilizers, henceforth, is an
advantage for the huge arid and semiarid regions all over the globe (Gull et al. 2014).
Finger millet, scientifically named as Eleusine coracana and commonly known as
“Ragi,” is one of the most important cereal crops of the tropical and semiarid regions.
Finger millet is an important, but underutilized crop in semiarid and tropical regions
of the globe owing to its better pests and diseases resistance, great adaptation to
varied environmental range. It also has good production yield, able to tolerate
substantial salinity level, compared to other crops, the growing season is short. It
is waterlogging resistant and tolerates drought. During growth period, it requires
little input and with ever increasing planet population and lessening water sources,
therefore, is a significant crop for future human consumption (Taylor and
Emmambux 2008; Gupta et al. 2017).
In India, the Karnataka state is leading in finger millet production and accounts
for up to 58% of its worldwide production; however, only very few Indian are aware
about its nutritional importance and health benefits. In the context of climate
unpredictability, the crop has become very important and is providing nutritional
as well as fodder security and can be helpful in addressing the issue of nutrition
deficiency and contribute toward sustainable food security. There are enormous
possibilities to process finger millet grains into value-added products and beverages
in underdeveloped and developing nations. Moreover, finger millet is suggested for
abdominal (stomach) patients, since it does not contain gluten.
Rhizosphere, the biologically active and productive region of soil adjoining the
plant root, is the region for finger millet plant-microbe interaction. The
microorganisms interact in a variety of ways and it could be as follows: pathogenic
(negative), symbiotic (positive), or neutral. Microorganisms associated with finger
millet rhizosphere have been studied for their beneficial impact upon the finger millet
plant growth, nutrient uptake, also as bio-controlling agent and for stress mitigation
(Singh et al. 2004; Bakker et al. 2013).
To comprehend the dynamics within rhizospheric region and the effect on finger
millet plant traits by action of associated microbes, one should study the
accompanying microbiome. In this chapter, we have discussed the nutraceutical
facets, beneficial aspects of bacteria and fungi on finger millet plant growth, nutrients
acquisition, and stress mitigation.
17 Diversity and Function of Microbes Associated with Rhizosphere of Finger Millet. . . 433
17.2 Nutraceutical Value of Finger Millet
United States National Academics describes finger millet as a “potential super

cereal” among all the major cereals for being most nutritious (National Research
Council 1996). The minerals and micronutrients content of finger millet is higher
than major cereal grains including wheat and rice. It is considerably rich in calcium,
dietary fibers, and phenolic compounds (Gupta et al. 2017). Nutritive value of finger
millet is summarized in Table 17.1.
Along with its nutritional significance, the crop is also acknowledged for its
usefulness in management of various physiological disorders; regular incorporation
of whole finger millet grain in the diet can be beneficial against the risk of diabetes
(type II), heart disease, gastrointestinal disorders, and other ailments. The gluten-free
property of this wonderful grain makes it attractive and appealing to gluten-sensitive
people. Various nutraceutical features of finger millet are depicted in Fig. 17.1.
17.3 Microflora and Finger Millet
The rhizosphere of finger millet harbors a diversity of microbes which directly or

indirectly influence the plant growth, development, and adaptation. The microbial
activities in this area are influenced by rhizo-secretion and residential microbiome
interactions. The microflora which shows a wide diversity and beneficial role in the
rhizosphere consists mainly of bacteria and fungi. These microorganisms can pro-
vide many benefits to the plant such as nutrition uptake and transport, disease and
drought resistance etc. Existence and persistence of efficient microbes in the
rhizospheric soils could sustainably enhance the finger millet productivity, because
these microorganisms have great potential to work as biofertilizer and biopesticide
(Kumari et al. 2019).
Eleusine coracana (Ragi), as discussed earlier, is widely grown in varied agro-
climatic conditions, is adaptable to diverse environmental conditions (nutrient defi-
cient soil, low precipitation, high desiccation rates, drought, and challenges), and
provides food and fodder security. Due to these challenges, the yield losses globally
are expected to reach up to 30 % of the global production by 2025 and drought could
be one of the main reasons (Nanda et al. 2010). Drought stress affects the physio-
chemical mechanism and various biochemical reactions which include increased
production of reactive oxygen species (ROS) and reduced production of chlorophyll
content which is in response to several biotic and abiotic stimuli. These physiologi-
cal changes affect the secondary metabolites production and their accretion.
Arbuscular mycorrhizal fungi (AMF) association with host plant has gained atten-
tion due to their role in mitigation of water stress. The AMF plants are more tolerant
than non-AMF plants. Under stress conditions, mycorrhized hyphae or extra radical
mycelium (ERM) excretes a glycoprotein, glomalin which improves soil structure
and increases water and nutrient uptake capacity. In recent years, this symbiotic
association of rhizospheric fungi for plant growth-promoting attributes, nutrient
acquisition, and disease mitigation in finger millet crop has been studied (Goss
434
Table 17.1 Composition and concentration of finger millet grain nutritional compounds
Non-
Essential essential
amino acids amino acids
Minerals and trace (g/100 g (g/100 g
elements (mg/100g) Vitamins (mg/100g) Proximate composition (per 100g) Phenolic compounds protein) protein)
Calcium 350 Thiamine 0.42 Protein (g) 7.7 Total phenol (mg/100g) 102 Phe 6.2 Asp 7.9
(mg)
Phosphorus 283 Riboflavin 0.19 Fat (g) 1.5 Protocatechuic acid 23.1 His 2.36 Glu 27.1
(mg) (μg/mg)
Potassium 408 Niacin (mg) 1.10 Crude fiber (g) 3.6 Gentisic (μg/mg) 61.5 Ile 5.1 Ala 8.0
Sodium 11 Vitamin E 22.0 Carbohydrate (g) 72.6 P- Hydroxy benzoic 8.9 Leu 13.5 Arg 5.2
(mg) acid (μg/mg)
Magnesium 137 Total folic 18.3 Energy (Kcal) 336 Vanillic (μg/mg) 15.2 Lys 3.7 Cys 1.6
acid (μg)
Iron 3.9 Total dietary fiber (% of 19.1 Caffeic (μg/mg) 16.6 Met 2.6 Gly 4.8
crude fiber)
Manganese 5.94 Syringic (μg/mg) 7.7 Thr 5.1 Pro 6.7
Molybdenum 0.102 Coumaric (μg/mg) 56.9 Val 7.9 Ser 6.9
Zinc 2.3 Ferulic (μg/mg) 387.0 Tyr 3.6
Cinnamic (μg/mg) 35.1 Trp 1.3
Source: Sood et al. (2019), Gopalan et al. (1989), Chandra et al. (2016), and McDonough et al. (2000)
R. Choudhary et al.
17
PREVENT OBESITY
ANTIULCERATIVE
• Tryptophan
• Fibers (Lowers appetite)
BONE HEALTH
NATURAL RELAXANT
• Calcium
• Prolamins, (Strengthening
globulins, bones)
albumins CBPs
ANTI-DIABETES (TYPE II)

ANTIOXIDANT FINGER MILLET • Phytochemical, dietary
• Phenolic compounds (Eleusine coracana) fibers (Lowering glycemic
index)
• Flavonoids and tannins
LOWERING BLOOD CHOESTROL

ANTICANCER
• Lecithin and methionine
• PTs (protease inhibitor) (Eliminates excess fat from liver)
• RBI (Ragi beneficial • Threonine
inhibitor) (Hinders fat formation)
ANTIMICROBIAL ANEMIA
• Phenolic compounds • Iron
Diversity and Function of Microbes Associated with Rhizosphere of Finger Millet. . . 435
Fig. 17.1 Nutraceutical aspects of finger millet (Source: Chandra et al. (2016); Devi et al. (2014) and Kumar et al. (2016a, 2016b))
et al. 2017; Kamal et al. 2015; Ahanger et al. 2014). Fungal species belonging to the
genus Glomus has drought tolerant abilities, and they also promote plant growth,
nutrient accumulation, gaseous exchange, transpiration conductance under water
stress conditions (Kamal et al. 2015). In the rhizospheric zone, rhizobacteria and
AM fungi interact with each other synergistically and this association enhances the
beneficial attributes of PGPR for plants (Bianciotto et al. 2009).
Soil structure and composition also plays a major role in the diversity of
rhizospheric microflora such as endospore forming, aerobic bacteria belonging to
Bacillus species which are abundantly found in high altitude regions. The
rhizospheric zone of Eleusinecoracana harbors high abundance of Bacillus and
Paenibacillus spp. A number of bacterial species belonging to genera Pseudomonas,
Serratia, Stenotrophomonas, and Streptomyces, and fungal genera Ampelomyces,
Coniothyrium, and Trichoderma are also well associated with the finger millet plant,
and they are involved in plant growth promotion, nutrient uptake, and hormone
stimulation (Dheeman et al. 2017).
Bacteria have the capability to regulate the growth of other organisms, and this
property is observed in the soil ecosystem and can be used for crop protection and
enhanced crop production. Free living, aerobic, endospore forming rhizobacteria
have the potential to be used in sustainable and intensive agriculture (Dheeman et al.
(2019). The biocontrol activity is due to the enzyme chitinase produced by Pseudo-
monas fluorescens which deforms the cell wall of Ragi blast fungus
Pyricularia grisea resulting in killing of the pathogen (Radjacommare et al. 2004).
Plant hormones IAA, ethylene, cytokinins, and gibberellins produced by plant-
associated microbes play significant role in modulating the plant growth and devel-
opment under normal and stress conditions. Microorganisms which produce these
beneficial hormones have the potential to protect plants against biotic and abiotic
stresses (Berg 2009).
The inappropriate use of chemical fertilizers and pesticides is an environmental
hazard and need to be replaced by safe substitute for use in sustainable agriculture
(Leach and Mumford 2008). Effective, competent, and proficient microbes can be
the safe substitute of chemical fertilizers and pesticides. Figure 17.2 shows the
diverse role of microbes toward finger millet plant growth promotion as well as
about stress extenuation.
17.4 Microbial Diversity in Finger Millet
Rhizosphere microbial community composition is often distinct with different plant

species. This is also true of finger millet rhizosphere (Kumari et al. 2019). This
microbial diversity is because of finger millet plant rhizo deposition or root exudate,
and also the physical and chemical properties of the soil of a particular place also
decide the microbial community. Different types of rhizobacteria, PGPF, and
arbuscular fungi (AF) have been found to be associated with finger millet rhizo-
sphere. Association of such beneficial microbes in finger millet rhizospheric zone
has a positive effect on plant and also on the environment (Lernoud and Willer
17
PGPF
AMF
PGPR
Im
pa
c
to
n
ho
st
Enhanced plant growth
p
la
nt
• Nitrogen fixaton
• Phytohormone production
Root hair • Phosphate solubilization
• Stress resistance
Rhizoplane • Resistance to
phytopathogens
Rhizosphere • Nutrient uptake
• Increased plant yield
Root exudate
• Biocontrol
Root cap
Fig. 17.2 Microbial role in finger millet rhizosphere for regulating growth
2016). In the rhizospheric zone, associated microorganisms utilize a large portion of

the root exudates (carbohydrate, amino acids, organic acid etc.) which promote their
proliferation and activity in the rhizosphere, since these plant excreted metabolites
not only accelerate the survival of microorganisms, but also offer nourishment to
them. A wide diversity of microorganisms in the rhizospheric zone of
Eleusine coracana interacts with the host plants and plays a significant role in
plant health and development. Numerous mechanisms are involved in augmentation
of plant growth and acquisition of nutrients by rhizo-microbes and provision of
resistance against plant pathogens (Berg 2009).There is a wide array of micro- and
macroorganisms in the finger millet rhizosphere which carry out diverse activities
and contribute to enhancing crop production.
17.4.1 Bacterial Association with Finger Millet Rhizosphere
Bacteria associated with rhizosphere play an important role in enhancing finger

millet productivity, adaptation, and resistance to biotic and abiotic stresses.
Rhizospheric bacteria exhibit several plant growth-promoting attributes which are
expressed by either direct or indirect mechanisms (Fig. 17.3) which ultimately
govern plant growth and development (Upadhyay et al. 2018). These bioinoculants
are widely used in sustainable agriculture, but less information is available about the
beneficial rhizobacteria associated with finger millet rhizosphere. Bacillus, Pseudo-
monas, Azospirillum sp. are some of the common rhizo-bacteria which promote
Eleusine coracana growth (Santhaguru and Santharam 2012). These bacterial
genera have the potential to produce secondary metabolites, phytohormones, and
also have phosphate solubilization ability. Pseudomonas spp. are prevalent in finger
millet rhizosphere and provide resistance to phytopathogens as well as contribute/
assist in plant growth under normal and several stress-related conditions.
Blast disease of finger millet caused by Pyricularia grisea cause 50-100 % yield
loss has been managed by Pseudomonas sp. strain MSSRFD41. This bacterial strain
produces antifungal substances, hydrolytic enzyme, and also siderophore. Applica-
tion of this bacterial strain resulted in inhibition of P.grisea growth. The bacteria
caused fungal hyphae deformation. Interestingly, finger millet seeds inoculated with
this strain (MSSRFD41) had better germination level, higher seedling vigor index,
and increased root and shoot length as compared to the control seeds (Sekar et al.
2018).
Kumari et al. (2019) isolated 111 rhizobacteria from finger millet grown at
different locations. Forty bacterial isolates displayed biocontrol effect against
Pyriculariagrisea, while 38 showed antagonism against Rhizoctoniasolani (Banded
blight disease) and 30 isolates exhibited antagonistic property against both the
disease-causing agents. The antifungal compounds produced by rhizobacteria help
them to control the fungal infection. Thirty bacterial isolates produced siderophore
and solubilized P; 18 isolates produced enzymes β, 1-4 glucanase, and amylase. All
the above-mentioned properties of 111 rhizobacteria resulted in better finger millet
growth and nutrient acquisition.
17
Biofertilizer Biological control • Phoduction of

• Phoduction of antibiotics, antifungal
sderophore compounds,sideroph
• Phosphate ore,HCN and lytic
solubilization enzymes
Improved crop • Acquired and
• Insoluble quality and yield Better soil health
potassium induced systemic
solubilization resistance (SAR &
ISR)
• Biological • Volatile organic
nitrogen fixation compounds synthesis
and liberation
DIRECT INDIRECT
MECHANISM MECHANISM
• Phoduction of PGPR • ACC deaminase
IAA,cytokinin, activity
gibberellin • Low erhylene level
• Phoduction of Insoluble/organic phosphate ISR responses • Antioxidant
abscisic acid enzymes
• Phoduction of • Proline and
Solubilized phosphate Prevention of plant diseases
etylene quaternary amine
biosynthesis
Phytohormone production Tolerance to stress
Fig. 17.3 Direct and indirect mechanisms of plant growth-promoting rhizobacteria in finger millet plant
It has been reported that efficacy of PGPRs with respect to finger millet plant
development and growth relies upon the distribution, diversity, and population
dynamics in different areas. Members of Bacillus genus and their significance in
finger millet sustainable production have been studied and documented by many
researchers. Moreover, their eco-friendly biocontrol potential has considerably
increased over the past several years (Kumar et al. 2011; Agarwal et al. 2017).
Autochthonous and allochthonous properties and chemotactic colonizing properties
of Bacillus sp. associated with finger millet rhizosphere have been studied. Based on
the BS (Bonitur Scale) and VSD (Venn Set Diagram), two efficacious Bacillus
sp. strains, viz. Bacillus pumilus MSTA8 and Bacillus amyloliquefaciens
MSTD26, were isolated from rhizospheric soil. The application of these isolates
showed increased seed yield up to 37.87% and 16.45 harvest index (HI) in field and
laboratory trials (Dheeman et al. 2019). Bacillus spp. association has significant
impact on the productivity of finger millet crop. Inoculation of bacteria to finger
millet resulted in stress tolerance, secretion of extra cellular enzymes, signal
molecules, phosphate solubilization, siderophore production etc. (Dheeman et al.
2017). The Bacillus isolates inhibited Sclerotium rolfsii in finger millet crop by
secreting extra cellular enzyme chitinase and β-1, 4- glucanase which degraded the
cell wall of the causative fungus. Both the isolates possessed excellent plant growth-
promoting attributes and also produced secondary metabolites which were helpful in
crop protection and production (Dheeman et al. 2019). In another study, Singh and
Goel (2015) reported that Chryseobacterium sp. PSR 10 inoculation resulted in
enhanced plant growth, chlorophyll content, nitrate reductase in Eleusine coracana
(VLM-149), and solubilized phosphate under pot experiment. The PSB (phosphate
solubilizing bacteria) can also promote the growth of finger millet by providing more
phosphate to the plant, therefore, the PSB could be an imperative approach in finger
millet for enhancement of growth and yield. PGPR not only provides disease
resistance to the host plant, but also their association is capable of reducing the
effect of abiotic stress (Fig. 17.4). Chandra et al. (2018a) evaluated the efficacy of
ACC deaminase (1-aminocyclopropane-1-carboxylic acid) producing drought-
resistance bacteria (three isolates) on finger millet plant under stressed and
non-stressed conditions. These potential isolates possessed gene (structural gene
for enzyme ACC deaminase) and were able to reduce the plant stress hormone
ethylene level. These isolates were identified as Pseudonomas spp. Along with stress
mitigating property, inoculated bacterial strains improved plant growth and
nutritional content in leaves of finger millet as compared to control plant under
stressed and non-stressed conditions. During pot trials, bacterial isolates also
improved the antioxidant activity that protected plant from drought induced oxida-
tive impairment. According to the authors, they were the first to report the drought
tolerance effect of ACC deaminase producing bacteria on Eleusine coracana.
Reports on drought tolerance bacteria and their positive impact on host plants in
managing drought (other than finger millet) have also been documented in many
studies (Ahmad et al. 2011; Kumar et al. 2016a, 2016b).
ACC producing PGPB strains are to benefit the plant either as single inoculator in
consortia. Several drought tolerant rhizobacteria (Variovorax sp., Achromobacter
17
Arbuscular mycorrhizal fungi ABIOTIC STRESSES

ROLE OF PGPM
(in abiotic stress) Plant growth promoting rhizobacteria
Reducing electrolyte leakage and cellular

damage and enhancing post chilling
recovery
Increased evapotranspiration,
Redox modification / acidification
DROUGHT reduced germination, cell division
and photosynthesis rate
Lowering soil pH and increasing
metal sequestration Nutrient imbalance, disturbed
SALINITY ion homeostasis, membrane
damage
Inducing morphological changes in cell
structure and expression of heat shock water deficiency, elevated
proteins HEAT evaporation and deactivation of
photosynthetic enzymes
Balancing ion flux, reducing foliar Free radical formation, cellular

COLD damage, reduced photosynthesis
accumlation of toxic ions, elevated
nutrient uptake and decreased metal Eleusine coracana rate
toxicity
HEAVY Cytoplasmic enzyme inhibition,

METALS oxidative stress, protein and
Increasing relative and absolute water content and
apoplastic water fraction and reduced transpiration
cellular structure damage
Fig. 17.4 Schematic representation of abiotic stress effect upon Eleusine coracana and its mitigation through plant growth-promoting microorganisms
spp., Pseudomonas spp., and Ochrobactrum sp.) have plant growth-promoting

features. The synergistic effect of several drought tolerant rhizobacteria (Variovorax
sp., Achromobacter spp., Pseudomonas spp., and Ochrobactrum sp.) and their plant
growth-promoting features, uptaking nutrients and water have also been reported in
literature. The elevated level of cellular osmolytes and antioxidant enzymes and
reactive oxygen species (ROS) scavenging enzymes help in elimination of free
radicals and prevent damage to cell membrane and genetic material which ultimately
boosts the stress adaptation in the plant (Chandra et al. 2020). Chandra et al. (2018b)
focused on the plant growth, antioxidant efficiency, and nutrient composition
(sodium, potassium, calcium, phosphate, and nitrogen) of finger millet (var.
VL-149) plant under varied stressed and non-stressed conditions. They observed
that both type of inoculations, i.e., single or in consortia formulation had a remark-
able effect on root elongation, shoot length and shoot/root dry weight as compared to
the non-treated Eleusine coracana plants. Microbial consortium of Azotobac-
ter chroococcum (nitrogen-fixing bacteria), Bacillus megaterium (phosphate
solubilizing bacteria), and Pseudomonas fluorescens (PGPB) showed varied positive
effects (plant growth) on finger millet plant (Swapna and Brahmaprakash 2013).
Similar strategy of microbial combinations (Azotobacter chroococcum, Bacillus
megaterium, and Pseudomonas fluorescens) was used by Gangaraddi et al. (2020)
to evaluate their effect on the development and growth of Eleusine coracana plant
under greenhouse conditions. It was observed that finger millet plant height, leaf
size, chlorophyll content, and nitrogen uptake were more with application of con-
sortium as compared to control. Thanuja and Ambika (2010) screened plant growth-
promoting potency of five bacterial strains (RPHG1, RPHG2, RPHG3, RPHG4, and
RPHG5) isolated from rhizoplane of Holostemmaada-kodien (an endangered medic-
inal plant) on finger millet root, shoot length, and wet biomass at seedling stage. Out
of five isolates, only one (RPHG5) isolate was found to improve plant growth.
Abiotic stress is becoming a serious threat to crop production in many areas. Soil
salinity is one of the factors which is affecting the crop yield. Use of microorganisms
could mitigate the effect of soil salinity (Shrivastava and Kumar 2015). Figure 17.5
shows the rhizospheric effect of PGPM on nutrients uptake.
In another interesting work, Rai (1991) isolated three salt tolerant strains (STR1,
STR2, and STR3) of Azospirillum brasilense from the rhizosphere of
Eleucine coracana in calcareous saline soil and examined their tolerance capability
at varied concentrations of salts. Only one bacterial strain (STR1) was found to be
effective against higher salt concentration and showed credible growth of finger
millet plant. Table 17.2 exemplifies the different bacterial species showing various
mechanisms, which influences the different plant growth aspects.
17.4.2 Fungi Association with Finger Millet Rhizosphere
Fungi and plants symbiotic association has been known for years. Finger millet’s
rhizosphere has many diverse functional fungi (mycorrhizae), and their biotic
interaction under the soil plays a crucial role in the agricultural productivity.
17
PGPF
AMF
Increased root
PGPR Rhizospheric
Finger millet plant surface area
Fe
P
Zn
P
Cu
Zn
Mn
Cu
Mn
Fe
P
P
Mn
Cu Zn Mn
P
Mn Zn Cu
Zn
Zn
a Fe b
Fig. 17.5 Application of PGPM increases rhizospheric surface area and enhanced nutrient uptake. (A) Plant root without PGPM, unable to uptake nutrient, (B)
PGPM (rhizobacteria, arbuscular mycorrhizae fungi, and plant growth-promoting fungi) application and mycorrhizal network helps in enhanced root surface
area and improved nutrient uptake
Table 17.2 Plant growth-promoting bacteria, their mechanism and effect on finger millet plant
Bacterial species Mechanism Effect on plant References
Pseudomonas sp. Production of antimicrobial 1. Blast Sekar et al.
secondary metabolites and disease (2018); Ali
hydrolytic enzymes management, et al.
Biosynthesis of high growth (2009)
molecular weight protein promotion
molecules 2. Drought
stress tolerance
Bacillus sp. Nitrogen fixation, formation Yield Dheeman
of stress resistant endospores, improvement, et al.
secretion of extracellular foot rot disease (2019)
enzymes,
Chryseobacterium sp. Phosphate solubilization Increased plant Singh and
growth Goel
(2015)
Azospirillum brasilense Reduced activity of nitrate Salt tolerance Rai
reductase and nitrogenase, (1991);
increased uptake of K and Ca Hamdia
ions et al.
(2004)
Streptomyces cinnamomeous P solubilization and Improved P Krishna
mineralization content and et al.
plant growth (1982)
Arbuscular mycorrhizal fungus (AMF) is one of the contributing agents, which assist
the plant in nutrient uptake, promotes growth, and also helps in phosphorus mobili-
zation (Miyasaka et al. 2003; Srivastava et al. 2014). The omnipresent nature of
AMF and extra radial mycelium abundance in the soil is helpful in nutrient transport
even in harsh environment (i.e., low rainfall, drought). Srivastava et al. (2014)
isolated Glomus intraradices and Glomus etunicatum from the rhizosphere of
Eleusine coracana and examined their effect (root and shoot dry weight, mycorrhi-
zal infection, and phosphorus proportion) upon nine genotypes of finger millet. Both
species significantly promoted plant growth and nutrient uptake, and all the exam-
ined parameters ranged higher in AMF inoculated plants than non-inoculated plants
except two cultivars (named as 11 and 21). Glomus intraradices inoculation resulted
in higher shoot dry weight and shoot P content in all finger millet genotypes as
compared to G. etunicatum, although both the strains showed improved phosphorus
content. Arbuscular mycorrhizae application has also been reported to be beneficial
to crops in nutrient and water uptake under abiotic stress conditions. During water
stress condition, plant undergoes varied physiological changes (i.e., reduced chloro-
phyll content, low accumulation of secondary metabolites, oxidative damage of
biomolecules, and elevated level of ROS). Rhizophagus intraradices was found to
mitigate drought stress and induced tolerance when inoculated in finger millet
seedlings. Under severe drought stress, R. intraradices colonization with finger
millet rhizosphere led to significantly increased plant growth (7%), P and
chlorophyll amount (29%), higher amount of proline, soluble sugar, flavonoid (16 %
in roots), phenol content in leaves, 25% elevated level of ascorbate in leaves,
reduced lipid peroxidation, and higher level of glutathione (GSH 182%) as com-
pared to non-mycorrhizal control plants (Tyagi et al. 2018).
Common mycorrhizal network (CMN) formed by AM fungi and their root
colonization in Eleusine coracana and Pigeon pea showed interconnecting associa-
tion. During an experiment under water stress, AMF transferred the water from water
logged pigeon pea to water restricted finger millet roots (Singh et al. 2019; Saharan
et al. 2018).
In another report, Glomus mosseae found in the rhizospheric zone of finger millet
proved to facilitate acquisition of nutrients, P, N, and water, which resulted in
healthy growth of the host plant (Ramakrishnan and Bhuvaneswari 2014).
In another study, the varied effects of different VAM fungi (vesicular arbuscular
mycorrhizal fungi) on plant growth parameters of five finger millet genotypes were
investigated. Inoculation of Glomus caledonicum resulted in highest response on
finger millet genotype HR-374. This genotype showed enhanced P, N, Zn, and Cu
content in shoots, highest plant biomass, and mycorrhizal root colonization (Tewari
et al. 1993). Table 17.3 exemplifies the AMF species mechanism and effect on plant
growth aspects.
17.4.3 Synergistic Effect of Dual Inoculation (Bacteria and Fungi)

on Finger Millet
In plant rhizosphere, the microorganism population interacts positively or negatively

with each other. Similarly arbuscular mycorrhizal fungi (AM fungi) act synergisti-
cally with other microorganisms, and their combined inoculation has been found to
be beneficial for overall plant growth. Bama and Ramakrishnan (2010) found that
the dual inoculation of AMF and bacteria (Azospirillum) contributed in improving
crop growth and yield. Arbuscular mycorrhizal fungi elevated the phosphorus
mobilization to the plant, while Azospirillum stimulated nitrogen and other plant
growth attributes, which resulted in 36.8% higher yield in finger millet cultivar.
Krishna et al. (1982) reported that the phosphorus content and growth of finger
millet were enhanced when they inoculated the plant with VAM Glomus fasciculatus
and Streptomyces cinnamomeous. When these two microbes were inoculated indi-
vidually, they performed well in terms of enhancing plant growth and nutrient
acquisition as against dual inoculation.
In many studies, it has been reported that dual inoculation of fungi and bacteria in
rhizospheric area promotes plant growth and yield (Krishna et al. 1982; Bama and
Ramakrishnan 2010; Ramakrishnan and Bhuvaneswari 2014). Kamal et al. (2015)
isolated 104 bacterial strains and 96 actinomycetes from six different rhizospheric
zones of finger millet and among all the strains, they found Streptomyces labedae
(SB-9), Streptomyces flavofuscus (SA-11), Pseudomonas poae (KA-5), and Pseu-
domonas fluorescens (KB-7) to be more promising. The crop was inoculated singly
as well as dually with bacteria and fungi (Glomus intraradices) under water stressed
Table 17.3 Mechanism of AMF and their effect on finger millet plant
Fungal species Mechanism Effect on plant References
Glomus intraradices Hormone Plant growth, nutrition Srivastava
production, P uptake, higher shoot et al. (2014)
solubilization growth and shoot P
content
Glomus etunicatum Production of Plant growth, nutrition Srivastava
antifungal uptake, elevated et al. (2014)
metabolites, cellular phosphorus content
deformations in
fungal hyphae
Rhizophagus intraradices Minerals uptake and Increased plant growth, Tyagi et al.
transportation, P and chlorophyll (2018)
stress mitigation amount, higher amount
of proline, soluble sugar,
flavonoid, phenol
content in leaves, higher
elevated level of
ascorbate in leaves,
reduced lipid
peroxidation, and higher
level of glutathione
Glomus mosseae Nutrients P, N, water, and nutrients Ramakrishnan
mobilization acquisition and
Bhuvaneswari
(2014)
Glomus caledonicum Nutrients Enhanced P, N, Zn, and Tewari et al.
mobilization Cu content in shoots, (1993)
more plant biomass and
mycorrhizal root
colonization
Glomus fasciculatus Phosphate Enhanced phosphorus Krishna et al.
solubilization and content and plant growth (1982)
mineralization
and irrigated conditions. Among all the consortia, combination of S. labedae (SB-9)
with Glomus intraradices and P. poae KA-5 with Glomus intraradices significantly
enhanced higher plant height. Association of P. poae with P. fluorescens,
G. intraradices, and S.labedae with Streptomyces flavofuscus, G. intraradices
resulted in better performance under water stressed and normal conditions. All the
strains produced higher level of secondary metabolites. Glomus intraradices and
P. poae KA-5 consortium inoculation also resulted in higher content of antioxidants,
superoxide dismutase, and accumulation of proline in the leaves of finger millet
under water stressed environment.
The combined interaction of AMF (G. mosseae) and bacteria (A. chroococcum)
promoted plant growth via many mechanisms including nitrogen and phosphorus
mobilization in low fertility soils. Chandana and Venkataramana (2019) first
reported the interaction and synergy of phosphorus solubilizing bacteria
A. chroococcum and AMF G. mosseae on in finger millet crop. Azotobacter assists
Table 17.4 Effect of AMF, phosphate solubilizing bacteria on finger millet plant
Treatment Effect on plant (Eleusinecoracana)
AMF P solubilization and mobilization
AMF + Azospirillium brasilense Higher P acquisition, increased plant growth attributes,
spore number
AMF + Bacillus polymyxa Increased P uptake capacity of plant
AMF + Azospirillium brasilense Increased plant growth attributes, root colonization rate,
+ Bacillus polymyxa spore number, higher N and P content and uptake in roots
and shoots
in N fixation and hormone production, whereas AM fungi were helpful in P mobili-

zation, the consortia resulted in enhanced grain production, and their application can
reduce the application of N and P fertilizer up to 25-50 % without any affect upon
grain yield.
In another study, dual inoculation of Azospirillum and arbuscular mycorrhizae
fungi resulted in enhanced plant biomass of Eleusine coracana (Bama and
Ramakrishnan 2010). Ramakrishnan and Bhuvaneswari (2014) studied the effect
of inoculation of single or combined form of Glomus mosseae,
Azospirillium brasilense, and phosphate solubilizing bacteria Bacillus polymyxa on
finger millet. The output of the experiment is described in Table 17.4.
The beneficial fungi act synergistically with other microorganisms present in the
rhizosphere, including endophytic microbes. Rhizophagus intraradices has been
reported to associate symbiotically with endophytic fungus Piriformospora indica
in drought stress conditions and enhanced drought tolerance capacity of the finger
millet plant. Both the strains worked individually as well as in the association and
enhanced the secondary metabolites, chlorophyll content, antioxidant enzymes, and
reduction in lipid peroxidation, electrolyte leakage, malondialdehyde, and hydrogen
peroxide in finger millet plant (Tyagi et al. 2017).
17.5 Conclusion
Soil is regularly losing its fertility due to industrialization and environmental

stresses. Restoration of soil to usher in sustainable agriculture is of global concern.
Indiscriminate application of chemical fertilizers and pesticides has contributed to
deterioration of soil health and hence reduced the nutritive value of crops. Exploita-
tion of beneficial rhizospheric microbial population of finger millet could reduce the
application of chemical inputs to a certain extent. The popularization and use of
competent and effective microbes not only will pave the way toward sustainable
agriculture, but also to have a clean and green ecosystem. Consequently, dynamic
rhizospheric interactions with potential microbes will lead to a positive effect upon
the crop yield, and therefore, these microbes can be employed as biofertilizers/
biopesticides/plant stimulators. This will result in healthy and sustainable
agriculture.
17.5.1 Future Aspects
Various types of abiotic and biotic stresses affect the finger millet plant survivability,
growth attributes, and productivity. By expressing stress tolerant proteins, the crop
can sustain various stress conditions by modulating the biological and physiological
features. An alternate solution of stresses challenge in finger millet plants is to use
potent microbial approaches. This approach will certainly prove advantageous in
refining the present research scenario of finger millet. The employment of nutrient
mobilizing, stress tolerating consortium of plant growth-promoting microbes may be
applied to boost plant growth under abiotic and biotic stress condition. These
beneficial microbiome will stimulate finger millet plant growth by modulating
phyto-hormones, improving nutrition uptake, producing metal chelating
siderophore, and improving the antioxidant system. Induction of acquired systemic
resistance and induced systemic resistance during diverse stresses could also be
another approach by microbes. On the other hand, application of AM fungi might
play a role in enhancing the water and nutrients supply under stressful conditions.
Therefore, application of competent microbes has huge potential to resolve future
food security issues and also helps in maintaining the soil health. Finally, identifica-
tion and characterization of molecular mechanisms responsible for various finger
millet plant-microbe-interactions is significant to exploit these beneficial microbes in
agro-ecosystems.
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Diversity and Community Structure
of Arbuscular Mycorrhizal Fungi 18
in the Rhizosphere of Salt-Affected Soils
R. Krishnamoorthy, R. Anandham, M. Senthilkumar, and

Tongmin Sa
Abstract
Soil salinity is one of the major problems in agricultural production. Since saline
soil occupies more than 7% of the total land surface, it is expected to increase in
next few years due to the many environmental factors and anthropogenic
activities. Plant itself develops many adaptation mechanisms to overcome soil
salinity stress. Among many adaptation mechanisms exhibited by plants,
establishing symbiotic association with Arbuscular Mycorrhizal Fungi (AMF)
is economically more beneficial to the plants. AMF mitigate plant salinity stress,
and it has many adaptation mechanisms to survive under stress conditions. AMF
species belongs to the order Glomerales was documented to be dominated in the
salt-affected soil. Species of Glomerales order was reported to survive in various
environmental stress conditions. Glomerales diversity was found higher in salt-
affected soils compared to other three orders namely Diversisporales,
Archaeosporales, and Paraglomerales. Use of autochthonous (native), habitat
R. Krishnamoorthy (*)
Department of Crop Management, Vanavarayar Institute of Agriculture, Coimbatore, Tamil Nadu,
India
Department of Environmental and Biological Chemistry, Chungbuk National University,
Cheongju, Chungbuk, Republic of Korea
R. Anandham
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil
Nadu, India
M. Senthilkumar
Agricultural College and Research Institute, TNAU, Eachangkottai, Tamil Nadu, India
T. Sa
Department of Environmental and Biological Chemistry, Chungbuk National University,
Cheongju, Chungbuk, Republic of Korea

23, https://doi.org/10.1007/978-981-15-9154-9_18
454 R. Krishnamoorthy et al.
adopted (salt stress tolerant) AMF species may be selected to establish symbiotic
association with plants to improve growth and yield of plants in salt-affected soils.
Keywords
Arbuscular mycorrhizal fungi · Diversity · Glomerales · Soil salinity
18.1 Introduction
Soil salinity is one of the important abiotic stresses which play a significant role in
reducing plant growth and agricultural production worldwide. Out of 1.5 billion ha
of arable land in the world, 77 million ha are declared as unsuitable for plant growth
because of the soil salinity (Miransari 2016). Presences of salt in soil affect plant
physiological conditions due to higher accumulation of sodium and chloride ion in
plants. Effects of soil salt on plant growth are explained by many authors (Horneck
et al. 2007; Evelin et al. 2009; Ding et al. 2010). Plant adopts many mechanisms to
alleviate salt stress; among the mechanisms adopted by plants, establishing symbi-
otic relationship with arbuscular mycorrhizal fungi (AMF) is considered as one of
the effective mechanisms.
AMF are obligate symbiont which has the association with more than 80% of the
terrestrial plants. AMF adopt different mechanisms to help the plant to overcome salt
stress (Evelin et al. 2009). Even though AMF alleviate salt stress in plant, high
salinity is found harmful to AMF fitness, spore germination, and community struc-
ture. Soil salinity is one of the important abiotic factors which shapes soil AMF
community structure. AMF naturally occur in salt-affected soil might have devel-
oped adaptation mechanisms to overcome salt stress and perform better under salt-
affected soils. This chapter provides an overall view on AMF and how it mitigates
salinity stress in plants? What is the impact of salt on AMF colonization and
community structure?
18.1.1 Arbuscular Mycorrhizal Fungi (AMF)
In Greek, the term mycorrhiza means fungus root (mykes ¼ fungus, rhiza ¼ root),
which was first coined by A.B. Frank in the year 1885. Based on the fugal associa-
tion and structures, mycorrhizal is grouped into seven types viz., ectomycorrhiza,
endomycorrhiza, monotropoid, ericoid, arbutoid, orchid, and arbuscular mycorrhizal
fungi (AMF). Among these seven types of mycorrhiza, ectomycorrhiza and
endomycorrhiza (AMF) are more abundant and studied extensively (Allen et al.
2003). AMF are obligate symbiotic fungi which establish a relationship with plant
roots of more than 80% of the terrestrial plant species (Smith and Read 1997). In
earlier times, these endomycorrhizae are called as vesicular arbuscular mycorrhizal
(VAM) which was changed to AMF because of the members of this group do not
form vesicles (storage organ of mycorrhiza). AMF spores are called
18 Diversity and Community Structure of Arbuscular Mycorrhizal Fungi in the. . . 455
Fig. 18.1 Signaling, colonization, and nutrient uptake by AMF, (a), (b), (c), and (d) illustrate
stages of AMF colonization and nutrient transport
chlamydospores; spore germination, signaling between AMF and plants, penetrating

of hypha into plant roots, colonization, and nutrient exchange are illustrated in
Fig. 18.1.
This symbiotic relationship has a very long evolutionary history of more than
400 million years, which involves the plant and AMF signaling molecules. For
successful establishment of the symbiotic relationship, plant secretes the
Fig. 18.2 Arbuscules and vesicles of the AM fungi in plant roots
strigolactones, which trigger AMF spore germination, with response to plant signal-
ing molecules AMF produces the myc factor (lipochito-oligosaccharides) that helps
in the communication and successful establishment of the symbiotic relationship
(Akiyama et al. 2005; Maillet et al. 2011). In line with this, plant activates the
common symbiosis signaling pathway (CSSP) to accommodate the fungi in plant
roots and leads to calcium spiking in the roots. AMF attached to the roots surface by
the structure called hyphopodium, followed by formation of pre-penetration appara-
tus (PPA) occurs in the epidermal cells and leads to the cortical cells (Oldroyd 2013).
PPA helps in the formation of inter, intracellular hypha and arbuscules in the cortical
cells. Arbuscules are the site of nutrient exchange between plant and fungi
(Fig. 18.2). Arbuscules are finger like structure, based on the formation it grouped
into Arum and Paris. In Arum type, the hyphae and arbuscules grow along the
longitudinal intercellular air channels between the walls of the root cells, whereas in
paris type, arbuscules are formed in the intracellular spaces of the plant roots (van
Aarle et al. 2005). Vesicles are developed to accumulate the excess nutrients
absorbed by the fungal hypha (Fig. 18.2). Vesicles are formed by the hyphal
swelling, and these are formed in inter- or intracellular spaces of the plant cells.
18.1.2 AMF Mediated Plant Growth Promotion
AMF plays a key functional role in crop production and ecosystem sustainability.
These fungi colonize the plant roots and extend the hyphae beyond rhizosphere soil,
and the extended region is called as mycorrhizosphere (Fig. 18.2). Compared to
rhizosphere region, mycorrhizosphere region has more microbial activities.
Mycorrhizosphere helps in enhancing plant nutrient absorption, water retention
efficiency (Bedini et al. 2009), bio-control ability, improved secondary metabolite
synthesis, tolerance to abiotic stress, degradation of environmental pollutants
(Gamalero et al. 2009), and phosphate uptake. The beneficial role of AMF on soil
health is highly important for sustainable agriculture (Barrios 2007).
18.2 Soil Salinity and its Impact on Plant Growth
Soil salinity is defined as the concentration of water dissolvable salts extracted from
soil (Richards 1954). The water extractable salt contains cations (Na+, Ca2+, Mg2+,
and K+) and anions (Cl, SO42+, HCO3, CO32 and NO3) (Tanji 2002). Electrical
conductivity of the soil saturation extract was mostly used to quantify the salinity
(ECse). ECse is reported as milliSiemens/cm (mS/cm) or deciSiemens/m (dS/m,
equivalent to mmhos/cm). Soil salinity affects the plant in three ways one is creating
ionic imbalance in the cells because of the shortage of essential nutrients (N, P, K)
compared to salt ions. Second is drought stress, and third is ion toxicity (Evelin et al.
2009). Salinity also increases the production of reactive oxygen species (ROS) in
plant, which causes the oxidative damage in cells (Ding et al. 2010). These processes
ultimately affect the photosynthesis, osmotic potential of the cell, growth, and
protein synthesis. Naturally, plants have developed the intrinsic mechanisms like
production of ROS scavenging enzymes, increase aquaporin channels, and ion
homeostasis (Munns 2005).
United States department of agriculture classified the soil based on the soil ECse
as mentioned in Table 18.1 along with the plant response to each salt class. Salt
tolerant plants can be grouped into two types one is excluders and another is
includers. Excluders prevent excessive uptake of salt ions, while includers take up
more salt ions and store outside the cytosol (Munns 2002). Mechanical removal
(Horneck et al. 2007) of salt from soil or development of salt tolerant crops
(Wei-Feng et al. 2008) is economically not feasible, whereas use of microorganisms
like arbuscular mycorrhizal fungi may improve plant establishment and growth
under salt stress in economically feasible manner.
18.2.1 Salt-Affected Ecosystem
Establishing crops in salt-affected ecosystem (reclaimed lands) is gaining huge

attention in many parts of the world particularly in Asian countries. For the estab-
lishment of crops and restoration of vegetation in salt-affected reclaimed land, AMF
play important role. Many successful studies were conducted to enhance plant
growth in salt-affected soil by using AMF (Talaat and Shawky 2014;
Table 18.1 Soil salinity classification (USDA) and plant responses

Salinity Hazards for crop
class ECsea growth Plant responses
Non 0–2 Very low Negligible
Slightly 2–4 Low Restricted yield of sensitive crops
Moderately 4–8 Medium Restricted yield of many crops
Very 8–16 High Only few tolerant crops yield satisfactorily
Extremely >16 Very high Only few salt tolerant forage species grow
satisfactorily
a
Electrical conductivity of the soil saturation extract
Krishnamoorthy et al. 2016; Ma et al. 2019). Even though AMF enhancing plant
establishment and growth in salt-affected soil, their diversity and community
changes with different level of salt are still not well explained. Our group has done
a detailed survey of AMF root colonization and the community change of AMF
specie with respect to the soil salinity in one of the largest reclamation site named
Saemangeum which is located in South Korea (Krishnamoorthy et al. 2014).
Saemangeum is one of the reclamation sites which added 283 km2 of land and
118 km2 of lake to South Korea’s total geographical area. This reclamation site was
made by constructing a sea dike on the estuary of yellow sea for 33.9 km long, and
water was evacuated. The land areas were planned to use for multiple purposes like
agriculture, industry, tourism, global business, scientific research, new and renew-
able energy, ecological environment, and residential districts. Among these, higher
percentages of area are allotted for agricultural (30.3%). However, establishment of
crops in saemangeum reclaimed land is more challenging (Ryu et al. 2010). Soil in
this area has contained high amount of salt and low level of nutrients, which are not
suitable for crop cultivation (Yang et al. 2008).
18.2.2 Mitigation of Salt Stress by AMF
AMF have the ability to enhance the plant capacity to overcome the salt stress by
improving nutrient absorption, maintaining ionic balance, protection of enzyme
activities, accumulation of osmolytes, and facilitating water uptake (Fig. 18.3).
Under salt stress plant face problem in acquiring sufficient amount of water, a
process in which aquaporins participate (Luu and Maurel 2005). Aquaporins are
water channels, which regulate passive movement of water from soil to plant cells. In
plants, aquaporins are divided into four groups like plasma membrane intrinsic
proteins (PIPs), tonoplast intrinsic proteins (TIPs), noduline like intrinsic proteins
(NIPs), and small and basic intrinsic proteins (SIPs). Aroca et al. (2007) reported that
mycorrhizal inoculation increased aquaporin gene expression under salinity stress;
gene expression shows positive correlation with leaf relative water content. Soil
salinity significantly affects nutrient uptake especially phosphorus, because phos-
phate ion precipitate with Mg2+, Ca2+, and Zn2+. Salinity also interferes with
nitrogen uptake and utilization by interfering nitrogen metabolism. Higher Na+ in
soil leads to suppress the uptake of K+ ions, where Na+ competes with K+ for uptake
through common transport systems. AMF mitigate salinity stress by selective uptake
of nutrients from soil and prevent the toxic ion entering into the plants. AMF are
proved to have appositive influences on plant nutrient uptake under salt stress
(Sharifi et al. 2007). Hajiboland et al. (2010) demonstrated that inoculation of
Rhizophagus intraradices significantly increases N, P, and K uptake in tomato
under salt stress.
Proline, free amino acids, soluble proteins, and sugars are organic solutes, which
are involved in maintaining osmotic balance. Sheng et al. (2011) reported that
mycorrhizal inoculation increases the sugars, organic acid to overcome salt stress.
Similarly, the stress proline synthesis in leaves was decreased in mycorrhizal
Fig. 18.3 Effect of soil salinity on plants and salt stress alleviating mechanisms of AMF
inoculated plants under high salinity. Soil salinity affects the chlorophyll content by
affecting the uptake of Mg ions, which is important in the synthesizing specific
enzyme responsible for chlorophyll synthesis. Chlorophyll content of the plants can
be increased by mycorrhizal inoculation (Kumar et al. 2010). Reactive oxygen
species like singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl
radical are produced during normal metabolism. Ding et al. (2010) reported that
salinity stress increases the production of ROS in the cell compared to cell under
normal metabolism. These ROS in cells are scavenged by superoxide dismutase
(SOD), catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase, gluta-
thione peroxidase, and some of the non-enzymatic compounds like carotenoids,
glutathione, tocopherols, and ascorbic acid also scavenge oxygen radicals. Mycor-
rhizal inoculation can increase SOD, CAT, and APOX in plants grown under high
salt level (Abdel Latef and Chaoxing 2011).
18.3 AMF Phylogenetic Position and Classification
Before formation of Glomeromycota phylum, arbuscular mycorrhizal fungi were

placed in the phylum Zygomycota under the order Glomales. AM fungal hyphal
characters are similar to those of Zygomycota; however, 16S rDNA analysis showed
Table 18.2 Phylogenetic position and classification of AMF

Phylum
Glomeromycota
Class
Glomeromycetes
Orders (4) Families (12) Genera (34)
Glomerales Claroideoglomeraceae Claroideoglomus; Dominikia; Funneliformis;
Glomeraceae Glomus; Kamienskia; Oehlia; Rhizophagus;
Sclerocystis
Septoglomus
Diversisporales Acaulosporaceae Acaulospora
Diversisporaceae Corymbiglomus; Desertispora; Diversispora;
Otospora
Redeckera; Tricispora
Gigasporaceae Bulbospora; Cetraspora; Dentiscutata;
Gigaspora; Intraornatospora; Paradentiscutata;
Racocetra; Scutellospora
Pacisporaceae Pacispora
Sacculosporaceae Sacculospora
Archaeosporales Ambisporaceae Ambispora
Archaeosporaceae Archaeospora; Palaeospora
Geosiphonaceae Geosiphon
Paraglomerales Paraglomeraceae Innospora; Paraglomus
Pervetustaceae Pervetustus
unknown incertae sedis Entrophospora
affiliation
http://www.amf-phylogeny.com/amphylo_taxonomy.html (updated May 2018)
clear separation from the Zygomycota phylum and formed new phylum
Glomeromycota. Detailed information of the history of AMF classification and
phylogenic grouping is reviewed by Sturmer (2012). As it is first discovered,
AMF species are described based on the sporocarp, based on this first classification
was published by Gerdemann and Trappe (1974) with one order (Endogonales) and
four genera. Later Morton and Benny (1990) changed the order name to Glomerales
from Endogonales and included two more genera, which results in six genera under
the order Glomerales. New phylum of Glomeromycota was formed by Schuβler
et al. (2001) based on the available rDNA sequences of AMF. This new phylum
Glomeromycota consists of four order and nine genera. Few years later, Sieverding
and Oehl (2006) included two new genera (Kuklospora and Intraspora) in
Entrophosphora family. Schußler and Walker (2010) further updated the
Glomeromycota with 18 genera under 11 families in total. However, this classifica-
tion was not accepted by all due to the lack of live specimen availability for all
18 genera. AMF phylogeny is still updating based on the availability of the cultures
and their sequences. So far, 34 genera are accepted and are presented in Table 18.2.
18.3.1 Impact of Soil Salinity on AMF Symbiosis
Guo and Gong (2014) reported that soil salinity affects the AMF root colonization in
a salinized south coastal plain of Laizhou Bay, China. Recently, Parihar et al. (2019)
showed the negative correlation between soil EC and AMF spore count in saline
alkaline soils of India. Another negative correlation between soil salinity and AMF
colonization in rice plant was recently reported by Parvin et al. (2019). Impact of soil
salinity on AMF root colonization and AMF species dominance was given in
Table 18.3. Our study conducted in Saemangeum reclaimed land also revealed the
AMF root colonization reduction with increasing soil salinity level of the native
plant species (Krishnamoorthy et al. 2014).
18.3.2 Soil Salinity on AMF Community Changes
Two decades ago, spore morphology of AMF was used as an important tool to find
out the species name and diversity analysis. However, with the technology develop-
ment many novel techniques are implemented successfully in diversity analysis.
Among the molecular techniques, Terminal restriction fragment length
polymorphisms (T-RFLP), Denaturing gradient gel electrophoresis (DGGE), and
high-throughput sequencing are widely used in AMF diversity analysis.
Recently, Zhang et al. (2019) reported the community structure of the AMF in
Songnen Plain, which is the largest saline-sodic area in China with the help of high-
throughput sequencing techniques. Glomerales order was found to dominate in the
saline-sodic degraded grassland compared to other AMF orders. However, increas-
ing soil salinity concentration showed negative correlation to the AMF diversity
In our study, we have used salt-affected Saemangeum reclaimed soil for AMF
diversity analysis using T-RFLP technique (Krishnamoorthy 2014). In this study,
soil samples were collected from different salinity level and grouped into five
different classes based on USDA classification (Table 18.1). Total DNA from soil
sample was isolated using Power soilTM DNA isolation kit (MO Bio Lab., Inc,
USA). And the isolated DNA was amplified with two rounds using different sets of
primers. In First round of PCR amplification, primer LR1/FLR2 was used, and for
second round, FLR3/FLR4 was used to amplify the large subunit (LSU) of AMF.
For easy detection of sequences in T-RFLP analysis, FLR3 and FLR4 primers were
fluorescently labeled at 5’ end with FAM and HEX, respectively. Sequence size of
Terminal Restriction Fragment (T-RF) was determined using ABI 3130 DNA
sequencer (Applied biosystem, USA) with ROX 500 (Applied biosystem) as a
standard. T-RF size of 40–400 bp only used to analyses AMF diversity.
FLR3/FLR4 primers used in our study were proved to amplify the LSU of
Glomus, Funneliformis, Acaulospora, Gigaspora, Entrophospora, and
Scutellospora. Number of terminal restriction fragments (T-RFs) obtained from
AluI and MboI digestion ranged from 11 to 96 and 5 to 31, respectively. Of the
T-RFs obtained from AluI digestion, 37% of the fragments were not observed in soils
462
Table 18.3 Dominance of arbuscular mycorrhizal fungi in salt-affected soils

Salinity level Colonization with
EC (dS/m)/% Crop/soil Country increased salinity Dominant AMF species/order References
1.6–25 dS/m Rice root and soil Bangladesh Decreased Glomerales Parvin et al.
(2019)
0.02–2.62 dS/ Soils of Leymus chinensis China NG Glomus, Rhizophagus, Septoglomus and Zhang et al.
m dominated grass land Funneliformis (2019)
0.05–0.85 dS/ Plant root and soil India Decreased Rhizolglomus fasciculatum, Parihar et al.
m Funneliformis mosseae (2019)
0.1–12.8 dS/m Seepweed, couch grass, China NG Glomerales Cui et al. (2016)
and corn
0.03–28 dS/m Arthrocnemum sp. plant West Decreased Gigasporaceae, Glomaceae and Afaf et al. (2015)
root and soil Algeria Acaulosporaceae
0.1–5.4 dS/m Plant root and soil India Decreased NG Mohan and
Menon (2015)
1.82–4.95 dS/ Tamarix articulata Vahll Algeria Decreased Funneliformis mosseae, Bencherif et al.
m rhizosphere Funneliformis geosporum, (2015)
Funneliformis coronatum, Rhizophagus
fasciculatus and Gigaspora gigantea
0.6–31.5 dS/m Plant roots and coastal South Decreased Funneliformis caledonium, Krishnamoorthy
reclamation land soil Korea Funneliformis mosseae and Rhizophagus et al. (2014)
proliferus
1–18.5% Plant root and soil sample China Decreased G. intraradices, G. fasciculatum and G. Guo and Gong
irregulare (2014)
NG—Not given
R. Krishnamoorthy et al.
Fig. 18.4 (a) Abundance of terminal restriction fragments (T-RFs) and AMF ribotype corresponds
to fragment size of Alu1 digestion. (i) Non, (ii) Slightly, (iii) Moderately, (iv) Very, (v) Extreme
saline. (b) Abundance of terminal restriction fragments (T-RFs) and AMF ribotype corresponds to
fragment size of Mbo1 digestion. (i) Non, (ii) Slightly, (iii) Moderately, (iv) Very, (v) Extreme
saline
Fig. 18.4 (continued)

with ECse more than 4. Interestingly, 21% and 4% of the fragments were found in
salt level ranges from <2 to 8 and <2 to 16 ECse, respectively. In addition to this,
12% of the unique fragments were observed in all salt classes, 10% of the fragments
observed in soils with ECse > 8, and remaining 26% of the fragments were
inconsistent. AMF richness was found to be high in moderately and slightly saline
conditions for AluI and MboI digestion. Species richness showed negative correla-
tion (r2 ¼ 0.1178, P ¼ 0.025) to soil salinity, in contrast to richness and diversity
indices which were not significantly different among salt classes.
Abundance of T-RF ranges from 40 to 400 bp is presented in Fig. 18.4, and
abundance pattern was found to change with respect to salt classes. Possible ribotype
of 50 T-RFs from AluI and MboI digestion was identified using MiCA (Fig. 18.4a,
b). Funneliformis constrictum (304 bp), Glomus sp. WUM3 (305 bp),
Funneliformis caledonium (307 bp and 308 bp) were found only under non saline,
slightly and moderately saline conditions. Interestingly, Funneliformis mosseae
(183 bp), Rhizophagus proliferus (152 bp), Glomus sp. MUCI43207 (182 bp), and
Funneliformis geosporum (187 bp) ribotypes are found in most of the salt classes
(at least four). Two species belong to the family Gigasporaceae [Scutellospora
sp. hr9 (125 bp); Gigaspora rosea (127 bp)] and one uncultured glomeromycete
(55 bp) were found only in soil samples with ECse more than 4. Abundance of
Scutellospora sp. hr9 and uncultured glomeromycete was increased as salt level
(ECse) increased above 4. In addition to this, MboI digestion revealed the presences
of Funneliformis coronatum (105 bp) only in high salt levels (>8 ECse) (Fig. 18.4b).
Interestingly, Rhizoglomus fasciculatum was found in the strongly saline alkaline
condition while Funneliformis mosseae was well distributed under slightly (22.99%)
to moderately saline alkaline condition (35.78%) of saline soil found in Varanasi,
India (Parihar et al. 2019).
T-RFLP-based community structure analysis revealed that ribotype pattern dif-
fered with respect to soil salinity level. Out of 100% ribotypes obtained, only 12%
were observed in all samples and these ribotypes might have an adaptation mecha-
nism to overcome salinity stress. But remaining 88% percent of the ribotypes vary
with respect to soil salinity. Ribotypes with lack of tolerance mechanism were only
present in low saline soil (ECse < 8) and disappeared in elevated salinity levels
(Fig. 18.4a, b). Particularly, F.caledonium was found to be dominated only in low
saline soil. This species might not have an adaptation mechanism to overcome high
soil salinity. Similarly, dominance of Funneliformis genus in rhizosphere soil of
black locust (Robinia pseudoacacia) plant grown in the salt-affected area of China
(Sheng et al. 2019). Contradict to this, dominance of F. caledonium in yellow river
delta was reported, where the soil salinity was 40 dS/m (Wang et al. 2004).
Ribotypes present in high salinity level might have developed a tolerance mecha-
nism to salt stress, and this could possibly suppress the sensitive ribotypes.
Verbruggen et al. (2012) showed that only tolerant AMF species could survive in
environmentally stress conditions by suppressing sensitive ones. Competition
between tolerant and sensitive ribotypes may lead to the succession process, where
sensitive ribotypes are suppressed and tolerant ribotypes are dominated. Due to this
process of succession, the richness and diversity indices have not changed severally
with respective to soil salinity. Similarly, the allelopathic effect of invaded garlic
mustard plants on AMF community in a forest ecosystem has also been observed
(Barto et al. 2011).
Abiotic factors of soil can alter the diversity of AMF over multiple generations
(Verbruggen and Kiers 2010). In line with this, soil salinity has had an effect on the
AMF abundance and diversity of the salt-affected site. Similar to soil salinity, other
soil abiotic factors are also found to influence the abundance and functions of AMF
(Oliveira et al. 2010). Species prediction for particular T-RF using the microbial
community analysis III (MiCA) tool was an easy and cost-effective method to find
out AMF community changes with respect to soil salinity. This online tool has been
widely used in bacterial diversity studies (Yi et al. 2009), but has been limited to
AMF diversity studies, because of the low number of sequences in databank, with
respect to T-RF size of 28S rDNA. However, in the current study, we managed to
obtain species name for 50 T-RFs, which are belonged to three different genera
(Glomus, Gigaspora, and Scutellospora). Interestingly, 58% of the identified
fragments were belonged to Glomus and eight percent of the identified fragments
belonged to Gigaspora and Scutellospora in T-RFLP analysis.
The recent report by Parvin et al. (2019) also showed the occurrence of order
Glomerales in rice grown in the salt-affected soils of Bangladesh. This indicates the
importance of AMF symbiosis required by the plants to alleviate any kind of abiotic
stress. The soil used by Parvin et al. (2019) was affected by both salts and heavy
metals. In line with this, Wang et al. (2015) documented the dominance of
Glomerales in China’s wetland paddy field was noted, and Watanarojanaporn
et al. (2013) reported the same in Thialan’s paddy fields. These reports clearly
indicate the dominance and wider adoptability nature of Glomerales in
microaerophilic/anaerobic soil conditions present in the paddy region.
Our analysis and several other recent reports clearly indicate the dominance of
order Glomerales in higher saline conditions and well distributed in the soil,
irrespective of the salt concentration. Species of order Glomerales is known to be
well adapted to various environmental stresses, in particular to soil salinity.
Looking in to the results of many studies, the order Glomerales of AMF was found to
be predominant in salt-affected soil and their colonization formation was also higher.
The use of native adopted species of Glomerales order could therefore be more
effective in the establishment of vegetation in salt-affected lands. However, due to
the lack of live specimens for all AMF species described so far is one of the major
drawbacks in AMF phylogenic classification and proper naming of the species in the
diversity analysis studies. In the future, having the live specimen and their genetic
information for all the species placed in the phylogeny and proper nomenclature of
the genus with species will only provide the accurate and reliable information on
AMF diversity analysis studies.
Acknowledgements This work was supported by basic Science Research Program through the
National Research Foundation (NRF) funded by the Ministry of Education, Science and Technol-
ogy (2015R1A2A1A05001885), South Korea. RK is pleased to acknowledge Indian Council of
Agricultural Research (ICAR), New Delhi, India for the doctoral programme in their International
Fellowship (IF). RK's tuition fees and other expenses were also provided by ICAR-IF.
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Beta-Glucanolytic Soil Actinomycetes:
Diversity and Applications 19
Lekshmi K. Edison and N. S. Pradeep
Abstract
Screening of economically important actinomycetes from soil habitats is still in

the infant phase because a major part of soil diversity is still unexplored.
Understanding the environmental topography and biodiversity factors before
isolating a potential microorganism with industrial applications is important.
Exploration of unusual ecological niches for the searching of distinctive enzyme
producing microorganisms is extremely interesting, and also leads to the
advancements in high-throughput screening programs. Microbial β-glucanase is
well characterized and economically important enzyme produced by a range of
microorganisms. Actinomycetes have been extensively documented as a source
of β-glucanolytic enzymes. Amongst the broad range of genera, Streptomyces are
the most dominant group of enzyme manufacturers. Underexplored and unknown
habitations are vital resources of rare actinobacterial strains. This chapter deals
with the better understanding of beta-glucanase secreting actinobacterial diversity
and their application.
Keywords
Glycosyl hydrolases · Brewing industry · Streptomyces · Western Ghats
L. K. Edison
Microbiology Division, KSCSTE-Jawaharlal Nehru Tropical Botanic Garden and Research
Institute, Thiruvananthapuram, Kerala, India
N. S. Pradeep (*)
KSCSTE-Malabar Botanical Garden and Institute of Plant Sciences, Kozhikode, Kerala, India

23, https://doi.org/10.1007/978-981-15-9154-9_19
472 L. K. Edison and N. S. Pradeep
19.1 Introduction
Nowadays the industries are looking for novel microbial strains with the intention of
producing efficient enzymes to meet the enzyme requirements in a sustainable and
eco-efficient manner. Innovative enzyme production platforms from potential
microbes provide more choice and uncover previously unexploited enzyme sources.
Streptomyces is the largest genus of phylum Actinobacteria, well characterized and
recognized as fundamental unhampered sources of novel compounds with vast
industrial applications (Sathya and Ushadevi 2014; Mukhtar et al. 2017). Streptomy-
ces is well documented as the predominant reservoirs of several bioactive
compounds (Terkina et al. 2006). At present, the existing 75% of natural antibiotics
are made by Streptomyces species (Khattab et al. 2016).
According to previous reports, Streptomyces produces a variety of carbohydrases
that hydrolyse many soluble and insoluble polysaccharides (Chater 1993).
According to Wu et al. (2018), actinobacteria are considered as better candidates
for isolating β-glucan degrading enzymes; however, very little have been explored
for potential screening and production of β-glucanase. Some Streptomyces spp.,
namely S. rimosus (Beyer and Diekman 1984), Streptomyces sp. Mo (Kurakake
et al. 2013), S. torulosus (Park et al. 2012), S. sioyaensis (Hong et al. 2002) and
S. matensis (Woo et al. 2014), have been recognized as endo-β-1,3-glucanase
producers. The exo-β-1,4-glucanase, a cellulolytic enzyme, activities have been
reported in S. coelicolor A(3) (Lee et al. 2018), S. reticuli (Walter and Schrempf
1996a) and S. thermocerradoensis I3 (Carolina et al. 2015). Actinobacteria are
abundantly distributed in all kinds of soils. The population is the highest in soil
surface layer and progressively decreases with depth. Actinobacteria are typically
leading soil microbes (Hill et al. 2011). Mostly beta-glucan degrading actinomycetes
strains are found in soil habitats and highly sensitive to waterlogged and acidic pH
soil conditions. Glucanolytic actinomycetes strains are important re-cyclers of soil
organic matters (Holmalahti et al. 1994). Many beta-glucanase producing
actinobacteria show antagonistic activities to phytopathogens like Pythium spp.,
Fusarium spp., Colletotrichum spp., etc. (Hong et al. 2002). This chapter briefly
explains the diversity and applications of beta-glucanase producing soil inhabitant
actinomycetes.
19.2 Actinobacteria
The phylum Actinobacteria is recognized as the foremost taxonomic group amid the
currently recognized main lineages within the Bacteria domain (Ludwig et al. 2012).
The majority are free-living and are extensively disseminated in terrestrial and
aquatic environments (Macagnan et al. 2006). The name actinomycetes derived
from Greek words aktis or aktin for ray and mukes for fungi, since the hyphae
grow by the combination of tip extension and branching. Conventionally, they were
considered as an intermediate form between bacteria and fungi. Actinobacteria
superficially resemble filamentous fungi, since they produce mycelia and reproduce
19 Beta-Glucanolytic Soil Actinomycetes: Diversity and Applications 473
by sporulation. However, like bacteria, the cells are thin with peptidoglycan cell wall
and prokaryotic nucleoid; moreover, they are vulnerable to antibacterial agents
(Barka et al. 2016). The adequate exclusive features delineate the Actinobacteria
into ‘Kingdom Bacteria’. They are aerobic, sporulating, Gram-positive bacteria with
high GC-rich genome and characterized with the non-septate distinctive substrate
and aerial mycelium.
The actinomycetes are pervasive microbial group broadly dispersed in nature
such as soils, fresh and saline water, and also in the mid-air (Srinivasan et al. 1991).
The majority are soil inhabitants (Kuster 1968), although they are broadly dispersed
in diverse extreme habitats including deep-sea vent sediments (Colquhoun et al.
1998), deepest areas of Mariana Trench (Pathom-aree et al. 2006), Antarctic deep-
freezing soils (Moncheva et al. 2002) and also in deserts (Diraviyam et al. 2011).
Especially they are largely found in the superficial layer of soils with alkaline and
rich organic matter, and gradually decrease when increasing the depth (Takahashi
and Omura 2003).
The phylum Actinobacteria included in the fourth and fifth volumes of Bergey’s
Manual of Determinative Bacteriology represent the largest taxonomic units includ-
ing 5 subclasses, 6 orders and 14 suborders (Ludwig et al. 2012). All actinomycetes
are encompassed in the order Actinomycetales and divided into four families—
Actinomycetaceae, Streptomycetaceae, Mycobacteriaceae and Actinoplanaceae
(Williams et al. 1989). The two generally defined actinobacterial genera are Strepto-
myces and Micromonospora. The genus Streptomyces is massively documented as
the major reservoirs of natural bioactive products such as antibiotics, enzymes,
immunosuppressive agents, antitumor agents, etc. (Terkina et al. 2006).
In the actinomycetes, genus Streptomyces was first introduced in 1943 by
Waksman and Henrici (Williams et al. 1983). Concerned with the number and
species diversity, Streptomyces signifies the largest taxonomic unit in actinomycetes
(Bhattacharyya et al. 1998). They are Gram-positive, aerobic and non-acid fast
bacteria with around 70% GC content in genome (Dehnad et al. 2010). Currently
more than 500 species of the Streptomyces genus have been defined (Mohanraj and
Sekar 2013) and also unveils extensive phylogenetic spread (Aderem 2005). Strep-
tomyces is the only morphologically diverse actinobacteria which is found copiously
in soil habitats, plays a major role in carbon recycling by the production of hydro-
lytic exoenzymes. Due to the production of the spectacular diversity of secondary
metabolites, Streptomyces is considered as ‘chief competent chemists in nature with
boundless interest in industry and medicine’ (Hopwood 2007).
The actinobacterial members, especially Streptomyces, are the dominant
producers of innumerable antibiotics, secondary metabolites and industrial enzymes.
They have the ability to biotransform and bioconvert certain organic compounds,
urban and agricultural wastes into economically valued products. Streptomyces, the
promising reservoir of a diverse collection of industrially important enzymes,
actively participate in the putrefaction of various organic compounds and these
enzymes are widely exploited in food, leather, detergent, textile, pharmaceutical
and medical industries (Salwan and Sharma 2018).
To encounter the increasing demand for industrial enzymes, there is a need for
endless research and innovations to find novel highly efficient enzymes with cost-
effective and environment-friendly production. Further, the large accessibility of
genomic and proteomic data leads to the high-throughput production of high-value
enzymes from Streptomyces strains. Numerous genes responsible for the hydrolysis
of the polysaccharides recognized in Streptomyces genome indicate that microbes
are proficient producers of glycosyl hydrolases (GH) and decompose the complex
polysaccharides which is vital to carbon cycle of earth and animal nutrition
(Talamantes et al. 2016).
19.3 Beta Glucanase: A Glycosyl Hydrolase (GH)
Glycosyl hydrolases [(GHs) E.C.3.2.1.x] comprise enzyme groups, which hydrolyse

the glycosidic bonds within carbohydrate molecules or between carbohydrate and
non-carbohydrate molecules. They are actively researched groups of enzymes, have
distinguished applications in the food industry, biomass degradation, bioethanol
production, waste processing and also in pest control (Fulop and Ponyi 2015).
GHs have remarkable functional diversity and are extensively distributed prokary-
otic, eukaryotic, and also in Archaea with multiple copy number in each organism.
GHs are categorized primarily based on the substrate specificity and further classi-
fied according to the sequence similarities, catalytic mechanisms and hydrophobic
cluster analysis.
The first GH structure, resolved around 30 years ago, was hen egg-white lyso-
zyme. Currently, the GHs are grouped into 140 families (Cantarel et al. 2009; Abe
et al. 2017), deliver an understanding of the comparative structural features, evolu-
tionary relationships and mechanisms of action (Sathya and Khan 2014). The
updated list of GH families is available in Carbohydrate-Active Enzymes database
(CAZy). GHs have a segmental structure comprising a catalytic domain containing
active site residues and one or two non-catalytic domains which participate in
substrate binding, for example, carbohydrate binding domain (Davies and Henrissat
1995).
β-Glucanases are one among the major GHs, hydrolyse the insoluble or partially
soluble β-glucan substrates and release D-glucose through acid catalysis by proton
donor and a nucleophile or base. As in typical GHs, they have multi-domain
architecture. They generally act as exo- and endo-hydrolases. The endo–βglucanase
randomly cleaves the internal β-glucan chain and releases short oligosaccharides.
The exo-β-glucanase acts on the non-reducing terminals of polypeptide chains and
releases glucose units. According to the cleavage of glycosidic bonds, additionally,
they are categorized as β-1,4-glucanases, β-1,3-glucanases, β-1,2-glucanases,
β-1,6-glucanases, β-1,3-1,4-glucanases, etc. (Dake et al. 2004; Abe et al. 2017).
The β-1,4-glucanases are a common cellulosic type enzyme, whereas others
recognized as non-cellulosic types. Microorganisms like fungi, bacteria and plants
actively synthesise extracellular or intracellular enzymes. This is significant in
choosing specific substrates for the assay of these groups of enzymes. Till now, no
β-specific glucanases have been reported in the Archaea (Bauer et al. 1996). Based
on similarities in amino acid sequences the β-glucanases are categorized into several
GH families in the Carbohydrate Active enZymes (CAZy) (http://www.cazy.org/
Glycoside-Hydrolases.html, accessed on 10-04-2020) database.
The β-glucanases have appreciable ecological implication and commercial usages
in well-established biotechnological industries, for instance, in brewing and wine
production, animal feed industry, coffee processing, waste treatments, textile indus-
try, biofuel production and also in agriculture (Annamalai et al. 2016a). In brewing
industry, it efficiently degrades the β-glucan contents in cereal grains, thereby
increasing the wort filtration rate and reducing the chance of β-glucan precipitation
and also decreasing the mash viscosity and turbidity (Celestino et al. 2006). During
winemaking, the addition of β-glucanase improves colour extraction, must
clarification, skin maceration, filtration, and ultimately wine quality and stability
factors (Singh et al. 2007). In poultry and animal feed enzyme industry, cereal
β-glucan rich feed produces a viscose digesta, which impede in nutrient release
and absorption. Hence the addition of β-glucanases in barley rich animal feed
reduces the anti-nutritive effects of β-glucan, thereby increasing digesta viscosity,
nutrient release and feed passage rates (Choct 2001; Mathlouthi et al. 2002). In the
textile industry, it is used for cotton softening, denim finishing, colour care and
cleaning (Cherry and Fidantsef 2003). In agriculture sectors, the β-glucanases are
used as a biocontrol agent since it is capable of degrading cell wall of plant
pathogens, weaken the seed endosperm, and enhances seed germination, and also
helps for preparation of plant and fungal protoplasts applicable in various research
purposes (Bhat 2000).
19.4 Beta-Glucanolytic Actinomycetes: Diversity
The screening of β-1,3-glucan degrading microorganisms was carried out by

collecting the bulk soil samples from P. cocos cultivation areas of Liu’an City,
Anhui Province, China. P. cocos is a fungus with 90% β-glucan by dry weight which
grows near the pine tree roots. The high-throughput sequencing identified 18.64% of
isolates were actinobacteria and β-1,3-glucanase activity has been evaluated among
the actinomycetes. Out of 58 isolated actinomycetes strains, only 11 indicated the
ability to degrade glucan. Here, ten isolates were recognized as the genus Strepto-
myces through 16S rRNA gene sequence analysis. Those were S. capoamus,
S. cellostaticus, S. coelescens, S. cinerochromogenes, S. viridochromogenes,
S. indiaensis, S. olivogriseus, S. filipinensis and Kitasatospora phosalacinea
(Wu et al. 2018).
The actinomycetes strain Streptomyces sp. EF14 isolated from the rhizosphere of
potato showed antagonistic activity against root rot disease caused fungus
Phytophthora fragariae var. rubi (a pathogen of raspberry root rot). The strain has
hydrolytic effect on β-1,3-, β-1,4- and β-1,6-glucans by producing β-1,3-glucanase,
β-1,4-glucanase and β-1,6-glucanase (Valois et al. 1996). Actinobacteria isolated
from herbal vermicompost and chickpea rhizosphere soils observed their activities
Table 19.1 List of actinobacteria producing different beta-glucanases

Actinomycetes strains Beta-glucanase enzymes Reference
Streptomyces albogriseolus 1,3-Beta-glucanase, 1,4-beta- Van Zyl 1985
glucanase
Streptomyces gancidicus Exoglucanase, endoglucanase Jeffrey and Azrizal
2007
Streptomyces viridobrunneus Endoglucanase Da Vinha et al.
SCPE-09 2011
Streptomyces drozdowiczii Endoglucanase de Lima et al. 2005
Streptomyces reticuli Exo-1,4-beta-glucanase (Avicelase) Walter and
Schrempf 1995.
Streptomyces sp. EF-14 1,6-Beta-glucanase Fayad et al. 2001
Streptomyces rimosus l,3-Beta-glucanases Beyer and Diekman
1984
Streptomyces murinus l,3-Beta-glucanases Koki et al. 1978
Streptomyces matensis Endoglucanase and exoglucanase Prasad et al. 2014
Streptomyces Exo-1,4-beta-glucanase (Avicelase) El-Naggar et al.
viridiochromogenes 2011
Streptomyces sioyaensis Endo-1,3-beta-glucanase Hong et al. 2002
Streptomyces sp. G12 Endo-1,4-beta-glucanase Cecchini et al. 2018
Streptomyces angustmyceticus Beta-l,3-glucanase Wonglom et al.
NR8-2 2019
Cellulomonas biazotea, Endo-beta-l,4-glucanase Thayer et al. 1984
Cellulomonas cartae
Micromonospora melanospora Endo-beta-l,4-glucanase, exo-beta- Eida et al. 2012
l,4-glucanases
Nocardiopsis aegyptia, Endo-beta-l,4-glucanase, exo-beta- El-Sersy et al. 2010
Streptoverticillium l,4-glucanases
on plant growth promotion in chickpea. Among the 89 actinomycetes screened,

4 strains showed as most promising to produce β-1,3-glucanase which helps in
antagonism against fungal pathogens of chickpea (Sreevidyaa et al. 2016).
According to Javmen et al. (2012), Actinomyces rutgersensis 88 have the ability to
produce laminarinase (β-1,3), gentibiosinase (β-1,6), licheninase (β-1,3; 1,4) and
β-glucanase (β-1,3; 1,6), thus confirmed as the promising candidate for efficient
β-glucan extraction from Saccharomyces cerevisiae yeast cell walls (Javmen et al.
2012). The production of different beta-glucanases from different actinobacteria is
shown in Table 19.1.
19.5 Western Ghats in Kerala: Excellent Resource

of Beta-Glucanolytic Soil Actinomycetes
Western Ghats regions in India are one of the most incredible places containing
treasurable natural resources of the earth (Gadgil 1979). It has a protected and well-
preserved unique biodiversity. The Western Ghats in India signifies geomorphic
Streptomyces althioticus Streptomyces cinereoruber Streptomyces djakartensis Streptomyces drozdowiczii

TBG-MR17 subsp. cinereoruber TBG-AL14 TBG-AL19
TBG-AL13
Streptomyces tanashiensis Streptomyces violascens Streptomyces zhihengii Streptomyces racemochromogenes

TBG-CH22 TBG-MR3 TBG-MR7 TBG-NR3
Fig. 19.1 Diverse Streptomyces isolates from Western Ghats regions with β-glucanase activity
features of enormous importance with a unique ecological niche, exclusively with

mountain ranges starting from central Maharashtra and extending to the extreme
southern point of Kerala. It is a tremendously distinguished biodiversity hot spot of
gorgeous flora and fauna and is recognized by UNESCO as a World Heritage Site
(Ruckmani and Chakrabarti 2011). Moreover, the dynamic rich biodiversity, nearly
all regions are enduring unexplored, uninterrupted and are massively treasured with
prospective microorganisms. Hence the Western Ghats areas are also mentioned as
microbial biodiversity treasure houses (Nampoothiri et al. 2013).
Currently, the investigation for isolating unique and effective microbial strains
from the unexplored ecological habitats everywhere in the world remains to be an
important part of the research. Microbial studies focussed on extreme environments
can be exploited to produce innovations in enzyme technology that may become
future preface of green biotechnology. The uniqueness in environmental condition
certainly affected the Streptomyces distribution (Mitra et al. 2008; Jagan Mohan
et al. 2013). As a result of extensive screening of actinomycetes populations from
these natural and well-treasured habitats have been commercially exploited (Arasu
et al. 2008; Balachandran et al. 2015; Rao et al. 2015).
Study conducted for exploring β-glucanase enzymes producing actinobacterial
diversity from the 10 diverse unexplored unusual and ecological habitats in Western
Ghats regions of Kerala have led to the identification of 127 morphologically
dissimilar actinomycetes strains (Fig. 19.1). Predominantly, the selected regions
were unexploited, unique natural forest and mountain areas, viz. Munnar, Chinnar,
Nelliyampathy, Agasthyarkoodam, Neriyamangalam, Anamalai, Wayanad,
Kulathupuzha, Palode and Marayoor. The soil from Western Ghats areas have
enormously exploited for isolating the microorganisms in connection with
industrial-level production of economically important enzymes (Nampoothiri et al.
2013). Rhizosphere samples from mountain and forest regions are vast domicile of
taxonomically different actinomycetes strains particularly Streptomyces sp. The rich
recalcitrant biopolymers present in these soil samples make them energetically
involved in the forest nutrient turnover (Bontemps et al. 2013).
The study also envisioned to screen and quantify different β-glucanase enzymes,
endo-β-1,3-glucanase (Laminarinase) and exo-β-1,4-glucanase (avicellase or
cellobiohydrolase) from the isolated soil actinomycetes strains. Microcrystalline
cellulose or Avicel was used as a substrate for the screening of exo–β1,4-glucanase.
Previous reports suggested Avicel is the specific substrate for cellobiohydrolases or
exo-acting β-1,4-glucanase (Florencio et al. 2012; Annamalai et al. 2016b).
Exoglucanase secreted by Streptomyces reticuli capably utilizes Avicel (crystalline
cellulose), when Avicel is supplied as a sole carbon source (Wachinger et al. 1989;
Walter and Schrempf 1996b).
Out of 127 total strains isolated from Western Ghats soil samples, 106 Streptomy-
ces strains (83% of total strains) showed exo-β-1,4-glucanolytic activity and merely
79 strains (62%) exhibited endo-β-1,3-glucanolytic activity. Based on the obtained
enzymatic index values, 14 strains with exo-β-1,4-glucanolytic activity and 8 strains
with endo-β-1,3-glucanolytic activity were designated for quantitative evaluation of
enzyme production using submerged fermentation. Actinomycetes strain Streptomy-
ces althioticus TBG-MR17 exhibited highest exo-β-1,4-glucanase production
(95 U mL 1) and Streptomyces cinereoruber sub sp. cinereoruber TBG-AL13
showed maximum endo-β-1,3-glucanase production (219 UmL 1), after 5 days of
incubation (Edison and Pradeep 2020).
19.6 Conclusion
Western Ghat’s ecological habitats are diverse and almost unexplored. Microbial
populations from these distinctive niches remain rich reservoirs of valuable
metabolites and prospective to deliver a wide range of applications beneficial to
humanity. Exclusively fast-emerging enzymes prerequisite in different magnitudes
demand an urgent need to discover actinobacteria as a treasured resource of market-
able enzymes. Studies showed that Western Ghats soil ecosystems are peculiar
habitats for auspicious actinomycetes diversity with exceptional β-glucanase activ-
ity. Features such as large assortment of rich woodland areas, varying climatic
environments and fewer divergences in soil type with low acidic to alkaline pH
and less electrical conductivity are comparatively favourable for the distribution of
largest actinobacterial population with high β-glucanase activity.
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Microbial Diversity of Chickpea
Rhizosphere 20
Balram Sahu, Deep Chandra Suyal, Pramod Prasad, Vinay Kumar,
Anup Kumar Singh, Sonu Kushwaha, P. Karthika, Annand Chaubey,
and Ravindra Soni
Abstract
Chickpea (Cicer arietinum L.) is the third most important food legume in the
world; its annual production is nearly about 11.5 million tons with an estimated
land area of 14.56 million hectares. The chickpea is a multifunction crop and has
huge nutritional value. From a microbiological point of view, it is a legume crop
and harbor rhizobia in its root in the form of nodules. The chickpea also has a
diverse microbial population including both bacterial and fungal species. These
microbial communities especially bacterial genera play an important role in its
growth and protection. In this chapter, we will discuss these microbial
communities and their role in chickpea growth.
Keywords
Chickpea · Rhizosphere · PGPR
B. Sahu · A. K. Singh · S. Kushwaha · P. Karthika · R. Soni (*)

Department of Agricultural Microbiology, College of Agriculture, Indira Gandhi Krishi
Vishwavidyalaya, Raipur, Chhattisgarh, India
D. C. Suyal
Department of Microbiology, Eternal University, Baru Sahib, Himachal Pradesh, India
P. Prasad
Regional Station, ICAR-Indian Institute of Wheat and Barley Research, Shimla, Himachal Pradesh,
India
V. Kumar
ICAR-National Institute of Biotic Stress Management, Raipur, Chhattisgarh, India
A. Chaubey
Banda University of Agriculture and Technology, Banda, Uttar Pradesh, India

23, https://doi.org/10.1007/978-981-15-9154-9_20
484 B. Sahu et al.
20.1 Introduction
The word rhizosphere represents the surrounding regions of the rhizosphere which is
under the direct influence of the roots. The term was first introduced by Hiltner
(1904). This zone is considered, a zone of high microbial activity which is greatly
influenced by the root and its exudates (Pinton et al. 2001; Soni et al. 2016, 2017;
Suyal et al. 2015b, c). This region affects the biological, chemical, and physical
activities of the surrounding soil (Saharan and Nehra, 2011; Kumar et al. 2019).
Rhizosphere can be distinguished into ecto- and endo-rhizospheric regions. The term
endorhizosphere is generally describing a multilayered microenvironment, which
includes a mucoid layer on the root surface, the epidermal layer of the root tissue
including the root hairs and the cortical cells. Whereas, the ectorhizosphere is the soil
surrounds the root surface up to a few millimeters (Goel et al. 2017). However, it is
not easy to make a clear-cut boundary between rhizosphere soil and bulk soil, but a
usual separation of the rhizosphere and bulk soil can be obtained by shaking the
roots manually. Soil adhering to the root surface is the rhizosphere soil. Also, plants
may have a strongly adhering layer of root hairs. This layer is referred to as
rhizosheath, and also consists of mucilage material, microorganisms, and soil
particles (Goel et al. 2017; Joshi et al. 2017; Liu et al. 2019; Basirat et al. 2019).
The plant rhizosphere is an important soil ecological environment for plant-
microbe interactions. It involves several microorganisms interacting in different
manners viz. associative, neutralistic, parasitic or symbiotic (Suyal et al. 2014a;
Dash et al. 2019). These interactions depend on the type of microbes, plant defense
system, and soil nutrient status as well as the environment (Verma et al.2013; Giri
et al. 2015; Goel et al. 2017). Furthermore, the rhizosphere of leguminous crops
presents a unique ecological niche in which nodule formation is an important factor
of the metabolic activities; however, other inoculated microorganisms/PGPRs are
known to improve plant vigor and yield, especially in chickpea (Valverde et al.
2006; Verma et al. 2012, 2013; Joshi et al. 2019). PGPR and abiotic stress resistance
have been reported in several crops, including soybean, chickpea, and wheat (Kumar
et al. 2014, 2018; Ngumbi and Kloepper 2016; Goel et al. 2018; Rawat et al. 2019).
The application of PGPR has been reported to enhance the growth and development
of chickpea and other crops under natural field conditions (Rokhzadi et al. 2008;
Suyal et al. 2015a; Rajwar et al. 2018). These PGPRs are known to exhibit multiple
traits including HCN production (Joseph et al. 2007; Suyal et al. 2014b; Shukla et al.
2015). Jain et al. (1999) and Rudresh et al. (2005) have documented the advanta-
geous effect of PGPR towards chickpea plant growth and productivity.
20.2 Microbial Diversity of Chickpea Rhizopshere
20.2.1 Chickpea Rhizosphere
Chickpea (Cicer arietinum L.) is the world’s third most important food legume,
cultivated on about 11.5 million hectares in tropical, subtropical, and temperate
regions of the world. In context to feed nutritious food to the burgeoning human
20 Microbial Diversity of Chickpea Rhizosphere 485
population, pulses play a significant role, as these are rich in vitamin, mineral, and
protein (protein tablets). However, their production and productivity are still very
low in India. It contributed around 45% of the total annual production of the pulses
in the country. In the world scenario, 75% of total pulses are produced by India.
Among the states, Rajasthan, Maharashtra, UP, and Andhra Pradesh have
contributed around 14, 10, 9, and 7% of the pulses, respectively.
Chickpea is among the most grown legumes (Romdhane et al. 2007). It is known
to maintain symbiosis with Mesorhizobium ciceri (Khan et al. 2006). This crop plays
an important role in organic farming by adding the atmospheric nitrogen in the soil
through biological nitrogen fixation under nutrient limiting conditions. Ali et al.
(2015) have investigated the diversity of chickpea-associated rhizospheric
microorganisms which belonged to Bacillus, Chryseobacterium, Enterobacter,
Pantoea, Pseudomonas, Rhizobium, and Sphingobacterium. Moreover, 16S
rDNA-based analysis of chickpea endophytes revealed the dominance of Firmicutes
and Proteobacteria. Further, Actinobacteria, Enterobacter, and Pseudomonas were
among the most frequent endophytes. The proteobacterial members belonged to
Enterobacter, Klebsiella, Kosakonia, Rhizobium, and Pantoea. These were followed
by the members of Pseudomonadaceae and Xanthomonadaceae. Brígido et al.
(2019) and Maheshwari et al. (2019) have documented the lower abundance of
Klebsiella, Kosakonia, Leifsonia, Rhizobium, and Staphylococcus than Bacillus,
Enterobacter, Pseudomonas, and Stenotrophomonas. Similarly, 240 Bacillus strains
were screened by Singh et al. (2014a, b, c) based on their ability to produce indole
acetic acid, hydrolytic enzymes, and siderophores besides phosphate solubilization
potential. A similar study was carried out by Sreevidya and Gopalakrishnan (2017)
in which they have investigated the role of Bacillus species in the growth promotion
of chickpea. Pandey et al. (2019) have isolated chickpea-associated plant growth-
promoting strains of Azotobacter chroococcum, Bacillus pumilus, Bacillus subtilis,
and Pseudomonas aeruginosa. In another study, Joseph et al. (2007) have isolated
150 bacterial strains of Azotobacter, Bacillus, Pseudomonas, and Rhizobium from
the rhizosphere of chickpea. Now a days, metagenomics has emerged as a potential
tool to explore the agriculturally important microorganisms and their genes for
sustainable agricultural developments (Suyal et al. 2019a, c). It has also shown its
importance for investigating the diversity and community structure of rhizospheric
microorganisms (Goel et al. 2017, 2018). Some of the rhizobacteria reported from
chickpea rhizosphere are summarized in Table 20.1.
20.2.2 Rhizobial Species
Nitrogen is an important limiting nutrient for plant growth. Almost 78% of the gas
components in the environment are represented by nitrogen in molecular form (N2).
The Rhizobium is the main input to the fixation of symbiotic nitrogen in pulse crops.
Krusell et al. (2005) stated that there is a specific organ called nodules, those results
from rhizobial infection through symbiotic nitrogen fixation in legumes. Rogers
et al. (2009) recorded that a better atmospheric CO2 may be accepted to support the
486 B. Sahu et al.
Table 20.1 List of some chickpea rhizobacteria and their function in rhizopshere
Rhizobacteria Properties References
Stenotrophomonas maltophilia (with Nodulation, leghaemoglobin Abd-Allaa et al.
Rhizobium and VAM ) content, nitrogenase activity (2019)
during salt stress
Bacillus pumilus, B. subtilis, Growth promotion and biocontrol Sharma et al.
B. licheniformis, B. safensis, B. cereus traits (2019)
Azotobacter chroococcum, B. subtilis, PGPR and stress tolerance Pandey et al.
Pseudomonas aeruginosa, and (2019)
Bacillus pumilis
B. altitudinis, Pseudomonas Plant growth-promoting traits Baliyan et al.
chlororaphis (2018)
Serratia marcescens, Pseudomonas Antagonistic activity Palmieri et al.
fluorescens, Rahnella aquatilis, and (2017)
Bacillus amyloliquefaciens
Pseudomonas geniculata Plant growth-promoting traits Gopalakrishnan
et al. (2015)
P. aeruginosa P and K uptake Ahemad and
Kibret (2014)
P. aeruginosa Plant growth-promoting traits Yadav and
B. megaterium, Azotobacter antagonistic activity Verma (2014)
chroococcum
Lysinibacillus fusiformis Antifungal activity Singh et al.
(2013a, b)
Bacillus, Pseudomonas, Azotobacter, Plant growth-promoting traits Joseph et al.
Rhizobium (2007)
Pseudomonas striata Phosphate solubilizing bacterium Meena et al.
(2010)
Erwinia herbicola and Enterobacter Nitrate-solubilizing ability Rangeshwaran
agglomerans et al. (2008)
N2-fixation and growth of symbiotic N2-fixing crops when grown in the nonappear-
ance of any ecological constraints like drought or low temperature, nutrient defi-
ciency, and increasing CO2 levels may offer several defense mechanisms from
drought-induced and N2-fixation decrease (Suyal et al. 2017; Jeyakumar et al.
2020). Several cold-adapted PGPR are known to perform biological nitrogen fixa-
tion under cold stress conditions and contribute towards agricultural sustainability
(Suyal et al. 2017,2019b; Soni et al. 2015).
Furthermore, chickpea has been considered as a restrictive host for nodulation by
rhizobia (Fig. 20.1). Chickpea is reported as a restrictive host for rhizobial nodula-
tion (Suneja et al. 2016). Only two species of Mesorhizobium, i.e., M. ciceri and
M. mediterraneum are known to nodulate this plant (Nour et al. 1994, 1995).
However, Laranjo et al. (2004) and Rivas et al. (2007) have reported several other
species of Mesorhizobium which may nodulate chickpea effectively.
Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium are known to
possess chickpea-associated symbiovars viz.ciceri. This symbiovar has been
Fig. 20.1 A chickpea plant

nodulated with native rhizobia
documented in several Mezorhizobium species, i.e., M. amorphae, M. ciceri,

M. huakuii, M. loti, M. mediterraneum, M. muleiense, M. opportunistum, and
M. tianshanense (Nour et al. 1994, 1995; Rivas et al. 2007; Laranjo et al. 2008;
Alexandre et al., 2009; Rogel et al. 2011; Laranjo et al. 2008). Further, inoculation of
chickpea with Rhizobium leguminosarum bv. ciceri has known to improve salinity
tolerance in the plants (Ogutcu et al. 2010).
20.2.3 Root-Associated Bacterial Endophytes of Chickpea
Majority of the research conducted in chickpea related to plant growth-promoting

rhizobacteria (PGPR) included isolation of root and root nodule-associated
rhizobacteria. However, bacterial endophytes were also recovered from chickpea
root endosphere (Chhabra and Sharma 2019; Giri and Dudeja 2019). On the basis of
colonization, rhizobacteria are placed in three main groups, i.e., bacteria present in
rhizosphere soils, rhizoplane, and root endopshere (Goel et al. 2017). Kumar et al.
(2013) isolated endophytic bacterial isolates from roots and nodules of legume crops
namely chickpea, field pea, and lucerne. Moreover, endophytic bacteria from root
488 B. Sahu et al.
and root nodules of chickpea plant were characterized by several scientific groups
(Saini et al. 2015; Egamberdieva et al. 2017). It was found that the inoculation of
these bacterial alone or in combination with Mesorhizobium-enhanced plant growth,
nodulation, and nitrogen- fixing parameters in chickpea. Similar to the other
rhizobacteria, endophytic isolates were also characterized from chickpea nodules
having antagonistic properties against chickpea pathogen F. solani causing root rot
(Bahroun et al.2018).
More recently, Brígido et al. (2019a) characterized bacterial endophytes from
chickpea root tissues grown in different soils. The diversity of endophytes showed
that the common bacterial genera were Proteobacteria, Firmicutes, and
Actinobacteria, with Enterobacter and Pseudomonas. Isolated endophytes were
also showed plant growth promoting activities including IAA and ammonia produc-
tion and also exhibited tolerance to salt and manganese (Mn) The functional
characterization of Pseudomonas sp. and Kosakonia sp. showed improved chickpea
mesorhizobia symbiotic performance under control and stress conditions (Brígido
et al. 2019b). Similarly more endophytes like Bacillus sp., Pseudomonas sp.,
Staphylococcus sp., Pantoea sp., Enterobacter sp., and Mixta sp. have been
characterized for plant growth promoting and antagonistic properties (Mukherjee
et al. 2020).
20.2.4 Actinobacteria of Chickpea
Actinomycetes are also an important components of chickpea rhizosphere microbial

populations and are useful in soil nutrient cycling. They are spore forming gram
positive bacteria, and generally characterized with substrate and mycelia growth.
Most of the actinobacteria isolated from chickpea rhizosphere (including rhizoplane
and root endosphere) belong to genus Streptomyces. Further, Streptomyces sp.
isolated from chickpea rhizosphere were reported for its antagonistic properties
against chickpea pathogens F. oxysporum f. sp. ciceris, M. phaseolina, and
S. rolfsii (Sreevidya et al. 2016). Similarly, Vijayabharathi et al. (2018) isolated
219 endophytic actinobacteria from chickpea rhizosphere and other parts of chick-
pea. Best three belongs to Streptomyces genus and have shown for antagonistic
potential against Botrytis cinerea, causal organism of Botrytis grey mold (BGM)
disease, in chickpea.
20.2.5 Chickpea Rhizosphere Fungi
Every plant has both fungi and bacteria in its rhizosphere along with some other
microbial groups. As per available reports, approximately 50 pathogenic fungal
genera inhabit chickpea rhizosphere but only few are causing serious harm to the
plant and cause various severe diseases. These pathogenic fungi are Ascochyta
rabiei, Fusarium oxysporum, F. solani, Sclerotium rolfsii, Rhizoctonia solani,
Phytophthora megasperma, Pythium ultimum,Operculella padwickii, Sclerotinia
Sclerotiorum (Nene and Reddy 1987). Out of these, Sclerotium rolfsii (collar rot),
F. oxysporum f. sp. ciceris (Vascular wilt), and Rhizoctonia bataticola (dry root rot)
are accountable for causing diseases in different stages (Gupta and Sharma 2015).
Mitra (1931) firstly reported the dry root rot disease in chickpea. Earlier it was
known as Rhizoctonia wilt and later it was named as dry root rot. It is caused by
necrotrophic fungi R. bataticola. This disease generally appears around the
flowering and podding stage. Disease destructs the lateral roots and caused extensive
rotting (Khaliq et al. 2020). In India, one of the major chickpea disease, i.e., fusarium
wilt (FW), caused by fungus, F. oxysporum f. sp. ciceris can cause 10–15% yield
annual loss (Sharma et al. 2016).The fungus go into the vascular system of the
chickpea plant via the roots system. Similarly, collar rot of chickpea is another
disturbing soil-borne fungal disease which causes 10–30% yield loss (Maurya et al.
2008). Further, one of the major pulse producing region in central India is the
Bundelkhand. This region comprises seven districts of Uttar Pradesh and six districts
from Madhya Pradesh state of India and Chickpea is one of the major pulse crop of
this area (Kumar et al. 2017). However, the production in this region does not
commensurate with the acreage. The main cause for this situation is infections by
several soil-borne fungal pathogens (Trivedi et al. 2017). Mainly, fusarium wilt, dry
root rot, collar rot, stem rot (Sclerotinia sclerotiorum), are major culprit behind the
yield loss of chickpea in this region (Arya et al. 2019). Further, it was reported that
rhizosphere fungi reduce the productivity of chickpea but there are some plant
growth-promoting fungi also inhabiting chickpea rhizosphere (Bazghaleh et al.
2015). El Hazzat et al. (2018) explored fungal diversity in the chickpea rhizosphere
which is represented by 22 species of mycorrhizal fungi.
20.3 Functional Properties of Chickpea Rhizobacteria
The chickpea forms nodules with symbiotic association with Mezorhizobium spe-
cies. This symbiont is the main nitrogen fixer for chickpea plants. In this section we
will discuss other chickpea rhizobacterial members having plant growth properties
other than nitrogen fixation.
It has been observed that Chickpea inoculation with PGPR and phosphorous-rich
compost have improved the nodulation, growth, and yield of the crop (Shahzad et al.
2008). The study conducted by Midekssa et al. (2016), demonstrated that PSB
isolates from chickpea rhizosphere belong to different bacterial genera:
Acinetobacter, Bacillus, Brevibacillus, Burkholderia, Chryseomonas, Enterobacter,
Empedobacter, Pseudomonas, Ralstonia, Sphingomonas, and Stenotrophomonas.
Similarly, diversity and community structure of Chickpea rhizospheric phosphate
solubilizing microorganisms were documented by various scientific groups (Peix
et al. 2001; Kundu et al. 2009; Singh and Tejo Prakash 2012; Solanki et al. 2018).
490 B. Sahu et al.
Further, phosphate solubilization potential of M. ciceri and M. mediterraneum

strains isolated from Chickpea was also documented (Parmar and Sindhu 2013).
The application of PGPR with P-enriched compost in an integrated manner is known
to improve the growth and yield of chickpea (Shahzad et al. 2008). According to the
previously reported work, strains isolated from C. arietinum are the best solubilizers
of phosphorus in liquid medium (Halder et al. 1990; Nour et al. 1994, 1995; Tomer
et al. 2016, 2017). Neelam and Meenu (2003) were isolated high tricalcium phos-
phate solubilizing capability of Pseudomonas sp. from the rhizosphere of Trigonella.
Furthermore, fungal species were also reported from chickpea rhizosphere having
P-solubilization ability. The most abundant genus is the Glomus. Likewise, Asper-
gillus, Penicillium, and Trichoderma isolates were also recovered from chickpea
rhizosphere soil and screened for plant growth promoting activities in chickpea plant
(Yadav et al. 2011). These fungal isolates were potent P solubilizer and IAA
producer.
20.3.2 Biotic Stress Management
Further, Kumar et al. (2016) isolated 28 different Bacillus sp. from the chickpea
rhizosphere. This Bacillus sp. has antagonistic properties against chickpea wilt
caused by Fusarium oxysporum. Similarly, Rangeshwaran and Prasad (2000)
obtained a rich population of P. fluorescens isolates and which can fully control
the chickpea wilt. Finding similar to these two studies have already been reported
one decade back by Landa et al. (1997) that rhizospheric bacteria from chickpea
mainly Bacillus and Pseudomonas sp. were best biological agents to control chick-
pea wilt caused by F. oxysporum. However, it was also reported that the Bacillus
species isolated from the Chickpea rhizosphere also helpful in controlling root rot in
chickpea itself (Singh et al. 2013a, b). In one more interesting study, it was revealed
that these rhizobacteria are synergistically supported by native Mesorhizobium
isolates in providing this antagonistic property (Kumari and Khanna 2020). There-
fore, some workers also used microbial consortia of chickpea rhizosphere microbes
to cure Fusarium wilt in chickpea (Palmieri et al. 2017). The microbial consortium
made from the chickpea-associated bacterial strains B. amyloliquefaciens,
P. fluorescens, Rahnella aquatilis, and Serratia marcescens is known to control
Fusarium more efficiently in comparison to the individual applications of the
respective strains. Furthermore, a consortium of chickpea rhizobacteria Pseudomo-
nas, Trichoderma, and Rhizbium also controls the biotic stress of collar rot (Singh
et al. 2013a, b). Similarly, four chickpea rhizobacteria, namely B. cereus,
Achromobacter xylosoxidans, B. thuringiensis, and B. subtilis were also helpful in
suppressing root rot disease in chickpea caused by F. solani (Egamberdieva et al.
2017). Some of the rhizobacteria having antagonistic properties are summarized in
Table 20.2. Not only bacteria, actinobacteria but also fungal antagonists like
T. harzianum, T. viride, and A. niger were isolated from the chickpea rhizosphere
(Srivastava et al. 2015).
Table 20.2 List of chickpea rhizosphere bacteria and their antagonistic behavior to phytopathogenic fungi
20
Effective against
Genera Species Pathogen Disease Reference
Bacillus Bacillus sp Fusarium oxysporum f. sp. ciceris, Chickpea wilt Landa et al. (1997)
Fo. phaseoli, and Fo. melonis
B. subtilis Fo. ciceris race 1 Chickpea wilt Singh et al. (2014a, b, c)
B. subtilis F. solani Chickpea wilt Singh et al. (2014a, b, c)
B. subtilis Macrophomina phaseolina Dry Root Rot of Chickpea Singh et al. (2014a, b, c)
Bacillus subtilis Fo. ciceris Karimi et al. (2012)
Bacillus sp. Fo. ciceris Chickpea wilt Landa et al. (2001)
Bacillus subtilis Fo. ciceris Chickpea wilt Fernández-Herrera et al.
(2019)
Pseudomonas P. fluorescens Fo. ciceris Chickpea wilt Landa et al. (1997)
P. cepacia Phytophthora megasperma f. sp. root rot of chickpea Myatt et al. (1993)
medicaginis
Microbial Diversity of Chickpea Rhizosphere
P. fluorescens Pm.medicaginis root rot of chickpea Myatt et al. (1993)

Pseudomonas sp. isolate NBRI9926 Fo. ciceris Chickpea wilt Nautiyal (1997)
Pseudomonas sp. isolate NBRI9926 Rhizoctonia bataticola Dry root rot in chickpea Nautiyal (1997)
Pseudomonas sp. isolate NBRI9926 Pythium sp. Seed rot in chickpea Nautiyal (1997)
P. putida, P. aeuroginosa Fo. ciceris Chickpea wilt Karimi et al. (2012)
P. putida, P. alcaligenes, Pseudomonas M. phaseolina Dry Root Rot of Chickpea Akhtar and Siddiqui (2009)
isolate (Ps28)
Pseudomonas sp. Fo. ciceris Chickpea wilt Landa et al. (2001)
Streptomyces S. maltophilia Verticillium dahliae wilt of oilseed rape Landa et al. (1997)
Rhizobium Rhizobium sp. isolate NBRI9513 Fo. ciceris Chickpea wilt Nautiyal (1997)
Rhizobium sp. isolate NBRI9513 R. bataticola Dry root rot in chickpea Nautiyal (1997)
Rhizobium sp. isolate NBRI9513 Pythium sp. Seed rot in chickpea Nautiyal (1997)
Paenibacillus Paenibacillus sp Fo. ciceris Chickpea wilt Landa et al. (2001)
491
Stenotrophomonas Stenotrophomonas sp. Fo. ciceris Chickpea wilt Landa et al. (2001)
492 B. Sahu et al.
20.3.3 Abiotic Stress Management
PGPR rhizobacteria are well known to possess multiple traits. Malik and Sindhu
(2011) have revealed that chickpea-associated Pseudomonas were able to produce
indole acetic acid (IAA) significantly when supplemented with L-tryptophan in LB
broth. Furthermore, the production of IAA by the chickpea-associated rhizobacteria
is known to contribute to plant growth promotion as identified through the green
fluorescent protein (GFP) reporter gene (Fierro-Coronado et al. 2014). Also, some
chickpea root-soil bacteria like Azotobacter, Bacillus, and Pseudomonas also proven
to protect the plant in drought conditions by producing ACC deaminase enzyme
(Pandey et al. 2019). This enzyme cleaves the 1-aminocyclopropane-1-carboxylic
acid (ACC) which is the precursor for ethylene production and ethylene is responsi-
ble for the early maturity of the plant during stress. Furthermore, Bacillus species are
well-known biological agents to improve the plant health and plant protection from
abiotic stresses (Radhakrishnan et al. 2017). A salt loving B. subtilis promotes
chickpea seed germination and plant growth by increasing the production of some
photosynthetic pigments, sugars, proteins, and some osmolytes like proline, glycine,
betaine, and choline, in salt-stressed chickpea crop (Qurashi and Sabri 2013).
Similarly, a consortium of three Bacillus species, i.e., Bacillus subtilis, Bacillus
thuringiensis, and Bacillus megaterium induced metabolic and physiological
changes in chickpea plant during drought conditions. They changed several amino
acids, amines, sugar alcohol, sugars, organic acids, fatty acids, and other intermedi-
ate compounds (Khan et al. 2019a, b). Further, Arbuscular mycorrhizal fungi (AMF)
has been reported to improve growth of chickpea under drought stress (Hashem et al.
2019). In addition, growth enhancement and levels of protein, Fe, and Zn were also
reported found in mycorrhizal chickpea (Pellegrino and Bedini 2014).
20.4 Microbial Consortia and Chickpea Growth
In another study, Nag et al. (2018) screened 86 isolates from the chickpea rhizo-
sphere, out of which 16 on Yeast extract Mannitol agar, 40 from the nitrogen-free
medium, 29 from media used for Azosprillum isolation. One PSB was also obtained
on selective culture media. In this study, they found that the potential chickpea
rhizobacteria were tested for their effect on chickpea seedlings. The results revealed
that rhizobacteria are performing better as compared to control (Fig. 20.2). Similarly,
the performance of consortium (Fig. 20.3) was observed in the pot experiment (Porte
et al. 2017). In continuation, Kushwaha (2020) in his master’s thesis conducted a
field experiment using one of the best consortiums originally isolated from the
chickpea rhizosphere. He observed that consortia performed better in all recorded
parameter including grain yield (Fig. 20.4). However, not only bacterial consortia
but also bacterial and fungal consortium also enhances the nutritional value in
chickpea plants (Yadav et al. 2017).
Inoculated Rhizobacteria isolated from Chickpea Rhizosphere
Uninoculated Chickpea
Seedlings
Fig. 20.2 Effect of Chickpea rhizobacteria on chickpea seedlings
Control
Control Control
100% NPK 75% NPK Consortium + 100% NPK
Fig. 20.3 Effect of chickpea rhizobacterial consortium on performance of chickpea in pot

experiment
Not only PGPR activities, chickpea rhizobacterial consortia also used to reduce
abiotic stress in the plant. In a report, the synergistic effect of B. amyloliquifaciens
and P. putida was observed in mitigating drought tolerance in chickpea (Kumar
et al. 2016). Khan and Bano (2018) used plant growth regulators salicylic acid
(SA) in combination with chickpea rhizobacteria viz. B. subtilis, B.thuringiensis,
and B.megaterium for phytoremediation of heavy metals. These PGPRs further
increased the accumulation of heavy metals and macronutrients in chickpea shoot
and the rhizosphere. They recommended these PGPR for phytoremediation in
chickpea. Nonetheless, chickpea root-soil bacteria are also used for bioremedia-
tion of chromium (Shreya et al. 2020). A similar application has been done to
improve the growth and yield of chickpea under salinity stress by Riaz
et al. (2019).
494 B. Sahu et al.
Fig. 20.4 Performance of chickpea after inoculation with rhizobacterial consortium in field
condition
Acknowledgments The author RS acknowledge the financial assistance provided by the

Chhattisgarh Council of Science and Technology, Raipur for his research cited in this chapter.
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The Rhizosphere Microbiome and Its Role
in Plant Growth in Stressed Conditions 21
Bhrigu Bhuyan, Sourav Debnath, and Piyush Pandey
Abstract
The rhizosphere is a region where microorganisms exist within the soils and grow
in direct influence of the plant root system. Rhizosphere plays a vital role in the
nutrient cycling process and also protects plants against different environmental
stresses such as salinity, flooding, pathogens, temperature, contaminants, etc.
Root exudates are released by plant roots and it has a significant role in shaping
the microbiome. The microbial community offers the functions which contribute
in improving plant growth and health, even in stressed environments. Plant-
microorganisms interactions and the protection of plant health and productivity
in a stressed environment yet remain poorly understood. This chapter summarizes
recent knowledge of rhizospheric microorganisms in the process of plant-
microbiome interactions in the stress environment to improve plant growth
employing current omics techniques such as metagenomics, metaproteomics,
metatranscriptomics. These approaches, along with strategies such as stable
isotope probe (SIP), and bioinformatics tools provide insights into integrated
functional harmony between plant and microorganisms for enhanced plant
growth in stressed environments.
Keywords
Rhizosphere · Microbiome · Root exudates · Omics · Stable isotope probe (SIP)

engineering
B. Bhuyan · S. Debnath · P. Pandey (*)

Department of Microbiology, Assam University, Silchar, Assam, India

23, https://doi.org/10.1007/978-981-15-9154-9_21
504 B. Bhuyan et al.
21.1 Introduction
In 1904, Hiltner described the term “rhizosphere”, which is a hotspot in the soil near
root surface, having maximum microbial activity under the influence of plant root
(Hrynkiewicz and Baum 2011). In natural habitats, microorganisms live in a com-
munity that forms a complex environment called microbiome, and similarly, in
rhizosphere habitats, it forms a rhizosphere microbiome (Philippot et al. 2013).
The rhizosphere microbiome consists of diverse types of microorganisms which
are influenced by the roots (Tan et al. 2017). In rhizosphere, the microbiomes density
is relatively very higher than the bulk soil (Watt et al. 2006). In different biotic- and
abiotic-stressed conditions, the rhizospheric microorganisms play a major role in the
cycling of nitrogen, phosphorus, or other minerals and provide resistance to plants
(Bender et al. 2016; Lladó et al. 2017). The tripartite interactions among soil, plant,
and microbes in the soil habitat (Antoun and Prevost 2005) are significant, helpful,
unhelpful, or impartial that controls the plant growth (Lau and Lennon 2011).
There are large numbers of microbes such as bacteria, algae, fungi,
actinomycetes, etc., present in the rhizosphere. Out of all microorganisms, bacteria
are the most dominate organisms in the rhizosphere, in terms of abundance. Some of
these bacteria promote the growth of plants and known as plant growth-promoting
rhizobacteria (PGPR) (Kloepper et al. 1980). Some distinct species of genera such as
Serratia, Pseudomonas, Azospirillum, Bacillus, Klebsiella, Burkholderia, etc., are
some of the known PGPR (Joseph et al. 2007). The organic compounds released
from roots of plant have role in selection of the bacteria which contribute for the
growth of the plants (Lynch 1990), and create a selective environment (García et al.
2001). The constituents present in root exudates are determined by the physiological
status of plants along with other factors, and act as chemo-attractant for soil microbes
(Kang et al. 2010).
Rhizosphere microbiomes affect plant productivity by manipulating plant physi-
ology through underground complex interactions (Schnitzer et al. 2011).
Egamberdieva et al. (2017) reported that beneficial interaction between rhizospheric
bacteria Pseudomonas and Rhizobia improves growth of the plant Glycyrrhiza
uralensis (a plant used in food manufacture) in salt- stressed environment. In
different stressed environments, the function of rhizosphere microbiome for devel-
opment of plant growth is an important phenomenon and there is possibility of
microbiome engineering to achieve desired effects. Most of the earlier efforts have
been focused on individual microbes-mediated plant growth, while the role of total
microbiome had been greatly ignored. The functional capabilities of microbiome
communities need to be deciphered to design the advance strategies (Bashiardes
et al. 2018). For such application, the focus should be on developing main
characteristics of crop productivity such as availability of nutrients, soil fertilization
ability, etc (Syed Ab Rahman et al. 2018). Therefore, Amrani et al. (2015) suggested
that different “omics” approaches should be introduced with the soil to improve
microbiomes in rhizosphere. Haas et al. (2017) have explained role of “omics”
techniques to know about the community of microbiomes. The decreasing price in
sequencing, combined with sequencing technologies and development of powerful
21 The Rhizosphere Microbiome and Its Role in Plant Growth in Stressed Conditions 505
computational technique have given great number of information about microbiome

studies associated to rhizosphere (Fierer 2017). Here, in this chapter, the rhizosphere
has been explained with elaborated discussion on topics such as—the importance of
the microbiome in rhizosphere, dynamics of the microbiome in rhizosphere in stress
environment, the effect of root exudates (RE) in shaping microbiome in stress
environment, the effect of PGPR in microbiome under stress environment,
microbiome impact on plant growth in stress environment, “omics” approaches
and combinations with stable isotope probe (SIP) along with bioinformatics tools
with the perspective to utilize rhizosphere microbiome for improved plant growth
under stress conditions.
21.2 The Idea of Rhizosphere Microbiome
The culture-independent techniques have exposed the complexity of the microbiome

associated with rhizosphere are greatly underestimated for its functions. High
throughput sequencing technologies revealed that only marginal (up to 5%) number
of species has been cultured. Exploration of higher taxonomic abundance and
characteristics of the microbiome are challenging, but new pipelines are generating
it with 16S rRNA gene sequence data, which are improving the insight in the
rhizospheric environment (Edgar 2013; Gonzalez et al. 2019). In a metagenomics
study of rhizosphere, Alzubaidy et al. (2016) reported that rhizospheric microbiomes
have higher number of microbial reads as compared to bulk soils. In another
metagenomics study of rhizosphere of the graminoid, De Angelis et al. (2009)
reported that relative abundance of (147 of 1917 taxa) bacteria were detected in
comparison to bulk soil. In the past, many studies of rhizosphere microbiomes
depicted that the most dominant phylum of bacteria is the Proteobacteria
(De Angelis et al. 2009; Peiffer et al. 2013). For example, Weinert et al. (2011)
reported that in the metagenomic study of the rhizosphere of potato in field soil, 2432
operational taxonomic units belonged to Proteobacteria, followed by Firmicutes,
Actinobacteria, Bacteroidetes, and Acidobacteria. Also, in another study, the most
dominant phyla Bacteroidetes, Proteobacteria, Actinobacteria in rhizosphere
microbiome of Arabidopsis thaliana in natural soils under controlled conditions
were reported (Bulgarelli et al. 2012).
Diversity of molecular techniques to examine the genetic information in the
rhizosphere have been reported by Barret et al. (2011) who concluded that it is
obvious that the majority of our present knowledge at genetic and functional
expression level for the rhizosphere has been dependent on reporter gene studies
and this has permitted only a few species of the microbiome to be distinguished for
their surroundings in terms of different functions. Innovative strategies have been
designed, such as promoter-trapping technology that allowed recognizing Pseudo-
monas fluorescens genes that were upregulated in the rhizosphere, and almost,
20 such genes were identified (Rainey 1999). Though, with the recent development
of metagenomics and metatranscriptomics, larger data sets are now available to the
researchers to study the dynamics of functional genes in complete microbiome.
Different characteristics at genetic level between the microbiome of rhizosphere and

normal soils have been compared, and it was noted that 93.2% of total genes were
revealed in the rhizosphere, whereas 32.0% were revealed in the normal soils, that
indicating the functional advantage of rhizosphere for different plant growth
mechanisms. Rhizosphere microbiome comprises, genes of bacteria, archaea, and
fungi as 5390, 103, 246, respectively and in normal soils, it was reported as 1849,
38, 84, respectively. Hence, this represents the larger prosperity of microorganisms
in the rhizosphere soil as compared to normal soils (Li et al. 2014).
21.3 Dynamics of the Microbiome in Rhizosphere in Stress

Environment
The rhizosphere is dynamic and their component gets changed with time and other
factors. Different biotic as well as abiotic factors and roots exudates help in shaping
the rhizospheric environment as per their requirement to inhabit pathogenic
microorganisms and affect their activities. The microbial communities shaping up
depend upon various criteria which are described in the sections below.
21.3.1 Crop Rotation Changes Microbiome
Most of the paddy fields after it is reaped are kept empty till the arrival of a new
season. To utilize the time and space, different crops are tested to produce and add
nutrients to the field. One such is the cultivation of maize, after rice. It has been
observed that in a particular type of soil, loss of moisture results in cracks, which
might develop as a result in loss of important substances such as organic carbon and
other nutrients. Cabangon and Tuong (2000) reported that the application of straw as
a cover in field is seen to reduce the above adverse effects with improving the
physical structure of the soil including the nutrient content. It has been reported that
the application of straw has a capability to alter the microbial communities of the
area (Asari et al. 2007; Shrestha et al. 2011; Conrad et al. 2012). Shrestha et al.
(2011) found that during flood condition which turns anaerobic, rice straw decom-
position follows up with increased production of compounds such as acetate,
formate, and H2 as by-product and eventually produce methane which plays instru-
mental role in altering the microbial communities in the field. With the increase in
methanogens, other bacteria such as Clostridium, Sphingobacteria, Cyanobacteria,
and Bacilli had also been identified in the community (Rui et al. 2009). Lee et al.
(2012) reported emergence of many hydrogenotrophic and acetoclastic
methanogens during nonflooded conditions and the bacteria such as Alpha-, Beta-,
and Gamma-Proteobacteria, Actinobacteria, and Bacillus were not detected
(Conrad et al. 2012; Lee et al. 2012).
21.3.2 Phosphorus Shapes Microbiome
Soil with low phosphorus (P) content is spread all over the globe. It is a macronutri-
ent that helps in plant growth. It is the reason for limiting the productivity of most
agriculture-based lands (Von Uexküll and Mutert 1995). The addition of phosphorus
from external sources results in only 10–25% uptake for the plants (Johnston et al.
2014). Lambers et al. (2006) and Shen et al. (2011) reported that roots which firstly
come into contacts with the soil, change morphologically and physiologically, i.e., to
facilitate the uptake of P or increased uptake of carboxylate, proton, and discharge of
soil-related P as reported by Rengel and Marschner (2005), Pang et al. (2010), Wang
et al. (2010), Mehraet al. (2017). An alternative strategy such as use of PGPR, takes
part in increasing the P availability for plants (Richardson et al. 2009; Richardson
and Simpson 2011). Seeling and Zasoski (1993) reported microbes assist in transfer
of orthophosphate ions into the soil, thus aiding transport of organic P (Seeling and
Zasoski 1993), and also by induction of metabolic processes that solubilize and
mineralize P from sparingly available forms of soil inorganic and organic P
(Richardson et al. 2009). A study of soil bacterial communities under low P on
soybean plant, reported that with enrichment of bacterial community, the availability
of P also increased. At phylum level, Proteobacteria was most abundant and in
genus level Pseudomonas was most dominant, followed by Dechloromonas,
Massilia, and Propionibacterium (Tao et al. 2019). Thus, it can be said that changes
take place in community level which can be advantageous for the plant to thrive.
Bender et al. (2016) introduced arbuscular mycorrhizal fungus in corn field to note
its potential in promoting growth of the crop. Further, the authors reported that when
native fungi are less abundant, then the introduction of new fungi can have a positive
effect on growth and yield of crops. It can also be positively correlated with the
availability of total P, which is present in the soil, i.e., the higher level of P in soil, the
better the inoculated fungi will bond with the roots and establish the network
around it.
21.3.3 Saline Stress Shapes Microbiome
Saline stress is a gigantic problem in today’s world, as it directly affects productivity.

The germination of the seed does not take place due to the increased salinity (Ungar
1995; Coleman-Derr and Tringe 2014). Therefore, it’s always challenging to find the
plant varieties that grow well in saline regions and the compatible PGPR microbes
associated with it (Yuan et al. 2016). It has been found that the PGPR are very
important in establishing the plants in the stress environment (Kohler et al. 2009) and
also ameliorate various environmental stresses (Akhtar et al. 2015; Upadhyay and
Singh 2015; Wang et al. 2016a, b). Tiwari et al. (2011) reported that Arthrobacter
sp., Bacillus pumilus, Halomonas sp., Nitrinicola lacisaponensis, and Pseudomonas
mendocina were able to manage 2–25% of saline stress. Etemadi et al. (2020)
studied the indulgence of scrub Cressacretia and the associated comparative micro-
bial community analysis of rhizospheric and bulk soil, it was seen that in rhizosphere
Bacillus, Planctomyces, Halomonas, Jeotgalibacillus and in bulk soil

Paenisporosarcina, Bacillus, Planctomyces were abundant. In genus
Saccharospirillum, Marinobacterium, and Halomonas were more abundant in
rhizospheric soil than the bulk soil differing much from bulk soil. Therefore, it can
be said that according to the need or under influence of various conditions, the
microbiome tries to shift to cope with the immediate environment. Vanegas et al.
(2019) studied the fungal community in mangroves under saline environment. The
researchers reported that saprotrophs are most abundant including mycorrhizae
namely Amorosia, Aspergillus and Cystobasidium was the most abundant including
Trichoderma and Penicillium which allows them to colonize in the rhizosphere by
suppressing other groups of phytopathogenic fungi. Therefore, a factor like saline
conditions also takes part in shaping up the microbiome.
21.3.4 Invaders Shape Microbiome Dynamics
Due to the ongoing human activities and the daily wear and tear of the soil and
introduction of new plant species, it has been seen that other plants that were not
dominant previously in a particular area have suddenly emerged as the most domi-
nant by different biotic and abiotic factors. Some areas are resistant to such invaders
whereas particular sites had been easily invaded and such phenomena are not well
understood (Catford et al. 2012). The invasive species of plants which are distributed
in various geographical locations might have an influence on the microbial commu-
nity of the soil. The success story of such invasiveness can be mediated by the
absence of virulent pathogens, growth of pathogens, the presence of mutually
beneficial organisms, and the availability of nutrient resources. Nutrients such as
phosphate and other important substances can resist stress and maintenance of soil
systems are the keys to the success of the invading species (Van der Putten et al.
2007). The geological location and the time of introducing a plant are considered
essential for a successful plant-microbe interaction, which helps in bringing a
positive change to the microbial community. All these depend upon the immediate
situations, soil related features, type of flora, and microbial community of the soil
with reference to diversity and composition (Inderjit and Cahill 2015). In a study by
Rodríguez-Caballero et al. (2020) reported that the invasiveness of Carpobrotus
edulis and how it shapes the rhizosphere microbiome that the most abundant phylum
was Actinobacteria, followed by Proteobacteria and the existence of Bacteroidetes,
Firmicutes, Gemmatimonadetes, Acidobacteria, Chloroflexi were low in abundance.
Further, the author reported that Firmicutes, Bacteroidetes, and Proteobacteria were
higher in the native rhizosphere while invasive rhizosphere showed the abundance of
Verrucomicrobia and Acidobacteria.
21.4 Root Exudates(RE) Shape Microbiome to Improve Plant

Growth in Stress Conditions
The root is the most important part for attracting microbes. The tissues of root tips
are the first to interact with the soil and actively select microbes (De Angelis et al.
2005). Kawasaki et al. (2016) reported that distinct parts of root harbors different
type of microorganisms. Therefore, there are differences in tips and bases of roots in
terms of presence and availability of microbes which also vary in accordance with
the age, type of the roots. It was observed that members of Oxalobacteraceae were
abundant on nodal and seminal roots; Comamonadaceae population was rich on the
tip and base of nodal roots. Polyangiaceae (class Delta-proteobacteria) was seen on
the basal region of the nodal roots, while Sorangium reported highest in the region,
signifying a role that is entrusted to only a particular zone. For example, the mature
root zone harbors the community of microbes which are responsible for the degra-
dation of dead cells shedding from old root parts (Jones et al. 2009). It was found that
under stress condition, plants tend to recruit specific microbes with the help of RE
and microbes provide beneficial traits to the plants (Lundberg et al. 2012; Bulgarelli
et al. 2012; Edwards et al. 2015). Hooper et al. (2015) studied the role of RE in
different species of Desmodium, i.e., D. uncinatum, D. intortum, D. ramosissimum,
and D. incanum against Striga hermonthica. It was seen the RE were successful in
inhibiting S. hermonthica. Di-C-glycosylflavones, an important component of REs,
is known to responsible for antifungal activities (McNally et al. 2003). REs have
been reported for antifeedant activity against Brown Planthopper, Nilaparvata
lugens (Chul-sa et al. 2009) and corn earworm, Helicoverpa zea (Boddie) (Byre
et al. 1996). Also antagonistic activity of REs have been reported against Striga
hermonthica (Hopper et al. 2010), in addition to their role in assisting the coloniza-
tion of mycorrhizae (Akiyama et al. 2002). Vives-Peris et al. (2018) analyzed the
root secretion of citrus plant on rhizobacteria and got into conclusion that the
exudates from the plants which are exposed to stress (abiotic) are capable to
influence in a positive way, i.e., increase of bacterial load and also can be modified
accordingly with the stress type and genotype. Cesari et al. (2019) have studied the
role of RE during stress of restrictive water and found out that the external environ-
ment also plays an important role for shaping the pattern of the RE which then results
in attracting the beneficial bacteria to the root by changing the pattern of RE resulting
in increase of naringenin, oleic FA, citric acid, lactic acid, and the release of terpenes
which are known for antioxidant and antimicrobial property. In a study by Xiong
et al. (2020), the ability of RE to attract beneficial microbes for growth promotion
was studied and it was reported that the RE of Limonium sinense has ability to
promote the growth and chemotaxis effect to attract beneficial bacteria Bacillus
flexus KLBMP 4941. The components of the RE individually had positive role in
growth, mobility, chemotaxis, and colonization of root by the desired bacteria
having a positive role in increasing the photosynthetic ability of the plant and
other necessary mechanisms to support the plant growth required in saline condition.
Ludueña et al. (2017) investigated the activity of pqqE gene and pqq promoter in
Serratia species in presence of REs under low phosphate condition and it was found
that the activity of pqq promoter region has a diverse role based on the concentration,
nature of REs, and bacterial growth. Exudates are effective in increasing the expres-
sion of pqq genes and modifying it for better phosphate solubilization.
21.5 Application of PGPR Shape the Rhizosphere Microbiome

to Improve the Plant Growth in Stress Conditions
In stress environments such as salinity, flood, toxicity, pathogens, temperature,

natural and anthropogenic pollution, total microbiome including algae, bacteria,
fungi, protozoa, actinomycetes, etc., present in rhizosphere supports the plants by
promoting the colonization and growth of the plants. Therefore, the application of
these rhizosphere microbiomes is well evident in plant development (Bhattacharyya
and Jha 2012; Raza et al. 2016). Within microbiomes that are associated with the
rhizosphere, the bacteria are the important organisms that are responsible for plant
growth (Kaymak 2010). These PGPR assembles the plant growth-enhancing
chemicals because of efficient uptake of micronutrients consequently promoting
the plant health (Kumari et al. 2018; Khosravi et al. 2018). Hence, microbiomes
are significantly responsible for plant growth in stress conditions (Fig. 21.1).
Gupta et al. (2015) reported that PGPR plays an important role in several
applications of soils system to form it energetic for plant productivity in different
stress environments. The strong effects of PGPR are posed through root colonization
which increases the plant development, through fixation of nitrogen, production of
indole-3 acetic acid (IAA), phytohormones, production of siderophores, solubiliza-
tion of phosphate, and other such activities (Hayat et al. 2010; Ahemad and Khan
2012a). IAA production ability of bacteria is key attribute for plant development
because it stimulates the action of the enzyme 1-aminocyclopropane-1-carboxylate
(ACC) deaminase that degrades the precursor (ACC) of ethylene (hormone of plants
actively involved in plant development) and quantity of ethylene can control the
stress responses (Saleem et al. 2007; Hayat et al. 2010). Also, rhizobacteria can
increase the growth of root and root hairs which increases the resistance mechanism
to plant against environmental stress such as salinity, temperature, soil contaminants,
pesticide, pathogens attack (Russo et al. 2008; Tank and Saraf 2009; Ahemad and
Khan 2012a, b; Lugtenberg and Kamilova 2009). Zakry et al. (2012) reported that in
stress conditions, these PGPR promote plant development by improving the physio-
logical activity of the plant. The resistance capabilities of plants reduce the environ-
mental stresses by stimulating the production of the natural compounds and enzymes
which decrease the biotic and abiotic stresses (Zakry et al. 2012). Moreover, the way
of function of PGPR is diverse that depends on the respective plant (Garcia et al.
2015). For example, under salt stress, the PGPR Pseudomonas sp. and Acinetobacter
sp. promote the growth of host plant barley and oats, respectively (Chang et al.
2014). Sarkar et al. (2018) reported that two potential PGPR, Bacillus safensis and
Ochrobactrum pseudogrignonense, protect the host plant Triticum aestivum L. in
temperature- stress environment. Similarly, Oteino et al. (2015) reported that pre-
vention of tomato molt virus from the host plant Brassica juncia was done by
21
The Rhizosphere Microbiome and Its Role in Plant Growth in Stressed Conditions
Fig. 21.1 Microbiome and their positive effects in plant development under stress environment such as salinity, flood, toxicity, pathogens, temperature, natural
and anthropogenic pollution, etc., Rhizosphere microbiome activates various defense-related genes for improvement of plant growth. Root exudates and PGPR
511
have important role in shaping microbiome that is promoting plant growth. Molecular techniques and combination of stable isotope probe to know about the
function of rhizosphere microbiome in plant growth under stress conditions
Bacillus amyloliquefaciens and the efficient PGPR Bacillus subtilis prevents the
powdery mildew disease from the host plant Hordeum vulgare (Oyedele et al. 2014);
Paenibacillus polymyxa prevents the fungal disease from the host plant Sesamum
indicum (Ngumbi and Kloepper 2016).
21.6 Specific Rhizospheric Microbiome Improves the Plant

Under the stress environment, the whole microbiomes (Fig. 21.1 and Table 21.1) in
the rhizosphere, encourage the plant development and protect plants against different
harmful actions (Raaijmakers et al. 2009). The whole microbiomes in the rhizo-
sphere induce different mechanisms such as fertilization ability, development of
root, rhizodegradation, etc., that decrease the environmental stresses. The
rhizospheric microbes specifically bacteria, fungi, etc., play a critical role in increas-
ing the plant development in stress conditions that helps in plant productivity
(Dimkpa et al. 2009), whereas some microorganisms in rhizosphere indirectly
protect the plants from fungal diseases (Lugtenberg and Kamilova 2009).
21.6.1 Specific Microbiome Supports the Plant Growth in Abiotic

Stress Conditions
The function of microbiome associated with plants exposed in different abiotic stress
environments takes great importance in recent past (Souza et al. 2015). Earlier, it was
reported that several rhizospheric microorganisms play a great role in development
of plant growth in different types of abiotic-stress conditions (Table 21.1). For
example, the microorganisms belonging to the genera Trichoderma,
Bradyrhizobium, Bacillus, Rhizobium, Pseudomonas, etc., have been actively
involved in plant development in abiotic-stress conditions (Panlada et al. 2013;
Ahmad et al. 2015; Sorty et al. 2016).
One of the most important abiotic stresses is the soil salinity that is extremely
harmful for development and production system of plant. But the beneficial interac-
tion between halotolerant bacteria with the plant can promote the growth in salt-
stress conditions. Upadhyay et al. (2009) reported that 24 strains can tolerant
elevated levels (8%) of NaCl. Further, the author reported that the maximum number
of strains showed PGP attributes such as IAA production, siderophore production,
phosphate solubilization, nitrogen fixation, and Bacillus was the most revealed
genus in this environment. Jha et al. (2012) reported that microorganisms
Zhihengliuella sp., Pseudomonas spp., Brevibacterium casei, Vibrio sp., Halomonas
sp., Cronobacter sakazakii, and Rhizobium radiobacter can tolerant salt stress.
Low or high temperature is another abiotic stress that affects the plant growth.
Under low-temperature stress, microbial inoculants competently colonize the rhizo-
sphere region of the plant that develops their resistance mechanisms, leading to plant
growth. For example, Pseudomonas spp. and Burkholderia phytofirmans PsJN
Table 21.1 The rhizosphere organisms supporting the plant growth in biotic and abiotic-stress environment
21
Dominant phylum/
Stress Nature of stress Host plant micro-organism Functions References
Abiotic Salt Vitis vinifera Burkholderia, Bacillus Proline accumulation Barka et al.
stress 2006
Salt Phragmites australis Glomus fasciculatum Carbohydrates accumulation Al-Garni
2006
Osmotic stress Zea mays Bacillus megaterium Higher rate of water transfer, Marulanda
increase of ZmPIP isoforms related et al. 2010
to gene expression of root
Heat Triticum aestivum Bacillus Change in metabolites, lessens El-Daim
amyloliquefaciens, oxygen radicals, protects from heat et al. 2014
Azospirillum brasilence shock
Drought Triticum aestivum Bacillus thuringiensis To produce volatile organic Timmusk
AZP2 elements et al. 2014
Salt Glycine max Pseudomonas simiae Promotion of germination of seed Vaishnav
et al. 2016
Carbon dioxide Kandeliao bovata Proteobacteria, Degrade organic substrates Yin et al.
Crenarchaeota, 2018
Methanoseata
Various abiotic phenomena Populus deltoides Proteobacteria, Reduces stress Timm et al.
(CO2, drought & heavy metal Actinobacteria, 2018
toxicity) Verrucomicrobia
High temperature Grassland Actinobacteria and Adaptation Bei et al.
Proteobacteria 2019
Antibiotic Cyperus alternifolius Methylosinus, Degradation of Sulfonamide Man et al.
Methylotenera, 2019
Methylocaldum,
Methylomonas
(continued)
513
514
Dominant phylum/
Stress Nature of stress Host plant micro-organism Functions References
Continuous cropping Glycine max Xanthomonadales, Plant growth and maintenance Tian et al.
Bacillales, 2019
Chaetomiaceae,
Orbiliacaea
Saline and alkaline Cynara scolymus Nitrospira, Nitrospira: Nitrogen cycle, controls Yue et al.
Gemmatimonas the oxidation of nitrite in the soil 2020
Gemmatimonas: Carbon cycle
Salt Zea mays Kocuria rhizophila PGP attributes, regulation of Li et al.
phytohormone levels 2020
Salinity and drought Spinacia oleracea Proteobacteria Adaptation by encoding proteins Ibekwe
(Halomonas elongate) et al. 2020
Salt Cressa cretica Planctomyces, Various PGPR effects Etemadi
Halomonas and et al. 2020
Jeotgalibacillus
Agrochemical Dwarf wheat cultivar Verrucomicrobia, Production of specific hormone Kavamura
Planctomycetes, et al. 2020
Acidobacteria
Biotic Fungus and Nematode Wild Plants Bacillus, Enterobacter, PGP and antifungal effect El-Sayed
stress (Fusarium oxysporum, (Zygophyllum simplex and Pseudomonas et al. 2014
Sclerotinias clerotiorum, L., Peganum harmala L.,
Meloidogyne incognita) etc.)
Influence of plant Glycine max Proteobacteria, In growth of plant Liu et al.
Acidobacteria, 2019
Actinobacteria, and
Bacteroidetes
Change of soil Phaseolus vulgaris Proteobacteria, Nitrogen fixation and using Pérez-
Acidobacteria, and nutrients Jaramillo
Bacteroidetes et al. 2019
B. Bhuyan et al.
21
Fungal (B. cinerea, F. solani, Panax ginseng Meyer Bacillus Antagonistic activity Sun et al.
F. oxysporum, etc.) amyloliquefaciens 2019
Fungal (Fusarium oxysporum, Cucumis sativus Paenibacillus jamilae PGP and antifungal activity Wang et al.
Rhizoctonia solani, etc.) HS-26 2019
Bacteria (Ralstonia Chrysanthemum spp. Lysinibacillus sp., PGP and antibacterial activity Djaya et al.
solanacearum) Bacillus subtilis, and 2019
Pseudomonas
fluorescens
Fungus (Rhizoctonia solani, Arachis hypogaea L. Bacillus subtilis Better yield and fungicidal effect Ahmad
Sclerotium rolfsii, etc.) et al. 2019
515
promoting the growth of wheat grapevine plant, respectively, in low temperature

conditions (Egamberdiyeva and Höflich 2003; Barka et al. 2006; Yarzábal et al.
2018). PGPR can promote the plant growth in flooding conditions. For example, the
bacterial species Enterobacter cloacae and Pseudomonas putida enhanced growth
of Lycopersicon esculentum plant in flooding conditions (Grichko and Glick 2001).
In worldwide, contamination is an important abiotic factor that greatly affects the
plant growth. Rhizoremediation has become the most suitable method of bioremedi-
ation for contaminated soils. It is the process of degradation of contaminants in the
rhizosphere by mutual association of plant and microorganisms (Kuiper et al. 2004).
For example, Kotoky and Pandey (2019) reported that inoculation of soil bacilli
Bacillus flexus S1I26 and Paenibacillus sp. S1I8 could degrade polyaromatic
hydrocarbons in the rhizosphere of Melia azadirachta plant. Similarly Cruz-
Hernandez et al. (2012) reported that inoculation of fungal sp. Lewia could degrade
the hydrocarbons in the rhizosphere of Festuca arundinacea plant that ultimately
help in promoting the plant growth in abiotic-stress environment.
21.6.2 Specific Microbiome Supports the Plant Growth in Biotic

Stress Conditions
The rhizospheric microorganism protects the plant from various biotic stresses
(Table 21.1). For example, it can trigger killing or removal of soil-borne pathogens
during infection by different mechanisms such as antibiosis, quorum sensing that
disturbing virulence, resistance capacity, elemental and nutritional competition
(Duffy et al. 2003; Chan et al. 2011; Druzhinina et al. 2011; Raaijmakers and
Mazzola 2012; Schenk et al. 2012). Brakhage and Schroeckh (2011) reported that
the rhizospheric bacteria and rhizospheric fungi provide antibiotic metabolites which
can reduce the virulence capacity of pathogenic microbes. For example,
Trichoderma sp. and Agrobacterium sp. (Kim et al. 2006; Druzhinina et al. 2011).
Microbiomes in rhizosphere supply the volatile organic compounds that are the
metabolites which can help in plant development by attacking the pathogens. For
example, the genus belonging to the bacteria Pseudomonas sp., Bacillus sp., Serratia
sp., etc., antagonizes the fungal pathogens through the volatile organic compounds
(Vespermann et al. 2007; Jamalizadeh et al. 2010).
Dede et al. (2020) reported that the Actinomycetes protect the host plant Olive
through various PGP and antifungal activity from the pathogenic bacteria Staphylo-
coccus aureus. Similarly, Bacillus subtilis CKT1 protect the host plant Solanum
lycopersicum L. through PGP and antifungal activity from the fungal pathogen
(Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum) (Walia
et al. 2013).
21.7 Modern Molecular Sequencing Techniques: To Know

the Functions of the Rhizospheric Microbiome in Plant
To recognize the function of the microbiome in rhizosphere of plants growing in

stress conditions is challenging due to complex multidimensional interactions and
diversity involved. Therefore, to understand the role of the microbiome in plant
growth, different novel molecular techniques (Fig. 21.1) have employed to know the
taxonomic and functional analysis, physiology, metabolism of microbiome
associated with the plant growth in stress conditions. “Omics” approaches, i.e.,
metaproteomics, metagenomics, metatranscriptomics are advanced molecular
techniques to understand and analyze the role of the microbiome under stress by
determining the microbial diversity in the rhizosphere (Rastogi and Sani 2011; de
Castro et al. 2013).
21.7.1 Metaproteomics
Metaproteomics is a technique that deals with the protein study of environmental

samples. This protein content takes a key role in stress responses that showing the
phenotypic traits and thus, this protein study deals with the expression of protein in
samples (Keller and Hettich 2009; Kosova et al. 2015). Singh and Nagaraj (2006)
reported that this proteomic study depends on new bioinformatics tools, separation
methods such as two-dimensional polyacrylamide gel electrophoresis, etc. Earlier,
metaproteomics studies of the plants such as Oryza sativa; Lactuca sativa and
microorganisms in the soil system have been reported pertaining to the stress
responses (Wang et al. 2011; Moretti et al. 2012).
In metaproteomics study, Renu et al. (2019) optimized the metaproteomic extrac-
tion protocol from maize rhizosphere and analyzed the functional characteristics of
microbial communities and further identified expressed proteins matched by the
microbial species and provides relevant information about system functioning, its
microbiome, and ecology of maize rhizosphere. In a proteomic study of vineyard
rhizosphere, the highest contributors of expressed proteins are the genus of the
bacteria such as Streptomyces, Bacillus, Bradyrhyzobium, Burkholderia, and Pseu-
domonas that showing regulation of metabolic processes (activities of the phospho-
rus) (Bona et al. 2019). In another metaproteomics study of rice plant, Wang et al.
(2011) reported that most significant phyla were Actinobacteria, Proteobacteria, and
Firmicutes that expressed protein contents in rhizosphere (Wang et al. 2011).
21.7.2 Metagenomics
Metagenomics confers information of the community of microorganisms by

analyzing analysis of nucleotide sequences that are collected from environments. It
is a technique, in which the genomic contents of a whole community of
microorganisms are sampled to explore the microbial community analysis and to

monitor for special traits or DNA sequences (Hugenholtz and Tyson 2008; Desai
et al. 2012). This technique provides an investigation of microorganisms at the
genetic level and will help us to know the functions of microbes (Kimura 2006;
Cuadros-Orellana et al. 2013). Metagenomics approach is very supportive to under-
stand the bacterial community of rhizosphere that helps in plant growth in stressed
environment. Earlier, Nikolic and Schwab (2011) studied the metagenomic analysis
of bacterial endophytes colonizing field-grown Solanum tuberosum L. plant and
reported that ACC deaminase genes (acdS) were found in bacterial endophyte that
helps in plant growth. Liljeqvist et al. (2015) studied the metagenomic analysis of
acid mine drainage stream biofilm situated at a depth of 250 m below the surface in
the Kristineberg mine, northern Sweden, where temperature is low (6–10 C) and
identified the most abundant microbes that are similar to the psychrotolerant acido-
phile, Acidithiobacillus ferrivorans as well as other Acidithiobacillus species, an
Acidobacteria-like species, and a Gallionellaceae-like species. Further functional
characteristics indicated that cold-shock proteins, the pathways responsible for the
formation of an antifreeze protein and compatible solutes that help in growth in
low-temperature conditions.
In a metagenomics study of rice plant, Sessitsch et al. (2012) reported that
endophytic bacteria at a functional level induce plant growth in stress environment
by enhancing the resistance mechanisms. Using metagenomic analysis, Bell et al.
(2014) reported that soil pollutants can shape the microbial community in rhizo-
sphere. Also, these metagenomic studies of rhizosphere help us to know about
unknown microbial strains that may be beneficial for degrading pollutants that
may have unidentified degrading pathways.
21.7.3 Metatranscriptomics
Metatranscriptomics is a technique that deals with the study of microbial gene

expression of individual microorganism or microbiome by sequencing of mRNA
transcripts collective from microbial communities (Urich et al. 2008; Moran et al.
2013). Generally, the reads of metatranscriptomics are mapped to specialized
databases using alignment tools, such as Bowtie2, BWA, and BLAST. After
mapping, the results are annotated using different resources, such as Swiss-Prot,
KEGG, COG and then analysis is carried out which is based on the objective of the
study (Aguiar-Pulido et al. 2016). Earlier, transcriptomics technique has been
employed to study the microbiome of different plants and contaminated environ-
ment. In a transcriptome study of symbiotic relationship of rapeseed
Stenotrophomonas rhizophila, Alavi et al. (2013) reported that spermidine is the
growth regulator in stress conditions that helps promoting the rapeseed growth. In
another study of transcriptomics of cucumber, Li et al. (2014) reported that
Bvu-miR13 regulates WD repeat proteins that are tolerant to environmental stresses.
Safdarian et al. (2019) studied the transcriptomic analysis of wheat plants, influenced
by rhizobacteria Arthrobacter nitroguajacolicus in salt-stress conditions and
reported upregulation of many genes that actively participates in cell differentiation

and metabolism followed by promoting the plant growth in salt stress. The authors
reported that the relative abundances of phenylpropanoid synthesis were most
dominant with high number of genes present in root inoculated with efficient
bacteria under salt-stress conditions (Dixon et al. 2002).
21.7.4 Introduction of Stable Isotope Probe (SIP) with Molecular

Biology Techniques
Combination of stable isotope probe (SIP) with molecular biology techniques is one
of most useful modern approaches to determine the microbe-microbe interactions,
i.e., the community structure and composition with the relatable functions and the
various metabolic process taking place within the community (Bressan et al. 2009).
This SIP connects the elements to an organism’s genome and then organisms utilize
the labeled isotope into their nucleic acids that increase the density of nucleic acids
(Radajewski et al. 2000). The example of isotope atoms are 15N, 13C, 18O, 13CO2
(Rasche et al. 2009; Haichar et al. 2012, 2016; Angel et al. 2018). This SIP has wide
applications in microbiota of rhizosphere and other environmental samples to
recognize the novel enzymes and genes of microorganism that is responsible for
rhizoremediation and other metabolic process (Haichar et al. 2016). In a study of
potatoes, SIP 13C, has been used to recognize the bacterial organisms (Rasche et al.
2009). In another study of SIP, Haichar et al. (2012) reported that using DNA and
RNA-SIP techniques under 13CO2, that provide an information of microorganism
that actively incorporate the root exudates and mRNA-SIP techniques help in
examine the control of bacterial genes expression by root exudates. In other study
of SIP, Fortunato and Huber (2016) used RNA-SIP combined with other molecular
methods to identify the chemolithoautotrophic microbes situating in the deep-sea
vents. Similarly, Gülay et al. (2019) used combined DNA-RNA SIP method that
figures out the responsible microorganism which carry out nitrification in the
biofilters of groundwater Hanajimaa et al. (2019) took advantage of the SIP by
using 13C-labeled E. Coli that is used to find out the groups of bacteria which take
nutrition from the dead bacteria.
21.8 Conclusion and Prospects
Rhizosphere microbiomes are sensitive to environments and remain dynamic.

Hence, needs attention with prospects of exploration in biotic- and abiotic-stressed
environments. The “omics” techniques give important information to recognize the
function of the rhizosphere microbiome in plant growth under stress environments.
These molecular approaches totally depend on different modern sequencing
technologies that help in detection of a vast number of microorganisms related to
individual function, information at genetic, transcriptomic, and proteomic level.
These techniques in such a way that provide information of root exudation are not
only activating the genes that are associated with the nutrient responses and motility
but also produce antibacterial and antifungal substances, participate in degradation
of contaminants as per the need. Therefore, for better understanding of rhizosphere
microbiome-mediated plant growth under stress environment, the current molecular
approaches and technology must be exploited more, to discover the explanations on
the function of the rhizospheric microbiome for plant growth and development under
stress environment.
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Rhizobacteria-Mediated Alleviation
of Abiotic Stresses in Crops 22
Priyanka Gupta and Manjari Mishra
Abstract
Crop production is often limited by varied abiotic stresses. Unlike animals, plants
do need a more finely regulated signaling system to withstand these natural
constraints, which otherwise negatively affect plant growth and productivity.
Since plants and the rhizospheric microbes are coevolved, the later is adapted
to continuously changing environmental factors and hence provide an excellent
source for the alleviation of abiotic stresses. Interaction of plants with microbes
under stress conditions lead to the activation of complex signaling machinery in
both plants as well as in microbes. The outcome of this cross signaling makes the
plant tolerant of aforesaid conditions. In the current chapter, the underlying
mechanisms of cross-tolerance conferred to plants by its microbial counterparts
have been summarized. This chapter focuses on plant responses to varied abiotic
stresses and the molecular changes taking place in response to the stress. Different
mechanisms and possible explanations by which soil microbes are known to
confer stress tolerance to different crop plants are also summarized here.
Keywords
Abiotic stress · Crop · Rhizobacteria · Omics · Osmoprotectant · Microbes
Priyanka Gupta and Manjari Mishra have been contributed equally to the chapter.
P. Gupta (*)
Department of Biotechnology, Maharashtra Education Society’s Abasaheb Garware College,
Savitribai Phule Pune University, Pune, Maharashtra, India
M. Mishra
Plant Stress Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi,
India

23, https://doi.org/10.1007/978-981-15-9154-9_22
532 P. Gupta and M. Mishra
22.1 Introduction
Agriculture is an important sector of the economy, especially in developing

countries. Meeting the demand to feed the exponentially increasing world population
from the limited available cropland is the major challenge for the crop biologists.
Discrete factors responsible for the worldwide decline in crop production can be
categorized into two broad categories: one is man-made factors and another is
environmental factors. The rapid transformation of the agriculture field into indus-
trial and urban land as well as exponentially increasing world population accounts
for man-made factors. On the other hand, environmental factors in the form of biotic
and abiotic stresses are among the most severe threats to crop production. Global
warming and climate change have imposed a negative impact on crop productivity.
These factors have exacerbated abiotic stresses in the form of drought, heat, cold,
and flooding (Wassmann et al. 2009; Braun et al. 2010). Apart from these, contami-
nation of agricultural land with excessive salts (salinity), and heavy metals, which
can also be clubbed into abiotic stresses, can cause severe loss (more than 50%) of
crop yield (Sahu et al. 2019). These abiotic stresses disturb the normal plant
physiology and metabolism. Interestingly phenotypic variations can be observed in
plants when exposed to the stresses, as mentioned earlier. The impact of these
stresses can vary from one crop system to another (Cramer et al. 2011; Mirouze
and Paszkowski 2011). However, plants do have a basic stress-responsive signaling
mechanism that works to fine-tune the physio-chemical system according to the
environment (Bahuguna et al. 2018; Gupta et al. 2019; Nongpiur et al. 2019). These
signaling mechanisms operate broadly at three hierarchical levels, namely stress
perception, signal transduction, and stress response (Hasegawa et al. 2000; Sierla
et al. 2013). Numerous molecules/carriers are involved in this whole process.
Sensors or the receptors are the first component of the stress signaling which senses
or perceives any change in the external environment (Osakabe et al. 2013; Nongpiur
et al. 2020). A vast number of receptors or stress sensors viz. histidine kinases, G
protein-coupled receptors (GPCR), serine/threonine kinases, transporters, etc., have
been identified (Kacperska 2004; Osakabe et al. 2013). Once the signal is perceived,
it triggers the downstream cascade such as the signaling network between Ca2+ ions,
ROS, and shuttle protein members which ultimately initiate signal transduction
(Nishida and Gotoh1993; Lee et al. 2007). These cascade mediators, in turn, are
responsible for the generation of effector molecules in the form of stress-related
transcription factors, chaperones, osmolytes, stress-responsive proteins, etc. (Verma
et al. 2013; Arbona et al. 2017; Gupta et al. 2017). Figure 22.1 illustrates a broad
outline of the stress signaling cascade in plants.
Crop biologists are employing various strategies to overcome the effect of abiotic
stresses on crop plants. Classical breeding techniques and modern transgenic tech-
nology are the two major approaches used for the development of stress-tolerant
crop plants. The classical method employs breeding high yielding crops with the
varieties having stress-tolerant trait and subsequently selecting the new one having
both the qualities. On the other hand, transgenic technologies employ the expression
of stress responsive genes for the development of stress- tolerant varieties. Apart
22 Rhizobacteria-Mediated Alleviation of Abiotic Stresses in Crops 533
Fig. 22.1 A broad signal transduction circuitries operative in plants under abiotic stresses. A
schematic representation of the events taking place under abiotic stresses in the plant cell. All
abiotic stresses are perceived by certain receptors that activate signal carrier molecules such as Ca2+.
Further activation of signaling mediated through other signal components, ultimately lead to the
tolerance against the stresses
from these methods, another approach that has become popular these days includes
the use of plant growth-promoting rhizobacteria (PGPR) for the mitigation of abiotic
stresses (Kang et al. 2014a; Meena et al. 2017; Kumar et al. 2018). The microbes are
the most abundant and fundamental creatures of life on earth and hence they have
become an integral part of the soil ecosystem. Therefore, they directly make
associations with different plant parts. The bacteria residing in the rhizosphere,
exert a beneficial effect on the plant by mobilizing essential nutrients from soil to
the plant; fighting against phytopathogens; alleviating the detrimental effect of
stress, etc. (Barassi et al. 2007; Odoh 2017). These bacteria are called plant
growth-promoting rhizobacteria (PGPR). These soil microbes have been widely
explored for decontamination of the soil from heavy metal toxicity (Khan et al.
2009). These PGPRs are being utilized for minimizing the detrimental impact of
abiotic stresses on crop plants too (Yang et al. 2009; Gupta et al. 2015). Since
microbes have coevolved in the same environment along with the plants, they too are
adapted to such harsh conditions. Various researches indicate that the plant-microbe
interaction can improve the overall tolerance of the plants against the environmental
hurdles. Therefore, this chapter gives comprehensive information on plant-microbe
interaction for the alleviation of abiotic stresses as well as various stress-dependent

components and pathways regulated by rhizospheric microbes.
22.2 Impact of Abiotic Stresses on Crop Plants
Plants utilize light energy, water, minerals, and carbon for running the normal cell
physiology and overall growth. Being sessile, plants do experience a plethora of
environmental stresses, and thus they must have more tightly regulated stress-
responsive machinery to survive under aforesaid circumstances. These environmen-
tal aberrations are in the form of soil salinity, drought, extremes of temperature (heat
and cold), and soil heavy metal toxicants. These factors interfere with nutrient
mobilization from the soil; disturb plant water balance; disrupt soil matrix composi-
tion; misbalance water holding capacity of the soil; decrease total plant yield; cause
ion toxicity in the plant cell (Alami et al.2000; Qadir and Schubert 2002). Initially,
the stress effects are observed only at the cellular level, while at the later stage
physiological changes are observed. Stresses also disturb the hormone balance of a
plant. Under drought stress, the plant cytokinin levels are reduced, while abscisic
acid (ABA) level is increased (Dobra et al. 2010; Nishiyama et al. 2011). The
elevated ABA level plays a very significant role in drought stress and regulates
many physiological changes in plant cells such as the closure of stomata (Liu et al.
2005; Daszkowska-Golec andSzarejko2013). Drought stress also leads to the accu-
mulation of reactive oxygen species (ROS) including superoxides, and peroxides
which cause cell membrane damage (Zhao et al. 2001; Wang et al. 2005). These free
radicals also damage the nuclear membrane and finally damage DNA. The negative
impact of drought also includes decreased photosynthetic efficiency, reduced leaf
size and water potential, delayed flowering, reduced productivity, and seed viability
(Xu and Zhou 2006; Osakabe et al. 2014). Drought stress is considered to be one of
the most severe abiotic stresses because it can lead to complete yield loss and also
interferes with the nutrient balance of the plant cell. It affects nitrogen metabolism by
targeting the activity of the key enzyme of the pathway, nitrate reductase as a result
of which mobilization of nitrate from the soil is reduced which in turn causes
nitrogen deficiency in plants (Caravaca et al. 2005). One of the mechanisms
employed by the tolerant plants to overcome the lethal effect of drought is the
modification in the root architecture which includes extended root length and higher
root surface area, to absorb more nutrients and water from the soil (Wu et al. 2013;
Rao et al. 2016).
Heat stress or high-temperature stress is another severe threat to crop plants.
Plants do require the optimum temperature to complete their life cycle. Deviation
from this optimum temperature can lead to malfunctioning of cellular processes and
consequently yield is compromised. Some of the key physiological changes
observed under heat stress are higher transpiration and evaporation rate, also severe
cellular and tissue water scarcity (Wahid et al. 2007). Reduction in photosynthetic
efficiency, reduced seed setting rate, reduced photorespiration, and lower seed
germination rate is also associated with high-temperature stress (Xu et al. 2014).
Thus, heat stress affects almost all the developmental stages of the plant. Extremes of
temperature also include a sudden drop in environmental temperature from optimum
growth temperature. This stress is termed as cold or chilling or freezing stress. Cold
stress is another severe abiotic stress which can lead to complete yield loss (Koini
et al. 2009). Cold stress affects plants by diverse mechanisms such as the formation
of ice crystal which in turn causes physio-chemical damage to the plant, photosyn-
thetic efficiency also decreases as CO2 fixation is compromised, and there is a
generation of ROS or free radicals, etc. Plants produce antifreezing proteins to
cope with the adverse effect of cold injuries (Theocharis et al. 2012; Janmohammadi
et al. 2015; Clemente-Moreno et al. 2020). Certain cold tolerant plants survive under
stress by accumulating osmoprotectants such as proline, mannitol, trehalose, glycine
betaine, etc. (Rathinasabapathi 2000; Chen and Murata 2002; Hasanuzzaman et al.
2019).
Similar to drought, salinity is also known to exert a lethal impact on the plant.
Highly saline soil can adversely affect the plant biomass as well as total yield. In
some cases, if the plant is exposed to salinity for a longer duration, the plant may die.
Salinity in the form of sodium, calcium, and magnesium salts of chloride, bicarbon-
ate, and sulphate can interfere with the mobilization of other essential nutrients from
the soil to the plants (Munns and Tester 2008). If the conductivity of soil becomes
equal to or more than 4 dS/m (40 mM NaCl concentration), the soil is considered to
be saline (Munns and Tester 2008). The physiological impact of high salt concen-
tration in the soil can be summarized as, a sharp decline in soil water potential which
in turn restricts water entry in the plant; nutrient imbalance because of excess Na+
ions in the rhizosphere competing with essential ion for the uptake; cellular toxicity
as caused by excessive Na+ ions in the cell leading to altered biochemical processes
(Boursiac et al. 2005; Aroca et al. 2012; Ambede et al. 2012; Gupta et al. 2019).
Therefore, it can be concluded that salinity causes both ionic as well as osmotic
stress and the combined effect of these stresses exerts a negative impact on plant
growth.
22.3 Mitigation of Abiotic Stresses Using Rhizospheric Microbes
Plant biologists are employing various strategies to combat these abiotic stresses. As
described earlier, breeding methods and transgenic technologies are the most com-
mon methods used for the development of stress-tolerant crop varieties. However,
these methods are time-taking and expensive too. The use of microbial inoculants is
the new tool devised by crop biologists for sustainable agricultural development.
Many soil bacterial isolates are termed as plant growth-promoting bacteria (PGPB)
because of their direct and indirect impact on plant growth (Kloepper and Schroth
1981). These PGP traits include solubilization of nutrients, synthesis, and secretion
of plant growth regulators such as IAA, organic acids, nitrogen fixation, production
of protective enzymes such as ACC (1-aminocyclopropane- 1-carboxylate) deami-
nase, glucanase, and chitinase, induction of systemic resistance, and production of
volatile organic compounds (Di Simine et al. 1998; Sharma et al. 2003; Mayak et al.
2004a;Mayak et al. 2004b; Saleem et al. 2007; Antoun and Prévost 2005). These
PGPRs have become an integral part of current agricultural practices to boost crop
production, improve the nutritional value, overcome the lethal effect of environmen-
tal stresses, and control phytopathogens (Glick et al. 1998; Yang et al. 2009; Yadav
et al. 2020). Various PGPRs belonging to distinct genus namely Acinetobacter,
Acetobacter, Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Bacillus,
Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium,
Gluconacetobacter, Pantoea, Pseudomonas, Ralstonia, Rhizobium, Serratia etc.,
have been identified (Lucy et al. 2004; Singh et al. 2019). Past research carried out
on these PGPRs shows that their close association with plant provides greater
tolerance against various abiotic stresses (Schlaeppi and Bulgarelli 2015;
Vandenkoornhuyse et al. 2015). Crop plants such as rice, wheat, soybean, tomato,
apple, and lettuce, when coinoculated with PGPRs, show better adaptation to abiotic
stresses (Meena et al. 2015; Etesami and Alikhani 2016). Figure 22.2 gives the
mechanism of stress tolerance by rhizospheric microbes. The detailed mechanisms
of tolerance provided by the microbial community have been summarized in the
upcoming section.
22.4 Mechanisms of Tolerance Provided by Soil Bacteria/

Rhizobacteria
PGPRs utilize various mechanisms to support abiotic-stress tolerance in plants.

These mechanisms are summarized in the subsequent section:
22.4.1 Mitigation of Stresses via Secreted Phytohormones
The rhizobial microbes are known to synthesize and secrete phytohormones such as
indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinin, and gibberellic acid
(Yang et al. 2009; Dimkpa et al. 2009; Kim et al. 2012). The phytohormones are
important biological components of plants and enable it to survive under normal as
well as harsh conditions. Therefore, the production of phytohormones by PGPRs
provides an indirect mode of survival under stress. IAA is an auxin that is known to
promote root growth. Egamberdieva and Kucharova (2009) have shown that inocu-
lation of IAA producing bacterial species to the plant can make the plant tolerant to
drought stress as it promotes the lateral root formation and hence facilitates more
water absorption from the soil. Similarly, Dimkpa et al. (2009) have shown that
Azospirillum inoculation could make the plant tolerant to low water stress via IAA
production. In another study, Lavandula dentate plants showed tolerance to drought
stress when coinoculated with IAA producing PGPR B. thuringiensis (Armada et al.
2014). Some PGPR produce volatile compounds such as nitric oxide which induces
the IAA biosynthesis pathway in the plant and, thereby promotes root growth under
drought stress (Creus et al. 2005; Molina-Favero et al. 2008). Apart from IAA, ABA
is another important stress hormone, whose elevated level plays a significant role in
22
Rhizobacteria-Mediated Alleviation of Abiotic Stresses in Crops
Fig. 22.2 Mechanisms operated by PGPRs to confer stress tolerance. A schematic representation of the various mechanisms utilized by the rhizobial
counterpart to provide tolerance against multiple abiotic stresses to the plants
537
abiotic stress signaling (Yamaguchi-Shinozaki and Shinozaki 1994; Gupta et al.

2017). Bresson et al. (2013) showed that a novel strain of Phyllobacterium
brassicacearum confers tolerance to Arabidopsis plants against osmotic stress.
The mechanism suggests that the inoculation of the bacterium can lead to higher
ABA levels in plants and, thus reduces the transpiration rate, thereby reducing the
water loss. A. brasilense is another PGPR which optimizes the growth of inoculated
Arabidopsis plants under drought stress (Cohen et al. 2008). Kang et al. (2014b)
have demonstrated that one strain of Pseudomonas putida produces gibberellin
making the soybean plants tolerant to drought stress. Cytokinin produced by Bacil-
lus subtilis assists the Platycladus orientalis plants to drought stress adaptation (Liu
et al. 2013). Certain species of Pseudomonas also secrete IAA and provide tolerance
against salt stress (Chang et al. 2014).
Gaseous phytohormone ethylene is widely distributed throughout the plant.
Ethylene plays a dual role in plant growth. At lower concentration, it acts as a signal
to promote vegetative growth of a plant, while at a concentration higher than the
threshold, it acts as a signal molecule to guide the plant to prepare for abiotic
stresses. Jackson (1997) has demonstrated the hyperaccumulation pattern of ethylene
under abiotic stresses. The elevated level of ethylene guides various morphological
as well as physiological changes in plants such as modification in roots. These
changes assist the plant to fight against external threats. Research carried out on
1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme confirms that ACC
deaminase produced by PGPRs plays a positive role in abiotic stress signaling (Glick
et al. 1998; Pandey et al. 2005). ACC deaminase cleaves ethylene precursor, i.e.,
ACC and consequently lowers down the ethylene level in the plant under stress
condition (Glick 2004). ACC deaminase is produced by diverse groups of Gram-
positive and Gram-negative bacteria (both rhizospheric and endophytic) (Jia et al.
1999; Pandey et al. 2005). Interestingly, ACC is present in the plant cell, while ACC
deaminase is microbial in origin. Therefore, ACC is secreted out as plant exudate
and taken up by ACC deaminase producing microbes (Glick et al. 1998). Under
drought stress, inoculation of Achromobacter piechaudii led to higher fresh and dry
weight in tomato plants (Mayak et al. 2004a). This PGPR produces ACC deaminase
which lowers the ethylene level in the plant leading to drought-stress tolerance.
Similarly, ACC deaminase producing PGPR Variovorax paradoxus leads to drought
tolerance to inoculated pea plants (Dodd et al. 2005). Arshad et al. (2008) have
demonstrated the beneficial effect of ACC deaminase producing bacterial strain in
the pot as well as field level. Likewise, inoculation of Pseudomonas fluorescens
significantly increased water absorption in the pea plant under drought stress (Zahir
et al. 2008).
22.4.2 Mitigation of Stresses via the Production of Osmoprotectants
Osmoprotectants or compatible solutes or osmolytes namely polyamines, proline,

betaine, sugar and amino acid derivatives, quaternary ammonium compounds, etc.,
play an essential role in abiotic stress regulation. Some soil microbial strains are
known to produce and secrete compatible solutes under stress condition which
facilitates plant survival under stresses. In an interesting study carried out by Shintu
and Jayaram (2015), inoculation of Bacillus polymyxa was found to confer tolerance
to drought stress in tomato plants due to the secretion of proline. Maize plants
accumulated higher proline when inoculated with Pseudomonas putida under
drought stress and hence maintained their leaf water potential (Sandhya et al.
2010). Gusain et al. (2015) have analyzed the effect of PGPR consortia comprising
of Pseudomonas jessenii, Pseudomonas synxantha, and Arthrobacter
nitroguajacolicus on contrasting rice genotypes (IR64 and Sahbhagidhan) under
drought stress. Surprisingly, treatment with PGPR consortium caused a higher
accumulation of proline leading to stress tolerance. However, the genotypes differ
in the extent of proline accumulation. Various other studies confirm the stress
tolerance in diverse plant groups due to hyperaccumulation of proline upon PGPR
inoculation (Sandhya et al. 2010; Ansary et al. 2012; Armada et al. 2014). Tolerance
to salinity stress was also observed in Brassica juncea upon inoculation with
Trichoderma harzianum which caused accumulation of osmoprotectants (Ahmad
et al. 2015). Apart from proline, various reports confirm the accumulation of amino
acids and sugars in the plant when coinoculated with PGPR under stress conditions
(Sandhya et al. 2010; Bano et al. 2013). Inoculation of B. subtilis and Klebsiella
variicola in Arabidopsis and maize, respectively leads to the accumulation of
glycine betaine leading to protection against water stress (Zhang et al. 2010; Gou
et al. 2015). A higher level of glycine betaine produced by many PGPRs also makes
plant tolerant to osmotic stress by regulating water loss mechanism in plants
(Nadeem et al. 2010). Sandhya et al. (2010) showed that plants inoculated with
B. subtilis and Pseudomonas showed tolerance to osmotic stress because of the
hyperaccumulation of glycine betaine. Researchers have also utilized modern tools
such as recombinant DNA technology where instead of using microbe in their
natural form, engineered PGPR strains are used which can synthesize specific
osmoprotectants. Suarez et al. (2008) used transgenic Rhizobium in which gene
encoding for trehalose-6-phosphate synthase is overexpressed leading to higher
trehalose accumulation in P. vulgaris conferring drought tolerance. Similar reports
are available for maize plants (Rodríguez-Salazar et al. 2009). Inoculation of maize
plants with genetically modified Azospirillum brasilense, which can synthesis and
accumulate higher trehalose, can confer drought tolerance to the inoculated plants
(Rodríguez-Salazar et al. 2009). One report confirms that salinity stress-induced
accumulation of trehalose in the soil bacterium Bradyrhizobium japonicum can
improve the soybean growth and nodulation under salinity stress (Sugawara et al.
2010). Dardanelli et al. (2009), showed that the application of Bradyrhizobium
strains also led to higher accumulation of trehalose in the bacterium which indirectly
supported the survival of peanut plants under salinity stress. Hence, these reports
give evidence that osmoprotectants produced and accumulated by the PGPRs play
an important role in providing stress tolerance to the crop plants.
22.4.3 Mitigation of Stresses by Maintaining Ion Homeostasis

and Detoxification
Plants require a balanced cellular ionic environment for the normal functioning of
bioprocesses inside the cytoplasm as well as in the nucleus. Exposure to prolonged
abiotic stresses leads to disturbance in the ion balance of the cell. To cope with the
external factors, plants need to maintain ion homeostasis by adjusting the expression
pattern of distinct transporters and ion channels in the cell. Interestingly, certain
PGPRs are identified, which are involved in maintaining ion homeostasis of the plant
cell by direct or indirect mechanisms. Work carried out by Ryu and colleagues
(2004) demonstrated that inoculation of a novel strain of Bacillus subtilis GB
03 leads to tissue-specific expression of HKT1, i.e., high-affinity K+ transporter
1, making the inoculated Arabidopsis plant tolerant to salinity stress. HKT1 are the
transporters that differentially maintain cellular K+ and Na+ levels. Under salt stress,
B. subtilis GB 03 emits VOC which in turn represses the expression of HKT1 in the
root, while promoting expression in the shoot. Consequently, Na+ levels are opti-
mally maintained in the plant (Ryu et al. 2004; Zhang et al. 2008). A similar
mechanism was proved using athkt1 Arabidopsis mutant plants where mutants
showed severe salt-stress phenotypes due to lack of functional HKT1 (Ryu et al.
2004; Zhang et al. 2008). Inoculation of PGPR consortium comprising of
P. syringae, Enterobacter aerogenes, and P. fluorescens helped the maize plants
in maintaining the high K+/Na+ ratio under salt-stress conditions, making the plant to
function normally even under salinity (Nadeem et al. 2007). However, the exact
mechanism is not clear. Mishra et al. (2009a) showed that Pseudomonas inoculation
to cold-stressed wheat plants can lead to higher accumulation of prolines, higher
chlorophyll content as well as higher K+/Na+ levels making the plant tolerant to cold
stress. Similar observations were made with a few other strains of Pseudomonas,
where wheat plant maintained lower Na+/K+ levels and showed tolerance to cold
stress (Mishra et al. 2009b). Work done by Ashraf et al. (2004) showed that
inoculation of a PGPR producing exopolysaccharide to wheat plant, conferred
tolerance to ionic stress as the former can cover the root zone and hinders the
movement of Na+ ions from soil to the plants. Likewise, salt-affected maize plants
inoculated with Azospirillum resulted in a higher K+/Na+ ratio and thus conferred
salinity tolerance (Hamdia et al. 2004).
Along with the ions, the excess of ROS generated due to abiotic stresses also
creates a problem for the cellular biochemical processes. Detoxification of this ROS
is another challenging factor for the crop biologists working in this area. Literature
confirms that inoculation of varied PGPR strains also led to better ROS management
strategies in stressed plants. PGPRs belonging to genus Bacillus are widely used for
ROS detoxification in the plants due to the availability of superoxide dismutase
(SOD), one of the key detoxifying enzymes. Oxidative damage caused by these ROS
molecules can be overcome by either enzymatic or nonenzymatic ROS detoxifica-
tion machinery in the cell (Kaushal and Wani 2016). To combat drought stress,
Gusain et al. (2015) have used PGPR consortium of P. jessenii, P. synxantha, and
A. nitroguajacolicus for treating contrasting rice genotypes. Interestingly, the
antioxidant enzyme machinery comprising of CAT, SOD, APX, and POD was
upregulated making the plant tolerant to drought. A similar kind of consortium
analysis was carried out on drought-stressed maize plants, where inoculation of
five Pseudomonas species upregulated ROS detoxifying enzyme machinery confer-
ring drought tolerance to the plants (Sandhya et al. 2010). Combination of PGPR and
arbuscular mycorrhizal (AM) fungus also led to drought tolerance in lettuce by
managing ROS as well as by the accumulation of proline (Kohler et al. 2008).
Various other reports confirm the activation of CAT, SOD or APX activity in
stressed plants upon PGPR inoculation (Baltruschat et al. 2008; Sun et al. 2010;
Heidari and Golpayegani 2011; Vardharajula et al. 2011). Table 22.1 gives the
details of diverse microbes used for the alleviation of abiotic stresses from crop
plants.
22.5 Omics Strategies for Cross-Tolerance
Plant-microbe interaction is a highly complex and interwoven system and affects

plant ecology, health, and productivity. Therefore a deep understanding of this
interaction will help to improve crop performance under adverse environmental
conditions. Interaction of microbe with plant root sensitizes distal or local plant
part at physiological, biochemical, and molecular levels. For dissecting deep inter-
action mechanisms and its interconnection with stress alleviation, multiomics
approach could be more promising to decipher changes at the genome, proteome,
and metabolome levels. Figure 22.3 gives a detailed outline of the omics strategies
employed for stress alleviation in the plant-microbe interaction.
22.5.1 Genomics Approaches
An optimum level of tolerance to abiotic stresses could be obtained through plant-

microbe interaction. Omics study will provide deep knowledge of the mechanism
behind plant-microbe interaction. The unusual decrease in the cost of DNA sequenc-
ing costs has shown the way to the formation of large-scale bacterial genome
collections. Several publically available genomic datasets of plant-associated bacte-
rial isolates are easily approachable. Genomes of bacterial isolates can be compared
to find out the specific gene or pathway correlating with the phenotype of interest and
their function could be predicted through their genetic manipulation. Currently,
several genomes of bacterial isolates from different plant environments have been
sequenced and compared to select the genes that affect general adaptation to plants
(Levy et al. 2018). This information could assist to have a close insight into the
mechanisms of established plant–microbe interactions. Growth-promoting and
stress-alleviating activity of T. atroviride on tomato is confirmed through the
degradation of IAA in the rhizosphere and ACC deaminase activity (Gravel et al.
2007). The presence of ACC deaminase in T. atroviride was checked through RNA
interference at genomic level (Viterbo et al. 2010; Kubicek et al. 2011). The
Table 22.1 List of diverse microbes having a role in plant-stress tolerance
542
Crop Abiotic
plants stresses PGPR used Mechanism References
Rice Drought P. jessenii, A. nitroguajacolicus Promotes plant growth Gusain et al. (2015)
Salinity PGPR Induction of the stress responsive plant gene Jha et al. (2014)
RAB18
Soybean Drought P. putida Higher gibberellin levels promote growth Sang et al.(2014)
Salinity Pseudomonas simiae Enhances the germination rate of soybean seed Vaishnav et al. (2016)
Pseudomonas koreensis Enhances K+/Na+ ratio Kasotia et al. (2015)
Glomus etunicatum Higher proline accumulation in root Sharifi et al. (2007)
Glomus intraradices Accumulation of carbohydrates Porcel and Ruiz-Lozano
(2004)
Wheat Drought Bacillus amyloliquefaciens, A. brasilense Enhanced expression of the stress responsive Kasim et al. (2013)
genes
Streptomyces pactum ABA signaling Li et al. (2019)
Pseudomonas spp. Protects from oxidative damage Chandra et al. (2018)
Bacillus thuringienesis Production of alginate Timmusk et al. (2014)
Agrobacterium fabrum, Bacillus ACC deaminase activity elevated Zafar-ul-Hye et al. (2019)
amyloliquifaciens
Salinity Glomus clarum, Glomus etunicatum Regulated K+/Na+ levels Daei et al. (2009)
Heat Bacillus amyloliquefaciens Production of HSPs and better ROS El-Daim et al. (2014)
management
Tomato Drought Azospirillum brasilense Phytohormone IAA production Molina-Favero et al. (2008)
Salinity Achromobacter piechaudii Reduced levels of ethylene, Increased biomass Mayak et al. (2004b)
under stress
Heat Curvularia proturberata Root colonization de Zelicourt et al. (2013)
Heavy Methylobacterium oryzae Nickel and cadmium uptake is reduced Madhaiyan et al. (2007)
metal
P. Gupta and M. Mishra
22
Maize Drought Rhizobium Accumulation of osmoprotectant and regulated Bano and Fatima (2009)
K+/Na+ levels
Salinity Azospirillum lipoferum Higher ABA levels induced by GA Cohen et al. (2009)
Osmotic Bacillus megaterium Induction in ZmPIP expression Marulanda et al. (2010)
stress
Pepper Drought B. licheniformis Expression of the stress responsive genes Lim and Kim (2013)
Salinity Burkholderia and Arthrobacter spp. Higher proline accumulation Barka et al. (2006)
Azospirillum brasilense and Pantoea High rate of photosynthesis del Amor and Cuadra-
dispersa Crespo (2012)
Lettuce Drought Bacillus spp. Higher plant growth Vivas et al. (2003)
Salinity Bacillus subtilis Phytohormone induced higher biomass Arkhipova et al. (2007)
Glomus intraradices ABA-mediated regulation Aroca et al. (2007)
Pea Drought Pseudomonas fluorescens Modification in root for more water absorption Zahir et al. (2008)
Variovorax paradoxus Phytohormone mediated higher yield Dodd et al. (2005)
Heavy Pseudomonas brassicacearum Nutrient uptake by root modification Safronova et al. (2006)
metal
Rhizobacteria-Mediated Alleviation of Abiotic Stresses in Crops
543
Fig. 22.3 Omics approaches to identify genes, proteins, and metabolites, involved in stress
alleviation through plant-microbe interaction. A representation of different strategies that help to
identify the impact of plant-microbe interaction on abiotic-stress tolerance
inoculation of Pseudomonas sp. AK-1 and Bacillus sp. SJ-5 to soybean results in
superior tolerance to salt stress due to proline accumulation and lipoxygenase
activity (Kumari et al. 2015). Proline accumulation in bacterial-treated plants was
also found due to overexpression of GmP5CS gene (Kim et al. 2007). Inoculation of
Pseudomonas brassicacearum, Rhizobiumleguminosarum with Brassica juncea
provides tolerance to zinc toxicity (Adediran et al. 2016). Zinc toxicity was mediated
by expression of acetyl transferases lacA and cysE genes (Downie 1989). There are
several reports relating to abiotic stress alleviation and plant-microbe interaction.
Metagenomics is an alternative approach to sequence plant microbiome
metagenome. High-throughput metagenomics sequencing is an exceptionally useful
tool for a better understanding of plant growth-promoting rhizobacterial
communities. Two types of ACC-deaminase genes (acdS), homologous to Pseudo-
monas fluorescens, for stress alleviation were detected from potato endophyte
through PCR analysis. Metagenomic libraries helped to find out the entire acdS
operon from uncultivated endophyte and discovered a transcriptional regulator gene
acdR at upstream of acdS (Nikolic et al. 2011). The metagenomics library also
helped to find out salt tolerance genes in uncultivable bacteria by growing at
inhibitory NaCl concentrations of 750 mM (Kapardar et al. 2010).
22.5.2 Proteomics Approaches
For the physiological metabolism and protein-protein interaction studies in plants

and microbes, proteomics has emerged as a powerful tool. Proteomics provides a
deep understanding of biological regulation through the identification of several

proteins responsible for stress alleviation. Ghabooli et al. (2013) reported that
P. indica mitigates the drought stress in barley by photosynthesis stimulation
releasing energy and higher antioxidant production. Wang et al. (2016) screened
the cDNA library of 4 M salt-stressed Dunaliella salina and found a novel gene
Ds-26-16. This gene provides salt tolerance in E. coli, Haematococcus pluvialis, and
tobacco through upregulating the transcription factors for salt tolerance. Microbes
are more responsive to stress conditions due to having diverse metabolic pathways.
Metaproteomics is the advanced version of proteomics that deals with multiple
metabolic interactions occurring simultaneously in an ecosystem. Currently,
Haloarchea and Halobacteria are getting more attention due to their ability to
grow in high salt conditions and their plant growth-promoting ability can be
employed for the alleviation of stresses in saline and sodic soils. Pseudomonas,
well known for hydrocarbon degradation ability, has been well characterized for
their plant growth-promoting ability through proteomics studies (Wang et al. 2015).
These studies assure that these species could be used for field applications under
diverse environmental conditions. The methylotrophic bacteria are also gaining
more attention due to their plant growth-promoting ability under various conditions
(Yim et al. 2013). The proteomic studies of this characteristic phyllosphere commu-
nity have helped to explore the proteins involved in survival mechanism under
relatively harsh environments. There are other reports also where proteomic studies
have helped to identify several proteins to create a boom in stress alleviation
strategies at the molecular level.
22.5.3 Metabolomics Approaches
Metabolomics includes all the metabolites elaborated by an organism in the present

environmental conditions which directly correlates with diverse metabolic pathways
inside the cell. It is, therefore, essential to know the metabolome of an organism both
in normal and stress conditions to find out the changes induced within the pathways
under stress condition. The metabolomic studies highlight the presence of various
bioactive chemicals in plant metabolome (Ketchum et al. 2003). This observation
shows a relationship with the identification of various molecules secreted by plants
to induce main biochemical pathways in colonizing microbes (Micallef et al. 2009).
Auxins produced by Trichoderma spp. to stimulate plant growth and stress allevia-
tion (Contreras-Cornejo et al. 2009). Two secondary metabolites, harzianolide and
6-pentyl-a-pyrone of Trichoderma were also observed to have auxin-like effects in
the etiolated pea stem (Vinale et al. 2008). The alteration in the rhizospheric
microbial molecular mechanisms is yet to be explored. Allen et al. (2009) reported
that it is not only the accumulation of a specific metabolite but also the changes in the
flux of several pathways which decides the stress tolerance. Rhizospheric bacteria
produce the plant growth-promoting biomolecules (cytokinins, gibberellins, etc.)
which serve the purpose (Robin et al. 2006). Sorty et al. (2016) reported that IAA
produced by different microbial species influences seedling growth of wheat under
saline conditions. Similarly, the phosphate solubilizing ability of strains of Bacillus

sp. has successfully improved the yield of fennel in semiarid saline soil (Mishra et al.
2016). Microbial siderophores have been reported to help in the uptake of iron to
plant roots and stress alleviation by heavy metal. The siderophores produced by
Pseudomonas fluorescens C7 successfully supplied the iron to Arabidopsis thaliana
(Vansuyt et al. 2007). Many microbes from the ecosystem represent interdependence
regarding the substrate utilization and metabolite exchange where cometabolism
plays an important role. Cometabolism involves concurrent oxidation of
nonsubstrate compounds with that of true substrates during bacterial growth. This
property of cometabolism has an important role in biochemical pathways involved in
the metabolism of polycyclic and polyaromatic compounds (Chauhan et al. 2008).
Metabolomics studies of such processes could be exploited to the sites where the
native ecosystems have converted to the stress conditions due to the accumulation of
xenobiotic and recalcitrant compounds. The quantitative metabolomics studies could
reveal the level of intervention by the stressor in the overall cellular homeostasis.
Sorty et al. (2016) reported the employment of microbial-originated biomolecules
under imitated conditions in the phyllosphere which demonstrate that live microbes
fail to express vital genes for PGP activity under stress conditions. Therefore, the
enhancement of the rhizosphere with the appropriate quantity of such metabolites
requires a thorough evaluation.
22.6 Conclusion
The current chapter focuses on plant-microbe interaction involved particularly for

surviving under abiotic stresses. Plants do respond to these stresses at the prelimi-
nary level but prolonged stress can cause major damage. Microbes have provided an
important tool for the mitigation of abiotic stresses in plants. In this chapter, we have
shed light on the various mechanisms of the stress tolerance provided by these tiny
living creatures to the plants. The rhizospheric microbes provide passive tolerance to
the stressed plants by various mechanism viz. production of phytostimulators and
osmolytes, ion detoxification, nutrient mobilization, induction of stress responsive
gene, etc. Reports from diverse research summarized in this chapter confirm that the
microbes provide tolerance against distinct abiotic stresses to various crop plants
including both monocots and dicots. Altogether, the native microbes provide an
excellent natural source for mitigating the abiotic stresses and thus directly contrib-
ute to the higher production without compromising the crop quality under marginal
conditions.
22.7 Future Prospect
Agricultural stability is the prime most requirement of crop biologists across the
world because all the other sectors such as health and economy are dependent on
agriculture. With global climate issues, increasing production, while maintaining
crop quality has become a challenging task for scientists. Extensive work has been
carried out to understand the plant responses to abiotic stresses and technologies to
mitigate these stresses. Potential of these innate rhizobial microbes have been
realized in the alleviation of the plant abiotic stresses too. In turn, the plant-microbe-
stress interaction studies have identified many novel pathways, gene circuitry,
biomolecules, and metabolites signal cascade and have broadened up the existing
knowledge of plant stress alleviation. However, these studies are at a level, where
the key components which are involved in the stress alleviation are identified, but the
knowledge about the exact mechanism is not there. Future studies must address the
following technologies for the betterment of crop systems: (1) Identifying the best
candidate PGPR or combination of PGPR consortium which can function for most
of the cropping systems and which can prove functionality at multiple abiotic stress
level, (2) Furthermore, extensive work needs to be done to dissect out the molecular
machinery and pathways involved the plant-PGPR interaction under stresses,
(3) Molecular analysis to fetch out the key stress regulatory genes from the bacterial
counterparts, (4) Utilizing the best candidate gene for the ectopic expression in the
sensitive plant varieties to make them multistress tolerant, and (5) Applying the
“omics” strategies to have a complete snapshot of the global transcriptome, prote-
ome, and metabolome changes in plant-microbe-stress interaction.
Acknowledgments Research fellowships received from Council for Scientific and Industrial
Research (CSIR) to PG and grants received from SERB-DST to MM are duly acknowledged. We
thank Dr. Sushil K. Sharma for giving us an opportunity to contribute a book chapter.
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Rhizospheric Microbes as Potential Tool
for Remediation of Carbofuran: An 23
Overview
Mohd Aamir Khan, Abhishek Sharma, Sonal Yadav, and

Satyawati Sharma
Abstract
The use of chemical pesticides, fertilizers, hybrid seeds, and modern irrigation
practices is an obligation for our agriculture to be healthy and to be able to feed
the vast population. More than half of the cultivable land in the Indian cropping
system depends on chemical pesticides, which leads to increased demand
and consumption of these pesticides. Carbofuran (2,3-dihydro-2,2-
dimethylbenzofuran-7-yl methylcarbamate) belongs to the methyl carbamate
group of pesticides extensively employed as nematicide and insecticide and is
one of the most consumed pesticides in India. It is highly persistent in the soil,
which leads to its leaching to groundwater and surface water, causing various
health hazards in humans and other communities. Thus, there is an imperative
demand for feasible approaches for the removal of carbofuran from soil and
water. Bioremediation technologies for pesticide removal have been used for
both on-field and off-field applications and are widely accepted across the world
because of their eco-friendly nature. The authors in the present chapter review the
current advancements on microbial remediation of carbofuran, its degradation
pathways, and mechanisms, including the role of enzymes and other factors.
Keywords
Pesticide · Carbofuran · Bioremediation · Ecofriendly
M. A. Khan · S. Yadav · S. Sharma

Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi,
India
A. Sharma (*)
Amity Food and Agriculture Foundation, Amity University, Noida, Uttar Pradesh, India
e-mail: asharma5@amity.edu

23, https://doi.org/10.1007/978-981-15-9154-9_23
558 M. A. Khan et al.
23.1 Introduction
The ever-growing population has triggered the transition from traditional practices to
modern science-based agriculture. The use of chemical pesticides, fertilizers, hybrid
seeds, and advanced irrigation practices is must for our agriculture to be strong and
supportive of feeding the vast population. As the availability of land is decreasing
day-by-day, the application of fertilizers and pesticides has become necessary to
meet the demand for food grains. Recent reports by Directorate of Plant Protection,
Quarantine and Storage (DPPQ&S), Ministry of Agriculture & Farmers Welfare,
revealed that total chemical pesticide consumption in the year 2018–2019 was
around 59,670 MT (Metric tonnes) technical grade (Government of India 2019a).
According to a report by DPPQ&S during 2018–2019, the total area under cultiva-
tion across India was ~167 million hectares. Out of the total cultivated area, ~52%
was under the use of chemical pesticides, ~8% under biopesticides, ~19% under
both chemical and biopesticide, and ~ 21% under no pesticides (Government of
India 2019b). So in Indian cropping systems, the significant portion of the cultivable
land is under the use of chemical pesticides, which leads to the increased demand
and consumption of indigenous as well as imported chemical pesticides. Among all
the Indian states, Maharashtra and Uttar Pradesh are major pesticide consuming
states with annual use of 11,746 MT and 11,049 MT during 2018–2019, respec-
tively. Both the states constitute more than a 38% share of the total pesticides
consumed throughout the year (Fig. 23.1). Other major pesticides consuming states
are Punjab, Haryana, and Telangana, which together share more than 24% of the
total pesticides consumed during 2018–2019 (Government of India 2019a).
Fig. 23.1 State-wise consumption of pesticides in India during 2018–2019 (Govt. of India 2019a)
23 Rhizospheric Microbes as Potential Tool for Remediation of Carbofuran: An. . . 559
In 2018–2019, 33.51 MT of imported carbofuran had been consumed in India,

which was fourth among the highest consumed imported pesticides after Profensos
(38.04 MT), Cartap hydrochloride (37.72 MT), and Imidacloprid (35.72 MT) (Gov-
ernment of India 2019c). Among the use of indigenous pesticides, 198.83 MT of
carbofuran was consumed during 2018–2019, which was categorized under the top
ten highly consumed indigenous chemical pesticides in India (Government of India
2019d). According to the DPR’s Pesticide Use Report, >130,000 pounds of
carbofuran (active ingredient) was used alone in California during 1999–2000. As
an outcome of excess and diverse use and discharge, carbofuran was frequently
detected in the surrounding water bodies, which posed potential health hazards (Seo
et al. 2007).
Carbofuran (C12H15NO3) is a crystalline white solid, which is highly stable at pH
seven or below, with the stability decreasing with increasing pH. It belongs to the
n-methyl carbamate class of pesticides, which are esters derived from N-methyl
carbamic acid (Fig. 23.2; Table 23.1). WHO has classified carbofuran as a highly
hazardous compound underclass 1b of Active Pesticide Ingredient Index (WHO
2010). Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate),
first registered in the United States in 1969, is one of the most consumed pesticides
Fig. 23.2 Chemical structure

of carbofuran (Source:
USEPA 2007)
Table 23.1 Physicochemical properties of carbofuran

2,3-dihydro-2,2-dimethylbenzofuran-7-yl
IUPAC Name, molecular formula methylcarbamate; C12H15NO3
Molecular weight 221.26
Melting point 150–152 C
Solubility Water-351 ppm
Acetone-150 ppm, Acetonitrile-140 ppm, Benzene-
40 ppm, Cyclohexane-90 ppm, Dimethyl formamide-
270 ppm, and Dimethyl sulfoxide-250 ppm
Vapor pressure 6 107 mm Hg (25 C)
Hydrolysis half-life (days) 27.7 (pH 7, 25 C)
Aqueous photolysis half-life (days) 7.9 103 (pH 7)
Soil photolysis half-life (days) 138 (27 C, pH 5.7)
Source: Rubin and Evert (2006)
in India. It is an n-methyl carbamate insecticide widely used in agriculture, forestry,

and horticulture, for protection against nematodes (USEPA 2007). It is mainly used
on crops such as potatoes, corn, canola, strawberries, and alfalfa (Chapalamadugu
and Chaudhry 1992). Target species include corn rootworm, chinch bugs, beetles,
green bugs, aphids, grasshoppers, lygus bugs, leafhoppers, weevils, nematodes,
wireworms, etc.
The carbofuran operates by inhibiting the acetylcholine esterase (AChE). It
thereby provokes the buildup of acetylcholine at the nerve cell junctions and the
receptors, conclusively increasing the charge in the nerves to fire continuously,
resulting in convulsions, tremors, and death (Ecobischon 1993; Hayes 2001).
Carbofuran on continued exposure can cause sweating, weakness, blurred vision,
abdominal pain, nausea and vomiting, muscle twitching, and loss of coordination
and may cause breathing to stop.
Carbofuran detoxifies in soil via several mechanisms, including microbial degra-
dation, hydrolysis, and photolysis. It is highly persistent in soil (t1/2: 26–110 days in
land) depending upon soil pH, moisture content, soil type, and the microbial
population. Carbofuran phenol, 3-hydroxycarbofuran, and 3-ketocarbofuran are
the major resultant products of carbofuran degradation (WHO 2004). The laboratory
study by Campbell et al. (2004) observed half-life (t1/2) of carbofuran in native and
Ottawa sand and reported 40 days as the carbofuran half-life. Soil half-life of
carbofuran in Bagan Datoh and Labu soil (Malaysian soil samples) was determined
at various field capacities. The autoclaved soil samples differed in soil moisture, and
the organic matter had carbofuran half-life of 147.5 and 301.37 days at full field
capacities at 30 C (Farahani et al. 2008).
Similarly, the degradation of carbofuran in water occurs via hydrolysis, microbial
decomposition, and photolysis. According to a report by Health and Family welfare
Canada, the carbofuran showed higher stability at lower pH with aqueous half-lives
as 1.0 (pH 8), 8.2 (pH 7), and 690 (pH 6) weeks (WHO 2004).
In a study entitled “Carbofuran in drinking water” (WHO 2004), it was reported
that humans on exposure to carbofuran following aerial applications had 0.7–2 mg/
day as maximum inhaled doses. Regarding acute toxicity tests, oral toxicity for
carbofuran in rats has LD50 values of 6–7.8 mg/kg. In comparison, acute dermal
toxicity of carbofuran in rabbits has an LD50 value of 4403 mg/kg of intact skin
(USEPA 2007). According to a study by FMC in 1976, human studies under
controlled exposure were conducted where male tests were orally subjected to
three different doses of carbofuran, i.e., 0.05, 0.1, and 0.25 mg/kg, and the symptoms
were observed at different time intervals. The signs and symptoms related to
exposure were found at a rate of 0.25 mg/kg after 3 hours of exposure. The
symptoms included salivation, dry mouth, abdominal pain, diaphoresis, nausea,
vomiting, and drowsiness. Similar symptoms were witnessed on dermal exposure
(4 mg/kg) of carbofuran after 2–3.5 h of exposure (Rubin and Evert 2006).
Farmers across the world use a broad range of pesticides and insecticides, which
makes it highly ticklish to develop a single protocol that can be applied universally
for the removal of these chemicals. Ion exchange resins and biosorbents employed
for toxicant removal are susceptible to environmental conditions. Other technologies
that are frequently used for the decontamination of pesticides include

photodegradation, chemical oxidation with ozone, combined ozone, and UV irradi-
ation, Fenton oxidation, biological degradation, coagulation, and adsorption (Gupta
et al. 2006).
23.2 Remedial Measures for Carbofuran
23.2.1 Physicochemical Treatments
The physicochemical treatments for carbofuran removal from contaminated samples

may include adsorption, absorption, advanced oxidation processes, etc. Adsorption
is the most efficient and widely applied treatment method and suitable for mass-scale
treatments. Several adsorbents have been used for the removal of contaminants from
polluted sites. Activated carbon (granules) is known to decontaminate potable water
as well as industrial effluents (Sotelo et al. 2002). Adsorption using natural waste
materials is economical and eco-friendlier as these processes produce a very lesser
amount of secondary effluents, even though the adsorption reaction is slow (Das
et al. 2014). Salman and Hameed (2010) explored banana stalks for the production of
activated charcoal to adsorb carbofuran from water. Researchers have developed
carbonaceous adsorbent materials from fertilizer and steel industry wastes that can
adsorb 50% of the pesticides within 15–30 mins of contact time (Gupta et al. 2006).
Chemical treatments such as advanced oxidation degrade hazardous and persis-
tent organic compounds into carbon dioxide with the production of OH radicals
(Ma et al. 2010). AOPs such as ultrasound, Fenton, ozone, and ultraviolet light are
generally used for the removal of pesticide from contaminated water. Tennakone
et al. (1997) treated wastewater contaminated with carbofuran at the rate of 222 mg/
L using a combination of UV/TiO2 processes at pH 2.8. The process resulted in
oxidation of 90% and the complete mineralization of carbofuran to carbon dioxide
within 6 h and 15 h reaction times, respectively. Researchers have also used
ultrasonic processes at ultrasound energy of 1800 W with the treatment efficiency
of 90% carbofuran oxidation within a reaction period of 30–60 mins when applied at
the concentration of 30 mg L1 (Hua and Pfalzer-Thompson 2001). In another study,
ozone oxidation was coupled with UV to treat carbofuran (100 mg L1)-
contaminated wastewater at pH 2 with 90% oxidation of the carbofuran within
50 mins (Mahalakshmi et al. 2007). Ma et al. (2010), by using a combined ultra-
sound and Fenton process, were able to degrade more than 99% carbofuran
(20 mg L1), out of which 46% of carbofuran was mineralized within 30 mins at
pH 3. Semiconductor oxides such as ZnO and TiO2 have also been used in
photocatalytic degradation of carbofuran, where complete mineralization was
observed within 5 h with TiO2 under optimal conditions. Four degradation products
identified by GC-MS were 2,3-dihydro-2,2-dimethylbenzofuran-7-yl carbamate,
2,3-dihydro-2,2-dimethyl-7-hydroxy benzofuran (carbofuran phenol), 2,3-dihydro-
2,2-dimethyl benzofuran, and 2,3-dihydrobenzofuran (Mahalakshmi et al. 2007).
Although physicochemical treatments are comparatively rapid than bioremedia-

tion, they cause detrimental effects to the soil, requires high energy, and costs inputs
than biological treatments (Foght et al. 2001).
23.2.2 Rhizospheric Microbes for Carbofuran Removal
The rhizosphere is a restricted zone of soil near the plant roots where numerous
microbial biochemical processes take place, including contaminant degradation. The
rhizosphere provides an increased supply of nutrients to the microorganisms in the
form of a wide range of organic compounds such as sugars, alcohols, and acids via
root exudates and mucilage, which leads to the increased microbial activity and
diversity (Hrynkiewicz and Baum 2012). Microorganisms have dominated the field
of bioremediation due to their ability to transform all forms of organic matter. The
significant proportion of bacteria in the rhizospheric zone comprises nonsporulating,
gram-negative bacilli followed by gram-positive bacilli and aerobic nonspore
forming bacteria.
The rhizosphere affords microhabitats for aerobic and anaerobic microorganisms
(Walton et al. 1994). The plants have been exposed to different natural as well as
anthropogenic toxicants, including halogenated aliphatics, aromatics,
nitroaromatics, chloroaromatics, pesticides, polychlorinated biphenyls (PCBs),
polycyclic aromatic hydrocarbons (PAHs), phthalate esters, and nitrosamines. The
degradation of these compounds using rhizospheric microbes has been extensively
studied. The fate of these compounds present in the rhizosphere depends upon the
interaction between three components of the rhizosphere, i.e., the microorganisms,
plant, and soil. Along with the potential pollutant degrading microorganisms, the
plant factors also play a significant role in the increased rate of pollutant removal
from rhizospheric sites. The plant factors include different organic and inorganic
chemicals in the form of root exudates, which vary with the plant species, the
physical morphology of the roots, which affects the bioavailability of the compounds
to the microbes, plant cell wall, and the uptake mechanisms. The different microbial
species for carbofuran removal are discussed below.
23.2.2.1 Microbial Degradation of Carbofuran

The remediation of carbofuran by rhizospheric microbes has been documented in
several reports. A gram-negative rod-shaped Burkholderia sp. PCL3 was isolated
from carbofuran-phytoremediated rhizosphere soil of rice. The isolate immobilized
on organic carriers was able to degrade carbofuran up to 200 mg L1 in basal salt
media within 30 days of incubation (Plangklang and Reungsang 2012). Peng et al.
(2008) isolated Paracoccus sp. YM3 from carbofuran contaminated sludge. The
Paracoccus sp. YM3 showed 78% carbofuran degradation efficiency after three days
of incubation, which was further enhanced to 88% by the addition of sucrose as a
carbon source. Sphingomonas paucimobilis (96–99% reliability) and Pseudomonas
aeruginosa (94.2% reliability) have been isolated from potato-cultivated soils
contaminated with different pesticides (Castellanos Rozo et al. 2013). A novel
carbofuran-degrading bacterium, Novosphingobium sp. strain FND-3, was able to

degrade 100 mgL1 carbofuran within 4 hours of incubation. The degradation
pathway suggested the transformation of carbofuran to carbofuran-7-phenol, which
hydrolyzed into 2-hydroxy-3-(3-methylpropan-2-ol) benzene-N-methylcarbamate
as an end product (Yan et al. 2007). Bano and Musarrat (2004) isolated a Pseudo-
monas sp. from agricultural soil, which completely degraded 100 ppm of carbofuran
from spiked soil within 40 days of incubation with a degradation constant of
0.035 day1. The controls were maintained with no bacterial inoculation, and no
significant degradation of carbofuran was observed. The isolate was also checked for
its plant growth-promoting potential and biocontrol activity with different PGPR
(plant growth-promoting rhizobacteria) biochemical tests and showed positive
results for IAA production, phosphate solubilization, siderophore production, etc.
Krishna and Philip (2011) studied the carbofuran degradation using
Chryseobacterium joostei (MTCC 9237) in soil bioreactor with different soil types
viz. sandy, clayey, red, and compost under aerobic and facultative anaerobic
conditions. The carbofuran degradation efficiencies observed in sandy, clayey, red,
and compost soil were around 86%, 61%, 59%, and 47% in aerobic and 92%, 77%,
75%, and 63% in facultative anaerobic conditions, respectively. Under aerobic
conditions, two different metabolites of carbofuran, namely, 3-ketocarbofuran and
3-hydroxycarbofuran, were detected, while in anaerobic conditions, only
3-ketocarbofuran was detected in soil fractions. The other carbofuran-degrading
microbial strains isolated in recent years have been listed in Table 23.2.
23.2.2.2 Fungal Degradation of Carbofuran

Fungi are the other most abundant group of microorganisms in soil exceeding the
bacterial biomass. Rhizospheric fungi benefit the plant by enhancing the nutrient and
water uptake, increased pathogen resistance, and tolerance to environmental stresses.
Several fungal species like Mucor ramannianus, Pichia anomala, Trametes
versicolor, Phlebia sp., and Lenzites betulinus have been reported for carbofuran
degradation (Seo et al. 2007; Yang et al. 2011; Ruiz-Hidalgo et al. 2014; Wang et al.
2019). Two different white-rot fungi Phlebia sp. and Lenzites betulinus degraded
~50% of carbofuran (80 mg kg1 soil) after an incubation period of 20 days.
However, the enhanced degradation rates were obtained after using the immobilized
cultures of the same fungal strains on wheat straw (Wang et al. 2019). In a study by
Seo et al. (2007), Mucor ramannianus was tested for the degradation of carbofuran
and one of its metabolites, i.e., carbofuran-7-phenol, and its fate was elucidated by
detecting the different degradation metabolites. It was observed from the MS
analysis of culture extract of M. ramannianus when incubated with 1 mM
carbofuran, and carbofuran-7-phenol produced different metabolites. The
metabolites formed from further degradation of carbofuran-7-phenol were detected
as 3-hydroxycarbofuran-7-phenol and 7a-(hydroxymethyl)-2,2-dimethyl
hexahydro-6H-furo[2,3-b] pyran-6-one or 2-hydroxy-3-(3-methylpropan-2-ol) phe-
nol. Pichia anomala strain HQ-C-01 resulted in 95.2% degradation of carbofuran
(50 mg/l) within 48 h by utilizing it as a sole source of carbon in minimal media,
Table 23.2 Common carbofuran-degrading microbial strains

Sl. Concentration
No. Microorganism tested Description Reference
1 Pseudomonas 200 mg l1 Spiked minimal salt Bano and Musarrat
sp. NJ-101 media (MSM) (2004)
2 Pseudomonas putida 50 mg l1 Spiked MSM and soil Gong et al. (2018)
50 mg kg1
3 Paracoccus sp. YM3 50 mg l1 Spiked MSM Peng et al. (2008)
4 Pseudomonas 200 mg l1 Spiked MSM Castellanos Rozo
aeruginosa et al. (2013)
5 Sphingomonas 200 mg l1 Spiked MSM
paucimobilis
6 Novosphingobium 100 mg l1 Spiked MSM Yan et al. (2007)
sp. FND-3
7 Novosphingobium 200 & Spiked phosphate Nguyen et al.
sp. KN65.2 500 mg l1 buffer saline (2014)
8 Klebsiella 20 & 30 mM Spiked MSM Kadakol et al.
pneumoniae (2011)
ATCC13883T
1.9 Pichia anomala 200 mg l1 Spiked MSM Yang et al. (2011)
HQ-C-01(yeast) 50 mg kg1 Agricultural soil
1.10 Burkholderia 20 mg kg1 Bioslurry phase Plangklang and
cepacia PCL3 sequencing batch Reungsang (2010)
reactor
5 mg kg1 Small field scale Plangklang and
(agricultural soil) Reungsang (2011)
11 Trametes versicolor 10 mg kg1 Spiked rice husk Ruiz-Hidalgo et al.
(2014)
12 Mucor ramannianus 1 mM Spiked potato dextrose Seo et al. (2007)
media
13 Bacillus cereus 100 mg l1 Spiked MSM Onunga et al.
Bacillus (2015)
thuringiensis
14 Chryseobacterium 200 mg kg1 Submerged soils in Krishna and Philip
joostei bioreactor (2011)
15 Cupriavidus sp. 400 mg l1 Spiked MSM Gupta et al. (2019)
ISTL7
leading to the formation of benzofuranol as degradation intermediate detected via

GC/MS analysis (Yang et al. 2011).
23.2.3 Pathways for Microbial Degradation of Carbofuran
Several intermediate metabolites during carbofuran degradation have been separated

and detected via LCMS and GCMS analyses. In a study by Yan et al. (2007),
Novosphingobium sp. FND-3 was found to degrade carbofuran by utilizing as a
Fig. 23.3 Proposed pathways for microbial degradation of carbofuran (Yan et al. 2007)
sole carbon source and resulted in the production of several intermediate compounds
detected by GCMS analysis. The peaks for carbofuran and one of its metabolite
carbofuran-7-phenol were observed at retention times of 9.36 and 6.39 mins,
respectively. The MS analysis of culture extract revealed the presence of metabolites
resulting from the cleavage of ether and ester bonds present in the carbofuran. The
metabolite with M+ peak at 239 m/z was formed possibly due to the addition of water
molecule, causing the cleavage of ether bond, which was further detected as
2-Hydroxy-3-(2-methylpropan-2-ol)benzene-N-methyl carbamate. Another metabo-
lite of M+ peak at 182 m/z detected at 8.37 mins was proposed to be 2-hydroxy-3-
(3-methylpropan-2-ol) phenol formed from hydroxylation of carbofuran-7-phenol.
The third compound detected with M+ peak at 237 m/z was found to be similar to the
fragment ion patterns of 4-hydroxycarbofuran and 5-hydroxycarbofuran, which are
formed via hydroxylation of the benzene ring (Fig. 23.3).
In another study, carbofuran degradation metabolites were detected during
carbofuran degradation by wild type and mutant strains of Sphingomonas sp. KN
65.2. A metabolite with molecular mass 343.1178 Da was detected while screening
the wild type strain for carbofuran degradation. However, the structure of this
metabolite was not identified. In the case of the mutant strain, seven different
types of metabolites were detected and, depending upon the type of different
intermediate pathways, were hypothesized for carbofuran degradation (Fig. 23.4).
The hydrolysis of carbamate resulted in the formation of carbofuran-7-phenol, while
the oxidation of carbofuran-7-phenol causes the cleavage of the furan ring, resulting
in the formation of 3-(2-hydroxy-2-methyl propyl)benzene-1,2-diol. Other
metabolites such as (3-(2-hydroxy-2-methyl propyl)cyclohexa-3,5-diene-1,2-
dione), 4((2E,4Z)-2,8-dihydroxy-8-methyl-6-oxonona-2,4-dienoic acid), and
5-(3-hydroxy-3-methylbutanoic acid) were hypothesized on the basis of molecular
Fig. 23.4 Pathways for carbofuran degradation by Sphingomonas sp. KN 65.2 (Nguyen et al.
2015)
formulae, MS/MS spectra, monoisotopic patterns, etc. One more metabolite was
proposed to be formed by a hydroxylation methyl group on the furan ring of
carbofuran (Nguyen et al. 2015).
23.3 Advanced Strategies for Enhanced Carbofuran

Degradation by Microbes from Contaminated Soil
23.3.1 Organic Amendments: Biostimulants for Soil Applications
Recently, the research interest in the field of bioremediation has been toward the
behavior of contaminants in the presence of added organic carbon sources
(Blackshaw et al. 2005). The degradation of such organic carbon sources releases
sugars and amino acids that cause enhanced microbial activity in the soil. Moreover,
contaminant mobility is also affected due to the increase in dissolved soil organic
matter (Cox et al. 2001). Sludge from different energy generation plants, including
hydrogen, ethanol, and methane, was used as a stimulator for the enhanced degrada-
tion of carbofuran. While bioaugmentation treatment showed the half-life of
16.63 days, stimulation from the sludge from the hydrogen power plant recorded
carbofuran half-life of 9.53 days. Interestingly, the best carbofuran degradation (t1/2:
3.22 days) was observed when both the biostimulation and bioaugmentation
approaches were applied together (Pimmata et al. 2013). Biomixtures containing
bioaugmented lignocellulosic biomass have also been developed for the purification
of pesticide-contaminated wastewaters. Trametes versicolor, a medicinal mush-
room, was grown in rice husk and evaluated for carbofuran degradation. The
combined treatment degraded 55.1% carbofuran within 34 days with a t1/2 of
29.9 days and resulted in the formation of 3-hydroxycarbofurans, a degradation

product (Ruiz-Hidalgo et al. 2014). The low cost and recycling of nutrients are prime
factors for the use of sludge, compost, and other agro-industrial residues as organic
carbon amendments to the soil (Majumdar and Singh 2007).
23.3.2 Immobilized Potential Microbes: Soil Inoculants for Onsite

Applications
One of the bottlenecks in the successful application of microbe-based remediation is

the dearth of an adequate mechanism that could promote the growth kinetics of
microbial metabolism in the remediation site (Ghosal et al. 2016). To overcome the
issue, we can formulate efficient microbes into an enduring and commercially
relevant product that shall help to sustain its growth in the unfavorable pesticide-
contaminated environment. The use of inert materials as carriers provides physical
support for microbial cells, resulting in better exposure to nutrients, moisture, and
air, which enhances the viability of the microbial cells (Mishra et al. 2001). To date,
not much use of the immobilized microbial cells has been documented for the
cleanup of carbofuran contaminated media. Table 23.3 lists a few examples of the
immobilization techniques being used in carbofuran removal. Recently, water dis-
persible granular formulations have been developed for the controlled release of
RDX-degrading Pelomonas aquatica. The granular formulation degraded 86.25%
RDX (135 μM) in the aqueous phase and 51.04% in soil microcosm studies. The
efficiency of the same formulated isolate was enhanced by stimulating it with
sucrose as a carbon source, which resulted in 77.57% RDX removal from soil
(Khan et al. 2020). Two specific white-rot fungi, Phlebia. sp. and Lenzites betulinus,
when immobilized on wheat straw, increased the degradation of carbofuran by 30%
compared to the unimmobilized system (Wang et al. 2019). Priyani et al. (2018)
isolated two different microbial strains DN1 and OR2 from slaughter waste houses,
which were further immobilized within sodium alginate and were checked for
carbofuran degradation. The encapsulated microbial isolates degraded carbofuran
to carbofuran phenol, which was also utilized as a source of carbon and energy.
Microbial cell immobilization may provide an enhanced survival rate by stabilizing
cells under a stressed environment, which enabled a rapid and efficient degradation
in comparison to free live cells (Moslemy et al. 2002).
23.4 Conclusion
Rhizospheric microbial remediation of carbofuran is a rapid, low cost, environment-

friendly approach, which is preferred over other physicochemical technologies.
Deciphering the mechanisms of degradation along with the pathways will provide
more insights into the effective cleanup of the contaminated environment. The
addition of exogenous carbon sources at the contaminated sites increases the micro-
bial activity, thereby enhancing the carbofuran degradation rates. Different organic
Table 23.3 Different immobilized microbial cultures used in bioremediation

Carriers for Active
S. No. immobilization Contaminant ingredient Reference
1 Wheat straw Carbofuran Phlebia. sp. Wang et al. (2019)
Lenzites
betulinus
2 Water dispersible granules RDX Pelomonas Khan et al. (2020)
(Soy flour) aquatica
3 Calcium alginate beads Carbofuran Laccase Wang et al. (2017)
Methyl extracted from and Sreenivasulu
parathion Lenzites and Aparna (2001)
betulinus
Bacillus sp.
4 Polyurethane foam, Agar, Carbofuran Klebsiella Kadakol et al.
Polyacrylamide, Alginate, pneumoniae (2011)
and bentonite-alginate- ATCC13883T
PAC
5 Corn cob Carbofuran Burkholderia Plangklang and
cepacia PCL3 Reungsang (2013)
6 Fly ash:soil:molasses Endosulfan Bacterial and Abraham and
(15:3:1) fungal Silambarasan
consortium (2014)
7 Zeolite Atrazine Pseudomonas Stelting et al.
sp. strain ADP (2012)
8 Plant fibers (Loofah Methyl Bacterial Moreno-Medina
sponge) parathion consortium et al. (2014) and
Carbendazim Pattanasupong et al.
(2004)
9 Sugarcane bagasse Anthracene Phanerochaete Mohammadi and
chrysosporium Nasernejad (2009)
10 Tezontle Propanil Bacterial Herrera-González
consortium et al. (2013)
11 Cloth sachet Lindane Actinobacteria Saez et al. (2012)
Agar
Calcium alginate
Silicone tubes
carbon sources can be explored for their effect on carbofuran removal from
contaminated soil and water systems. Also, formulating the microbial cultures
ensures their viability over more extended storage periods by providing them with
an inert surface for adsorption and nutrition. The choice of ingredients for the
development of microbial formulations is very significant. There might be a possi-
bility where a recipient might transform parent toxicant to another metabolite that
can act as xenobiotic to the microbe in the formulation. The challenge, however, is to
fabricate a commercially available and effective microbial formulation that shall lead
to the progress of an effective bioremediation process for recalcitrant compounds in
the environment.
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Trichoderma spp.: A Unique Fungal
Biofactory for Healthy Plant Growth 24
Hesham Ali El Enshasy, Kugan Kumar Ambehabati,
Siti Zulaiha Hanapi, Daniel J. Dailin, Elsayed Ahmed Elsayed,
Dalia Sukmawati, and Roslinda Abd Malek
Abstract
The increased global demand for food leads to the extensive use of chemical
fertilizers, which posed significant impacts and drawbacks on animal, environ-
mental, and plant resources. Therefore, much attention has been paid recently
toward the development of biofertilizers and biocontrol agents. Trichoderma is a
fungal species that exists in various ecosystems and has many beneficial effects
H. A. El Enshasy (*)
Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Johor Bahru,
Johor, Malaysia
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi
City of Scientific Research and Technology Application, New Burg Al Arab, Alexandria, Egypt
e-mail: henshasy@ibd.utm.my
K. K. Ambehabati · S. Z. Hanapi · R. A. Malek
Johor, Malaysia
D. J. Dailin
Johor, Malaysia
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi
E. A. Elsayed
Bioproduct Development Chair, Zoology Department, Faculty of Science, Kind Saud University,
Riyadh, Kingdom of Saudi Arabia
Chemistry of Natural and Microbial Products Department, National Research Center, Cairo, Egypt
D. Sukmawati
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Jakarta,
Jakarta, Indonesia

23, https://doi.org/10.1007/978-981-15-9154-9_24
574 H. A. El Enshasy et al.
on plant growth. They have both mycoparasitic and antagonistic potential.

Trichoderma is currently used to control pathogenic microbes and as active
enhancer for plant system defense mechanisms. It can also colonize root systems
and potentially affect their inherent metabolic activities through the production of
various metabolites. Moreover, Trichoderma can have a great influence in com-
bating different biotic/abiotic stress conditions surrounding plant vegetation. It
can also affect the solubilization of various soil nutrients, thus making them easily
consumable by plant. Recently, it has been found that Trichoderma is responsible
for the production and secretion of certain metabolites and enzymes that exert an
antipathogenic influence on different microbes found in soil. The current chapter
will focus on different aspects regarding Trichoderma including its mycoparasitic
and antagonistic potential.
Keywords
Trichoderma spp · Biotic stress · Abiotic stress · Antibiosis · Mycoparasitism
24.1 Introduction
Globally, extensive use of chemicals and fertilizers either in single or in complex

form has been increased manifolds due to the increased demand of food by
burgeoning population. Further, the extensive use of chemicals in agriculture
imposed numerous negative impacts on environment and animal and human health.
In order to overcome this issue, the usage of bio-/microbial fertilizer and
biopesticides has been introduced as the best alternatives to reduce/replace the
usage of chemical fertilizers. There is a range of soil-borne bacteria and fungi that
can colonize the plant roots that have beneficial impact on the plants. In addition to
the classical mycorrhizal fungi and Rhizobium, there are many other plant growth-
promoting rhizobacteria and fungi such as Piriformospora indica and Trichoderma
spp. that can stimulate the plant growth (Van Wees et al. 2008). They can form the
endophytic association, interact with the other microbes like mycorrhizal fungi, and
stimulate the production of phytohormones such as salicylic acid (Poveda et al.
2019). However, this process involves the molecular recognition between the two
partners through the signaling networks. Both Jasmonic acid (JA) and ethylene
(ET) are known for their ability to form the transduction molecules for the induced
systematic resistance (ISR). One of the common features of the ISR response to the
microbes is the priming for the enhanced defense. However, in the primed plants,
most of the times, defense response is accelerated when there is occurrence of attack
by the pathogens or herbivores, which causes the response to be much more lethal
and stronger to the attackers.
Trichoderma spp. is an avirulent plant symbiont ubiquitously present in many
agroecosystems. Some of the strains have ability to reduce the disease severity
caused by pathogens by direct mycoparasitism and/or indirectly through enhanced
ISR (Viterbo and Horwitz 2010). Due to the presence of root-derived nutrients and
24 Trichoderma spp.: A Unique Fungal Biofactory for Healthy Plant Growth 575
fungal prey, Trichoderma spp. establishes in the rhizosphere and shows positive
rhizosphere interactions with other plants (Druzhinina et al. 2011; Halifu et al.
2019). The potential strains appear to show their effects on plants in different
ways such as by increasing plant growth and nutrient uptake, which leads to
significant increase in the rate of seed germination as well as stimulates the plant
defense/resistance against biotic and abiotic stresses (Shoresh and Harman 2008). In
recent times, increasing number of studies have been conducted to reveal the
molecular basis of plant and Trichoderma interaction. For example, some compara-
tive studies have been done to analyze the genome sequence of the two established
biocontrol Trichoderma species viz. T. atroviride and T. virens. Both the species
show better picture on how mycoparasitism takes place (Kubicek et al. 2011).
Trichoderma spp. are used not only to control the growth of pathogen but also for
the enhancement of plant growth and contribute in the development of root system.
They also stimulate the defense system of the plant. Some of the strains on the other
hand are known to penetrate epidermis and colonizate the root surface. Besides the
root colonization, the Trichoderma strains produce secondary metabolites, which
can act as the plant growth regulators. For example, they produce gluconic acid,
citric acid, and oxalic acid, which reduce soil pH and thus solubilize phosphate,
minerals, and other micronutrients (Promwee et al. 2014; Li et al. 2015). Therefore,
Trichoderma plays three key roles in supporting plant growth: source of different
metabolites necessary for growth and nutrient solubilization, acts as biological
control agent using different mechanisms, and supports healthy plant growth under
biotic and abiotic stress (Fig. 24.1).
Fig. 24.1 Different functional effects of Trichoderma to support healthy plant growth
24.2 Root Colonization Mechanism of Trichoderma spp.
Different strains of Trichoderma colonize root systems of many crops. The fungus
has the capacity to colonize the roots of monocotyledonous and dicotyledonous
plants and alters the hormones, soluble sugars, phenolic compounds, water content,
photosynthesis rate, and transpiration processes, which results in significant changes
in the metabolism of the plant (Brotman and Lisec 2012). Trichoderma can colonize
broad range of hosts, and therefore, it can be used as an efficient strategic strain that
can enhance the immunity of the plant and create a good microenvironment for plant
growth and production. The plant-derived sucrose plays a vital role in facilitating
root colonization and coordinating the defense mechanisms and increases the rate of
photosynthesis (Harman et al. 2004).
Any fungal strain that promotes plant growth and provides protection must be
able to colonize the plant roots. The overall colonization processes involve the
recognition, adhesion to roots, and colonization on the plant roots and lastly show
strong response toward the metabolites produced by plant as a result of external
fungal invasion. However, in the case of Trichoderma, the adhesion process to the
root surface is mediated by the hydrophobins. “Hydrophobins are small (about
100 amino acids), cysteine-rich, hydrophobic proteins that are present in large
amounts in fungal cell walls, where they form part of the outermost layer (rodlet
layer); sometimes, they can also be secreted into the medium” (Linder et al. 2002;
Lienemann et al. 2013). The T. asperellum produces class-I hydrophobins, which
have been shown to support the colonization of the plant roots. The colonization
process involves attachment of the hydrophobins to the root surface and protects the
hyphal tips from the plant defense compounds. The swollenin is the expansion-like
protein with the cellulose-binding domain, which helps in recognizing the cellulose
and plays important role in plant cell modification and initiation of root colonization
(Morán-Diez et al. 2009). According to Samolski et al. (2012), T. harzianum
encodes the cysteine-rich cell wall protein, which plays significant role in adhesion
to the root of the tomato plants.
Trichoderma normally colonizes the root surface and causes some changes in
plant morphological features during the mycoparasitism when the hyphae start to
penetrate the root epidermis.
However, the penetration process of the root tissue is limited only to the first and
the second layers of the root surface. The root penetration is usually facilitated by the
secretion of certain hydrolytic enzymes such as cellulases and proteases. The
degrading enzymes of plant cell wall are also majorly involved during the active
root colonization. After 72 h of root colonization, yeast-like cells can be observed
together with the plant epidermal and cortical cell walls. According to Chacón et al.
(2007), the newly formed barrier consists of deposition of large amount of cellulose
and infiltrations of cellulose.
The typical reaction of the host toward Trichoderma can be found beyond the
sites of the potential fungal penetration and it does not induce the reinforcement of
the plant cell wall, which surprisingly is efficient for the restriction of the fungal
growth for the intercellular spaces of the epidermis and cortex. This prevents the
entry of the Trichoderma into the vascular stele. Besides that, the plants, on the other
hand, also show some reaction against fungal invasion simply by accumulation and
secretion of antimicrobial compounds, and therefore, the ability of fungal propagules
to colonize the plant root mainly depends on the ability of the strain to tolerate these
compounds. When it comes to Trichoderma, the resistance is associated with the
ABC transport system. The ABC system is one of the key factors for the multiple
interactions established by Trichoderma with the other types of microbes in
the environment (Ruocco et al. 2009; Liu et al. 2019). During this process, all the
phenolic compounds are removed from the plants (Chen et al. 2011) with the
phytoalexin production being reduced as well (Masunaka et al. 2011). On the
other hand, secretion of cysteine-rich protein in a small amount was detected in
both T. harzianum and T. atroviride. This protein has similar homologue with
avirulence protein known as Avr4. Due to the homologue similarity, the cysteine-
rich protein is expected to protect against plant chitinase with the same mechanism
like Avr4.
During the root colonization, the enzyme that degrades the cell wall to facilitate
fungal penetration is called swollenin (Brotman et al. 2008). This enzyme helps to
carry the cellulose binding module and can cause changes on the crystalline cellulose
structure of the plant cell wall (Eibinger et al. 2016). The protein has a set of
sequences, which is similar to another plant protein known as expansins. This
protein usually helps to cause expansion of the cell wall in plant roots and root
hairs. Due to its function of causing expansion, Trichoderma may increase the root
surface area once it is successfully established in the plant rhizosphere (Druzhinina
et al. 2011). Exchange of the molecular messages takes place during Trichoderma
root colonization, and it includes deposition of the fungal elicitors in the root cell
apoplast (Contreras-Cornejo et al. 2014a; Hermosa et al. 2012). However, the exact
molecular and biochemical mechanisms that take place during the fungal coloniza-
tion need in-depth investigation. According to Segarra et al. (2007), the same effects
were identified in cucumber plants when it was inoculated with T. asperellum T34
where the identified proteins were classified into four groups, which are related to
metabolism and energy, stress and defense, secondary metabolism, and protein
biosynthesis and protein folding structure.
24.3 Trichoderma in Biotic Stress Alleviation
Microbe-associated molecular patterns, which are also known as MAMPs, are

molecules that enhance defense response to initiate some sort of stimulation of
plant immune system in addition to the physical and chemical barriers that formed
in the early stages (Newman et al. 2013). This molecular pattern has the capability to
detect the domains or motifs with its conserved structural traits. The plant responses
initiated by MAMPs take place rapidly and transiently. The early stage of the
response caused by MAMPs generally involves several mechanisms such as move-
ment of ion fluxes across the plasma membrane, the initiation of the production of
reactive oxygen species (ROS), as well as the production of nitric oxide and
Table 24.1 List of MAMPs identified in different species of Trichoderma

Trichoderma
MAMPs species Activity References
Cellulase T. longibrachiatum Activate and heat denature the Martinez et al.
T. reesei cellulases that elicit melon by (2001)
activating the signaling pathway of Li et al. (2017)
SA and ET
Xylanase T. viride Evoke the synthesis of ET and the Rotblat et al.
Trichoderma spp. hypersensitive response in tobacco (2002)
leaf cells Ambehabati
et al. (2020)
18-mer T. virens Elicit the systemic defense in Viterbo and
peptaibols Trichoderma spp. cucumber plant against Chet (2006)
Pseudomonas syringae
20-mer T. viride Activation of JA and Sa synthesis in Engelberth
peptaibols lima bean et al. (2001)
(Alamethicin) Marik et al.
(2019)
Cerato- T. atroviride and Orthologues that can induce the Djonovic et al.
platanins T. virens defense response in maize and cotton (2006), Seidl
Sm1 et al. (2006)
Extracellular T. guizhouense Induction of ROS (H2O2) Xu et al. (2020)
protein accumulation and callose deposition
in maize
induction of ET biosynthesis genes. Different types of MAMPs have been identified

for the plant growth, namely, flagellin, but there are other compounds like specific
proteins, biosurfactants, antibiotics, and volatile compounds that also contributed to
the initiation of plant resistance mechanism (Newman et al. 2013; Zhong et al. 2017;
Xu et al. 2020). The list of some MAMPs identified in different species of
Trichoderma is given in Table 24.1.
Ethylene-inducing xylanase, also known as Xyn2 or Eix, is the very first
recognized Trichoderma MAMP, which is primarily produced by Trichoderma
species. This specific MAMP is produced as an elicitor in response to the specific
tobacco and tomato cultivation (Ron and Avni 2004). The Eix epitope recognized by
the plant consists of five surface amino acids. The fungus activates and heat
denatures the cellulases that elicit melon defense by the activation of the SA and
ET signaling pathways, respectively. There are some proteins produced by
Trichoderma, which are largely involved in root colonization and can also act as
MAMPs. According to Brotman et al. (2008), the swollen in the tissue initiates the
defense response in both cucumber leaves and roots and affords the local protection
from bacteria and fungi. Besides that, the SSCP cerato-platanin from T. virens and
Epl1 from T. atroviride will accumulate in the hyphae when the colonization
processes take place and start to act as MAMPs in cotton and maize plants (Seidl
et al. 2006). The glycosylation mechanism on the other hand helps to keep these
proteins in the monomeric form, which is needed to elicit the ISR response rapidly
(Vargas et al. 2008).
The chitin is an oligosaccharide that acts as elicitors in the activation of defense

responses in plants. It acts as scavenging agent and plays fundamental role in host
colonization (Awad et al. 2014). Since the plants can perceive chitin, they can
develop chitinase enzyme in order to release the fungal cell wall polymers. This
can trigger the defense response of the host. The mycotrophic activity of the
chitinase from the fungus can release chito-oligosaccharides, and this brings effect
toward the induction of defense mechanisms. On the other hand, certain types of
Trichoderma metabolites can exhibit antimicrobial effect at their maximum dose, but
at the same time, they act as MAMPs and auxin-like compound at the lower
concentrations. For example, compounds like 6-n-pentyl-6H-pyran-2-one (6PP),
harzianopyridone, and harzianolide activate the plant defense system and growth
when used at the low concentration (1 ppm) in tomato and pea (Vinale et al. 2008).
This scenario indicates that the developed response toward the fungus and their host
plants and defense mechanisms share the similar concept. Peptaibols can be defined
as the linear peptide antibiotic that consists of 5–20 amino acids generated by the
nonribosomal peptide synthase activity. Alamethicin is a peptaibol from T. viride,
which initiates synthesis of JA and SA in cucumber counter Pseudomonas syringae,
the leaf pathogen (Viterbo and Chet 2006). According to Luo et al. (2010), multiple
defense system is involved when the protection of tobacco plants against the tobacco
mosaic virus is initiated.
24.3.1 Trichoderma as Inducer for Defense Signaling Pathways
Trichoderma for the past few years has received less attention as plant resistance
enhancer. However, this was overcome with publication of more studies. One of the
well-known publications of De Meyer et al. (1998) explained the T. harzianum root
colonization mechanism and defense response in beans. Other research also shows
the penetration mechanism of T. asperellum in the root of cucumber and the
activation of systemic resistance mechanism (Yedidia et al. 1999). The proteome
and transcriptome of the plant leaves were systemically due to fungus root coloniza-
tion and the MAMP interaction. The triggering of the ISR takes place by the JA or
the ET signaling pathway, which is similar to the ISR induced by PGPR (Shoresh
and Harman 2008; Shoresh et al. 2005). This has been confirmed by other authors
such as (1) Segarra et al. (2007) confirmed that the MYBB72 converged when ISR is
triggered by PGPR and Trichoderma, (2) Korolev et al. (2008) investigated that JA
or ET induced by Trichoderma treatment led to enhanced resistance toward Botrytis
cinerea, and (3) according to Djonovic et al. (2007), Sm 1, cerato-platanin is
required for ISR induction by T. virens to fight against Colletotrichum graminicola
in maize.
However, several studies indicate that during the T. asperellum and cucumber
interaction, the defense response of the plant is highly dependent on time and
concentration of the inducer. During the first hour of contact between the two
partners, SAR-like response is observed and there is increase in the SA and peroxi-
dase activity. After the inoculation of the fungus, the systemic resistance of SA and
JA levels increased to high density. Similarly, Gallou et al. (2009) concluded that the
defense system caused by T. harzianum on potato was mainly depending on JA or
ET and SA signaling pathway. There are several findings that support the importance
of Trichoderma in signaling mechanisms (Ramirez-Valdespino et al. 2019). One of
the examples is inoculation of Trichoderma that can cause long-lasting effect on the
upregulation of SA gene markers in the plants that didn’t have any negative impact
from the pathogens (Singh et al. 2016a, b). However, if at all, the plant is challenged
by any pathogens, the pretreatment with Trichoderma can modulate the
SA-dependent gene expression (Singh et al. 2016b; Reacić et al. 2018). After the
infection, the induction of expressed gene is carried out through the JA signaling
pathway and caused an increase in the ISR mechanisms (Tucci et al. 2011; Singh
et al. 2019). Similar to that, a delayed and overlapping expression of defense-related
genes of both SA and JA or ET pathways was noticed when Arabidopsis roots were
colonized of by T. atroviride and wheat colonized by T. harzianum (Salas-Marina
et al. 2011; Singh et al. 2019). This happens against the biotrophic and necrotrophic
pathogens both locally and systemically.
24.4 Trichoderma in Abiotic Stress Alleviation
Besides being used for plant growth, certain species of Trichoderma can be applied
to increase the resistance of the plant toward abiotic and biotic stresses. According to
Chen et al. (2011), research studies conducted on different plants such as radish,
cucumber, and tomato indicate the presence of the fungus to enhance the plant
growth response toward the abiotic and biotic stresses. Harman et al. (2000) on the
other hand have concluded that Trichoderma also increases root development and
crop yield as well as fresh weight of the seedling and foliar area of the plant. Yedidia
et al. (2003) have confirmed that T. harzianum can enhance the uptake of nutrients in
the plants and improve plant growth, biomass accumulation, and other physiological
traits. In many cases, the genetic background of the plant can influence the outcome
of the plant-fungal interactions (Tucci et al. 2011). They confirmed that the genetic
background of tomato can affect the biocontrol agent, T. atroviride and
T. harzianum. The plant growth promotion activities of many Trichoderma strains
were found to be associated with the production of indole-3-acetic acid (Singh et al.
2019) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. According to
Kubicek et al. (2011), ACC deaminase consists of putative sequence, which can be
found in most of the Trichoderma genes. This enzyme plays significant role in root
elongation of canola plant (viterbo et al. 2010). RNAi silencing of the ACCD gene in
T. asperellum mutant strain has led to losing of the fungal ability to support root
elongation in canola seedlings. In other study, root colonization of maize by
Trichoderma is supported by the presence of sucrolytic enzymes. The presence of
invertase gene in T. virens (tvinv) supports hyphal growth through sucrose utilization
and also intensive root colonization. In addition, colonization of maize root by
T. virens increases photosynthesis rate as proved by the significant systemic increase
in carbon dioxide uptake rate in maize leaves (Vargas et al. 2009).
On the other hand, although the effect of Trichoderma on abiotic stress has been
well studied by several workers, the mechanisms on how the stress can be controlled
by the plant is yet to be discovered. Mastouri et al. (2010) have reported that treating
tomato seeds with T. harzianum fastens up the process of seed germination as well as
increases the seedling vigor by providing physical protection against the oxidative
damage. The amelioration of damage caused by the reactive oxygen species accu-
mulation in any stressed plants could be the well-known mechanism that happens
through any beneficial fungi and PGBR in which the plant increases its tolerance
against abiotic stresses. In general, salinity affects the plant growth and several
developments such as formation of lateral roots, primary root length, and root hair
induction, which all together reduce the total root surface (Robin et al. 2016). This
causes negative impact on the diameter of the shoot and the content of the chloro-
phyll (Contreras-Cornejo et al. 2014b). On the other hand, when the plants are
inoculated with Trichoderma, they show high tolerance toward the saline stress.
When the stress is too high, the fungi modify the saline stress. Most probably, this
can be done by the mechanisms initiated by the ABA accumulation, osmolyte-
proline, which is also known as L-Pro, and the accumulation of antioxidant ascorbic
acid. Brotman and Lisec (2012) also discovered the accumulation of L-Pro when
Arabidopsis thaliana was inoculated with T. asperelloides T203. It has also been
reported that Indole 3-acetic acid (IAA) plays important role in plant adaptation to
salt stress. Therefore, IAA producer microorganisms can enhance plant adaptation to
salinity stress by causing some modification on the plant hormone pathway (Waqas
et al. 2012; Korver et al. 2018; Singh et al. 2020). Other study showed that the
application of T. longibrachiatum T6 as growth-promoting biological agent can
enhance wheat plant tolerance to salt stress (Zhang et al. 2016). This effect was
carried out through the upregulation of gene set responsible for the expression of
antioxidant enzymes involved in abiotic stress defense system: catalase (CAT),
superoxide dismutase (SOD), and peroxidase (POD). A recent research also showed
that application of T. longibrachiatum T6 in wheat seedling increases plant tolerance
to salt stress by increasing IAA, ACC-deaminase, and ethylene synthesis (Zhang
et al. 2019). Other research showed that application of T. longibrachiatum T6 leads
to the induction of transcription of reactive oxygen species (ROS) scavenging
enzymes. This results in increase in the induction of glutathione s-transferase
(GST), thione reductase (GR), glutathione peroxidase (GPX), ascorbate peroxidase
(APX), and dehydroascrbate reductase (DHAR). In addition, it also enhances non-
enzymatic antioxidant defense system (Zhang et al. 2019).
The dual application of Trichoderma phytohormone as biocontrol and growth
enhancer on melon plants has been studied by Martinez-Medina et al. (2014). They
pointed out a strong relationship between the auxin induction in plant and the
presence of Trichoderma. In addition, the protective effect of this strain against
plant pathogens such as F. oxysporum is associated with the alternation of hormones,
abscisic acid, and ethylene induction in the plant. In general, the production of
growth hormones and other growth promoters is heavily affected by the environ-
mental growth condition of the Trichoderma spp. (Nieto-Jacobo et al. 2017).
24.5 Nutrient Immobilization and Solubilization
According to Hermosa et al. (2012), the growth of the plant as well as the produc-
tivity can be enhanced by solubilization of the mineral nutrients. In general, different
Trichoderma strains have been reported for their capacity to solubilize different
macro- and micronutrients from soil. This can be mediated through the production of
wide range of organic acids such as gluconic, citric, and fumaric acids, which reduce
the pH of soil, ease mineral solubilization, and thus increase nutrient plant consump-
tion and healthy growth. T. harzianum T22 can be taken as a good example, where
the particular stain can solubilize different types of plant nutrients, which can be the
limiting factor in plants in certain soils. Of different nutrient of soil, phosphate is one
of the essential elements which is needed for healthy plant growth. Phosphate is
present in soil mainly in insoluble form. It has been reported that different
Trichoderma strains were able to solubilize phosphate in soil and thus increased
availability for plant consumption (Sharma et al. 2013). The solubilization mecha-
nism involves the production of different type of organic acids and cultivation under
salt stress (Gaind 2016). However, exposure of T. asperellum Q1 to salt stress not
only increases phosphate solubilization but also increases phytohormone production
and thus enhances the growth of cucumber (Zhao and Zhang 2015).
However, the soil condition and the exposure of Trichoderma to abiotic stresses
can govern the phosphate solubilization capacity. Recent research showed that the
solubilization of tri-calcium phosphate (TCP) was increased when T. koningiopsis
grew under abiotic stress of draught and alkaline conditions (Tandon et al. 2020).
Other study also showed that Trichoderma strains such as T. harzianum,
T. viridescens, and T. citrinoviride can solubilize phosphate from tri-calcium phos-
phate and potassium from biotite concurrently (Nahidan et al. 2019). T. harzianum
increases the solubilization of essential macro- and micronutrients and mineral
uptake by tomato plant (Li et al. 2015; Singh et al. 2018). They also reported that,
in addition to phosphorus, T. harzianum strain SQR-T037 also increases Zn, Cu, and
Fe plant uptake by plant.
Trichoderma is well known for production of diffusible metabolites, which can
reduce the Fe(III) and Cu(III), which is controlled by the formation of Fe(II)-Na2-
bathophenanthrolinedisulfonic acid and Cu(I)-Na2-2,9-dimethyl-4,7-diphenyl-1,10-
phenanthrolinedisulfonic acid complexes. The solubilization mechanism of metal
oxides by Trichoderma spp. involves both the chelation and the reduction of iron.
This, however, can be useful to the plants as they solubilize the iron that cannot be
found all the time. In this nutrient solubilization process, the nitrogen signaling
always has some interaction with the other signaling networks in order to control the
growth and development of plant. In particular, T. asperelloides T203 boosts the
level of amino acid in any colonized plant. The level of amino acid is increased
mainly because of the allocation and continuous reuse of nitrogen. The plants that
have been inoculated with T203 always show increased level of amino acid, and this
can be the reason behind increased use of nitrogen (Brotman and Lisec 2012). In
addition to the above mechanisms of mineral solubilization in soil, some
Trichoderma strains are able to produce specific enzymes to ease phosphate
solubilization/mineralization such as phytase and phosphatase to enhance plant

phosphorus uptake. It was also reported that T. harzianum can produce thermal
stable acid phosphatase, which can extend the biotechnological application of this
strain as biofertilizer (Souza et al. 2016). More recent study also showed that
T. reesei can produce protein phosphatases, which can regulate the secondary
metabolite biosynthesis and cellulase enzyme production as well (Rodriguez-Iglesia
and Schmoll 2019).
24.6 Mycoparasitism
It has been reported that around 75 Trichoderma species have the ability to attack
and destroy the plant pathogenic fungi through mycoparasitic effect. Some of the
pathogenic fungi such as Botrytis cinerea, Fusarium spp., Alternaria alternata,
Rhizoctonia solani, and Bipolaris sorokiniana have the capability of causing such
damage to the plant (Singh et al. 2016a, b, 2019). The mycoparasitism process by
Trichoderma involves different steps initiated by host recognition followed by
hyphal attachment and coiling around the hyphae of the host (Harman et al. 2004).
To be more precise, it involves complex process that starts with tropic growth of the
biocontrol agent toward the host fungi followed by lectin-mediated coiling of
Trichoderma hyphae to the pathogen, which leads to complete inhibition of patho-
gen growth. Some of the antibiotics metabolites produced during this phenomenon
help to kill the pathogen. Table 24.2. shows the list of antibiotic metabolites that are
produced during this process.
In addition, the biocontrol agents have the ability to inhibit the production of
enzyme like pectinases from the pathogens, which facilitate plant tissue degradation.
Benítez et al. (2004) investigated the synergic effect caused by the interaction
between endochitinase of Trichoderma harzianum (gliotoxin), the hydrolytic
enzymes, and peptaibols on the germination of Botrytis cinerea. They reported
that Trichoderma virens strains, which are tentatively known as Q strains, can
produce gliotoxin. Gliotoxin is mainly produced by the gene clusters that have
NRP GliP as the core enzyme and normally is regulated by LaeA. This gene cluster,
in particular, is coregulated during the process of mycoparasitism, but it will not be
regulated in the presence of other fungi, which leads to the lack of gliotoxin
production (Mukherjee et al. 2012; Mukherjee et al. 2013). In addition, according
to Vinale et al. (2008), volatile metabolites have been reported to play vital role
during the mycoparasitism process. Crutcher et al. (2013) showed a list of volatile
compounds being produced by some of the Trichoderma strains during
mycoparasitism. However, the chemical profile of volatile compounds might be
varied and was highly strain dependent. For example, Trichoderma atroviride has
the capacity to produce 6-pentyl-α-pyrone, which was not reported in any other
strains. However, Trichoderma reesei releases very poor mixture of volatile
compounds as compared to any other strains. These differences in the capacity of
volatile compound production are directly related to effectiveness of different strains
as biocontrol agent.
Table 24.2 Antibiotics metabolites and enzymes produced by Trichoderma species during
mycoparasitism
Diversity Compound Effect Strain References
Pyrone 6-(1-Pentenyl)- Antifungal T. harzianum Claydon
2H-pyran-2-one activity against et al.
Aspergillus (1987),
fumigatus and Parker et al.
Penicillium (1997)
spp.
Nitrogen Harzianic acid Antimicrobial T. arundinaceum Vinale et al.
heterocyclic (HA) metabolites and T. harzianum (2013)
compound plant growth Mangaiello
regulator et al. (2018)
Trichothecene Trichodermin Phytotoxic T. brevicompactum Malmierca
effect et al. (2013)
Anthraquinone Pachybasin Increase the T. harzianum Lin et al.
coil of any (2012)
biological
control agent
against
Rhizoctonia
solani
Monoterpene β-Myrcene Regulate the T. virens Crutcher
expression of et al. (2013)
resistance
genes involved
in biotic and
abiotic stresses
Diketopiperazine/ Gliotoxin Antibacterial Trichoderma Vargas
NRP and antifungal virens et al. (2014)
activities Trichoderma spp. Bulgari
et al. (2020)
Hydrolytic Chitinases Break down Trichoderma spp. Tzelepis
enzymes fungal cell wall Trichoderma et al. (2015)
harzianum Ting and
Chai (2015)
Hydrolytic β-1,6-Glucanases Hydrolyze the Trichoderma spp. Druzhinina
enzymes cell wall of et al. (2011)
Botrytis
cinerea and
Phytophthora
citrophthora
Better understanding of Trichoderma role as biocontrol agent was obtained by

studying the molecular biology of the microbial interaction during the antagonistic
process. Mutant strain of Trichoderma atroviride that contains green fluorescent
protein gene (gfp) and glucose oxidase gene (gox) was used to determine the factors
that activate gene cascade of the biocontrol agent. However, expression of these
genes, which are primarily involved in the mycoparasitism, was mainly induced by
fungal cell wall hydrolysate and chitin. Ciliento et al. (2006) investigated the role of
Trichoderma ABC transporters during mycoparasitism. The data demonstrated that
the mycelia of the plant pathogen can induce the expression of specific ABC
transporter genes in Trichoderma species. However, confirmation was made when
production of knockout mutants exhibited slower growth rate as compared to wild
type strain. However, most of the cell wall degrading enzymes produced by
Trichoderma sp. have been purified and purified enzyme showed antifungal activity
toward wider range of fungal pathogens when tested alone or in combination with
other enzymes. On the other hand, the presence of different carbon sources enhances
the production of cell wall-degrading enzymes. However, better antifungal activity
has been reported by combining the purified protein with different types of synthetic
fungicides, which can affect the cell membrane integrity. A mixture of cell wall
degrading enzymes (of different lytic effects) can exhibit antimicrobial effect against
pathogen better than the treatment with synthetic pesticides alone (Lorito et al.
2006).
24.7 Antibiosis
The antibiosis process is mainly based on the biosynthesis of antimicrobial

compounds or hydrolytic enzymes that are produced during the infection process
and where competition for nutrients take places in the rhizosphere (Harman et al.
2004; Vinale et al. 2014). Although Trichoderma is able to produce a variety of
antibiotics, it is also able to produce antimicrobial primary metabolite such as
peptaibols (Mukherjee et al. 2013). Among the peptaibols, the class 11-, 14-, and
18-mer mainly contributed in pathogen control. It has been reported that the biosyn-
thesis of 18-residual peptaibols is regulated by nonribosomal peptide synthase gene,
which is the tex1 (Vinale et al. 2012). In the study of Bae et al. 2011, the fermenta-
tion broth of Trichoderma sp. exhibited antibiotic activity against pepper. They
observed that the broth inhibited the growth of pepper by about 31%. In addition, it
caused several damage to the pepper roots and the color of root tips became brown.
However, Trichoderma produces a variety of secondary metabolites with signifi-
cant biological activity. The fungal antibiotics can be divided into three main
categories viz. volatile antibiotics, water soluble compounds, and peptaibols. The
volatile antibiotics normally belong to the isocyanide derivatives, whereas koningic
acid can be one of the good examples for the water-soluble compound (Contreras-
Cornejo et al. 2014b). Peptaibols are homologous linear oligopeptides composed of
12–22 amino acids and are rich with α-aminoisobutyric acid (Aib). They have
usually acetylated N-terminus and amino alcohol group at the C-terminus rather
than amino acids.
The antimicrobial activities of Trichoderma compounds might undergo two
different mechanisms of action based on their chemical nature. High concentration
of antibiotics will be produced into soil when the production of low molecular,
nonpolar, and volatile compounds increases. This type of antibiotics exhibited long
distance activities in soil and can affect microbial community beyond the
rhizosphere zone. On the other hand, the polar antibiotics and peptaibols act near to
the proximity of the hyphae of the fungus and exhibit activity in short distance range.
According to Lorito et al. (2016), it has been demonstrated that fungal peptaibols can
prevent the activity of β-glucan synthase. The inhibition of glucan synthase
interrupts the buildup of cell wall of the pathogen, which can also disrupt the action
of β-glucanases.
However, the mode of action as well as the production of secondary metabolites
and their interaction with other compounds is yet to be studied fully for most of the
Trichoderma strains, although role of peptaibols is clear. The secondary metabolites
can be divided according to the producers into two groups viz. “Q” strains and “P”
strains. The P strain normally produces the compound named gliovirin, while the Q
strain produce antibiotic compound named gliotoxin. The differences between the
two are that gliotoxin has wide range of antibiotic activity, while the gliovirin is
limited because of the presence of specific potent inhibitor of the Oomycetes. The
production of gliovirin is interrelated to the efficiency of biocontrol of the P strain. In
some cases, in the presence of high carbon to nitrogen ratio, some strains produce
compound named viridiol, which acts as bioherbicide without any negative effect on
the other crops in the soil. The presence of this compound enables Trichoderma
strains to degrade the seed-emitted compounds, which promotes the germination of
pathogen. In general, the Q strain can enhance the phytoalexin synthesis, which can
help to improve the biocontrol efficiency by simply not being pathogenic to the
cotton roots (Howell 2002; Howell and Puckhaber 2005). On the other hand, the P
strain does not support the production of phytoalexin synthesis in cotton. However,
despite the study of Hanson and Howell (2004), about the phytoalexin synthesis in
cotton, the exact biochemical process involved during the synthesis is yet to be
discovered.
24.8 Conclusion
Soil fungi play critical and multifunctional roles in plant growth and stress tolerance
under different circumstances. In particular, strains belonging to Trichoderma spe-
cies are so far the most widely studied and used microbial inoculants in agriculture
and allied sectors. Their roles are not only limited to the conventional supply of
essential nutrients and prevention of diseases in plants but also to certain extent to
help plant adaptation to various environmental stresses and have the capacity to
produce several plant growth promoting substances. This ensures healthy growth of
plant with minimal use of chemical fertilizers and toxic pesticides. However, many
other functions and mechanisms of action are still under study. Therefore, further
research is needed to elucidate the complicated mechanisms of Trichoderma in
supporting healthy plant growth. This will help to develop new applications and
protocols for organic fertilization and prevention of plant diseases. This will help to
maximize the beneficial application of Trichoderma in field, supporting chemical
free fertilization practice, and improve agroeconomy of the current organic fertiliza-
tion strategies.
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Management of Sclerotium rolfsii Induced
Diseases in Crops by Trichoderma Species 25
Ratul Moni Ram, Rahul Singh Rajput, and Anukool Vaishnav
Abstract
Sclerotium rolfsii is a polyphagous pathogen, and its management is quite

difficult and uneconomical owing to its wide host range and long-term survival
in the form of hard resting structure, i.e., sclerotia. The pathogen is a threat to
most of the agricultural crops as it incurs heavy yield losses. Chemical control,
though, provides quick results, but it evokes concern relating to human health and
is non-eco-friendly. Indiscriminate use of chemical fungicides leads to accretion
of harmful chemical residues on the targeted crop, leading to serious health issues
of the consumers. Biological control of phytopathogens has emerged as an
attractive alternative to chemical control. Application of antagonistic microbes
is considered as one of the potential management strategies in integrated man-
agement approach owing to their eco-friendly nature. Among them, Trichoderma
species has been recognized as a potent biocontrol agent and is widely applied for
the management of soilborne pathogens. A number of Trichoderma-based
formulations, viz. Soilguard, Trichodex, and Trichojet, are commercially avail-
able in the market. Thus, biological management is a perfect alternative for
chemical control as it ascertains food security in an ecologically safe and sound
manner. Being a very popular biocontrol agent, Trichoderma spp. has a tremen-
dous ability in the management of various diseases induced by S. rolfsii. Several
researches carried out in the past are an explicit example of this. So the need of the
R. M. Ram (*)
Department of Plant Pathology, A.N.D.U.A & T, Kumarganj, Ayodhya, Uttar Pradesh, India
R. S. Rajput
Faculty of Agricultural Sciences and Allied Sciences, Rama University, Kanpur, Uttar Pradesh,
India
A. Vaishnav
Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University,
Mathura, Uttar Pradesh, India

23, https://doi.org/10.1007/978-981-15-9154-9_25
594 R. M. Ram et al.
hour is higher popularization of Trichoderma spp. for the efficient management

of S. rolfsii in an eco-friendly manner.
Keywords
Biocontrol · Formulation · Sclerotium rolfsii · Trichoderma
25.1 Introduction
Plant diseases are one of the major challenges to ecosystem stability and food
security for the soaring population. Due to the attack of pathogens both at pre- and
postharvest stages, the crop yields are severely affected. There are innumerable
pathogens, viz., Pythium, Phytophthora, Fusarium, Rhizoctonia, Sclerotinia,
Puccinia, Bipolaris, Erysiphe, etc., that causes heavy crop losses, but among them,
Sclerotium rolfsii is a major polyphagous pathogen attacking more than 500 plant
species (Singh et al. 2014). It is really a threat to all agricultural crops owing to its
broad host range. So the management of such a polyphagous pathogen is very
cumbersome. Though the chemical method is considered to be quite promising,
higher dependency on chemical pesticide use also imparted a major health and
environmental hazard, which ultimately paved the path to exploring eco-friendly
alternatives for minimizing this loss. At present scenario, the only possible protec-
tive measure that seems effective and promising for fulfilling the challenges of plant
disease management and soil fertility without affecting the sustainability of the
ecosystem and the environment is the use of antagonistic microorganisms.
Biocontrol using beneficial microbes, i.e., biocontrol agents (BCAs), is an
eco-friendly method, and in certain cases it seems to be the only available effective
biological option to combat plant diseases (Cook 1993). Some BCAs have also the
ability to enhance plant growth activity by improving nutrient acquisition through
siderophore production. Thus, biological control can be regarded as a safer, effec-
tive, and eco-friendly means to protect plants from phytopathogens and to manage
the ecological balance. Nature has gifted mankind a wide range of beneficial
microorganisms, among which Trichoderma is widely used as antagonistic fungi,
which possess potential ability for disease suppression and plant growth promotion,
along with other multifarious actions (Khan and Anwer 2011; Ram and Singh 2017).
Since the last few decades, Trichoderma spp. has become widely popularized and
has gotten global recognition as an effective antagonist for various phytopathogenic
fungi (Khan and Gupta 1998; Kubicek et al. 2011). Thus, it holds an immense
potential not just in crop improvement but also in maintaining agro-ecosystem
sustainability. The large number of Trichoderma-based formulations commercially
available in the market is a clear evidence of its effective results and widespread
popularity.
The current chapter primarily focuses on Sclerotium rolfsii and its biological
management through Trichoderma spp. and the various mechanisms employed by
Trichoderma spp. to overcome the phytopathogens. Furthermore, the various
formulations of Trichoderma and the associated constraints are also discussed.
25 Management of Sclerotium rolfsii Induced Diseases in Crops by. . . 595
25.2 Sclerotium rolfsii
Sclerotium rolfsii is a destructive polyphagous fungal pathogen that is a threat to a

wide range of agricultural and horticultural crops and can cause disease to more than
500 plant species in 100 families (Punja 1985; Sennoi et al. 2010). It usually causes
crown blight; damping off of seedlings, roots, bulbs, and tubers; stem canker; and
fruit rots in several other crops (Aycock 1966; Singh et al. 2014; Rajput et al. 2020).
In addition, it is also reported to cause significant losses as a postharvest pathogen in
storage and transit. The soilborne inoculum (sclerotia) is the prime factor in causing
infection and disease development (Mukherjee et al. 1993; Pacheco et al. 2016; Ray
et al. 2016; Shrestha et al. 2018). The pathogen is prevalent in subtropical, tropical,
and temperate regions around the world, especially in regions with high
temperatures and high moisture (Punja 1985; Lehner and Mizubuti 2017). The first
documentation of this fungus was made by Peter Henry Rolfs in connection with
tomato blight, which occurred in Florida (Aycock 1966). Incidence of S. rolfsii has
been reported in many countries across the globe, including countries in Europe, like
Belgium, Bulgaria, Spain, and Sweden, and in Asia, such as China, Japan, Pakistan,
and India (Fig. 25.1). In India, it has been reported from Arunachal Pradesh, Assam,
Bihar, Delhi, Haryana, Jammu and Kashmir, Meghalaya, Manipur, Madhya Pradesh,
Uttar Pradesh, and West Bengal (Mahato et al. 2017; Sajad and Jamaluddin 2017;
Dinesh et al. 2018; Sahu et al. 2019).
Sclerotium rolfsii can affect its hosts at any stage of growth, viz., from seedlings
to mature plants and even harvested produce (Sahu et al. 2019; Siddique et al. 2018).
This disease can cause approximately 30% crop loss in both field and greenhouse
conditions, and under severe condition it may reach up to 95% (Suriyagamon et al.
Fig. 25.1 Global distributions of Sclerotium rolfsii pathogen causing collar rot disease in tomato
crops (https://www.plantwise.org/KnowledgeBank/datasheet)
2018). Severely infected plants may show approximately up to 62% reduction in

fruit size and 84.3% reduction in fruit weight. High loss is observed when plants get
infected at early to mid-bloom stage as compared to plants infected near harvest
(Amin and Jampala 2018).
25.3 Management Practices Adopted for Control of S. rolfsii
This pathogen is a causative agent of southern blight, southern stem blight, and white
mold diseases in several crops. It causes rots in the crown, root, lower stem, and fruit.
Management strategies for the pathogen consist of cultural, physical, chemical, and
biological means of control. Managing collar rot through chemicals alone is not
satisfactory in view of environmental concerns and cost benefit ratio. Biological
control is regarded as one of the efficient management approaches that could achieve
good results (Biswas and Sen 2000; Sahni et al. 2008; Singh et al. 2013a; Dinesh
et al. 2018). Apart from single control practices, integrated disease management is
also being widely practiced. The prime idea behind integrating management
practices is its broad action spectrum. Often a consortium of biological, botanical,
and chemical methods is followed.
25.3.1 Biological Control
The indiscriminate use of chemical pesticides in order to minimize crop losses and
increase production has led to increased interest in searching for alternatives for
disease control. Biological control has been promoted as a sustainable approach for
crop production. In biological control, antagonistic microorganisms are used to
manage the population density of phytopathogens. These antagonistic
microorganisms, often referred as “BCAs,” compete with pathogens for nutrients
and space, leading to the inhibition of pathogen growth. They also secrete antibiotic
substances or other active metabolites that directly attack pathogens and either
reduce or completely inhibit their growth. In addition, antagonistic microorganisms
are also able to induce defense response in plants, which enhances plants’ immune
system and helps to fight attacks on pathogens (Rajput et al. 2019a). The most
studied microorganisms with antagonistic activity are Bacillus, Pseudomonas in
bacterial genera and Trichoderma, Paecilomyces lilacinus, Pochonia
chlamydosporia, Aspergillus, and Gliocladium virens in fungi (Mukherjee et al.
1995; Rangeshwaran and Prasad 2000; Vaishnav et al. 2014; Jain et al. 2014,
2018; Singh et al. 2013b, 2014).
25.3.1.1 Trichoderma Species

Trichoderma species are free-living, cosmopolitan hyphomycete fungi abundant in
diverse root ecosystems, especially those enriched with organic matter (Singh et al.
2009). Besides acting as antagonists against soilborne phytopathogenic fungi, they
also act as plant growth promoters. They are also known to induce resistance in the
hosts by mediating signal development in the host for the induction of local as well
as systemic resistance against some foliar pathogens (Singh et al. 2020; Shores et al.
2005). It is evident from numerous reports that the formulation of Trichoderma spp.
is quite popular in crop cultivation for growth promotion attributes. Trichoderma
spp. has high colonizing ability with plant roots rendering them rhizospheric com-
petent. Their higher root-colonizing ability directly facilitates enhanced uptake of
nutrients. Trichoderma influences plant growth through several means, i.e., stimula-
tion of phytohormones, induced defense network, solubilization of nutrients, and
increased uptake, coupled with suppression of phytopathogens (Ram and Singh
2017). The metabolic products of Trichoderma spp. have vivid applications not
only in agriculture but also in other sectors, viz., pharmaceuticals, beverage,
cosmetics, etc. (Ram et al. 2016; Keswani et al. 2014). Apart from biocontrol,
numerous species of this genus possess the ability to degrade hydrocarbons,
polysaccharides, as well as xenobiotic chemicals extensively used in the agriculture
and allied sectors (Harman and Kubicek 1998; Harman et al. 2004).
25.3.1.2 Historical Background of the Taxonomy of Trichoderma

The history of Trichoderma dates back to the time when Persoon (1794) mentioned
it in his fungal classification. Apart from Trichoderma, Persoon’s classification also
contained several other fungi, viz., Mucor, Puccinia, Ascobolus, and few other slime
molds (Klein and Eveleigh 2002). However, Bisby (1939) was the pioneer who
proposed T. viride as a species of Trichoderma. Till 1969, all identified strains of
Trichoderma belonged to T. viride as per Bisby’s classification (Druzhinina and
Kubicek 2004). In 1969, Trichoderma species were divided into nine “species
aggregates,” namely, T. aureoviride Rifai, T. longibrachiatum Rifai, T. harzianum
Rifai, T. pseudokoningii Rifai, T. piluliferum Rifai, T. polysporum Rifai, T. hamatum
Bain, T. koningii Oudem, and T. viride, based on the concept of “aggregate” species
designed by Rifai. From 1984 onward, Bissett initiated to reclassify Rifai’s aggre-
gate of species and indicated the drawbacks in Rifai’s theory for distinguishing
Trichoderma species (Chaverri and Samuels 2004). Merely five of Rifai’s aggregate
species above mentioned (T. harzianum, T. pseudokoningii, T. piluliferum, T.
longibrachiatum, and T. polysporum) contained less number of species, whereas
other aggregates compiled large number of species (Chaverri and Samuels 2004). An
attempt was made in 1991 by Bissett, who subdivided the genus Trichoderma into
five sections, which were Trichoderma, Longibrachiatum, Hypocreanum,
Pachybasium, and Saturnisporum (Druzhinina and Kubicek 2004). However, with
passage of time, advanced molecular techniques, especially sequence analysis of
internal transcribed spacer (ITS) region, restriction fragment length polymorphisms
(RFLP), and random amplified polymorphic DNA (RAPD), came to light which
brought a revolution in the morphology-based taxonomy of Trichoderma (Dodd
et al. 2002). Kindermann et al. (1998) was the pioneer in the molecular identification
of different Trichoderma spp. The precise identification of Trichoderma and
Hypocrea spp. has been possible with the development of tools such as TrichOKEY
and TrichoBLAST; both have online access at http://www.isth.info/ (Druzhinina
et al. 2005; Kopchinskiy et al. 2005). Furthermore, phenotype microarrays are also
being utilized for the classification of new species, which analyze the carbon use
patterns for 96 carbon sources (Bochner et al. 2001).
25.3.1.3 Biological Control Using Trichoderma Species

Trichoderma species are among the most promising fungal antagonists being exten-
sively used in disease suppression and act as effective antagonist against various
soilborne pathogens (Chet et al. 1981; Singh et al. 2009; Ram and Singh 2017). They
are key producers of cell-wall-degrading enzymes which target pathogenic fungi, the
phenomenon that makes them best suited for biological control in agriculture (Woo
et al. 2006). The fungus parasitizes phytopathogenic fungi through antibiosis,
mycoparasitism, competition for nutrients, and induction of resistance. Moreover,
it leads to the production of various secondary metabolites which aid in disease
resistance against phytopathogens. These prominent features make them the most
popular fungal biocontrol agent.
The biocontrol capability of Trichoderma was first reported by Weindling (1932),
who studied the role of T. lignorum in the biocontrol of Rhizoctonia solani, causing
disease in citrus seedlings. Since this pioneer work, several literatures have reported
on successful biocontrol by Trichoderma spp. Among different species of
Trichoderma, T. harzianum, T. virens, and T. viride are the most popular ones
exhibiting biological control (Singh et al. 2009). They efficiently control root rots/
wilt and foliar diseases in a wide range of crops and are antagonistic to a number of
soilborne fungi like Pythium, Phytophthora, Sclerotinia, Sclerotium, Rhizoctonia,
Fusarium, Macrophomina, etc. and even the root knot nematode Meloidogyne spp.
However, the first report on its mycoparasitism ability was made by Cole-Smith et al.
(1971), who through microtome sections demonstrated that medulla of infected
sclerotia of Sclerotium delphinii was wholly replaced by hyphae of T. hamatum on
agar plates. Likewise, Henis et al. (1978) reported mycoparasitism of Trichoderma
spp. against S. rolfsii, where within the infected fungal sclerotia chlamydospores
were produced abundantly in place of conidia. With passage of time, various
Trichoderma spp. have been found to demonstrate antagonistic effects against
S. rolfsii in different crops. Various Trichoderma spp. used for the management of
S. rolfsii disease in different crop plants is listed in Table 25.1.
Owing to their inherent property as plant growth promoters and BCAs, these
fungi have been widely studied and commercially marketed as biopesticides,
biostimulators, as well as soil amendments (Harman 2000; Lorito et al. 2004;
Khan and Mohiddin 2018). Currently in the market, the commercial products of
Trichoderma are available in various forms. Trichoderma-based products are boom-
ing in the agricultural market with more than 250 formulated products registered
worldwide, which alone occupy 60% of the biofungicide market (Singh et al. 2012).
All these products are being sold to farmers for disease control and in turn enhance
their yield (Woo et al. 2006).
Table 25.1 Fungal biocontrol agent Trichoderma used for the management of Sclerotium rolfsii in
different crops
BCAs Crop/study type References
T. lignorum Citrus Weindling (1934)
T. harzianum Ryegrass Wells et al. (1972)
T. harzianum Groundnut Backman and Rodriguez-
Kabana (1975)
T. harzianum Bean Elad et al. (1980)
T. harzianum Sugar beet Upadhyay and Mukhopadhyay
(1986)
T. harzianum Snap bean Papavizas and Lewis (1989)
T. viride Tomato Wokocha (1990)
T. harzianum Chickpea Kaur and Mukhopadhyay
(1992)
T. koningii Tomato Latunde-Dada (1993)
T. harzianum Sugar beet Abada (1994)
T. harzianum, T. virens Chili Jinantana and Sariah (1998)
T. parceramosum, Rice Cuevas et al. (2001)
T. pseudokoningii
T. harzianum Mung bean Mishra et al. (2000)
T. harzianum Tomato Dutta and Das (2002)
T. koningii Tomato Tsahouridou and
Thanassoulopoulos (2002)
T. viride Groundnut Manjula et al. (2004)
T. harzianum, T. virens Mint Singh and Singh (2004)
T. harzianum Groundnut Karthikeyan et al. (2006)
T. harzianum Chickpea Maurya et al. (2008)
T. harzianum Sunflower, mung bean Yaqub and Shahzad (2008)
T. harzianum (ITCC-4572) Groundnut Ganesan et al. (2007)
T. harzianum, T. viride Eternity plant (Zamioculcas Jegathambigai et al. (2010)
zamiifolia)
T. harzianum Faba bean Abdel-Kader et al. (2011)
Trichoderma spp. Groundnut Bagwan (2011)
Trichoderma spp. Chilli Joshi et al. (2010)
T. harzianum Tomato Singh et al. (2013b)
T. harzianum Artichoke (Helianthus Sennoi et al. (2013)
tuberosus L.)
Trichoderma spp. Cucumber, bottle and bitter Kotasthane et al. (2015)
gourd
T. harzianum, T. viride, Lentil Kushwaha et al. (2018)
T. virens
T. harzianum Tomato Suriyagamon et al. (2018)
Trichoderma sp. T76 12/2 Snake fruit (Salacca zalacca) Wonglom et al. (2019)
and lettuce
T. harzianum Barley Faruk (2019)
Table 25.2 List of different formulations of Trichoderma species available in India (Modified from Khan and Mohiddin 2018)
600
S. No. Biopesticide Antagonist Target pathogen Crop Source

1 Antagon TV Trichoderma viride Pythium, Phytophthora, Pulses, vegetables, oilseeds Green Tech, Agroproducts, Rajaji Road,
Macrophomina Coimbatore
2 Monitor WP Trichoderma spp. Sclerotinia sclerotium, Pythium, Pulses, vegetables, sugarcane, Agriland Biotech Pvt. Ltd., 36 Prince
Fusarium, Rhizoctonia potato, cotton, spices, oilseeds, Industrial State Mota-Metipi Baroda
fruits (Guj.)
3 Trichostar Trichoderma Sclerotium, Pythium, Pulses and vegetables GBPUA & T, Pantnagar
harzianum Rhizoctonia, Fusarium
4 Trichoderma Trichoderma spp. Fusarium, Sclerotium, Pythium, Sugarcane, pulses, vegetables Innovative Pest Control Lab, Bangalore
Rhizoctonia
5 Monitor Trichoderma spp. Sclerotium, Pythium, Fusarium, Pulses and vegetables Agricultural and Biotech Pvt. Ltd.,
Rhizoctonia Gujarat
6 Phule Trichoderma spp. Sclerotium, Rhizoctonia, Pulses and vegetables Department of Plant Pathology, MPKV,
Trichokill Pythium, Fusarium Rahuri
7 Gliostar Trichoderma virens Sclerotium, Pythium, Fusarium, Pulses and vegetables GBPUAT, Pantnagar
Rhizoctonia
8 Biowilt-X Trichoderma Macrophomina, Pythium, Pulses and vegetables Dept. of Plant Protection, AMU, Aligarh
harzianum Phytophthora
9 Ecoderma Trichoderma Sclerotinia, Phytophthora, Potato, cotton, spices, sugarcane, Margo Biocontrol Pvt. Ltd., Bangalore
harzianum + T. viride Pythium, Fusarium, Rhizoctonia fruits, pulses, vegetables
10 Trichogourd Trichoderma Sclerotinia, Pythium, Fusarium, Pulses, vegetables, oilseeds Anu Biotech Int. Ltd., Faridabad
harzianum Rhizoctonia
11 Funginil Trichoderma viride Sclerotinia sclerotium, Pythium, Pulses and vegetables Crop Health Products Ltd., Industrial
Fusarium, Rhizoctonia Area, Meerut Road, Ghaziabad
12 Defense Trichoderma viride Macrophomina, Sclerotinia, Pulses and vegetables Wockhardt Life Science Ltd., Mumbai
Pythium, Fusarium, Rhizoctonia
13 Bioderma Trichoderma Sclerotinia sclerotium, Pythium, Pulses, oilseeds, vegetables, Biotech International Ltd., India
viride/T. harzianum Fusarium, Rhizoctonia sugarcane, potato, fruits
14 Ecofit Trichoderma viride Pythium, Fusarium, Pulses and vegetables Hoechst Schering AfgroEvo Ltd., India
R. M. Ram et al.
Rhizoctonia, Macrophomina
25.4 Mechanism of Action
There are various mechanisms through which Trichoderma spp. imparts its antago-
nistic and plant growth promotion traits. The various mechanisms employed by
Trichoderma spp. for biocontrol is depicted in Fig. 25.2.
25.4.1 Mycoparasitism
Mycoparasitism is regarded as one of the most typical mechanisms exhibited by

Trichoderma species for the management of phytopathogenic fungi (Howell 2003;
Vinale et al. 2008). This process involves a chemotrophic growth of the antagonist
on the host, followed by attachment and coiling around the host hyphae (Chet et al.
1990; Woo and Lorito 2007). The lysis of the hyphal cell walls of pathogens is
carried out by various enzymes, such as chitinases, proteases, and β-1, 3-glucanases
(Cruz et al. 1992; Khetan 2001). β-1, 3-glucanases have the ability to degrade the
cell wall and inhibit the growth of host mycelium and spore germination (Lin et al.
2007). However, proteases usually degrade the cell walls and pathogen hyphal
membranes of the host. The mycoparasitic activity of Trichoderma has been
reported by many researchers against several pathogens (Mustafa et al. 2009;
Khan et al. 2011a, b, c; Kotze et al. 2011). Several physiological and biochemical
factors facilitate the process of mycoparasitism. Omann et al. (2012) explicated that
G-protein-coupled receptor Gpr1 plays an important role in mycoparasitism. Simi-
larly, Kumar et al. (2010) recognized the role of mitogen-activated protein kinase
Fig. 25.2 A schematic representation of the action mechanism employed by Trichoderma spp.
against different phytopathogenic fungi (Source: Singh et al. 2012)
(MAPK) in biocontrol activities. Deletion of the MAPK gene affects the biocontrol
potential of T. virens, and the mutants were reported to be less efficient (Mukherjee
et al. 2012).
25.4.2 Antibiosis
Antibiosis usually refers to the inhibition of targeted microorganisms by volatile

compounds or antibiotics produced by the antagonist (Irtwange 2006; Viterbo et al.
2007; Haggag and Mohamed 2007). Trichoderma species is also among several
biological control agents producing various types of antibiotics (Lewis et al. 1989;
Handelsman and Stabb 1996). Weindling (1934) reported gliotoxin produced by
T. lignorum, which possess lethal properties against Rhizoctonia solani and
Sclerotinia americana. Later on, gliotoxin-producing fungi were considered to be
Gliocladium virens instead of T. lignorum (Webster and Lomas 1964).
Similarly, volatile compounds like 6-pentyl-2H-pyran-2-one (6-PP) produced by
T. atroviride play a vital role in Trichoderma–host-pathogen interactions (Lorito
et al. 2004). Apart from rhizospheric ones, a few endophytic species of Trichoderma,
such as T. amazonicum, T. taxi, T. evansii, T. martiale, T. theobromicola, and
T. stromaticum, also possess biocontrol property and can protect plants from
phytopathogens through modulation at omics level (Bailey et al. 2006; Bae et al.
2009; Druzhinina et al. 2011). However, not all of them are registered as BCAs,
although they are commercially available as plant growth promoters.
25.4.3 Competition
Trichoderma competes with other pathogens for niche and nutritional requirements
(Wells 1988). Competition allows the biocontrol agent to displace the pathogen from
the targeted zone. Trichoderma spp. shows excellent competition with other fungi
for food and nutrients in the rhizospheric zone (Chet et al. 1990; Irtwange 2006). It
was reported that T. viride displaces Chondrostereum purpureum, the silver leaf
pathogen of plum trees, by competing for nutrients (Corke and Hunter 1979).
Similarly, T. harzianum T35 checks the growth of Fusarium oxysporum by compet-
ing for both nutrients and rhizosphere colonization (Tjamos et al. 1922).
Trichoderma spp. is known to be an efficient producer of siderophore, which
quenches iron from the soil and thus renders it unavailable for the pathogen. It
modifies the rhizosphere through soil acidification, which becomes unsuitable for the
growth of the target pathogen (Benitez et al. 2004). Apart from iron and zinc,
competition for carbon is also a deciding factor in determining the antagonism
potential of different strains of Trichoderma spp. (Sivan and Chet 1989, Viterbo
et al. 2007). However, few strains have the ability to colonize their rhizospheric zone
throughout their lifetime. In a study, it was found that maize plant treated with
T. harzianum strain T-22 had a twofold increment in root development in compari-
son with control (Harman et al. 2004). Secondary metabolites such as koninginin A
and 6-pentyl-alphapyrone act as plant growth regulators (Cutler et al. 1986, 1989).
However, citric and gluconic acids lower soil pH and facilitate solubilization of
micronutrients and other mineral components (Harman et al. 2004; Benitez et al.
2004; Vinale et al. 2008).
25.4.4 Induced Resistance
Often to defend plants from pathogen invasion, BCAs induce local and systemic
resistance in host plants to counter the attack of the pathogen. It emerged as a vital
tool by which selected BCAs build up their defense against a broad range of
phytopathogens (Ram et al. 2015). Plants usually generate induced resistance as a
result of an interaction by a pathogen, an insect, upon the colonization of the roots by
BCAs or even after treatment with a specific chemical (Singh et al. 2012). The first
report of induced resistance with T. harzianum strain T-39 disclosed that on soil
treatment, bean leaves were given induced resistance against diseases caused by
pathogens such as B. cinerea and C. lindemuthianum (Bigirimana et al. 1997).
Jasmonic acid and ethylene are the principal components of induced systemic
resistance. Certain strains of Trichoderma penetrate root tissues and induce a chain
of biochemical and morphological alterations to trigger resistance responses in the
host (Singh et al. 2011; Singh et al. 2013b). Induced resistance has been reported to
be effective in several monocots and dicots, against infection caused by fungi
(Phytophthora spp., R. solani, Alternaria spp., Colletotrichum spp., B. cinerea,
Magnaporthe grisea, etc.) and bacteria (Pseudomonas syringae, Xanthomonas
spp., etc.), and even in some viruses like cucumber mosaic virus (CMV) (Waghunde
et al. 2016).
25.5 Biological Management of S. rolfsii by Trichoderma Species
A lot of work has been done by various researchers on the management of Sclero-
tium rolfsii by different Trichoderma species on a wide range of crops. The
experiments were carried out through either the sole application of Trichoderma or
in combination with any fungicide, botanical agent, or other bioagents.
Muthamilan and Jeyarajan (1992) reported that seed pelleting method with
T. harzianum (5 109 conidia ml 1) is the best approach for the management of
root rot caused by S. rolfsii in groundnut. Application of T. harzianum at 60 g kg 1
of soil leads to a reduction in stem rot of groundnut caused by S. rolfsii of up to 83%
(Biswas and Sen 2000). However, Biswas and Sen (2000) observed that in pots,
application of T. harzianum inoculum to soil and seed dressing at sowing time
exhibited reduced disease incidence. The Sclerotium wilt of groundnut was effec-
tively reduced to 92.58% when soil was treated with T. harzianum at 10 g kg 1
(Patibanda et al. 2002). Manjula et al. (2004) reported that parasitization of the
mycelium of S. rolfsii attacking groundnut by T. viride pq 1 is due to the production
of extracellular chitinase. T. harzianum effectively controlled S. rolfsii, which causes
foot and root rot of brinjal (Islam 2005). Radwan et al. (2006) documented that
T. harzianum and T. hamatum were most effective against S. rolfsii and inhibited
mycelial growth by 79%. Karthikeyan et al. (2006) reported that T. harzianum,
T. viride, and P. fluorescens were effective against the growth of S. rolfsii (Sacc.) in
stem rot of groundnut. T. viride inhibited mycelial growth of the pathogen by
69.40%, while P. fluorescens exhibited 64.40% inhibition. S. rolfsii causing
damping off in tomato and other vegetable crops can be effectively controlled by
T. harzianum. In a study conducted by Okereke and Wokocha (2006), about
52–62% disease reduction was achieved on application of the latter.
Trichoderma species have also been used in commercial enzyme productions,
like production of cellulases, hemicellulases, proteases, and β-1, 3-glucanase (Verma
et al. 2007). T. harzianum has antagonistic activity against S. rolfsii as it produces
antibiotic substances such as harzianone, viridin, trichosetin, gliotoxin, glioviridin,
trichodermin, dermin, etc. (Eziashi et al. 2007; Ghildiyal and Pandey 2008).
Trichoderma spp. also illustrated antagonistic potential against S. rolfsii,
B. cinerea, and Fusarium oxysporum f. sp. ciceris under in vivo conditions
(Anand and Reddy 2009).
Rekha (2012) reported that different Trichoderma sp. reduced mycelial growth
and the formation of sclerotial bodies of S. rolfsii in tomato. Similarly,
Samsuzzaman et al. (2012) stated that tomato seedling treated with T. harzianum
recorded reduced mortality upon infection with S. rolfsii in soil. Moreover, it also
increased plant height and the production of tomato in treated plants. Thilagavathi
et al. (2012) reported that vermicompost-based bioformulations of Trichoderma spp.
reduced root rot disease of sugar beet caused by S. rolfsii, facilitating yield incre-
ment. Deepika et al. (2014) reported that the culture filtrate of T. harzianum PBT
23 at 50% significantly inhibited the mycelial growth of S. sclerotiorum, R. solani,
S. rolfsii, F. oxysporum, and B. cinerea at a range of 20.6–48.9%. Islam et al. (2016)
documented three Trichoderma strains (T. harzianum TR05, T. virens TR06, and
T. asperellum TR08) from Bangladesh which acted as potential BCAs against
tomato collar rot under greenhouse conditions. They found that with
T. asperellum, disease incidence was reduced; the effect was more consistent
when the biocontrol agent was inoculated in the seed and applied to the soil.
Basumatary et al. (2015) selected six fungal species, viz., Penicillium sp.,
Curvularia sp., Aspergillus niger, T. harzianum, T. viride, and Fusarium spp., as
bioagents against S. rolfsii. The maximum percentage of growth inhibition of
S. rolfsii was reported with T. harzianum (77.39%), followed by T. viride
(76.54%). Elshahawya et al. (2016) reported ten Trichoderma species and seven
fungicides which were found effective against pathogens such as Rhizoctonia solani,
F. solani, F. oxysporum, Macrophomina phaseolina, and Sclerotium species.
T. harzianum and T. atroviride were found to be most promising against S. rolfsii
in chickpea. The mutant strain of T. harzianum and T. atroviride inhibited pathogen
growth by 82.9% (Singh et al. 2016). Similarly, Ramzan et al. (2016) tested
15 bioagents antagonistic to S. rolfsii in mung bean, among which T. harzianum,
Bacillus cereus, B. subtilis, T. virens, P. fluorescens, and Micrococcus varians were
found effective, whereas Bacillus subtilis and T. harzianum were most efficient.
Kushwaha et al. (2018) evaluated the efficacy of Trichoderma spp. against

S. rolfsii causing collar rot of lentils. The antagonistic ability of three BCAs, i.e.,
T. harzianum, T. viride, and T. virens, was evaluated against S. rolfsii under in vitro
conditions. All of them have shown the ability of inhibiting the pathogen in vitro.
Inhibition percentage was found to be highest in T. harzianum (63.60%), followed
by T. virens (51.5%) and T. viride (50.85%) post 72 h of incubation. However, in
contrast, T. viride recorded the highest sclerotial reduction (91.31%), followed by
T. harzianum (84.92%) and T. virens (84.29%) post 15 days of incubation. In a
similar study, Bastakoti et al. (2017) reported that S. rolfsii was inhibited nearly up to
100% by three isolates of the Trichoderma sp.
Dinesh et al. (2018) conducted experiments to check the efficacy of various
Trichoderma species against the collar rot pathogen in tomato in vitro. Percent
inhibition was observed and recorded for all Trichoderma spp. against S. rolfsii.
Percent inhibition ranged from 13.02 to 63.02 within different species. Among the
different species studied, T. asperellum recorded maximum pathogen inhibition
(63.02%).
Rajput et al. (2019b) documented that Trichoderma spp. can effectively manage
collar rot disease in tomato. Four Trichoderma strains, namely T. harzianum
BHUP4, T. viride BHUV2, T. pseudokoningii BHUR2, and T. longibrachiatum
BHUR5, were applied through seed biopriming method. Tomato plants primed
with T. pseudokoningii BHUR2 were found to be less infected as compared to
other bioprimed plants upon being challenged with S. rolfsii. It may be ascertained
that T. pseudokoningii BHUR2 primed tomato plants enhanced the antioxidative
activity of enzymes, including phenylalanine ammonia-lyase (PAL), peroxidase
(POx), superoxide dismutase (SOD), and total phenol content (TPC), in response
to S. rolfsii infection.
Apart from being an important soilborne pathogen causing collar rot, seed rot,
seedling decay, damping off, and related disorders, S. rolfsii is also reported to cause
significant losses as a postharvest pathogen in storage. It is reported to cause
postharvest losses of up to 35% in elephant foot yam and ginger, 30% in carrot
and lady’s finger, and few in other crops (Dasgupta and Mandal 1989). Mukherjee
and Raghu (1997) reported that Trichoderma spp. have the potential to inhibit the
growth of S. rolfsii and consequently prevent the rotting of beets, carrots, elephant
foot yam, and bitter gourd under postharvest conditions.
25.5.1 Trichoderma in Consortium
Trichoderma spp. is effective even when it is applied singly, but in most cases it is
frequently combined with other beneficial microbes, botanicals, or chemicals in
order to enhance its effect. In this regard, also several works have been attempted
by scientists, and the results obtained were quite satisfactory.
Upadhyay and Mukhopadhyay (1986) reported that integration of a
pentachloronitrobenzene (PCNB)-tolerant isolate of T. harzianum with PCNB
improved disease control. Under field conditions, a combination of T. harzianum
and PCNB lowered the incidence of sclerotium root rot (76% disease control) and
increased root, foliage, and sucrose yield in sugar beet. Dutta and Das (2002)
documented that soil application of Trichoderma spp. during transplanting reduced
disease incidence and enhanced root and shoot dry weight (g/plant) and yield
(g/plant) in tomato. Application of T. harzianum resulted in minimum disease
incidence and enhanced root and shoot biomass, followed by T. viride and
T. koningii, as compared to control. Reduction in sclerotia production of the patho-
gen by different Trichoderma isolates was earlier reported by Prasad et al. (1999).
Charitha Devi and Reddy (2003) evaluated five isolates of Trichoderma sp. and
Pseudomonas sp. against S. rolfsii, the causal agent of groundnut root rot, and
reported that T. harzianum showed maximum (66.6%) pathogen inhibition. Simi-
larly in another experiment, the combination of T. harzianum and Rhizobium was
found to promote plant growth and increase seed production in groundnut. Ganesan
et al. (2007) performed integrated management of S. rolfsii in groundnut using a
combined application of T. harzianum (ITCC-4572) and Rhizobium. The results
were promising as the bioagents caused reduced disease incidence and increased
yield. However, a combined soil application of T. viride, seedling treatment, and soil
drenching with tebuconazole (0.05%) resulted in a complete control of the collar rot
of tomato in pot (Banyal et al. 2008).
Singh et al. (2012) conducted a study in order to check the efficacy of a microbial
consortium consisting of Trichoderma (THU0816), Rhizobium (RL091), and fluo-
rescent Pseudomonas (PHU094) in imparting defense responses in chickpea against
S. rolfsii causing collar rot. The results suggested that the triple microbial consortium
acted in a synergistic manner and provided maximum disease control compared to
double and single application. Singh et al. (2013b) used a double microbial consor-
tium of Trichoderma spp. and Pseudomonas spp. in tomato against S. rolfsii and to
assess the synergistic effect of compatible isolates for disease management and plant
growth promotion. Rajendraprasad et al. (2017) conducted experiments with
24 isolates representing two different species, T. harzianum and T. viride, and
12 different B. subtilis and P. fluorescence species. The combination of potential
T. harzianum 1 and P. fluorescence proved to be most effective in increasing
germination and in reducing pre- and postemergence collar rot in tomato when
inoculated with S. rolfsii. Similarly, Mahato et al. (2017) reported that soil mulching
+ neem seed cake and T. harzianum as soil amendments gave maximum disease
reduction and yield increase in the same crop.
The biocontrol property of Trichoderma spp. against S. rolfsii is mainly attributed
to its ability to produce various metabolites like α-glycosidase, β-xylosidase,
glioviridin, β-glucosidase, cellobiohydrolase, viridian, chymotrypsin, N-acetyl
β-glucosaminidase, trypsin, chymoelastase-like proteases, etc., along with its other
modes of action, such as mycoparasitism, rapid substrate colonization, competition,
and induction of defensive responses in plants.
25.6 Bioformulations of Trichoderma Species
Generally, the bioformulations are classified according to their physical state, i.e.,
dry or liquid formulations. Dry formulations include powder and granules, while
liquid formulations comprise oil, suspension, and emulsion (Singh et al. 2014,
2016).
Trichoderma-based formulations are registered as biofungicide in several
countries, viz., India, the UK, Switzerland, New Zealand, France, Sweden, Belgium,
the USA, and Chile, with few pending regulations of other countries. About
970 microbial-based biopesticide products are registered with the Central
Insecticides Board and Registration Committee (CIBRC) till date (http://cibrc.nic.
in/bpr.doc) under sect. 9(3B) and 9(3) of the Insecticides Act, 1968, Government of
India. The Central Insecticides Board and Registration Committee (CIBRC) is the
main regulatory body under the Insecticide Act 1968, which also covers
biopesticides. A review of the status of biopesticides showed that India has the
capacity to produce 1850 MT of Trichoderma formulations per annum, while the
requirement is 22,038 MT with a market value of Rs 260 crores, which indicates that
there is a requirement of more Trichoderma formulations (Sriram et al. 2013). The
number of biopesticide production units in India has exceeded 410, out of which
130 belong to the private sector (Singhal 2004; Singh et al. 2012; Desai et al. 2016).
Indian Council of Agricultural Research (ICAR) institutions, along with state agri-
cultural universities (SAUs), are taking initiatives for the research and production of
microbial biopesticides. The number of biocontrol research laboratories and produc-
tion units is continuously increasing day by day.
25.7 Challenges in Bioformulations of Trichoderma Species
Although a large number of research publications are published every year about
Trichoderma, the registration of Trichoderma for microbial biopesticide is limited.
There are several challenges in Trichoderma bioformulations, including inconsis-
tency in results, limited shelf life, and low awareness and popularization at farmer
level. The shelf life of the bioformulation is a principal point that determines the
success of the commercialization of its products. A short-lived shelf life of
Trichoderma spp. in any formulation can create a vital issue for the development
of commercial formulations. Further research has to be done in this field to enhance
the shelf life and existence of Trichoderma spp. in any formulation. Moreover, there
are lots of rules and regulations associated with the registration of microbial
biopesticides, and sometimes the registration cost exceeds the production cost. So
the government should charge a substantial amount from manufacturers and con-
struct committees to fasten the registration process.
Several experiments have been conducted by various researchers to enhance the
shelf life of Trichoderma-based products (Navaneetha et al. 2015). Th-10 isolate
from T. harzianum was successful in controlling Fusarium wilt of banana. It was
found that the dried banana leaf is best suited for growth with high density of
propagules of T. harzianum, but adding jaggery to the leaves increased its multipli-
cation, which resulted to an increase in shelf life of more than six months as the
stored substrate (Thangavelu et al. 2003). The survivability of Trichoderma spp.
conidia can increase in alginate pellet formulation when further supplemented with
10% cellulose (Shaban and El-Komy 2001). Kolombet et al. (2008) studied the
effect of different modifications in formulating T. asperellum, which includes the
addition of starch and trace amounts of copper, along with reduced pH. This gave a
shelf life of six months from the developed formulation. The formulation of
bentonite-vermiculite, however, maintained the colony-forming unit of
T. harzianum for eight weeks and also enhanced the shoot weight of melon plants
and gave resistance against Fusarium wilt disease (Martínez-Medina et al. 2011).
Prasad and Rangeshwaran (2000a) reported that the efficacy of T. harzianum could
be enhanced by the addition of wheat bran-kaolin granular formulation for
controlling seed rot and damping off of chickpea caused by R. solani. The damping
off ranged between 10 and 15% in granule-treated plots, whereas in other treatments,
51–57% disease incidence was observed. Similarly in another experiment, Prasad
and Rangeshwaran (2000b) stated that the use of molasses-yeast medium (MYM) for
the mass culturing of Trichoderma instead of brewers’ yeast medium gave satisfac-
tory results. In the same year, Prasad and Rangeshwaran (2000c) used three carrier
materials, i.e., talc, kaolin, and bentonite to check their effect on the shelf life of
T. harzianum in providing protection against R. solani of chickpea cv. Annigeri
under greenhouse conditions. Seed treatment with kaolin and talc formulation was
found to be most effective in retaining viable spores of bioagents up to 120 days.
The addition of chitin in the production media and also talc formulation of
T. harzianum can increase the shelf life of the formulation further by two months
(Sriram et al. 2009). Al-Taweil et al. (2010) reported that the shelf life of
Trichoderma can be increased by using alginate and paraffin oil formulations. The
addition of glycerol in the culturing media as osmoprotectant can also increase the
shelf life of T. harzianum formulations. In an experiment, it was found that the shelf
life of formulations was increased to 7–12 months by the addition of 3–6% glycerol
in the culture media, in comparison to formulations without glycerol having a shelf
life of about 4–5 months (Sriram et al. 2011).
Further, immobilization of microorganism is an effective method for improving
and enhancing shelf life with field efficacy. Microencapsulation, an important
immobilization technology, increases the shelf life of microorganisms in comparison
with other types of formulations with control delivery of microbes and also enhances
their field application. Trichoderma conidia spray dried and microencapsulated with
various sugars such as molasses, sucrose, or glycerols can extensively increase the
survival percentage of conidia even after drying. T. harzianum SQRT037 conidia
formulated with organic fertilizers exhibited enhanced control of Fusarium wilt
compared to the treatment, which includes a formulation made of only conidial
suspension, in cucumber (Yang et al. 2011). Muñoz-Celaya et al. (2012) also
reported that microencapsulation of T. harzianum conidia in a 1:1 ratio blend of
maltodextrin–gum Arabic polymeric matrix can give 11 folds higher conidia sur-
vival in comparison with nonencapsulated conidia after spray drying.
25.8 Conclusion
Sclerotium rolfsii is a polyphagous pathogen attacking nearly 500 crop species of

wide range of variety. The pathogen is really rampant in tropical, subtropical, and
warm temperate regions across the globe. Antagonistic fungi such as Trichoderma
spp. have immense potential in managing S. rolfsii, and a noteworthy research has
been done to manage the pathogen in different crops by applying Trichoderma. The
antagonistic property of Trichoderma is mainly due to its specific action
mechanisms, such as mycoparasitism (direct attack on the pathogen), antibiosis
(secretion of antibiotics and VOCs), competition (reduction of nutrient availability
for others), and induction of systemic resistance. A concern is always raised about
the application of Trichoderma in the field. Different types of formulations have
been developed for the application purpose of Trichoderma, such as dry, liquid,
granules, and many more. Despite being one of the most popular biopesticides, it still
has not been widely accepted by the farming community as certain factors are
responsible for its widespread acceptability. Moreover, the registration of
biopesticides is a cumbersome task, which also limits their manufacturers. So the
future thrust should be to popularize this magnificent BCA at farmer level and to
make its registration process simpler and cheaper. In addition to it, further researches
should be carried out to enhance the shelf life and effectiveness of such microbial
formulations.
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Biotic Stress Management in Horticultural
Crops Using Microbial Intervention 26
R. Umamaheswari, N. R. Prasannakumar, S. Sriram, Sushil K. Sharma,
M. S. Rao, and M. K. Chaya
Abstract
Soils are naturally gifted with beneficial microflora and fauna that innately help
the natural process of pest, disease, and nematode suppression. The diversity of
the soil biota in many ways helps to maintain the stability and promote antago-
nistic potential, which decides soil health and crop productivity. Utilizing this soil
biota for environmentally safe crop productivity is the utmost need of the hour,
especially in horticultural crops, which are consumed afresh. Environmental
concerns over conventional pesticides have led to increasing demand in the use
of biological control agents, especially the microbial biopesticides, for production
of safe horticultural produce. This chapter throws light on array of microbes that
can be effectively deployed for managing the biotic stress due to diseases, pests,
and nematodes in horticultural crops; their modes of action, commercial aspects,
and problems to be addressed for successful large scale adoption.
Keywords
Biotic stress · Horticultural crops · Insect pests · Plant diseases · Plant parasitic
nematodes
R. Umamaheswari (*) · N. R. Prasannakumar · S. Sriram · M. S. Rao · M. K. Chaya

Division of Crop Protection, ICAR-Indian Institute of Horticultural Research, Bengaluru,
Karnataka, India
e-mail: UMA.MAHESHWARI@icar.gov.in
S. K. Sharma
ICAR—National Bureau for Agriculturally Important Microogranisms, Maunath Bhanjan, Uttar
Pradesh, India
ICAR—National Institute of Biotic Stress Management, Baronda, Raipur, Chhattisgarh, India

23, https://doi.org/10.1007/978-981-15-9154-9_26
620 R. Umamaheswari et al.
26.1 Introduction
Horticultural crops that succumb to biotic stress induced by agents like viruses,
bacteria, fungi, insects, and nematodes are a major cause of pre- and postharvest
losses. Protected and hi-tech horticultural technologies mainly rely on continual
usage of chemical pesticides for reducing the assessed 45% crop loss due to agents of
biotic stress, which amounts to Rs. 290 billion annually (Kulkarni 2015). Excessive
applications of synthetic fertilizers and pesticides have disrupted the environmental
balance, and there is a rising public demand on organic food, which is residue free
and safe for consumption. Hence, there is a paradigm shift in the efforts of scientists,
policymakers, and stakeholders to focus more on eco-friendly and sustainable
management options. Biological control especially with microbial interventions
had emerged as the promising alternative to chemical pesticides by effectively
suppressing the target pathogen or pest population without causing any economic
loss in the crop. Several bacteria, fungi, viruses, and protozoa are commercially
exploited as microbial biopesticides for biological control of insect and noninsect
pests, plant pathogens, and plant parasitic nematodes. The most widely used micro-
bial biopesticide is the entomotoxic bacterium Bacillus thuringiensis (Bt), which
occupies 90% of the biopesticide market (Kumar and Singh 2015). At present,
biopesticides contribute to 5% of total pesticide market and are projected to outpace
the chemical pesticide market in near future with annual growth rate of more than
15% (Marrone 2014).In this chapter, we shall deal in detail about the different
microbes that can be effectively deployed for management of biotic stress induced
through diseases, pests, and nematodes; their mechanism of action against the target
pests and diseases, mass production and commercialization; mode of delivery, and
problems to be addressed for large-scale adoption of microbial biopesticides in
horticultural ecosystems.
26.2 Microbial Interventions for Plant Disease Management
Plant pathogens causing serious diseases have evolved along with the host plants.
The population explosion demands increased food production with enhanced crop
productivity as area available for cultivation is limited. Intensified crop production
and monocropping practices favor more disease incidence. Chemical control of plant
diseases is relatively a short-term measure and results in pesticide residue issues and
environmental issues. Further, the chemical control adds input cost, which cannot be
afforded by farmers in developing and under-developed countries. Biological con-
trol involves management of plant, pathogen, disease-suppressive microbes, i.e.,
biocontrol agent, and environment.
Fungi in the genus Trichoderma and bacteria in the genera of Pseudomonas and
Bacillus are of increasing interest as bioprotectants against plant diseases. Various
plant diseases in different crops are controlled by using these fungal and bacterial
antagonists. Many products based on fungal and bacterial antagonists have been
developed worldwide including India.
26 Biotic Stress Management in Horticultural Crops Using Microbial Intervention 621
Different types of pathogens affect different parts of the plants, viz., roots, stems,
flowers, leaves, fruits, etc. and result in various kinds of diseases, viz., root rot, wilt,
downy mildew, powdery mildew, rust, smut, leaf spot, anthracnose, etc. As there is
diversity in the plant pathogens, there is need for diverse biocontrol agents to
tackle them.
26.2.1 Root Pathogens
Soil-borne pathogens affect the root system. Both bacteria and fungi cause root
diseases. The bioagents effective against root pathogens are generally antagonists
with mycoparasitism or antibiosis. There are few cases of induced systemic
resistance also.
Among antagonistic fungi, Trichoderma and Gliocladium species are widely
studied. Trichoderma and Gliocladium spp. are successfully used to control root
rot, damping-off, and wilt diseases incited by R. solani, S. rolfsii, Macrophomina,
Fusarium spp., and Pythium spp. in crops like chickpea, cotton, sunflower, peanut,
tomato, beans, pea, soybean, potato, sugarbeet, sugarcane, maize, citrus, and many
other crops. Well-documented bioagents show mycoparasitism against P. ultimum
and P. vexans, and Talaromyces flavus, Coniothyrium minitans and Sporodesmium
sclerotivorum against Sclerotium and Rhizoctonia. Saprophytic nonpathogenic
Fusarium species has been used for the management of wilt diseases caused by
Fusarium species. The nonpathogenic Fusarium species not only compete for
nutrition and space but also induce systemic resistance. Bacillus subtilis and sapro-
phytic Streptomyces species have been used to control potato scab caused by
Streptomyces scabies. Bacterial bioagents especially Pseudomonas spp. inhibit the
root pathogens by antibiosis and siderophores, which interfere with the iron uptake.
Agrobacterium radiobacter effectively controls crown gall, which is caused by
A. tumefaciens, largely through the action of the antibiotic agrocin 84. Efficacy of
A. radiobacter was threatened when strains of A. tumefaciens resistant to agrocin
84 were isolated from galls of A. radiobacter treated plants. Hence, it is important to
understand the genetic basis of antibiotic resistance so as to design better strategies
for biocontrol through genetic manipulations.
Fluorescent pseudomonads (FPs) belonging to the genera Pseudomonas produce
water-soluble fluorescent pigments in glycine amended substrate. These bacteria
inhibit major soil-borne and foliar fungal pathogens, viz., Rhizoctonia solani (blight
diseases of cotton and rice), Gaeumannomyces graminis var. tritici (take all of
wheat), Pythium spp. (rot/damping off of wheat, cotton, maize, cucumber, bean,
and sugar beet), Fusarium oxysporum (wilts of cotton, cucumber, radish, tomato,
bean, chickpea, pigeonpea, flax, banana, and carnation), Ralstonia solanacearum
(potato wilt), Sclerotium rolfsii (root rot of groundnut, sunflower, and bean),
Aphanomyces euteiches (pea root rot), A. cochlioides (sugar beet root rot), and
Thielaviopsis basicola (tobacco root rot). These bioagents are ubiquitous in rhizo-
sphere of crop plants; but only a few of them are effective antagonists. Hence, the
selection of proper strain is very much essential in developing biological control
strategy. They should be applied as seed treatment as these bacteria establish well in
rhizosphere when they are introduced at germinating seedling stage.
26.2.2 Foliar and Above Ground Pathogens
Disease symptoms on stem portions include decay, die back, and cankers on forest
and orchard trees. Foliar diseases include rust, mildews, leaf spots, and blight.
Diseases on fruits include fruit rot, anthracnose, canker, etc. Common bioagents
identified for stem diseases include antibiotic producing bacteria such as B. subtilis
and Agrobacterium and fungal antagonists like Fusarium, Cladosporium,
Trichoderma, and hypovirulent strains of the pathogen Cryphonectria parasitica.
The microbes most frequently recorded as saprotrophs on surfaces of crop plants
include fungi like Trichoderma spp., Gliocladium spp., Aureobasidium pullulans,
Cladosporium spp., and yeasts such as Cryptococcus spp. and Sporobolomyces spp.
Botrytis leaf spot in onion was suppressed by Gliocladium roseum. Botrytis leaf and
flower blight of many ornamental plants and fruit crops were effectively controlled
by Trichoderma spp. Powdery mildew disease in grapevine and cucumbers can be
controlled by Ampelomyces quisqualis, Tilletiopsis, and Sporothrix yeasts.
Beneficial bacteria in phylloplane are Pseudomonas, Chromobacterium, and
Klebsiella. Pseudomonas spp. and Streptomyces spp. have been shown to inhibit
Botrytis rot in lettuce. Experiments with fluorescent pseudomonads have shown their
ability to suppress leaf pathogens like Pyricularia oryzae (rice blast), Xanthomonas
oryzae pv. oryzae (bacterial blight of rice), Sarocladium oryzae (sheath bight of
rice), rice tungro virus (tungro), Septoria tritici (leaf spot of wheat), Puccinia
recondita (wheat rust), Xanthomonas axonopodis pv. malvacearum (blackarm of
cotton), Erwinia carotovora (soft rot of vegetables), Cercosporidium personatum or
Cercospora personata (late leaf spot of groundnut), Colletotrichum lagenarium
(cucumber anthracnose), cucumber mosaic virus (mosaic), Pseudomonas syringae
pv. lachrymans (angular leaf spot of groundnut), Pseudomonas savastanoi
pv. phaseolicola (halo blight), Xanthomonas axonopodis pv. cyamopsidis (bacterial
blight of cluster beans), Botrytis cinerea (apple gray mold), Erwinia amylovora (fire
blight of apple), Puccinia carthami (safflower rust), Phoma betae (sugar beet leaf
spot), Tobacco necrosis virus, Alternaria sp. (tobacco leaf spot), and Cercospora
moricola (mulberry leaf spot).
Fire blight of rosaceae plants caused by Erwinia amylovora is an important flower
disease. Nonpathogenic E. herbicola could control this disease in combination with
P. syringae. This could be achieved mainly by competition for food and space.
The fruit diseases can also be managed using fungal bioagents. Bioagents, viz.,
Gliocladium roseum, Penicillium sp., Trichoderma viride, and Colletotrichum
gloeosporioides, were as effective as fungicides in suppressing B. cinerea on
strawberries and grapes.
26.2.3 Postharvest Pathogens
Postharvest diseases, which can be responsible for 10–50% loss of production, have
received considerable attention. Numerous reports have indicated the suppression of
postharvest diseases in fruit crops by species of Aureobasidium, Trichoderma,
Debaryomyces, Sporobolomyces, Enterobacter, Bacillus, and Pseudomonas.
Much of the research work on biological control of plant diseases is confined to
antagonism under lab and greenhouse conditions, which includes antibiosis, compe-
tition, and mycoparasitism. The efficacy of bacterial antagonists under farmer’s field
conditions is dependent upon several factors. Hence, successful field applications
can be done by selecting proper strains of the antagonist, using effective dosage of
the inoculant, following suitable delivery system, developing formulations for long-
term storage and transport, and applying them at the proper time.
26.2.4 Mode of Action of Biological Control Agents against Plant

Diseases
Major modes of action of bioagents are competition for space and nutrition,
mycoparasitism, antibiosis, and induced systemic resistance. Mycoparasitism
includes coiling of hyphae of the pathogen and penetration by haustoria and lysis.
Hydrolytic enzymes like β-1,3-glucanase, chitinase, protease, and lipase produced
by the antagonists help in the mycoparasitism.
Lectins (glycoproteins) produced by some soil-borne pathogenic fungi also play a
role in recognition (Upadhyay and Mukhopadhyay 1986; Barak et al. 1985; Baker
and Chet 1982; Elad et al. 1983). Antibiotics production is well known in
Trichoderma species (Lifschitz et al. 1986). Ghisalberti et al. (1990) reported two
toxins produced by T. harzianum isolates, which are found to be most effective
against take-all of wheat caused by Gaeumannomyces graminis f.sp. tritici.
Gliotoxin production by Trichoderma virens (¼ Gliocladium virens) is well
documented. Competition is an indirect mechanism by which Trichoderma and
other bioagents exclude the pathogens at infection site. Different modes of action
may act synergistically, resulting in inhibition of the pathogen growth.
26.2.5 Induction of Systemic Resistance (ISR)
Some Trichoderma strains are potent inducers of plant resistance like responses.
These responses are systemic and are termed as induced systemic resistance (ISR).
Unlike systemic acquired resistance (SAR) responses elicited by chemical inducers,
ISR induced by biocontrol agent is not associated with plant cell necrosis and does
not cause phytotoxicity. Xylanase from Trichoderma spp. is reported to be respon-
sible for ISR in cotton, tobacco, grapevine, etc. Induction of resistance includes
enhanced production of terpenoids or other phenolic compounds related to Shikimic
acid pathway or expression of hydrolytic enzymes in the treated plants. Gliotoxin
produced by T. virens is also known to induce systemic resistance. Seedling dip with
cell wall glucan elicitors produced by Trichoderma isolates could reduce the infec-
tion by Phytophthora capsica in chilies (Sriram et al. 2009).
Besides inhibiting the pathogen growth, many fungal bioagents, including
Trichoderma species, promote the plant growth also by the production of required
hormones or increased nutrition uptake and enhanced growth.
26.2.6 Mass Production Technology for Biocontrol Agents of Plant

Pathogens
26.2.6.1 Mass Production of Fungal Bioagents

For a successful bioagent, the mass production is crucial. The easier their production
process, the more is the acceptance by commercial producers. Fungal bioagents can
be produced by both solid-state and liquid-state fermentation methods.
Solid fermentation is an easier method for small production units. Solid substrates
used vary from cereal grains to agricultural wastes or by-products from other
biological processes. The dry spores produced from solid-state fermentation have
better shelf life than the formulations produced using liquid fermentation. The
hydrophobins present in the dry spores provide better desiccation tolerance to them.
Due to the advances made in the liquid fermentation technology, it has also been
successfully used in the production of bioagents. In India, industrial production of
Trichoderma using inexpensive media such as molasses and brewer’s yeast for
large-scale production is very common.
26.2.6.2 Mass Production of Bacterial Antagonists

Mass production of antagonistic bacteria involves growing the microorganisms in
the appropriate medium using rotary shakers or fermentors. Bacteria require ade-
quate nutrient supplements for proper growth. Hence, while looking for cheaper
alternatives, one cannot do the same as in mass production of fungi.
Usually, King’s B medium (broth) is ideal for mass production of antagonistic
bacteria; however, other media like Tryptic Soya Broth or Nutrient Broth can also be
used. It is better to choose the medium that provides optimum growth.
Bacteria are multiplied usually using rotary shakers (duplicating wrist arm action)
in large flasks (5 liters) under incubated (28 C) or room temperature (25 C)
conditions for 48 h. The incubation time can be chosen based on growth curve of
the selected antagonist. The harvested bacteria should always be in the active phase.
26.2.6.3 Fermentation
For large-scale mass multiplication (industrial scale) of bacteria, fermentors with
large volume capacity can be employed. Both batch type and continuous type
fermenters are available. However, only batch type fermenters are commonly used
in India. All other parameters such as inoculum load, pH, and temperature of the
substrate are regulated so as to obtain the optimum levels of fermentation continu-
ously. Fermentor raw materials like sugarbeet, potato, sweet potato, tapioca, apples,
raisins, grapes, sweet corn, rice, blackstrap molasses, sorghum, wheat, barley,
malted cereals, honey maple, etc. have been used to grow bacteria. Since our aim
is to get maximum viable cells quickly, we must standardize the conditions before
mass multiplication.
26.2.7 Development of Formulations of Bioagents
26.2.7.1 Development of Formulations of Trichoderma spp.

The desirable features of any bioagent formulations are market demand for that
product, industry friendly production process, suitable to long distance transport,
better shelf life with retention of desirable number of viable biological units, and cost
friendliness (Churchill 1982; Lisanski 1985).
Solid substrate fermentation-based formulations are directly applied or used to
amend the organic matter used for the field application. But solid-state fermentation
cannot be extended to large-scale production due to problems in contamination.
They have huge volume and need more space though the formulations have better
shelf life. The liquid-state fermentation needs short production time, less chances of
contamination, and products amenable for different types of formulations. The
carrier material may be inert or a food base or a combination of both.
26.2.7.2 Formulation of Antagonistic Bacteria

Talc is usually used as carrier, and its pH is adjusted to 7.0 using calcium carbonate.
Ten g of carboxymethyl cellulose (CMC) per kg of carrier is used as adherent. The
inoculum culture of the antagonist (containing a minimum population of
9 108 cfu/ml) is mixed with the sterile carrier (400 ml/kg) and air dried. Seeds
are coated with a thin layer of 1% CMC and mixed with the respective formulation/
s at the rate of 4 g/kg seed.
26.2.8 Delivery of Biocontrol Agents used Against Plant Pathogens
Delivery of bioagents can be done by different methods, viz., seed treatment,

seedling dip, soil amendment, soil drench, aerial spraying, wound dressing, applica-
tion using fluid drill, etc.
Seed priming is the best method for the delivery of many fungal and bacterial
antagonists against many seed-borne and soil-borne pathogens like Pythium,
Phytophthora, Fusarium, Rhizoctonia, etc. that cause damping off, wilt, and root
rot (Callan et al. 1990; Cliquet and Scheffer 1997). Soil application of bioagents is
normally recommended as enrichment of organic matter and then used for the
application in field. Completely decomposed farm yard manure or neem cake is
the commonly used substrate for enriching with Trichoderma or Pseudomonas. Soil
drench or seedling dip is more preferred with the bacterial bioagents. Seedling dip at
10 g per liter for 2 min with formulations of bacterial bioagents like Pseudomonas or
Bacillus provides better protection against most of the soil-borne pathogens. Though
aerial spraying is possible with Trichoderma formulations, success has been reported
with bacterial bioagents as aerial spray for many foliar diseases (Elad and Kirshner
1992). Biological control of foliar pathogens using bacterial antagonists will neces-
sarily depend upon the establishment and survival of the antagonists on the leaf
surface, which has a competitive environment. Addition of nutrients like yeast
extract or glucose (jaggery solution) into the inoculum spray has shown to enhance
the survival of the antagonists. The talc-based formulation is at the rate of 1kg/ha as
foliar spray. However, when used as wound dressing, there was good protection
from wounds on shrubs and trees applied at pruning, in advance of decay fungi
(Papavizas 1985).
26.2.9 Compatibility with Fugicides
Wilt and root-rot complex of chickpea, lentil, and pigeon pea were successfully
managed by integration of T. harzianum or T. virens with Vitavax (Patibanda et al.
2002). Integration of fertilizers or herbicides with biocontrol agents to control plant
diseases has also been attempted. Bacterial biocontrol agents are compatible with
most of the fungicides. Even fungal biocontrol agents like Trichoderma spp. are
insensitive to fungicides like carboxins, oxycarboxins, metalaxyl, tricyclazoles,
carpropamid, host defense inducers (e.g., benzothiadiazole), etc. Therefore, they
could be integrated easily. Integrating biocontrol agents with reduced doses of
fungicides seems to be a very promising way of controlling pathogens with minimal
interference with biological equilibrium. This would not only reduce the use of
fungicides but also improve the efficacy of a biocontrol system with reduced cost
and lessen the chances of development of fungicide-resistant strains of the pathogen.
Replacement of tridemorph as soil drenching can be possible with soil application of
T. virens at 1 kg/palm along with neem cake and FYM for the management of stem
bleeding disease of coconut. Integration of biocontrol agents and fungicides for seed
treatment is very effective even against high population of fast growing pathogens
like Rhizoctonia solani in soil. Under such a condition, biocontrol agent alone may
not be very effective as by the time it gets activated in soil, pathogen is able to
penetrate the host. Integration of fungicide with biocontrol agents helps in early
protection by fungicide and later protection by biocontrol agent. Fungicide also
provides congenial environment to insensitive biocontrol agent to multiply and
colonize spermosphere and rhizosphere by suppressing other microbes sensitive to
the fungicide.
26.2.10 Compatibility with Biofertilizers
Since several biofertilizers are recommended for seed treatment in pulses and oil
seeds, delivering of biocontrol agents along with biofertilizers will give a single
solution approach for both growth promotion and disease control. Studies conducted
at TNAU indicated that both Trichoderma and Azospirillum were compatible and
enhanced the biomatter production in addition to the control of root rot in sesamum
and groundnut. T. viride was compatible with Rhizobium in treated seeds as
evidenced from number of colonies of both organisms growing in vitro.
26.2.11 Biological Control of Bacterial Diseases using

Bacteriophages
Bacteriophages are naturally available in environment and can be isolated from soil,
water, plants, animals, and human body. Phages are self-replicating and self-limiting
as they replicate only as long as the host bacterium is present and are quickly
degraded in the absence of host (Kutter 1997). Bacteriophages are nontoxic. Humans
consume bacteriophages everyday without getting harmed (Jackson 1989). Though
phage particles are detected in organs and tissues such as brain and blood stream, no
serious side effects have been reported (Balogh et al. 2003).
They are host specific and can be combined with other biocontrol agents such as
PGPR and other antagonists (Tanaka et al. 1990). Production of phages is not
expensive. They can be stored for years at 4 C. If prepared in cocktails, they can
be used for wide range of strains and development of new strains can also be
checked.
Phages are to be applied early in the morning before dawn or late in the evening.
Otherwise, virus particles perish in daytime. There is need to develop formulations
and to standardize the delivery system. In future, the antibodies for the bacterial cell
wall receptors can be expressed in phages to enable specific and planned target of
phage to desired surface on bacterial host cell (Cao et al. 2000).
Saccardi et al. (1993) observed that fruit spot incidence on peaches could be
controlled by biweekly spray of phage suspension effective against Xanthomonas
campestris pv. pruni. Jackson (1989) developed H-mutants (phages that possess the
ability to lyse bacterial strains that are resistant to the parent strain while still
possessing the ability to lyse the wild type bacterium) for the management of
Pseudomonas syringae in bean cull. Flaherty et al. (2000) also used the H-mutants
for the control of bacterial spot in tomato. Flaherty et al. (2001) found that foliar
application of phage mixture with wide host range mutants reduced the leaf spot
disease in geranium caused by X. campestris pv. pelargonii. Use of mixture of host-
range mutant bacteriophages as twice-weekly sprays could reduce the disease
severity of bacterial spot of tomato by 17 percent (Flaherty et al. 2000,) while
chemical control could give only 11 percent reduction.
Svircev et al. (2002) isolated 51 phage isolates of Erwinia amylovora, the causal
agent of fire blight of apple. They could distinguish 6 phage types among them based
on molecular characterization of the phages using PCR and restriction endonuclease
digestions. Out of these 51 isolates, 23 isolates could suppress the bacterial incidence
on pear fruit surface.
26.3 Microbial Interventions for Insect Pest Management
Insects are also being infected with a variety of disease causing microorganism like
bacteria, viruses, fungi, and protozoans and cause natural mortality. These organisms
are capable of infecting and causing disease epizootics on insects naturally under
certain conditions that resulted in collapsing of an entire pest population. Such
organisms have been scientifically studied and extrapolated in management of insect
pests as biological control agents through introductory or inundative applications.
First time successful large-scale control was studied in 1879 on beet weevil,
Bothynoderes punctiventris using Metarhizium anisopliae in Russian Ukraine
(Metchnikoff 1879). Since these are already occurring in nature, they are safe to
environment and recommended to use as eco-friendly pest management strategies in
horticultural ecosystem.
26.3.1 Entomopathogens
The pathogens that infect and cause significant mortality in insects are called
entomopathogens. They are viruses, fungi, bacteria, protozoans, and
entomopathogenic nematode (EPN) (Fig. 26.1). Some successful microbial
entomopathogens used in management of insect pests on horticultural crops are
discussed below.
26.3.1.1 Viruses
Insect viruses may believe to have evolved 350–400 million years ago. They exhibit
great structural diversity as viruses could infect other form of life. They appear to be
Fig. 26.1 Infection routes of entomopathogens. (Picture courtesy: Surendra 2017)

with envelop (capsid) or sometimes nonenvelop with double or single stranded DNA
or RNA in their genome. Capsid of the virus plays a significant role in infection to
living host cells. Some are known to produce occlusion bodies consisting of virions,
which are usually embedded with protein matrix (Robert and Linda 2014). Though a
few viruses are found to be unique to invertebrates, most of the cases, they share
genetic and structural similarities with viruses of vertebrates (Dhaygude et al. 2019).
Not all of them could have been extrapolated to be used as biopesticdes to control
insect pests, but a few are very successful, e.g., NPV (Nuclear polyhedrosis virus)
and HaNPV-Helicoverpa armigera NPV. Only the baculoviruses from
baculoviridae have been used extensively in pest management. These viruses are
very diverse with dsDNA, circular, supercoiled genome with 80–180 kb size. The
members of the groups are NPV and GV (Granulosis virus), which are specific to
arthropods. The occlusion bodies are usually polyhedral with a crystalline matrix
consisting of ‘polyhedrin’ protein in the case of NPV, whereas for GV, they will be
granule or capsules with ‘granulin’ protein (Kari et al. 2013).
Based on the number of nucleocapsids per envelope, the NPV is categorized into
multiple nucleopolyhedrosis virus (MNPV) and single nucleopolyhedrosis virus
(SNPV). In lepidopteran viruses, both MNPV and SNPVs are found, but in other
insect order, only SNPVs are found. Likewise, GVs are also characterized into single
or multiple GVs (George 1992). The entomopathogenic viruses are highly specific
and limited to host range such that only one species or related genera is present in a
family.
26.3.1.2 Mode of Action

The entomopathogenic viruses are stomach poisons. The insect has to ingest the viral
particles through the food. Upon ingestion, the polyhedral bodies dissolve in alkaline
pH of the insect gut and release the viral particles inside the insect gut. Lipid protein
membrane around the virus fuses with plasma membrane of the gut wall cells and
releases nucleocapsids into the cytoplasm. The nucleotide transports virus DNA into
the nucleus of the cell, and expression of virus gene begins. Multiplication of the
virus will take place rapidly and finally infect many tissues and organs, primarily the
fat body, epidermis, and blood cells of the insect by filling with virus particles
(virions) (Kalawate 2014; Mazzone 1985; Granados and Williams 1986).
26.3.1.3 Symptoms of Virus Infection in Insects

The affected insect usually discolors, is shiny and fragile, loses its appetite,
regurgitates, and looks stressed with lethargic. The HaNPV-infected larvae usually
drop down from the tip of the leaf or top of the plant substrate. This type of behavior
of the larvae crawling top of the canopy is generally because of requirement of more
oxygen and also to keep its inmates away from further infection. The baculovirus
infected insect larvae generally die from 3 to 12 days after infection depending on
inoculums, temperature, and the larval instar at the time of infection.
26.3.1.4 Formulations Available, Commercialization

of the Viral Bioagents
Baculovirus-based biopesticides are being used as one of the components in
integrated pest management (IPM) program. These are very safe to nontargeted
organisms as they show very specificity of infection to target hosts. Baculoviruses
have been described in over 600 insect species. There are four genera in the family
Baculoviridae, viz., Alpha baculovirus (Lepidopteran-specific NPVs),
Beta baculoviruses (Lepidopteran-specific GVs), Gamma baculoviruses
(Hymenopteran-specific NPVs), and Delta baculoviruses (Dipteran-specific
NPVs). However, baculoviruses have been reported from a variety of diverse
invertebrate species. Prominent insect hosts are Diptera, Hymenoptera, and Lepi-
doptera. In addition, vast genetic variations within host insect species were observed
in baculoviruses. The NPV from Autographa californica (AcMNPV) is one of the
most extensively studied virus.
The viruses are very effective if applied at neonate stage. The NPV and GV are
the two viruses studied extensively on many insect pests. HaNPV and SlNPV
(Spodoptera litura) are being produced and available in the markets. Likewise,
GV has been successfully used to manage diamondback moth, Plutella xylostella,
apple codling moth, Cydia pomonella, sugarcane shoot borer, Chilo infuscatellus,
and coconut rhinoceros beetle, Oryctes rhinoceros. Some commercial baculovirus
formulations available in the market have been listed in Table 26.1.
26.3.2 Entomopathogenic Fungi
These are microorganisms that can penetrate the insects directly through exoskeleton
and produce spores all over the body, which results in death of the insect (Fig. 26.2).
The spores produced on the infected insects could disperse naturally, infect other
individuals of the population, and thus create epizootics. There are several stages of
infection that consist of contact of the fungal spore on the exoskeleton of the target
insects, which includes lytic enzymes, secondary metabolites, and adhesins
Table 26.1 Commercial baculovirus formulations

Product Name Manufacturer Baculovirus Pests
Spod-X Thermo Trilogy SeNPV Beet armyworm
GemStar Thermo Trilogy HzNPV Heliothis/Helicoverpa
Elcar Novartis CpNPV Codling moth
Madmex Andermatt Biocontrol, Switzerland
Granusal Behring AG, Werke, Germany
Caprovirusine NPP, France
VPN Agricola El Sol, Brazil AgNPV Velvetbean caterpillar
Gusano Thermo Trilogy AcNPV Autographa californica
Spodopterin NPP, France SlNPV Spodoptera litura
Adopted from: Kalha et al. (2014)
Fig. 26.2 Entomopathogenic fungal species. (Picture courtesy: Surendra 2017)

produced by EPF. Due to their high effectiveness and epizootic formations, the fungi
are commonly used as biopesticides in organic farming.
Fungal diseases are noticed in Lepidoptera (larvae), Homoptera (aphids,
whiteflies, cicadas, and scale insects), Hymenoptera (bees), Coleoptera (beetles),
and Diptera (flies and mosquitoes). Some commercially exploited entomopathogenic
fungi are as follows:
1. Beauveria bassiana—This fungus penetrate the host insects’ body through inges-
tion of food or in contact with the host cuticle. Multiplication takes place inside
the insect body and produces toxins such as beauvericin and bassianocide. The
toxin produced by B. bassiana results in paralysis of the host insects and
ultimately kills the insects within four or five days. This is generally being used
to control sucking pests and caterpillars.
2. Lecanicillium lecanii—This fungus has been used to control mainly sucking
pests, viz., mealy bugs, aphids, thrips, whiteflies, brown plant hopper, scale
insects, etc.
3. Metarhizium anisopliae—Green muscardine fungi are used to control coconut
rhinoceros beetle, groundnut cut worm, rice brown plant hopper, diamond back
moth, and early shoot borer, top shoot borer, and internode borer of sugarcane.
4. Nomuraea rileyi—A potential insect-infecting fungus that causes epizootic death
in various insects. It is used to control pod borers, cut worms, cabbage borers, etc.
5. Hirsutella thompsonii—White septate of the fungus becomes green or purple-
gray to purple upon sporulation. These fungi are used to control different hoppers
and bug pests, whiteflies, red mites, etc.
6. Isaria fumosorosea (¼Paecilomyces fumosoroseus)—It has got a wide host range
and is successful in controlling yellow and red mite, whiteflies, etc.
26.3.2.1 Mode of Action of Entomopathogenic Fungi

The extracellular toxins and enzymes like proteases and chitinases help the conidia
to penetrate the exoskeleton of the insect. The conidia germinate and occupy all the
cells of the body. During process of interaction, insect starves and dies due to the
toxins produced by the fungi. The infected larvae loose appetite, discolor patches on
the integument, and undergo partial paralysis, body becomes hardened, and condia
can be seen projecting outward the body of infected insect.
26.3.3 Entomopathogenic Bacteria
Aristotle described in his writings about the sickness of honey bee (Apis mellifera)
that was commonly what we call it as “foul brood”. Subsequently, differentiation
between pebrine and flacherie diseases of silkworm (Bombyx mori) by Louis Pasteur
in eighteenth century made noteworthy contributions to the knowledge of infectious
processes of bacteria in insects. Later on, Kirby and Bassi made tremendous
contribution to insect pathology and shown the way about application of microbes
in the field of entomology.
Since decades, a lot of work on isolation, characterization, and demonstration of

numerous bacteria have been carried out in both field and laboratory conditions.
Most of these bacteria belong to the families, viz., Pseudomonadaceae,
Enterobacteriaceae, Lactobacillaceae, Micrococcaceae, and Bacillaceae. They have
been categorized as spore-forming (Bacillus spp., Paenibacillus spp., and Clostrid-
ium spp.) and nonspore-forming (Pseudomonas, Serratia, Yersinia, Photorhabdus,
and Xenorhabdus). Based on gram staining, they are again classified into gram-
positive (Bacillaceae and Lactobacillaceae) and gram-negative (Pseudomonadaceae
and Enterobacteriaceae). Among them, Bacillus thuringiensis (Bt), B. sphaericus,
B. cereus, and B. popilliae have been explored much in developing microbial control
agents. The main route of infection is oral, i.e., through ingestion.
Most of the commercially available products are gram-positive bacteria from
genus Bacillus because of long shelf life and stability. However, efforts are also
being made to improve the shelf life of gram-negative bacterial pesticides. For
example, Bacillus and Paenibacillus are pathogenic to coleopteran, dipteran, and
lepidopteran insects. Likewise, Bacillus thuringiensis subsp.
aizawai, B. thuringiensis subsp. kurstaki (for lepidopteran caterpillars),
B. thuringiensis subsp. israelensis, B. thuringiensis subsp. sphaericus (mosquito
larvae), and B. thuringiensis subsp. tenebrionis (coleopterans) are more effectively
deployed in the management of several insect pests.
Major breakthrough in using bacterial pathogens as biopesticides was through
identification of Bt in 1901. Its gram-positive, rod-shaped, spore-forming bacteria
found in soil were isolated in 1901 and named in 1911. First commercial Bt was used
in USA in 1958. They disrupt the midgut membrane of insects and hence are placed
under group IRAC 11. They are highly specific to insects, and therefore, identifica-
tion of specific strains is very crucial.
26.3.3.1 Mode of Action of Bacillus thuringiensis

The B. thuringiensis (Bt) cells usually produce spores and toxins, which are called
delta endotoxin. The commercially products contain this spore, which becomes
bacterial cells inside the insect. Upon ingestion of the Bt by the insect, the delta
endotoxin gets activated in the insect’s gut by enzymes and alkaline (basic)
conditions. The endotoxin ruptures the cell walls of the gut, and bacterial cells
enter the body. Infected insects discontinue feeding in a few hours and die within
few hours to weeks (frequently 2–3 days). Different strains of Bt have different
endotoxins and kill the insects accordingly. The endotoxin is safe to human as the
human’s gut Ph is usually acidic where the endotoxin will not be activated. The type
of responses caused by Bt toxins in lepidopteran caterpillar is discussed below:
Type I response—Once delta toxin is ingested, paralysis of midgut occurs in a few

minutes. Symptoms of type I response include arrest of feeding, increase in pH of
the hemolymph, regurgitation, diarrhea, and sluggishness. General paralysis and
septicemia occur in 24–48 hours, resulting in the death of the insect. This type of
response is commonly found in silkworm, tomato hornworm, and tobacco
hornworm.
Table 26.2 Bt strains registered under Insecticide Act in Indiaa

Bt subspecies Strain/serotype Formulation type
Bt kurstaki (i) Strain A-97, serotype H-3a 35 WP
(ii) Strain DOR-Bt-1, serotype-(3a, 3b, and 3c) 0.5% WP
(iii) Strain HD-1, serotype 3a, 3b 3.5% ES
(iv) Serotype 3a and 3b, Strain Z-52 –
Bt galleriae Strain R 1593m, serotype 3a 1.3% FC
Bt israelensis (i) Strain 164, serotype H-14 WP
(ii) Strain VCRC B-17, serotype H-14 Slow release granules WP
(iii) Strain VCRC B-17, serotype H-14 12 AS
(iv) Serotype H-14 5 AS
(v) Strain VCRC B-17, serotype H14 5% WP
(vi) Serotype H-14
a
Adopted from Ramanujam et al. (2014)
Table 26.3 Commercial products of Bt and their usagea

Bt sub sp. strain Trade name Usage
Kurstaki Able, Bactospeine, Condor, Costar, CRYMAX, Cutlass, Lepidoptera
Dipel ES, Bactimos L, Futura, Lepinox, and Thuricide
Aizawai Florbac, Agree, Design, and Xentari Lepidoptera
Kurstaki SA-12 Costar Lepidoptera
Kurstaki Foil and Raven Lepidoptera
Coleoptera Thuricide, Biobit, Dipel, Foray, Javelin, and Vault Lepidoptera
kurstaki HD-1
a
Adopted from: Kalha et al. (2014)
Type II response—The same as above except there will be no general paralysis.

Septicemia occurs within 24–72 hours. This type of response is commonly found
in loopers, alfalfa caterpillar, and cabbage butterfly.
Type III response—Midgut paralysis is followed by cessation of feeding, but insect
moves actively without general paralysis. Mortality occurs in 48–96 hours, and
higher mortality could be seen if spores are ingested instead of delta endotoxin.
This is common in Mediterranean flour moth, corn earworm, gypsy moth, and
spruce budworm.
Type IV response—The neonates or the young once are highly susceptible, whereas
the late instars are quite resistant to infection. After ingestion of delta, endotoxin
midgut paralysis occurs followed by cessation of feeding. Insect could move
actively without encountering paralysis. Mortality occurs in 72–96 or more hours.
Higher mortality occurs if spores are ingested. Cutworms and armyworms are
examples for this category.
In India, several Bt strains are registered under Insecticide Act (Table 26.2) and
many commercial Bt formulations are available in the market (Table 26.3).
26.3.4 Bioefficacy of Entomopathogenic Microbes Against Pests

of Horticultural Crops
Reddy et al. (2019) reported that spraying M. anisopliae was on par with chemical
thiamethoxam 25 WG in effectively reducing Scirtothrips dorsalis population
(82.287 to 84.24%) and berry damage due to thrips in grapes. M. anisopliae was
also effective against onion thrips and Thrips tabaci by causing 58% reduction in
insect population and 49.12% increase in onion yield (Visalakshy and
Krishnamoorthy 2012). Swathi et al. (2019) reported the highest mortality (80%)
of the South American tomato moth, Tuta absoluta by Beauveria bassiana under
in vitro with LC 50 value of 1.15 107 spores ml1.
In bitter gourd, Soumya et al. (2017) evaluated several biocontrol agents and
found that Bacillus thuringiensis, Nomuraea rileyi, and Beauveria bassiana gave
better control of melon borer, Diaphania indica. Application of oil formulation of
M. anisopliae with 1 107 spores/ml effectively lowered thrips incidence (3.6 per
plant) and increased capsicum yield (Anon 2020). Avery et al. (2013) demonstrated
the efficacy of Isaria fumosorosea in mitigating the polyphagous Madeira mealybug,
Phenacoccus madeirensis in plants pre- and post-shipping.
26.4 Microbial Interventions for Nematode Management

in Horticultural Crops
Plant parasitic nematodes are one of the major biotic stress factors inflicting suc-
cessful crop production, quantitatively and qualitatively. Besides causing direct
crops losses, they also predispose the roots to development of disease complexes
involving other soil-borne pathogens. According to Sasser and Freckman (1987), a
global average of 12.3% crop losses is attributed due to nematodes in 40 major crops,
estimated at USD 173 billion, annually (Elling 2013). In India, through All India
Coordinated Research Projects on Nematodes, an average crop loss of 21.3%
amounting to Rs. 102,039.79 million (1.58 billion USD) annually is attributed due
to nematode damage; the losses in 19 horticultural crops alone remain at
Rs. 50,224.98 million (Kumar et al. 2020). There is always a great demand for
environmentally friendly microbial technologies to ensure residue free horticultural
crop production.
26.4.1 Fungal Bioagents for Nematode Control
Nematophagous or nematode destroying fungi are referred to as a diverse fungal

group that possesses the capability to attack, parasitize, and digest nematodes (eggs,
juveniles, and adults) for nutritional benefit. They produce trapping structures,
spores, appressoria, and toxins to infect nematode hosts (Mankau 1980; Lopez-
Llorca et al. 2008). Arbuscular mycorrhizal (AM) fungi also play a beneficial role in
nematode biocontrol (Sikora and Sitaramaiah 1980; Suresh and Bagyaraj 1984).
Though several fungi are reported to effectively parasitize nematodes, only a few
are commercially exploited as successful BCAs. In France, commercial formulation
of Arthrobotrys robusta is marketed as Royal 300® for control of mushroom
nematodes and A. irregularis is sold as Royal 350® for managing root knot
nematodes in tomato (Cayrol 1983). In U.S.A, Myrothecium verrucaria is marketed
as DiTera® by Valent Biosciences Corporation (Moosavi and Askary 2015). In
India, the three commercially successful nematophagous fungi that are widely
available as bionematicides are Purpureocillium lilacinum, Pochonia
chlamydosporia, and Trichoderma spp. (T. harzianum and T. viride).
26.4.1.1 Purpureocillium lilacinum (=Paecilomyces lilacinus)

Purpureocillium lilacinum, earlier known as Paecilomyces lilacinus, is a ubiquitous,
facultative fungal parasite that is effective against root knot, reniform, and cyst
nematodes (Lysek 1976; Jatala et al. 1979) (Fig. 26.3).
26.4.1.2 Mode of Action of Purpureocillium lilacinum

on Phytonematodes
P. lilacinum penetrates the eggs, proliferates inside, and hinders the juvenile devel-
opment inside the egg (Khan et al. 2006). Its hyphae enter into the adult females of
sedentary nematodes through the anus and vulva (Jatala et al. 1979). After penetra-
tion, the hyphae grow rapidly and destroy the juveniles inside the eggs. Later, they
produce a large number of conidiophores and move toward the adjacent eggs.
This fungus has been reported to produce peptidal antibiotics like leucinostatin,
lilacin, and paecilotoxin (Arai et al. 1973; Mikami et al. 1989). Many lytic enzymes
produced by this fungus like serine protease, chitinase, and glucoamylase facilitate
the rupture of the egg shell (Bonants et al. 1995; Mittal et al. 1995).
In commercial markets, these fungi are available as Wettable powder and liquid
formulations (Davies and Spiegel 2011). Some commercial formulations of
P. lilacinum are available in the Indian and global market as furnished in Table 26.4.
Fig. 26.3 Profuse growth of

P. lilacinum on egg mass of
M. incognita
Table 26.4 Some commercial products of Purpureocillium lilacinum

Biocontrol
fungi/strain Trade name Company Country References
P. lilacinum BIOCON Asiatic Manila, Timm (1987), Davide (1990)
Phil. Strain Technologies Philippines
No.1 Incorporation
P. lilacinum Soybean China Liu et al. (1996)
PL 251 Root
Bioprotectant
P. lilacinum BIOACT® PROPHYTA Germany Davies and Spiegel (2011),
PL 251 WG Kiewnick (2004), Atkins
BIOACT® et al. (2005), Wilson and
WP Jackson (2013)
P. lilacinum Melocon® Certis U.S.A. Wilson and Jackson (2013)
PL 251 WG
P. lilacinum Nemachek Several Copping (2004), Moosavi
Paecil countries and Askary (2015)
Paecilo
PL Plus
Green
Nemagon
P. lilacinum Yorker Agriland India Askary (2015)
IIHR Pl2 Biotech
Limited,
Gujarat
P. lilacinum Nematofree International India Askary (2015)
IIHR Pl2 Panacea
Limited
P. lilacinum PAECILO® Agrilife India Askary (2015)
IIHR Pl2
P. lilacinum GMAX Greenmax India Askary (2015)
IIHR Pl2 BIOGUARD Biotech
P. lilacium Bio- T. Stanes & India
Nematon company
limited, Tamil
Nadu
P. lilacinum Utkarsh Utkarsh India
IIHR Pl2 Nematoz P Agrochem
Pvt.Ltd,
Gujarat
Paecilomyces Mysis Varsha India
lilacinus IIHR Biosciences,
Pl2 Telangana
Paecilomyces Bioniconema Nico India
lilacinus IIHR organics,
Pl2 Gujarat
Paecilomyces Niyanthran Multiplex, India
lilacinus IIHR Bangalore
Pl2
Paecilomyces BoomNemo Devi biotech India
lilacinus IIHR Pvt. Ltd,
Pl2 Tamil Nadu
26.4.1.3 Bioefficacy of P. lilacinum on Plant Parasitic Nematodes

in Horticultural Crops
P. lilacinum has been successfully used to manage root knot nematode in several
horticultural crops, viz., tomato (Rao et al. 2012), banana (Devarajan and Rajendran
2001), betelvine (Jonathan et al. 1995), carrot (Sivakumar 1998), groundnut (Patel
et al. 1998), and potato (Jatala et al. 1980). It has also been reported to control
Tylenchulus semipenetrans in citrus (Manzoor et al. 2002), Radopholus similis in
banana (Devarajan and Rajendran 2001), and G. rostochiensis and G. pallida in
potato (Seenivasan et al. 2007).
Combined treatment of P. lilacinum and Pseudomonas putida was carried out,
and M. incognita was reduced by 66% and Fusarium oxysporum f. sp. gladioli by
57% in gladiolus (Sowmya and Rao 2012). Rao et al. (1998) reported that root dip
treatment of brinjal with P. lilacinum grown in neem cake extract resulted in least
root knot index due to M. incognita.
26.4.2 Pochonia chlamydosporia (=Verticillium chlamydosporium)
Pochonia chlamydosporia is a ubiquitous fungus that possesses the ability to

parasitize the eggs and females of root-knot and cyst nematodes (Wilcox and
Tribe 1974; Kerry 1990) (Fig. 26.4). Nagesh et al. (2007) reported its parasitism
on burrowing, citrus, and reniform nematodes.
26.4.2.1 Mode of Action of Pochonia chlamydosporia

P. chlamydosporia produces appressoria, which adheres and later penetrates inside
the egg shell. The mycelia proliferates inside the egg and parasitizes completely
(Esteves et al. 2009). Secretion of serine proteases (Segers et al. 1994), VCP1
protease (Segers et al. 1996), chitinase, collagenase (Huang et al. 2010),
Fig. 26.4 (a) Hyphae of P. chlamydosporia (verticillate nature). (b) Chlamydospores

Table 26.5 Some commercial formulations of Pochonia chlamydosporia

Biocontrol fungi/
strain Trade name Company/Country References
P. chlamydosporia KlamiC® Cuba Fernández-Larrea (2012),
var. catenulate Davies and Spiegel (2011)
P. chlamydosporia Xianchongbike China Mo et al. (2005), Davies and
Spiegel (2011)
P. chlamydosporia Green Nemafree Greenlife Biotech,
IIHR Vc-3 India
P. chlamydosporia NEMATOFREE M/s International
IIHR Vc-3 + Panaacea, India
P. chlamydosporia Brahma Putra Allwin Industries,
IIHR Vc-3 India
phomalactones (Viaene et al. 2006), and subtilisins (Segers et al. 1999) helps in
hydrolyzing the egg shell and enhancing the pathogenicity.
P. chlamydosporia formulations are successfully commercialized in Europe,
America, Africa, and India. Oil emulsion formulation containing mycelium, conidia,
and chlamydospores of P. chlamydosporia IPP-21 was commercialized in Italy for
some years with registration-free permission (Manzanilla-Lopez et al. 2013). Kerry
(1988) developed granular formulations and was successful in growing fungal
hyphae of P. chlamydosporia on alginate granules. Some of the commercial
formulations of P. chlamydosporia available in global and Indian market are
furnished in Table 26.5.
26.4.2.2 Biocontrol Potential of P. chlamydosporia in Managing

Nematodes of Horticultural Crops
Dhawan and Singh (2010) observed 96% parasitization of M. incognita eggs by
P. chlamydosporia under in vitro. Soil application of P. chlamydosporia effectively
reduced root knot nematode population in okra (Chaya and Rao 2012), brinjal (Rao
et al. 2003), and tuberose (Rao et al. 2004) and increased the crop yields.
26.4.3 Trichoderma spp.
Many species of Trichoderma like T. harzianum, T. viride, T. longibrachiatum,

T. koningii, T. pseudokoningii, and T. virens have been reported to be promising
BCAs of several plant parasitic nematodes and associated disease complex (Khan
et al. 1997; Sankaranarayanan et al. 1998; Nagesh et al. 2001).
26.4.3.1 Mode of Action of Trichoderma spp.

T. harzianum is a potential parasite of root-knot, cyst, and other plant parasitic
nematodes (Dos-Santos et al. 1992; Askary 2008). It produces several antibiotics
and toxins that protect the plant from nematodes (Di-Pietro 1995; Santosh et al.
2005). It effectively colonizes the plant roots and acts as a physical barrier
preventing nematode invasion (Inbar et al. 1994).
The hyphae of Trichoderma penetrate eggs and larvae of phytonematodes, and
activity of enzymes like chitinase, protease, and glucanase helps in parasitizing the
host (Haran et al. 1996; Haggag and Amin 2001). Rajinikanth et al. (2016) proved
that chi18-5 gene of T. viride played a key role in the parasitization of the
nematode eggs.
Induction of systemic resistance in host plants due to enhanced activity of defense
enzymes like peroxidase, polyphenol oxidase, catalase, phenylalanine ammonia
lyase, and chitinase in T. viride-treated green gram facilitated the host to resist
M. incognita invasion (Umamaheswari et al. 2004). Enhanced nutrient solubilization
and uptake in T. harzianum-treated plants increase the tolerance of the host plants to
pathogen attack (Altomare et al. 1999).
26.4.3.2 Commercialization of Trichoderma spp.

Several commercial formulations of Trichoderma spp. are available in the global and
Indian markets (Table 26.6).
However, based on the oligonucleotide barcodes and morphological characters,
Sriram et al. (2013) reported that T. viride widely used in India is T. asperellum.
26.4.3.3 Biocontrol Potential of Trichoderma spp. Against Plant

Parasitic Nematodes in Horticultural Crops
Trichoderma spp. are reported to exhibit biocontrol activity against a diverse group
of plant parasitic nematodes (Eapen and Venugopal 1995). Grace et al. (2019)
reported that culture filtrates of T. harzianum recorded 94.53% J2 mortality of
M. incognita under in vitro and soil application of vermicompost (2 tons/ha)
enriched in liquid formulation of T. harzianum (5 l/ha) that exhibited maximum
increase in yield and minimum population of M. incognita in tomato. Rao et al.
(2003) reported that combined application of T. harzianum and P. lilacinum in
papaya nursery recorded minimum root-knot index of M. incognita and highly
vigorous seedlings of papaya.
26.4.4 Arbuscular Mycorrhizal (AM) Fungi
AM fungi are obligate root symbionts that protect the host plants from abiotic and
biotic stress including plant parasitic nematodes (Vos et al. 2012). Baltruschat et al.
(1973) were the first to show that the plants preinoculated with AM fungi, Glomus
mosseae, were less susceptible to root knot infection.
26.4.4.1 Mode of Action of AM Fungi Against Phytonematodes

As AM fungi and plant parasitic nematodes occupy the same ecological niche in the
roots, there is a direct competition for space and nutrients (Hussey and Roncadori
1982). Higher nutrient uptake also results in increased root growth and branching,
which could counterbalance the suppression of root growth by plant parasitic
Table 26.6 Some commercial formulations of Trichoderma spp

Biocontrol
fungi/strain Trade name Company Country References
T. lignorum Mycolab Laboratorios Laverlam Columbia Woo et al.
(2014)
T. harzianum Trichobiol Control Biologico Integrado; Columbia Woo et al.
Mora Jaramillo Arturo Orlando- (2014)
Biocontrol
T. viride Trifesol BioCultivos S.A., Bogota Columbia Woo et al.
strain 2684 (2014)
T. harzianum Commander HTC Impex Private Limited India Biro-Stingli
Fungicide and Toth
(2011)
T. harzianum Ecosom-TH Agri Life India Woo et al.
SOM Phytopharma (2014)
(India) Limited
T. viride Ecosom-TV Agri Life India Woo et al.
SOM Phytopharma (2014)
(India) Limited
T. viride ANOKA K N Bio Sciences India Woo et al.
Private Limited, (2014)
Ranga Reddi
T. viride IIHR Bhoomika- Varsha Biosciences, Telangana India
Tv-5 1% W.P.
T. harzianum Hariz-1.15% Varsha Biosciences, Telangana India
IIHR Th-2 W.P.
T. viride IIHR Monitor Agriland Biotech Limited, India
Tv-5 Gujarat
T. viride Nicoderma Nico Organics, Gujarat India
IIHRTv-5
T. harzianum T-harz Nico Organics, Gujarat India
IIHR Th-2
T. harzianum Safe root Multiplex, Bangalore India
IIHR Th-2
T. viride Boom Devi Biotech Pvt. Ltd, Tamil India
IIHRTv-5 Derma Nadu
nematodes (Elsen et al. 2003). Colonization of AM fungi also alters the composition
of root exudates, which could hinder the host finding ability, hatching, and motility
of plant parasitic nematodes (Schouteden et al. 2015).
26.4.4.2 Biocontrol Potential of AM Fungi Against Plant Parasitic

Nematodes
Application of G. mosseae with neem cake effectively reduced Radopholus similis
population in banana (Reddy et al. 1997). G. mosseae and G. manihotis reduced root
knot nematode population in papaya (Jaizme-Vega et al. 2006), and G. mosseae and
G. margarita reduced the gall index in tomato (Siddiqui and Akhtar 2007).
Combined application of G. fasciculatum and Gigaspora sp. reduced M. arenaria

population on potato and increased the plant growth parameters under field
conditions (Abd-El-Khair and El-Nagdi 2014).
26.4.5 Bacterial Bioagents for Nematode Control
Bacteria are abundantly present in soil, and there is always a possibility for
nematodes to be infected by some of these bacteria. Members of the genera
Pasteuria, Pseudomonas, and Bacillus have received much attention owing to
their excellent biocontrol potential (Tian et al. 2007). Based on the mechanism of
action, they are classified as obligate parasitic bacteria, antagonistic bacteria,
rhizobacteria, and Cry protein-forming bacteria.
26.4.5.1 Obligate Parasitic Bacteria, Pasteuria spp.

Pasteuria species are Gram-positive, dichotomously branched, obligate, endospore-
forming bacteria with septate mycelium that parasitizes plant-parasitic nematodes
(Sayre and Starr 1985; Bekal et al. 2001). P. penetrans parasitizes root knot
nematodes, P. thornei parasitizes lesion nematodes, and P. nishizawae parasitizes
cyst nematodes (Gives et al. 1999; Atibalentja et al. 2000).
When juvenile nematodes move in the soil, spores of P. penetrans gets attached
to their cuticle, which further penetrates the cuticle and proliferates inside the
developing nematode stages. Bacterial spores fill the host body completely and
halt nematode reproduction (Sayre and Wergin 1977).
Pasteuria spores can be stored in air-dried soil and roots for the extended periods
without apparent loss of infectivity (Mankau and Prasad 1977; Stirling and Wachtel
1980). However, its obligate nature, lack of mobility, and dependence on the
dispersal agent throw a major setback for its large scale commercialization. Nagesh
and Reddy (1998) and Swarnakumari et al. (2016) modified the methods of mass
production of Pasteuria penetrans under in vivo and glass house conditions and
achieved higher recovery of bacterial spores.
26.4.5.2 Antagonistic Bacteria

Soil-borne bacteria are known to produce diverse metabolic products during decom-
position of organic matter, and some of them accumulate in soils and influence
nematodes in various ways including antagonism to plant parasitic nematodes. Soil
bacteria like Clostridium butyricum, Desulfovibrio desulfuricans, Chromobacterium
sp., and Bacillus thuringiensis var. thuringiensis have been demonstrated to produce
butyric acid, hydrogen sulfide, cyanide, and exotoxins that are antagonistic to
nematodes (Hollis and Rodriguez-Kabana 1966; Prasad and Tilak 1972).
26.4.5.3 Rhizobacteria
Rhizobacteria are well studied for their biocontrol potential against plant parasitic
nematodes (Sikora 1992). Bacillus spp. and Pseudomonas spp. are the main domi-
nant rhizobacterial populations in soil that antagonize the phytonematode population
(Krebs et al. 1998). Several other rhizobacteria are also reported to exhibit antago-
nism against phytonematodes, as reviewed by Tian et al. (2007).
Bacillus spp.
Members of Bacillus group are often called as ‘microbial factories’ for their capa-
bility to produce a spectrum of biologically active molecules that are antagonistic to
diverse group of phytopathogens (Ongena and Jacques 2008). Bacillus spp. form
endospores that could withstand unfavorable conditions.
B. subtilis, the most studied rhizobacterium in this group, produces several
antimicrobial compounds (Gray et al. 2006). Kavitha et al. (2012) reported
B. subtilis strains producing antibiotics, surfactin, and iturin that suppressed egg
hatching and caused mortality of M. Incognita juveniles. Prabu et al. (2019) detected
several antibiotic genes, viz., iturin-A, iturin-C, iturin-D, bacilycin BacD, bacilycin
BacAB, surfactin, and nematicidal purL gene detected in the genome of
B. amyloliquefaciens IIHR Ba-2, and the crude antibiotic extract showed signifi-
cantly higher J2 mortality and greater suppression in nematode egg hatching of
M. incognita (Fig. 26.5).
Pseudomonas spp.
Pseudomonas spp. are omnipresent bacteria in agricultural soils, which possess
many desirable qualities that make them excellent candidates for commercial exploi-
tation. Pseudomonas fluorescens, the most studied bacterium of this group, report-
edly increased plant growth and reduced nematode damage caused by several key
nematode pests, viz., Globodera rostochiensis, Heterodera spp., Meloidogyne
incognita, Tylenchulus semipenetrans, Radopholus similis, Pratylenchus coffeae,
and Helicotylenchus multicinctus (Cronin et al. 1997; Oostendorp and Sikora 1989;
Fig. 26.5 Effect of cell-free culture filtrates of B. amyloliquefaciens IIHR BA2 on M. incognita
eggs: (a) Eggs in untreated control and (b) Distortion of cell contents in eggs treated with culture
filtrates (Prabu et al. 2019)
Santhi and Sivakumar 1995). Production of toxic metabolites and nematicidal

components was reported to reduce hatching and invasion of nematodes (Becker
et al. 1988).
26.4.5.4 Commercial Rhizobacterial Products as Bionematicides

Many rhizobacterial bionematicide products are available in the global market.
Burkholderia cepacia, commercially available as Deny, is effective against
phytonematodes (Meyer and Roberts 2002). In U.S.A., a commercial combination
product of Paenibacillus macerans and Bacillus amyloliquefaciens (Bio YieldTM
and Gustafson LLC) is developed as transplant mix to control nematodes in bell
pepper, strawberry, and tomato (Meyer 2003). BioNem, a commercial formulation
of Bacillus firmus, is used in Israel to manage root knot nematodes (Giannakou and
Prophetou-Athanasiadou 2004). Many commercial formulations of P. fluorescens
are available in India like Ecomonas, Biocure-B, Sudolin, Monas, and BioTreat.
26.4.5.5 Cry protein-forming Bacteria

Bacillus thuringiensis Cry6Aa and Cry55Aa toxins exhibited synergistic activity
against M. incognita (Peng et al. 2011).Chahal and Chahal (1991) reported drastic
inhibition in hatching of egg masses and death of second stage juveniles of
M. incognita by B. thuringiensis. Ignoffo and Dropkin (1977) reported toxic effect
of B. thuringiensis on Meloidogyne sp., Panagrellus sp., and Aphelenchus sp.
26.4.5.6 Other Soil Bacteria

Many other soil bacteria are also reported to produce nematicidal compounds.
Actinobacteria, Streptomyces avermitilis, produce avermectins, which are macrocy-
clic lactones and possess potent broad spectrum anthelmintic, insecticidal, and
nematicidal activities (Nair et al. 1995). Seed treatment of okra with avermectin
was effective against reniform nematode, Rotylenchulus reniformis (Jayakumar et al.
2005). Soil application of avermectin, Abamectin B1 commercialized under the
name Vertimec®, caused significant reduction in penetration of M. arenaria into
tomato roots (Cayrol et al. 1993).
26.4.5.7 Field Application of Microbials for Nematode Management

Strategic application of bionematicides as seed treatment, substrate treatment, or
nursery bed treatment and soil application in main fields after enrichment in compost
like Farm Yard Manure or vermicompost or deoiled neem cake are reported to
effectively manage nematodes in several horticultural crops. Farmers could fetch
20–25% increase in crop yield coupled with 82 to 90% nematode control in banana,
tomato, okra, chilies, capsicum, cucumber, gerbera, and carnations, both under open
fields and protected cultivation (Rao et al. 2015a, b, c, 2017).
26.5 Challenges Facing Commercialization of Microbial

Biopesticides in India
There are several challenges to commercialization of microbial biopesticides in

India. Researchers, policymakers, funding bodies, industries, and marketing
agencies should join hands together to address these issues.
(i). Gap in awareness and information among the end users
The lack of awareness and knowledge about the microbial biopesticides and their
proper usage among the farmers is one of the major challenges facing the successful
use of biopesticides and its commercialization. Though the microbial biopesticide
packages carry detailed instructions regarding their storage and usage, farmers are
often not clear about their appropriate application methodologies. Sometimes, the
farmers lack the necessary skills for using these biopesticides in their farms.
(ii). Inconsistent field performance
Lack of stability in the efficacy of microbial biopesticides is also a reason for their
low reliability among the farmers. Since living microorganisms are the active
ingredients in these formulations, they are vulnerable to the vagaries of several
abiotic factors like temperature, moisture, pH, exposure to UV rays, and other soil
factors. It is also a popular practice among the farmers to mix the chemical pesticides
with biopesticides and apply them as a single spray, which is highly toxic to the
microbes and reduces the cell count. Lack of knowledge on the compatibility of
biopesticides with other agrochemicals also reduces the field performance of
biopesticides.
(iii). Poor shelf life and lack of quality in formulations.
Contamination and poor cell count are the major concern of the farmers in
commercial formulations. Sterile conditions have to be maintained to avoid contam-
ination, which is often difficult for a longer period. Contamination reduces the
population of useful microbes in the formulation and reduces its shelf life. This
results in poor quality of the products that perform inconsistently under field
conditions.
(iv). Inadequate quality check in commercial formulations
Nonavailability of good quality biopesticide products with sufficient microbial

cell count (cfu) is the major reason for their inadequate field performance and low
popularity among the farmers. The standards of the biopesticide formulations should
be met strictly, and government agencies should ensure the availability of quality
biopesticides in the markets. Stringent action should be taken on the scale of low
quality or spurious products available in the commercial market.
(v). Huge investment and lack of profit
Biopesticide industries have to invest a huge capital for selection of efficient

microbial strains, mass production, formulation, packaging, and storage and mainte-
nance of sterile conditions in all these stages. Generation of data on toxicology,
bioefficacy in different agroclimatic zones, and registration with CIB&RC also
needs a huge investment. Besides, this also involves high risks and less profit. All
these factors make the production of biopesticides a costly business, and hence,
companies come forward for their production only if there is a long-term profit.
(vi). Lack of guidelines for consortia formulations in India
In India, microbial biopesticides based on single microbial biocontrol agent are

legally certified for commercial production. However, the single biocontrol agent-
based biopesticides may not be effective in all kinds of soil environment and
agricultural ecosystems. This can be overcome by the combination of bioagents,
which can thrive in diverse environments and act on diverse pathogens through
multiple modes of action. Biopesticides containing combination of bioagents have
been approved in developed countries like USA for commercial production and use
in several agri-horticultural crops. Since these combination formulations have tre-
mendous potential in revolutionizing the entire plant protection system, there is an
urgent need for the registration of microbial consortium in developing countries like
India.
In India, the guidelines for registration and the minimum infrastructure to be
created by the manufacturers of entomotoxic bacteria, antagonistic bacteria,
entomopathogenic fungi, antagonistic fungi, and baculoviruses are formulated by
Central Insecticide Board and Registration Committee, Directorate of Plant Protec-
tion, Quarantine & Storage, Department of Agriculture, Cooperation & Farmers
Welfare under Ministry of Agriculture & Farmers Welfare and updated in their
website http://ppqs.gov.in/; http://ppqs.gov.in/divisions/cib-rc/guidelines?page¼1.
26.6 Conclusion
Microbial biopesticides possess great potential in sustaining crop and soil health
through multiple mechanisms. In spite of their overwhelming advantages, inconsis-
tent performance of applied microbes has proven to be a major obstacle mainly due
to lack of effective strains and quality products. One of the strategies to enhance the
efficacy is to combine two (or more) beneficial microbes in the formulation. Such
combinations have beneficial traits for more effective colonization of the rhizo-
sphere, more consistent expression under a diverse soil conditions, and antagonism
to a broad spectrum of plant pests or pathogens through different modes of action
than strains applied individually. Integration of biopesticide components with other
management options and interdisciplinary teamwork will also lead to more effective
control practices. Future research efforts in the line of nanotechnology-based
microencapsulation of microbes could increase their field efficacy and residual

action. Government should boost the start-ups in the form of loans and subsidies
to promote biopesticide industries for large scale production.
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Commercial Aspects of Biofertilizers
and Biostimulants Development Utilizing 27
Rhizosphere Microbes: Global and Indian
Scenario
A. John Peter, E. Leo Daniel Amalraj, and Venkateswara Rao Talluri
Abstract
The beneficial microbiomes associated with rhizosphere provide innumerable

benefits to plants and the environment. They collectively associate for
maintaining the health and nutrient dynamics of soil, sequestering/mobilizing
nutrients to plants, improving the plant growth, and defending them from biotic
and abiotic stress. Due to the modern cultivation practices and climate change,
sustenance and efficacy of beneficial soil microbiome have been deteriorated over
the years. It is, therefore essential to identify, mass multiply, and recharge
rhizosphere with the most effective and multifunctional beneficial microbiome.
Researchers have documented a sizeable number of plant beneficial
microbiomes for the past six decades. Producers have started manufacturing
and commercializing select microbiome successfully worldwide. Rules,
regulations, and nomenclature for commercializing them have differed from
country to country. Guidelines have been proposed for the use of beneficial
microbiome that is not covered under plant protection code. They were, however,
not strictly implemented in several countries due to their safety and green nature.
As the global biostimulant and biofertilizer market is expected to reach US$ 4.9
billion by the year 2025 with an annual growth rate of 11.46%, global regulatory
mechanisms for biostimulants and biofertilizers have become active since the
year 2018. The overall commercial potential of biostimulants and biofertilizers is
high in Europe, followed by North America, Asia Pacific, and Latin American
countries.
India and China have classified nutrient fixing/solubilizing/mobilizing
rhizobacteria as biofertilizers. Recently, regulatory guidelines are being framed
for commercializing biostimulants including beneficial microbiome. In this book
A. J. Peter · E. L. D. Amalraj · V. R. Talluri (*)

TNA Innovation Centre, Varsha Bioscience and Technology India Private Limited, Hyderabad,
Telangana, India

23, https://doi.org/10.1007/978-981-15-9154-9_27
656 A. J. Peter et al.
chapter, we have highlighted the rhizosphere beneficial microbe’s importance,

benefits, nomenclature, commercial potential, and regulations and the importance
for uniform global guidelines for product registration. The importance of
integrating biofertilizers with conventional fertilizers, introducing new microbial
consortia formulations, economics, and bottlenecks was also discussed.
Keywords
Microbiome · Biostimulants · Biofertilizers · Bioinoculants · Commercial ·
Rhizosphere · PGPR · Regulatory guidelines · Challenges · Biopesticides · FCO
1985 · Sustainable agriculture
27.1 Introduction
Rhizosphere microbiome consists of beneficial, harmful, and neutral

microorganisms present in and around the root zone of plants. Their profound
beneficial effects, viz., facilitating seed germination (Nelson et al. 2018), improving
vigor (Basra et al. 2005; Raja et al. 2017), root development and nutrient mobiliza-
tion (Wani et al. 2015), phytohormone production (Kloepper and Schroth 1978),
tolerance to abiotic stresses (Yang et al. 2009) and deleterious pathogens (Van Loon
et al. 1998; Mhatre et al. 2019), flowering (Lu et al. 2018), and overall growth and
development of plant (Khalid et al. 2004; Maiyappan et al. 2010), on plants are well
documented.
The introduction of modern cultivation practices, conventional pesticides, and
fertilizers has led to innumerable threats to the rhizosphere microbiome. They
drastically altered the soil microbial biodiversity, including a decrease in beneficial
plant probiotics population and their metabolic footprints in soil. Subsequently,
these changes have significantly affected soil organic carbon content, metagenomics,
pH, structure, respiration, enzyme/extracellular metabolite dynamics, and reduced
nutrient mobilization. The dynamic environmental changes have also adversely
affected the beneficial microbial population (Prashar and Shah 2016; Kumar and
Dubey 2020). Plants/crops have thus started showing less response to conventional
fertilizers with more nutrient deficiency symptoms and have become susceptible to
other biotic and abiotic stresses. Therefore, it has become necessary to revitalize the
soil by replenishing it with selected plant growth promoting and beneficial microbial
flora. Academicians and industries have identified cultivable rhizosphere
microbiome as plant probiotics. Ever-increasing plant probiotic requirements
brought out several commercially successful formulations. These formulations are
functionally successful due to their ability to improve crop production and protec-
tion, nutritional quality of the harvested produce, soil health, and reduced chemical
inputs along with degradation of toxic pesticide residues.
Commercially available plant beneficial rhizospheric microbiome is broadly
categorized as biopesticides, biofertilizers, and plant stimulants. Regulatory
guidelines have become stringent where beneficial microbes are used for plant
27 Commercial Aspects of Biofertilizers and Biostimulants Development Utilizing. . . 657
protection purposes. Microbes used for the purposes other than plant protection are
commercialized as biofertilizers/soil conditioners/biostimulants based on the respec-
tive country’s regulatory guidelines. For example, all the beneficial microbes that are
fixing/dissolving/mobilizing nutrients are included under Indian Fertilizer Control
Order, 1985 (FCO 1985) as biofertilizers in India. There is a very narrow functional
difference between biofertilizers and biostimulants. Nomenclature differs from
country to country as stimulants, biofertilizers, and/or soil conditioners. However,
they are becoming the inevitable tools for the next-generation promoters of sustain-
able agriculture.
Voluminous scientific research and field demonstrations in Europe, Asia Pacific,
America, Africa, and Middle East countries have confirmed that microbial-based
biofertilizers/biostimulants/soil conditioners are superior to conventional chemical-
based nutrients and stimulants. Several countries are encouraging farmers to
switchover or integrate these formulations with the existing practices. They also
assist biofertilizer manufacturers in meeting their domestic requirements.
The market growth of biofertilizers in the Asia Pacific is estimated at 10.8%
CAGR during 2020–2025 (Mordor Intelligence 2020) against the global market
growth estimation of 11.3% CAGR during 2019–2025 (Fortune Business Insights
2019). Among the Asia-Pacific countries, China is leading by occupying about
43.2% biofertilizer market share (Mordor Intelligence 2020). It promoted a pilot
plan to replace chemical fertilizers with biological formulations/organic fertilizers in
100 districts in China. The Government of India also promotes biofertilizers through
its various schemes like mission for sustainable agriculture, oilseeds, oil palm, food
security, and Krishi Vikas Yojanas. However, there should be a national mission
program to replace/integrate chemical fertilizers with biofertilizers intensively from
2020–2025 to reduce the burden of fertilizer subsidy and to increase the soil health
and crop productivity. India and several other countries should also formulate
simplified guidelines to regulate biostimulants and pave the ways for promoting
them among the farming communities.
This book chapter unfolds the commercial prospects of biofertilizers and
biostimulants, their importance to modern agriculture, recent advances, regulatory
framework, and their advantages over conventional fertilizers and plant stimulants.
27.2 Biostimulants
Biostimulants are the substances used in minute quantities to promote plant growth.
They are differentiated from nutrients and other soil amendments in terms of
quantities used for overall plant development. Biostimulants are not fertilizers as
they do not provide any nutrients directly to the plant. Their origin could be from
plants, animals, microbes, natural minerals, plant hormone chemicals and humus/
organic-rich soil derivatives.
Plant beneficial microbes present in and around the rhizosphere play a key role in
plant growth, development, and reproduction by interacting either directly or indi-
rectly regardless of the nutrient status of the plant (Behie and Bidochka 2014; Le
Mire et al. 2016). These associations extend from soil to interior of the plant cells
influencing the plant growth through nutrient recycling & mobilization, disease
resistance induction, abiotic stress tolerance, and fine tuning of other plant growth
regulating mechanisms (Ahmad et al. 2008). Microbial biostimulants, when applied,
can improve nutrient uptake efficiency by 5–25% and increase crop yield up to 10%
(European Biostimulants Industry Consortium 2011). They include bacterial
endosymbionts, plant growth-promoting rhizobacteria, and mycorrhizal/
nonmycorrhizal fungi. Few countries term some of the biostimulants as either
biofertilizers or soil conditioners.
27.3 Rhizospheric Bacteria
Rhizospheric bacteria modulate soil microenvironment around roots and enhance

nutrient availability as well as uptake by participating in biogeochemical cycles.
Based on their functionality and ecological diversity, they are broadly categorized
into (a) mutualistic endosymbionts and (b) mutualistic plant growth-promoting
rhizobacteria (PGPR). For example, Rhizobium resides in the root cells of legume
plants and helps in the fixation of nitrogen, while Azospirillum is multifunctional like
any other PGPR and exerts influence on the entire life cycle of the plants (Patrick du
Jardin 2015). PGPR-based biostimulants have long been commercially produced
and applied to various field crops (Köhl 2010; Pérez-Montaño et al. 2013) and to
horticulture crops (Reddy et al. 1999). Few of the PGPRs exhibit biocontrol activity
and protect plants from abiotic stresses (Bhattacharyya and Jha 2012).
PGPRs stimulate plant growth by producing plant growth regulators, including
various phytohormones viz., auxins, cytokinins, gibberellins, and ethylene (Calvo
et al. 2014). In addition, they can also increase the nutrient availability for the host
plants by nitrogen fixation, nutrient solubilization, production of volatile organic
plant growth promoting compounds, and iron sequestration (Bhattacharyya and Jha
2012; Calvo et al. 2014). They can also enhance crop tolerance to salinity and
drought (Upadhyay and Singh 2015). PGPRs can be used alone or in combination
with bacterial endosymbionts as microbial consortia to achieve perfect biostimulant
activity in plants. Microbial consortia exhibited a better biostimulant activity than a
single strain due to their increased genetic diversity, leading to better rhizospheric
colonization (Reddy et al. 1999). Some of the PGPR-based biostimulants that
enhance crop yield are given in Table 27.1 (Le Mire et al. 2016).
The efficiency of PGPR is dependent on several factors including plant species,
nature of soil, ecological conditions, and type of commercial formulations. With the
increasing scientific evidence on the advantages of PGPRs to the field crops, many
PGPR inoculants are being used as biofertilizers in traditional small-scale farms and
commercial agriculture systems throughout the world. Several leading
manufacturers across the globe commercialized brands having consortia and sole
organism formulations. A list of commercially utilized PGPR’s in Europe, North
America, and Asia is given in Table 27.2.
Table 27.1 Response of PGPR-based Biostimulants/Biofertilizers under field and green house
conditions for reducing chemical fertilizers
Crop growth
enhancement and
Field/ chemical fertilizer
Biostimulant Crops Greenhouse reduction level Reference
Burkholderia Rice Field Increased weight and Araújo et al.
vietnamiensis yield of traditional rice (2013)
AR112 compared to 100% N
chemical fertilization
K-Solubilizing isolate Wheat Greenhouse Recorded 51.46% Parmar and
HWP47 increase in root dry Sindhu
weight (2013)
Pseudomonas Wheat Greenhouse Improved N and P Islam et al.
aeruginosa uptake. Increased (2014)
chlorophyll content and
plant biomass under Zn
stress
Arthrobacter sp. and Wheat Greenhouse Increased tolerance to Upadhyay
Bacillus subtilis salinity and Singh
(2015)
Pseudomonas jessenii, Wheat Field Combining PGPR and Mäder et al.
Pseudomonas AMF increased the yield (2011)
synxantha, and a local up to 41%
AM
B. megaterium M3 & Wheat, Field and Single and combinations Çakmakçi
RC 07, Bacillus Barley Greenhouse of PGPR increased yield et al. (2014)
OSU-142, A.brasilense up to 40.4% in wheat
sp. 245, Paenibacillus and 33.7% in Barley
polymyxa RC05, when integrated with
B. licheniformis RC08, Nfertilizer
Raoultella terrigena,
and Burkholderia
cepacia
Rhizobium tropici Bean Greenhouse Increase of P (40%), N Tajini et al.
CIAT899 and Glomus (42%), nodule number (2012)
intraradices (70%), nodule mass
(43%), shoot dry weight
(24%), and root growth
(48%)
Bradyrhizobium sp. and Soybean Field and Increased grain yield by Marks et al.
concentrated Greenhouse 4.8% compared to the (2013)
metabolites from exclusive use of
Bradyrhizobium Bradyrhizobium
diazoefficiens sp. alone
Bacillus Tomato Greenhouse Combination of PGPR Adesemoye
amyloliquefaciens and AM reduced et al. (2009)
IN937a, Bacillus fertilizer use by 25%
pumilusT4, and Glomus without altering plant
intraradices growth, yield, and
nutrient uptake
(continued)

Crop growth
enhancement and
Field/ chemical fertilizer
Biostimulant Crops Greenhouse reduction level Reference
Azospirillum brasilense, Tea Field Combination of PGPR Babu (2020)
Bacillus megaterium, with 75% recommended
Frateuria aurantia, and fertilizers increased 8%
Pseudomonas sp. leaf yield. Improved the
quality of tea leaf
27.4 Rhizospheric Fungi
The coevolution of rhizospheric fungi with terrestrial plants led to the development
of various interactions with plant roots, including mutualistic symbioses and para-
sitism (Behie and Bidochka 2014). Beneficial rhizosphere fungi, viz., mycorrhiza,
Trichoderma, Purpureocillium lilacinum (Paecilomyces lilacinus), Beauveria
bassiana, Metarhizium anisopliae, Pochonica chlamydosporia (Verticillium
chlamydosporium) etc., are some of the commercially exploited microbes obtained
from the rhizosphere. Among the listed microbes, except mycorrhiza, rest of the
microbes are registered as biopesticides.
Arbuscular Mycorrhizal Fungi (AMF) readily establish symbiotic interactions
with the roots of majority of plant species and promote nutrition efficiency, water
balance, and protection against biotic and abiotic stresses. It is one of the most
important rhizospheric fungi, duly classified as biofertilizer/biostimulant. Commer-
cially, it is mass produced through the substrate-based production system,
aeroponics, and agrobacteria-mediated root culture techniques. Table 27.3 illustrates
the commercially exploited arbuscular mycorrhizal species through various culture
methods. However, Glomus sp. is predominantly used in India.
Some of the fungal endophytes like Trichoderma sp. also colonize plant roots and
help in nutrient transfer to the host. Trichoderma sp. produce toxic compounds
including viridin, peptaibols, gliotoxins, sesquiterpenes, and isonitriles, which
inhibit growth of other competitors. Trichoderma sp. act as mycoparasite through
the degradation of pathogen cell walls. They affect a wide range of pathogens that
include various genera, viz., Phytophthora, Colletotrichum, Fusarium, Botrytis,
Pythium, Rhizopus, Verticillium, Rhizoctonia, Sclerotium, Dematophora,
Fusicladium, and Helminthosporium. Rhizospheric fungi exhibit antagonism against
pathogenic fungi and promote plant growth through five mechanisms that include
the production of inhibitory compounds, inactivation of pathogen enzymes,
mycoparasitism, induced resistance, and competition for nutrients and space (Patrick
du Jardin 2015; Drobek et al. 2019).
Recently, the role of Purpureocillium lilacinum (Paecilomyces lilacinus) and
Pochonica chlamydosporia (Verticillium chlamydosporium) for the plant-parasitic
nematode control gained importance due to the ban of selected pesticide
Table 27.2 Commercialized and registered PGPR-based products in Europe, North America,
and Asia
Manufacturers Product name Active ingredient Crops
ABiTEP GmbH, FZB24®fl B. amyloliquefaciens Ornamentals and
Germany vegetables
Rhizovital B. amyloliquefaciens Field crops
42®
Agrinos, India iNvigorate® Azotobacter vinelandii and All plants
Clostridium pasteurianum
AGRO.bio BactoFil Azospirillum brasilense, Cereals
Hungary Kft., A10® A. vinelandii, B. megaterium,
Hungary B. polymyxa, and P. fluorescens
BactoFil Azospirillum lipoferum, Sunflower, potato,
B10® A. vinelandii, B. megaterium, and rapeseed
B. circulans, B. subtilis, and
P. fluorescens
Biovitis, France Cérès® Pseudomonas fluorescens Field &
horticultural crops
CCS Aosta Srl, Micosat F® B. subtilis BR 62, Paenibacillus Cereals, tomatoes,
Italy Cereali durus PD76, and Streptomyces sunflowers, beet,
sp. ST 60 and soybeans
Micosat F® Agrobacterium radiobacter AR Fruits, vegetables,
Uno 39, B. subtilis BA 41, and and flowers
Streptomyces spp. SB 14
Cleveland Biotech, AmniteA100® Azotobacter, Bacillus, Cucumber, lettuce,
UK Pseudomonas, Rhizobium, and tomato, and pepper
Chaetomium
Greenmax Gmax® PGPR Azotobacter, Phosphobacteria, Field crops
AgroTech, India and P. fluorescens
IAB (Iabiotec), Inómix® B. megaterium, Saccharomyces Cereals
Spain Biofertilisant cerevisiae, Azotobacter
vinelandii, and R.leguminosarum
Inómix® B. polymyxa (IAB/BP/01) and Cereals
Biostimulant B. subtilis (IAB/BS/F1)
Inómix® P. fluorescens, B. megaterium, Cereals
phosphore and Saccharomyces cerevisiae
Koppert Biological Panoramix Mycorrhiza, Trichoderma sp., Seed dresser
Systems and Bacillus sp.
Lallemand Plant Rhizocell ® B. amyloliquefaciens IT45 Cereals
Care, Canada GC
Lantmannen Amase® Pseudomonas azotoformans Cucumber, lettuce,
Bioagri Sweden tomato, and pepper
Mapleton Nitroguard® Azospirillum brasilense Cereals, seed rape,
AgriBiotec Pvt. NAB317, Azorhizobium sugar beet,
Ltd., Australia caulinodens NAB38, Azoarcus sugarcane, and
indigens NAB04, and Bacillus vegetables
sp.
TwinN® Azospirillum brasilense Cereals, seed rape,
NAB317, Azorhizobium sugar beet,
(continued)

caulinodens NAB38, and sugarcane, and
A. indigens NAB04 vegetables
Organica PGA® Bacillus sp. Fruits and
Technologies, vegetables
USA
Plant Health Care, Compete ® B. azotofixans, B. licheniformis, Field crops, tree
USA Plus B. megaterium, B. polymyxa, and nurseries
B. pumilus, and B. subtilis
Som Phytopharm AgriLife Paenibacillus azotofixans Field Crops,
India Limited, Nitrofix® Vegetables, and
India. Fruit crops
AgriVAM® Piriformospora indica Field Crops,
Vegetables, and
Fruit crops
K Sol B® Bacillus mucilaginous Field Crops,
Vegetables, and
Fruit crops
Mycorrhiza® Rhizophagus irregularis Field Crops,
Vegetables, and
Fruit crops
P Sol B® Bacillus megaterium Field Crops,
Vegetables, and
Fruit crops
RootamBio ® Paenibacillus azotofixans Field Crops,
Bacillus megaterium Vegetables, and
Bacillus mucilaginous Fruit crops
Bacillus mycoides
Si SOL B® Bacillus mycoides Field Crops,
Vegetables, and
Fruit crops
T. Stanes & Symbion®-K Frateuria aurantia Field crops and
Company Ltd., vegetables
India Symbion®-N Azospirillum, Rhizobium, Field crops and
Acetobacter, and Azotobacter vegetables
Symbion®-P B. megaterium var. Field crops and
phosphaticum vegetables
Valagro Heiko root Bacillus simplex VMC 30/29 Nursery,
Bioscience Private Vegetables, and
Limited, India Big plants
Heiko seed Bacillus megaterium VMC All crops
30/196
Varsha Bioscience Angkor Nature Azospirillum lipoferum, Bacillus Field crops,
and Technology megaterium var. phosphaticum, Vegetables, and
India Pvt. Ltd., and Frateuria aurantia fruit crops
India Omega® Bacillus sp., Streptomyces sp., Field crops and
Frateuria aurantia, and Vegetables
Azotobacter
(continued)

Rhizodyne® Azospirillum brasilense, Bacillus Field crops,
megaterium, Frateuria aurantia, Vegetables, and
and Pseudomonas sp. fruit crops
Primary source Le Mire et al. (2016); upgraded further until 2020
Table 27.3 Diversity of commercially exploited mycorrhizal species

Name of the arbuscular mycorrhizae Formerly known as
Acaulospora kentinensis and A. morrowiae –
Claroideoglomus claroideum Glomus claroideum
Claroideoglomus etunicatum Glomus etunicatum
Diversispora spurca –
Dentiscutata heterogama Scutellospora heterogama
Funneliformis coronatum Glomus coronatum
F. mosseae Glomus mosseae
Gigaspora decipiens and G. margarita –
Glomus deserticola, Glomus drummondii, and Glomus hoi –
Paraglomus majewskii –
Rhizophagus clarus Glomus clarum
R. diaphanum Glomus intraradices
R. irregularis Glomus intraradices
R. proliferus Glomus proliferus
formulations having extremely and highly hazardous active ingredients (Class 1a &
1b) in European Union and North and Latin America. These pesticides will be out of
shelves in India from 2020, which may result in the increased importance of these
two rhizospheric microbes.
27.5 Rhizospheric Endophytes
Microbes such as archaea, bacteria, protists, and fungi that colonize internal tissues
of the plants without causing any disease are called endophytes (Hardoim et al.
2015). A rhizospheric endophytic microbe inhabits the internal tissues of the root
region and symbiotically supports the plant by carrying nutrients from soil into
plants, regulating the plant growth by secreting various plant growth regulators,
increasing tolerance to various biotic stress, and enhancing disease resistance to
pathogens (Kandel et al. 2017). Some of the very important rhizospheric microbial
endophytes include Azospirillum brasilense, Bradyrhizobium japonicum,
Gluconacetobacter diazotrophicus, Methylobacterium mesophilicuma, Rhizobium
leguminosarum, Herbaspirillum seropedicae, Citrobacter., Bacillus megaterium,
Paenibacillus odorifer, Pichia guilliermondii, Chaetomium globosum, Periconia
macrospinosa, Rhinocladiella sp., and Coniothyrium aleuritis. Unculturable
microbes belonging to Proteobacteria, Actinobacteria, Bacteroidetes,

Gemmatimonadetes, and Firmicutes were reported to be plant endophytes of rhizo-
sphere origin. The deployment of microbial endophytes in the commercial agricul-
tural practices not only improves local microbial diversity but also helps to cultivate
crops with less fertilizers, fungicides, and insecticides, thereby reducing the input
costs and environmental pollutant levels generated in agriculture. A few of the
culturable endophytes like Azospirillum brasilense, Bradyrhizobium japonicum,
Gluconacetobacter diazotrophicus, and Rhizobium leguminosarum are already
commercialized as biofertilizers.
27.6 Microbial Biostimulants and Induced Systemic Resistance

in Plants
Understanding the mechanism of the biological activity of microbial biostimulants

and their molecular mode of action is an essential prerequisite for their commerciali-
zation. Both PGPR and PGPF are reported to produce elicitors called microbe-
associated molecular patterns (MAMPs).
MAMPs are essential structures of microbes that are recognized by pattern
recognition receptors (PRRs) present on the surface of plant cells. In plants, induced
immunity is one of the first lines of defense mechanisms. The prominent MAMPs of
bacteria are flagellin (Flg), elongation factor TU (EF-Tu), peptidoglycan (PGN),
lipopolysaccharides (LPS), and activator of XA21-mediated immunity (Ax21). The
fungal MAMPs include chitin, β-glucan, AVE1 peptide, and ethylene-inducing
xylanase (EIX).
Some of the MAMP-induced defense responses exhibited by the plants include
production of reactive Oxygen (ROS, oxidative burst) and Nitrogen species (NO,
Nitric oxide), antimicrobial compound induction, synthesis of pathogenesis-related
(PR) proteins, and plant cell wall alterations. These responses further initiate Ethyl-
ene and Jasmonic acid pathways in plants, resulting in induced systemic resistance
(Enebe and Babalola 2018). Though the mode of action of biostimulants on induced
defense mechanism of plants is scientifically proven, their mechanism is not given
importance while registering biostimulants. Induced plant defense against biotic and
abiotic stress due to microbial biostimulants is considered as secondary and a
byproduct of plant stimulants. Some of the prominent MAMPs and their
corresponding PRRs are given in Table 27.4.
27.7 Commercial Aspects of Biostimulants
Biostimulant products are becoming an indispensable part of inputs for sustainable

agricultural practices by the farming community throughout the world. In 2019, the
overall world biostimulant market was reported to be US $ 2.6 billion. It is expected
to reach around US $ 4.9 billion by the year 2025, with a compound annual growth
rate (CAGR) of 11.46% (David Beaudreau 2019). This forecast is further supported
Table 27.4 List of prominent MAMPs and their corresponding plant PRRs
S.
No. MAMPs Plant PRRs
1 Activator of XA21 (Ax21) XA21 and XA21D (Rice)
2 Avirulence on Ve1 tomato (Ave1) Ve1 (putative Tomato receptor)
3 Bacterial cold shock proteins (RNP1 motif) Csp15-Nsyl (Tobacco)
4 Bacterial superoxide dismutase (Sod) CsWAKL08 (Orange)
5 Beta-Glycan (GE) GEBP (putative receptor
Soybean)
6 Cellulose-binding elicitor lectin (CBEL) from Kinase BAK1(Arabidopsis)
Phytophthora
7 Chitin CeBip and CERK1 (Rice);
AtCERK1(Arabidopsis)
8 Elongation factor TU (EF-Tu; elf18/26) EFR (Arabidopsis;
Brassicaceae)
9 Flagellin (Flg; flg22) FLS2 (Arabidopsis)
10 Lipopolysaccharide (LPS) OsCERK1 (Rice)
11 Peptidoglycan (PGN) Lym1 and Lym3(Arabidopsis)
12 Pep-13 (An oligopeptide of 13 amino acids from P. BAK1, StSERK3A and
megasperma) StSERK3B(Potato)
13 Xylanase (EIX) EIX (Tomato)
Source from Mari-Anne Newman et al. (2013) and upgraded until 2020
by the increasing global market demand for the production of high-value crops under
varied climatic conditions. Worldwide, the biostimulant market is highly spread over
due to the presence of a large number of regional and global players. Some of the key
biostimulant manufacturers include Agrinos AS, Arysta Lifescience Corporation,
BASF SE, Bayer, Biostadt India Limited, Koppert B.V, ILSAS.p.A, UPL Limited,
Isagro, Italpollina, Syngenta, FMC Corporation, Adama, Acadian Seaplants,
Biovert, Sapec, and Valagro. However, only a few corporates are pursuing business
on microbial biostimulant formulations.
Europe dominates the biostimulant market share worldwide. Data on global
biostimulants’ market share for amino acids, sea weeds, humic, fulvic, microbials,
and other plant stimulants are given in Fig. 27.1 (David Beaudreau 2019).
The product profile of the global biostimulant industry is currently dominated by
acid-based biostimulant products followed by seaweed extracts. Microbial-based
biostimulants constitute about 10% of the total biostimulant market share. This also
includes biofertilizers as several countries have not differentiated between
biostimulants and biofertilizers. The global projected biostimulant product line for
the year 2020 is given in Fig. 27.2 (David Beaudreau 2019).
Even though the current percentage of microbial-based product line is less as
compared to the other products, because of increased product innovations, microbial
growth amendments are going to play a key role as biostimulant products in the
future.
Fig. 27.1 Projected biostimulants market share in the year 2020
Fig. 27.2 Projected global biostimulant product line in the year 2020
During the past few years, the majority of biostimulant manufacturing companies
worldwide are expanding their business into emerging markets by introducing high-
quality products at a lower cost through cost-effective production and R&D process.
In the coming years, with the popularization of biostimulant input benefits with
farming community, the global biostimulant industry is expected to reach its
pinnacle.
27.8 Plant Biostimulants and Regulations
In agriculture, registration of products (inputs) used is vital to safeguard their legal,

practical, and safe application. Biostimulants cannot be classified as fertilizers as
they do not provide nutrients. Similarly, biostimulants are not categorized as plant
protection products as they do not directly contribute to the control of plant pests and
pathogens. Due to this, biostimulants do not fit under any of the currently existing
pesticide or fertilizer regulatory frameworks. In the absence of a perfect definition
for biostimulants, registration procedure and the ensuing classification system
become irrational and create a hurdle to global trade and development.
In view of these difficulties in the classification of biostimulants, many countries
earlier developed different categories for registration of biostimulant products
designated as supplements, other fertilizers, plant conditioners, phytoforticants,
bioregulators, soil fertilizers, soil conditioners, etc. (Basak 2008; Traon et al.
2014; Torre et al. 2016). However, due to the recent developments in regulations
post 2018 globally, these compounds are being classified as biostimulants/
biofertilizers/soil conditioners.
In USA, the draft guidelines published by Environmental Protection Act (EPA) on
March 27, 2019, define plant biostimulants as “substances (chemical, biochemical,
or microbial) that enhance the crop yields by physiologically stimulating the plant”.
(U.S. EPA guidelines 2018; Keith A. Matthews 2019). EPA guidelines provide
useful clarification on the label claims for nonpesticide plant biostimulant products.
Some of the acceptable label claims in accordance with EPA under plant
biostimulant category include:
(a) Enhances nutrient assimilation of the plants,

(b) Promotes applied fertilizer efficiency,
(c) Increases nutrient uptake through natural sources,
(d) Corrects/alleviates/prevents/avoids plant nutrient-based deficiencies,
(e) Supports/helps/aids/enhances rhizospheric microbial populations,
(f) Enhances/supports/helps/aids/improves transformation of applied nutrients to
plant available form,
(g) Promotes overall nutrition in the plants,
(h) Enhances/improves/optimizes/helps/aids/supports higher root mass,
(i) Supports/helps/aids/enhances/improves soil conditions for healthy plant
establishment,
(j) Enhances/Improves/helps/support/aids tolerance to abiotic stress,
(k) Promotes/supports/enhances nutrient uptake,
(l) Shields/protects the leaves/plants from the effects of excess application of foliar
nutrients, and
(m) Reclaims/restores/recovers plants from sloppy management practices.
In Europe, plant biostimulant products can be marketed through following any

one of the two regulations: (a) The European Pesticide Law and (b) National
Regulations on Fertilizers. Article 2 of European pesticide law EC Regulation
no. 1107/2009 quotes that all product/s influencing the life processes of the plants
such as substances influencing their growth other than a nutrient are governed by
PPP regulations (European Fertilizer Regulation 2019; SEIPASA 2019). Therefore,
from a regulatory perspective, a majority of plant biostimulant products including
synthetic, natural substances, and microorganisms come under PPPs. However, the
major constraint to follow PPP route for plant biostimulant products is the lengthy
and costly procedures involved in following PPP regulations.
The introduction of plant biostimulant products through National Regulations on
Fertilizers in Europe could be relatively easy compared that of PPP route. However,
it is becoming increasingly difficult due to the marked differences in data
requirements for efficacy, toxicity, and ecotoxicity assessment of the European
member countries.
The current European law on EC fertilizers (Electrical Conductivity Fertilizer
regulation no. 2003/2003) strictly prohibits placing any other substance other than a
nutrient in the fertilizer act. In view of this constraint, and on the suggestions of
European Biostimulant Industry Council (EBIC), an amendment EC No. 1107/2008
regulation was introduced in the year 2019 in order to introduce substances, which
are not strictly nutrients but support plant growth. This amendment would come into
implementation only after two to three years.
In India, plant biostimulants are currently sold in the market in an unregulated
manner due to the lack of a proper definition and regulatory guidelines. The plant
biostimulant market in India is proliferating and is expected to reach US$ 180.95
million by the year 2023 with a CAGR of 16.49% (Research and Markets 2018),
especially microbial-based plant stimulants at 10.8% CAGR (Mordor Intelligence
2020). In view of the increasing demand for a proper legal definition and regulatory
guidelines for plant biostimulants, Department of Agriculture, Cooperation and
Farmers Welfare, Government of India, has proposed draft guidelines on plant
biostimulants in the year 2019. In the draft guidelines, an amendment was proposed
in the existing Fertilizer (Control) Order 1985 for inclusion of plant biostimulants
(Indian fertilizer control order 1985 and draft guidelines on FCO amendment 2019).
As per the new amendment (1) in class 2(h), the existing definition of “Fertilizer”
has been expanded to include plant biostimulant products. Accordingly, plant
biostimulants are defined as compounds, substances, and products including
microorganisms whose functions when applied to plants/seeds/rhizosphere are to
regulate and enhance crop physiological processes independent of the product’s
nutrient content and to improve input use efficiency, growth, yield, quality, and/or
stress tolerance. The biostimulants may include products of plants/animals or
microbial origin.
The primary function of the biostimulant products should be other than that for
pesticide use and nutrient source. Heavy metal and other contaminants, if any, may
be within the prescribed limits given under clause 20 C. Biostimulants notified under
subclause (i) shall be under any of the following categories including (a) Botanical
extracts, including seaweed extracts, (b) Biochemicals, (c) Protein hydrolysates and
amino acids, (d) Vitamins, (e) Cell-free microbial products, (f) Antioxidant,
(g) Antitranspirants, and (h) Humic and Fulvic acids and their derivations.
As per the draft guidelines 2019, any manufacturer/importer of plant biostimulant

products in India must apply to the Controller of Fertilizer in Form R along with
(a) Chemistry, (b) Bioefficacy, (c) Toxicity, and (d) Heavy metal limits of the
product to get those registered under plant biostimulant category.
27.9 Biofertilizers
Biofertilizers are the offshoot of biostimulants (Siddiqui and Mahmood 1999;

Burdman et al. 2000) with varied definitions and interpretations (Vessey 2003).
Microbial-based formulations and organic manure that positively are influencing the
plant growth and yield through various mechanisms are termed as biofertilizers.
Based on scientific study results, rhizosphere microbes like Azospirillum (Saharan
and Nehra 2011), Azotobacter (Kizilkaya 2009), and Rhizobium (Peoples et al.
1995) are considered as nitrogen-fixing biofertilizers. Considering that 80–85% of
phosphate fertilizers are immobilized and unavailable to plants (Goldstein 1986),
selected P-solubilizing rhizosphere microbes are approved as biofertilizers and
christened as P-solubilizers. Similarly, the ill effects of potassium fertilizers’ over-
dose on plants and their sequestration in soil (Silva et al. 2011; Vieira-Megda et al.
2015) necessitated the introduction of K-solubilizers as biofertilizers.
Ministry of Agriculture, Department of Agriculture and Cooperation, Govern-
ment of India, New Delhi, vide their order dated March 24, 2006, included
biofertilizers and organic fertilizers under section 3 of the Essential Commodities
Act, 1955 (10 of 1955), in Fertilizer Control Order, 1985. Compared to several
developed countries, India brought in a well-defined regulation for commercializing
biofertilizers and manures under the Fertilizer Control Order. It has permitted carrier
and liquid-based microbial biofertilizer formulations. The admissible commercial
formulations and standard prescriptions for microbial-based bio-fertilizers under
FCO (1985) are listed in Table 27.5.
The consortium of microbes has proved to be far more beneficial in terms of
efficiency and cost economics than single strains. The synergy of several microbes
without being antagonistic to each other while achieving the complicated objectives
for which they have been combined makes the consortia formulations unique.
Innumerable successful biostimulant formulations are having such microbial con-
sortia. Therefore, consortia of biofertilizer formulations are also permitted in India
under FCO (1985). A list of permitted biofertilizer consortia is given in Table 27.6.
All material shall pass through 0.15–0.212 mm IS sieve. With the introduction of
biofertilizers, India has partially reduced the bane of soil degradation from the
persistent use of chemical fertilizers. If properly implemented and commercialized,
biofertilizers can reduce the fertilizer load considerably and detoxify the soil. This
needs to happen since conventional chemical fertilizers harm beneficial microbes
(Vitousek et al. 1997), pollute the environment (Kumar et al. 2019), deplete the
natural resources, and damage the soil properties and geochemical cycles (Jafar and
Behzad 2016). Further, innovation and introduction of novel biostimulants/
biofertilizers are essential for sustainable agriculture. Therefore, possible newer
Table 27.5 List of approved bioinoculants in India under FCO (1985)

Quality standard prescribed as per FCO (1985)
Permissible
Recommended pH Quantity contaminant
Active (cfu per
ingredients* Form cfu g/ml) Strain feficiency
Phosphate solubilizing bacteria (PSB)
Bacillus Solidb 6.5–7.5 5 107/g Nil at 105 Phosphate solubilizing
megaterium Liquid 5.0–7.5 1 108/ Nil capacity shall be in the range
Pseudomonas ml of min. 30% when tested
striata spectrophotometrically.
Aspergillus sp. Solidb 6.5–7.5 5 107/g Nil at 105 Minimum 5 mm
solubilization zone in
prescribed media having at
least 3 mm thickness.
Nitrogen fixing bacteria (NFB)
Azospirillum Solidb 6.5–7.5 5 107/g Nil at 105 Formation of white pellicle in
lipoferum Liquid 5.0–7.5 1 108/ Nil semisolid N-free
Azospirillum ml bromothymol blue media.
brasilense Shall fix at least 10 mg N
fixation /g of malate applied
Rhizobium sp. Solidb 6.5–7.5 5 107/g Nil at 105 Should show effective
Liquid 5.0–7.5 1 108/ Nil nodulation on all the species
ml listed on the packet
Acetobacter Solidb 5.5–6.0 a
5 107/ Nil at 105 Formation of yellow pellicle
sp. g in semisolid N-free medium
Liquid 3.5–6.0 1 108/ Nil
ml
Azotobacter Solidb 6.5–7.5 a
5 107/ Nil at 105 The strain should be capable
chroococcum g of fixing at least 10 mg of
Azotobacter Liquid 5.0–7.5 1 108/ Nil nitrogen per gram of sucrose
vinelandii ml consumed
K-solubilizer
Frateuria Solidb 6.5–7.5 a
5 107/ Nil at 105 Minimum 10 mm
aurantia g solubilization zone in
Liquid 5.0–7.5 1 108/ Nil at 101 prescribed media having at
ml least 3 mm thickness
Zn-solubilizer
Pseudomonas Solidb 6.5–7.5 a
5 107/ Nil at 105 Minimum 10 mm
sp. g solubilization zone in
Liquid 5.0–7.5 1 108/ Nil at 101 prescribed media having at
ml least 3 mm thickness
Arbuscular mycorrhizae
Arbuscular Solidc 6.0–7.5 100/gm Not Total potential: 1200 IP/gm
mycorrhizal with min specified (determined by MPN method
fungi 60 spores/ with ten-fold dilution)
g
a
Generally used organism in India; but not restricted
b
All material shall pass through 0.15–0.212 mm IS sieve’
c
90% should pass through 250 micron IS sieve (60 BSS)
Table 27.6 Consortia of microbial formulations approved as biofertilizers under FCO (1985)
Quality standard prescribed as per FCO (1985)
Permissible
pH Quantity contaminant
Recommended (cfu per
Active ingredient Form cfu g/ml) Strain Efficiency
Bio-NPK Consortia
Organisms fixing Solid1 6.5–7.5 5 107/g Nil at 105 In consortia formulation,
N and or each quality of each culture
solubilizing P culture assessed individually as
and K 1 107/g specified for sole
Liquid 5.0–7.5 5 108/ Nil bio-fertilizers
ml or
each
culture
1 108/
ml
Bio NP, Bio NK, and Bio PK consortia
Formulation with Solid1 6.5–7.5 5 107/g Nil at 105 In consortia formulation,
NP, NK, and PK or each quality of each culture
fixing/ culture assessed individually as
solubilizing 1 107/g specified for sole
properties Liquid 5.0–7.5 5 108/ Nil biofertilizers
ml or
each
culture
1 108/
ml
biofertilizers and their combinations are given in Table 27.7. They suggested based
on credible scientific research and in-house studies carried out at Prof.
T.N. Ananthakrishnan Innovation Center, Hyderabad.
It is wise to include a few more beneficial rhizospheric microbes that are reported
to have biofertilizer activities. For instance, Azotobacter has diverse species, namely,
A. chroococcum, A. vinelandii, A. beijerinckii, A. nigricans, A. armeniacus, and
A. paspali. They are reported to fix nitrogen successfully in a wide array of crops,
viz., wheat, oat, barley, mustard, sesamum, rice, linseeds, sunflower, castor, maize,
sorghum, cotton, jute, sugar beets, tobacco, tea, coffee, rubber, and coconuts (Wani
et al. 2013). Similarly, species of Azospirillum such as A. lipoferum, A. brasilense,
A. amazonense, A. halopraeferens, and A. irakense have been reported to improve
the productivity of various crops (Sahoo et al. 2014). Commercial formulations in
India are only limited to selected organisms that are listed in Table 27.5.
Actinomycetes and their metabolites are thoroughly explored across the globe.
Introduction of secondary metabolites like Abamectin, Ivermectin, Spinosad, and
Milbemectin as insecticide/nematicide/miticides are the best example to prove the
Table 27.7 Proposed new biofertilizer formulations and related combinations

Proposed
bioinoculant Microbe Justification References
Sulfur oxidizing Thiobacillus sp. Increases soil available sulphate Kambam et al.
bacteria (SOB) and stabilizes soil pH (2014)
Magnesium Serratia sp. Recycles magnesium in soil Sumant et al.
oxidizing bacteria (2015)
(MoB)
Calcium Pseudomonas sp. Solubilizes complex calcium Gopinath et al.
solubilizing and converts into assimilatory (2015)
bacteria form
Silica solubilizing Bacillus sp. and Solubilizes silica in soil. Vasanthi et al.
bacteria Pseudomonas sp. (2018)
Methylotrophic Methylobacterium Phyllospheric plant growth Krishnamoorthy
bacteria radiotolerans promoter et al. (2018)
Bio NPKZn Bacterial consortia Fixes N and solubilizes & Babu (2020)
mobilizes P, K, and Zn
significance of Actinomycetes. They have strong antimicrobial potential and soil-

dominant saprophytic nature and are plant growth promoters (Franco-Correa et al.
2010) with immense biocontrol action against a range of phytopathogens (Wang
et al. 2013). They can produce phytohormones (IAA) and siderophores apart from
solubilizing phosphate and promoting plant growth (Jeon et al. 2003). Research
institutions/universities/industries need to pay attention to commercialize
Actinomycetes as biofertilizers/biostimulants.
Integration of organic manures, biofertilizers, biostimulants, and other slow-
released/controlled fertilizers with chemical fertilizers is the need of the hour to
safeguard the soil and increase crop productivity (Pandiselvi et al. 2017). An
ordinance by the Government of India to produce city compost by providing market
development assistance (Lok Sabha Report No. 34 2017) of INR 1500/ per metric
ton is also aimed to integrate compost with chemical fertilizers through fertilizer
marketing channel.
27.10 Market Demand & Global Opportunities
The global biofertilizer market is projected to reach USD 2.35 billion by 2022 at a
CAGR of 14.08% from 2015–2022 (Fig. 27.3). Another estimate suggested 11.3%
CAGR during 2019–2025 (Fortune Business Insights 2019). The increasing aware-
ness of consumers on clean energy, green cultivation, and safe food by consumers
coupled with an increase in fertilizer price, quality manufacturers, and area of
cultivation makes biofertilizers more appealing to growers and food consumers.
For example, the current price of 50 kg MoP is INR 950.00/ against the selling
price of INR 425.00/ during the year 2017. Importantly, integration of
Fig. 27.3 Global projected biofertilizer usage from the year 2012–2022
biofertilizers with reduced recommended dose of chemical fertilizers resulted in

healthy crops and soil with improved crop yields as compared to 100% use of
recommended conventional fertilizers. Government of India has reduced the propor-
tion of fertilizer subsidy from 3.03 billion US$ to 2.18 billion US$ against the total
budget allotted between the financial years 2016–2017 and 2020–2021.
27.11 Global Regulatory Guidelines for Biofertilizers
European Union (EU) has no set parameters and specific regulations for
biofertilizers. The regulation is a country based rather than EU based. However,
the draft of a new regulation for biostimulants has been finalized and expected to be
implemented from 2022. The draft guidelines include biofertilizers under
biostimulants as low-risk active ingredients. The registrant will be entitled to
15 years of registration validity and 13 years of data protection.
Poland considers biofertilizers as “growth stimulators” with a condition that it
does not pose threat to health of humans, animals, and environment. Spain, a leading
organic cultivating country, has no legislation on biofertilizers and allows local
administrators to regulate it. Italy identified mycorrhizae as “product with action
on the soil”. It is up to the associate countries to decide on implementing the EU
guidelines or keeping additional regulatory requirements in the year 2022. Currently,
it appears to be easier to register biofertilizers/biostimulants in several associate
countries than following EU guidelines.
Brazil regulates microbial biofertilizers as substance free from agro-toxic

chemicals, hormones, and stimulants. Though regulation has been framed way
back in 1980, the registration of products as biofertilizers is not visible.
United States of America has no legal definitions currently for the term
“biofertilizer”. The US National Organic Program (NOP) foresees only the possibil-
ity of using biological organisms for plant protection. EPA considers manures and
decomposed solid wastes as fertilizers. Biostimulant guidelines are framed under
Agriculture Improvement Act, 2018. It has considered formulations containing
Rhizobium, N-fixing bacteria, PSB, Selenium-solubilizing bacteria, and biofertilizer
consortia as biofertilizers/plant stimulants.
Republic of China has the national standard on the terminology of fertilizers by
including biofertilizers/soil conditioners. Biofertilizers/microbial fertilizers having
living microbial organisms should undergo microbial safety and product quality
standards. Product quality is estimated with living cells, carbon and water content,
pH, size of granules (for solid products), appearance, contamination, and validity
(Suh et al. 2006).
Australia places biofertilizers as biostimulants under fertilizer law for micro-
organisms and permits their combination with nutrients as fertilizers. The
Australian Pesticides and Veterinary Medicine Authority is authorized to register
biological agriculture products. The microbial biological formulations are governed
as per the Australian Biosecurity Act 2015.
Canada has a well-structured Canadian Food Inspection Agency (CFIA). It
defines procedures accepted by the industry for the registration of nonGMO
biofertilizers. It exempts full safety data required for product registration.
Formulations having Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium,
Bacillus subtilis, and Mycorrhiza are approved as biofertilizers.
India has enforced superior regulatory mechanisms for biofertilizers. It initially
brought manufacturing of biofertilizers under FCO (1985). Manufacturers need to
obtain a manufacturing license (Form F) from the respective state agriculture
departments. Commercialization is carried out by obtaining a marketing license
(Form A2) from the respective state department of agriculture. The Government of
India, however, removed the manufacturing license requirement in 2018. It obliged
manufacturers/marketers responsible for maintaining the quality of biofertilizers.
27.12 Potential for Biofertilizers and Biostimulants—Indian

Perspective
Modern agriculture used conventional chemical fertilizers abundantly over the years
to make it more productive and sustainable. However, their extensive use eroded soil
fertility by altering its physicochemical properties and microbial dynamics. Several
million tons of unused chemical fertilizer residues in the soil started posing serious
challenges to crop productivity and the environment. Biofertilizers and biostimulants
make the insoluble fertilizer residues into available forms and benefit the plants
either directly or indirectly (Bargaz et al. 2018). This is important to achieve the
Table 27.8 Information on revenue savings if biofertilizers are aggressively introduced in India
(2016–2021)
Savings if 25% biofertilizers are
Fertilizer subsidy allotted introduced
Billion % against total budget Billion % saving against Billion
Years US$ (Billion US$) US$ total budget INR
2016–2017 8.76 3.03 2.19 0.76 165.78
2017–2018 8.78 2.61 2.19 0.65 166.17
2018–2019 9.26 2.41 2.31 0.60 175.22
2019–2020 10.57 2.53 2.64 0.63 199.99
2020–2021 9.42 2.18 2.36 0.55 178.27
(BE)
Total 56.36 14.08 1066.47
BE, Budget Estimate
Note: US$ value is taken as Rs.75.64/ as per 4.5.2020 conversion rate
targeted food grain production of 321 million tons without further depleting the
feedstock/fossil fuels. Integrating biofertilizers and biostimulants with conventional
fertilizers is becoming essential to improve soil fertility, crop productivity, and
environment. Their long-term substitution may vitalize the small and marginal
farmers economically over sole chemical fertilizer usage (Venkataraman and
Shanmugasundaram 1992; Miransari 2011). It can also increase country’s economy
significantly if properly integrated with conventional fertilizers.
Abundant scientific studies across the world between 1995 and 2015 have proved
that biological fertilizers can reduce 20–30% of chemical fertilizer use. However,
until today, there are no constructive steps taken by the Governments across the
globe to integrate biofertilizers with conventional fertilizers effectively. Table 27.8
explains how India could have saved about INR 165.78–199.99 billions/annum, if it
had integrated biofertilizers with chemical fertilizers since 2016–2017.
Gradual reduction of saving from 0.76% to 0.55% against the total allotted budget
for the past five years indicates that India plans to reduce the burden of fertilizer
subsidy. When the budget makes fertilizer subsidy free, the cost of cultivation may
be doubled and burden the farming community. So, the Government should also
simultaneously focus on popularizing alternative and cost-effective fertilizer
ingredients. Biofertilizers are potential alternate sources that can be tapped fully
before detaching the subsidy.
Benefits of biofertilizers/biostimulants are not limited to economy alone. They
also mitigate biotic and abiotic stresses of the plants. Adaptation of crops/plants to
increased temperature, erratic rainfall, cold/heat waves, and elevated CO2 is the
contemporary challenge of global climatic change. For instance, plants had to cope
with 0.57 C increase in average temperature during the last 100 years in India. It is
likely to be challenged with 2.5 C by 2050 and 5.8 C by 2100. These factors are
likely to cause serious threat to crop growth, soil fertility, and water resources.
Introduction of ecto- and endorhizosperic microbes proved to help the plants to
mitigate these abiotic stresses through several mechanisms including modification of
plant response at gene level. Other rhizospheric microorganisms such as Pseudomo-
nas, Bacillus, Arthrobacter, Pantoea, Burkholderia, Rhizobium, etc. were reported
in several publications to enhance the tolerance of sunflower, maize, wheat, chick-
pea, groundnut, spices, and grapes to drought, salinity, heat stress, and chilling
injury under controlled conditions (AitBarka et al. 2006; Arshad et al. 2008).
Trifolium alexandrinum when inoculated with Rhizobium trifolii showed higher
biomass and increased number of nodulations under salinity stress condition
(Hussain et al. 2002). Pseudomonas putida RS-198 has been reported to enhance
seed germination, plant height, and weight of cotton when grown in alkaline soil. It
increased the rate of uptake of K+, Mg2+, and Ca2+and decreased the absorption of
Na+ (Yao et al. 2010). Calcisol produced by rhizobacters, viz., P. alcaligenes PsA15,
Bacillus polymyxa BcP26, and Mycobacterium phlei MbP18, provided tolerance to
high temperatures and salinity stress (Egamberdiyeva 2007). Achromobacter
piechaudii was shown to sustain and improve the quality of tomato and pepper
plants under 172 mM NaCl and water stress (Alavi et al. 2013). Interestingly, a root
endophytic fungus Piriformospora indica was found to defend host plant against salt
stress (Ansari et al. 2013). Combination of A. brasilense and AM enhanced plant
tolerance to various abiotic stresses (Joe et al. 2009). Similarly, P. putida or
B. megaterium and AM fungi were effective in alleviating drought stress (Marulanda
et al. 2009). Canola and barley plants were protected by P. fluorescens strain
169 (P.f.169) from the inhibitory effects of cadmium via IAA, siderophores, and
1-aminocyclopropane-1-carboxylate deaminase (ACCD) (Baharlouei et al. 2011).
Plants can thus be protected from a series of abiotic stresses including pollutants like
petroleum (Tang et al. 2010).
27.13 Commercial & Regulatory Challenges—

Guidelines vs. Ground Reality
Carrier-based biofertilizer formulations face innumerable challenges.

Manufacturers, while producing biofertilizers consider several predisposing factors,
viz., (a) Easy to use/transport, (b) Longer shelf life, (c) Consistent efficacy,
(d) Abiotic stress tolerance ability, and (e) Cost effectiveness. While formulating
guidelines, the Government has proposed suitable carriers like lignite, peat, humus,
peat soil, talc, and wood charcoal or similar material favoring organism growth with
the moisture content of 30–40%. Even a competent manufacturer cannot maintain
the moisture of formulation at 30–40% in case the carrier is other than lignite and
peat. Further, a better suitable carrier may not meet the prescribed standard of
0.15–0.212 mm IS sieve for NPK biofertilizers and 250 microns IS sieve for
Mycorrhiza. pH of the carrier is another important challenge that majority of
manufacturers face. Locally sourced carriers have pH ranging from 4.5–9. Impor-
tantly, biofertilizers are brought under Essential Commodity Act, 1955 on par with
conventional plant nutrients. Handling a biologically active formulation with differ-

ent variables is the fundamental challenge that biofertilizer manufacturers and
marketers undergo. Representations have been made to competent authorities to
drop particle size, moisture, and pH to meet the quality parameters.
Goods and Services Tax (GST) has broken the barriers of the federal system on
ease of doing business. Marketing agriculture inputs becomes a state subject. It is a
herculean task to get separate marketing permission from each state. It needs
passion, time, and liaison. Products like biofertilizers cannot afford such hardships.
So, the distribution restricts to mandal or district level. Corporates who come
forward to market also cannot get the permission unless the source of the manufac-
turer is approved in the respective states. It amounts to dual licensing for single
product. In the era of online medicine sale for human consumption with single
license, obtaining permission with 3 years validity from each state to market
biofertilizer is the major bottleneck.
New manufacturing and formulation technologies have emerged with increased
shelf life and varied carrier options. Processes like PGSS (Particle from Gas
Saturated Solutions) using carbon dioxide as supercritical fluid to obtain free-
flowing powder formulations, spray dried/freeze dried concentrates, desiccant-
based delivery system with better cell viability, alginate bead techniques etc. have
been developed in recent years. Regulatory frame works need to be reformed to
accommodate these new technological innovations.
Popularizing biofertilizers among the growers is a significant mission to be
considered for the next ten years. State department of agriculture often distributes
biofertilizers through subsidy schemes. Occasionally, predatory pricing hampers the
quality standards and brings negative opinion among farming community. More
active participation from state agricultural universities and ICAR affiliated research
institutions, state and central Government machineries, agri-input corporates, and
MSME manufacturers can popularize biofertilizers across India. Biofertilizer pene-
tration in the market despite regulations and government efforts to promote are still
in the nascent stage. Corporates selling conventional fertilizers should undertake
marketing of biofertilizers under their corporate social responsibility program.
Corporates have endorsed mycorrhiza and made them commercially successful.
However, it is only the tip of the iceberg.
27.14 Conclusions
Rhizosphere-based microbial plant biostimulants and biofertilizers have numerous

benefits to plants and the environment. Their commercial opportunities are less
tapped across the globe. Asia Pacific countries especially India and China have
formulated guidelines to commercialize biofertilizers. Regulatory guidelines have
also been proposed for microbial-based plant stimulants in developed and develop-
ing countries. Constructive business opportunities are visible from the year 2022 for
microbial-based biostimulants. In India, MSMEs need to equip themselves with
dossiers to meet the regulatory requirements across the globe. It is also important to
introduce new microbial active ingredients, consortia, and formulation technologies

with a better delivery system. Government has to play a key role by reducing license/
trade barriers across the federal system and increase the involvement of corporate
companies for integrating biofertilizers with conventional chemical nutrients suc-
cessfully. Microbial-based biostimulants and biofertilizers have all the essential
ingredients to become popular among the growers within a decade from now.
Acknowledgements The authors acknowledge the help rendered by Dr. V. Anitha, Director
(P&M), PJTSAU, Hyderabad, and Dr. P.S. Vimala Devi, Principal Scientist (Rtd), ICAR-IIOR,
Hyderabad, for their valuable feedback. Thanks are also due to Mr. R. Prasad Babu, Coordinator
(Regulatory Affairs), Varsha Bioscience, and Technology India Pvt. Ltd. for the suggestions and
support.
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Microorganisms for Sustainability 23

Series Editor: Naveen Kumar Arora

Pramod Kumar Sahu Harsh Vardhan Singh

Pawan Kumar Sharma

ISSN 2512-1901 ISSN 2512-1898 (electronic)

# Springer Nature Singapore Pte Ltd. 2020

unique mechanism of deploying microbes that confer tolerance to abiotic stresses

Uttar Pradesh/ Chhattisgarh Sushil K. Sharma

1 Microbial Interactions in the Rhizosphere Contributing Crop

9 Rhizosphere Microbes: Driver for Soil Health Management . . . . . 235

20 Microbial Diversity of Chickpea Rhizosphere . . . . . . . . . . . . . . . . 483

About the Editors

Sushil Kumar Sharma, National Agriculturally Important Microbial Culture Col-

Udai B. Singh, Plant–Microbe Interaction and Rhizosphere Biology Lab, ICAR-

of Mycology and Plant Pathology, Udaipur. He has published several research

Pramod Kumar Sahu, Plant–Microbe Interaction and Rhizosphere Biology Lab,

Harsh Vardhan Singh, Plant–Microbe Interaction and Rhizosphere Biology Lab,

Pawan Kumar Sharma, National Agriculturally Important Microbial Culture Col-

Important Microbial Culture Collection. He has published 46 research papers in

About the Series-Editor

Naveen Kumar Arora, Fellow of International Society of Environmental Botanists

Richa Agnihotri ICAR-Indian Institute of Soybean Research, Indore, Madhya

Hameeda Bee Department of Microbiology, Osmania University, Hyderabad,

Hesham Ali El Enshasy Institute of Bioproduct Development (IBD), Universiti

Sandhya Kollalu Department of Soil Science and Agricultural Chemistry, Univer-

Deepti Malviya Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-

J. P. Rai Department of Mycology and Plant Pathology, Institute of Agricultural

Sushil K. Sharma ICAR-National Bureau of Agriculturally Important

R. Umamaheswari ICAR-Indian Institute of Horticultural Research, Bengaluru,

Deepti Malviya, Udai B. Singh, Shailendra Singh, Pramod K. Sahu,

Rhizosphere is a hot spot where speciﬁc kinds of diverse microbial communities

Deepti Malviya and Udai B. Singh have contributed equally.

D. Malviya · U. B. Singh (*) · S. Singh · P. K. Sahu · K. Pandiyan · A. S. Kashyap · N. Manzar ·

# Springer Nature Singapore Pte Ltd. 2020 1

approaches to engineer rhizosphere in such way that it enables plant to enhance

(1) change in the physico-biochemical properties of niche (pH, EC, nutritional

1.2 Rhizosphere Structure and Function

surrounding the roots. This is referred to as rhizosphere effect. ATP-binding cassette

1.3 Microbe-Mediated Mechanisms of Plant Defence to Biotic

1.3.1 Phenolics and Plant Resistance

1.3.2 Induced/Systemic Disease Resistance

PRR-dependent signalling to facilitate acquisition of nutrients and to ensure dis-

1.4 Microbe-Mediated Mechanisms of Plant Defence to Abiotic

Environmental stress refers to adverse effects on plant growth and development. At

1.4.1 Generation of Reactive Oxygen Species and Their Effects

1.4.2 Site of Synthesis of ROS

1.4.3 Type of ROS Synthesized During Abiotic Stresses

Activation of O2 may arise by two different mechanisms: (1) absorption of sufﬁcient

1.4.4 Mechanisms to Scavenge ROS During Abiotic Stresses

1.4.5 Mechanisms of Plant Defence

screen in Xenopus laevis oocytes led to the recognition of a tobacco syntaxin,

1.4.6 MAPK Signalling

Plant mitogen-activated protein kinases (MAPK) receive signals from receptors or

1.5 Endophytes in Biotic and Abiotic Stress Management

Fig. 1.3 An overview of microbe-mediated modulation of physio-biochemical mechanisms of

Mechanism of action Endophyte Pathogen Host Reference

VOCs Endophytes Plant pathogens – Chung et al.

1.6 The ‘Holobiome’: Significance of Microbes in Host

In recent past, a series of studies have unveiled that an individual is actually an

1.7 Rhizosphere Engineering for Stress Management