Splat Book

Recent Trends in
Insect Pest Management

Volume - 7
Chief Editor
Dr. S.V.S. Raju
Professor, Department of Entomology & Agril. Zoology, Institute of
Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh,
India
Co-Editor
Kamal Ravi Sharma
Department of Entomology & Agril. Zoology, Institute of Agricultural
Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
AkiNik Publications
New Delhi
Published By: AkiNik Publications
AkiNik Publications
169, C-11, Sector - 3,
Rohini, Delhi-110085, India
Toll Free (India) – 18001234070
Phone No. – 9711224068, 9911215212
Email – akinikbooks@gmail.com
Chief Editor: Dr. S.V.S. Raju

Co-Editor: Kamal Ravi Sharma
The author/publisher has attempted to trace and acknowledge the materials

reproduced in this publication and apologize if permission and
acknowledgements to publish in this form have not been given. If any material
has not been acknowledged please write and let us know so that we may rectify
it.
© AkiNik Publications
Publication Year: 2022
Pages: 166
ISBN: 978-93-5570-261-6
Book DOI:
Price: ` 764/-
Contents
Chapters Page No.

Genome Editing Technologies and Its Applications in
1.
Entomology 01-18
(Sunitha SVL, Jethva DM, Harika B and Davaria PJ)
2. Remote Sensing Application in Crop Pest Management 19-32

(Tumma Mounika and Tapan Kumar Hath)
3. Management Tactics for Plant Parasitic Nematodes 33-51

(Babita Kumari, Sujata and Shweta)
4. Splat: A New Pheromone Dispensing Technology 53-75

(Venugopal U, Neelima K, Nagaraju MC and Manju KL)
5. Biocontrol Agent: A Promising Tool towards Sustainable

Agriculture 77-108
(Ushma Joshi, Mrunali Patel, Nehal Rami, Dhvani Patel and Kruti Dave)
6. Recent Scenario of Termite Infestation and Their Management 109-131

(Vijay Kumar, Umesh Chandra, Pradip Kumar Patel, Arvind Kumar and
Vishnoo Omar)
7. Role of Transgenic Plants in Pest Management 133-154

(Ajit Pandey, Aditya Patel, Rishi Nath Pandey and Arvind Kumar)
8. Role of Nano-Pesticides, Nano-Fertilizers and Nano Bio-

Sensors in Agriculture 155-166
(Vijay Kumar and Lalita Verma)
Chapter - 1
Genome Editing Technologies and Its
Applications in Entomology
Authors
Sunitha SVL
Ph.D. Scholar, Department of Entomology, Junagadh
Agricultural University, Junagadh, Gujarat, India
Jethva DM
Associate Research Scientist, Junagadh Agricultural
University, Junagadh, Gujarat, India
Harika B
M.Sc. Student, Department of Entomology, Agricultural
College, Bapatla, Andhra Pradesh, India
Davaria PJ
Ph.D. Scholar, Department of Entomology, Junagadh
Agricultural University, Junagadh, Gujarat, India
Page | 1
Page | 2
Chapter - 1
Genome Editing Technologies and Its Applications in
Entomology
Sunitha SVL, Jethva DM, Harika B and Davaria PJ
Abstract
Insects are the most diverse group of animals living on earth. They are
undoubtedly the most adaptable forms of life as their numbers far exceed
that of any other category of animal. Insect pests inflict damage to humans,
crops and farm animals. Insect pests cost billions of dollars in the form of
crop losses and insecticides usage every year globally. For reducing the
usage of insecticide scientists are developing other alternative
biotechnological approaches. In that transgenic (Bt) crops has great benefits
in insect management but soon after its implementation, the technology
started facing the major issue of insect resistance, leading researchers to start
pursuing advancements in newer biotechnological approaches to insect pest
management, such as genome editing technologies (ZFNs, TALENs and
CRISPR/Cas9). In that, the CRISPR/Cas9 system has attracted a lot of
attention due to its simplicity, efficiency, and versatility. This method is
being applied in almost all biological research areas and has a wide range of
applications. This review focuses on the applications of the genome editing
technology in entomology.
Keywords: CRISPR/Cas9, Genome editing, Insect and Pests
1. Introduction
In the 21st century, agriculture is decelerate by incessant challenges
from several abiotic and biotic stresses. Among the biotic stress, insect pests
being one of the foremost. Insects are responsible for the reduction in
potential yields resulting in stagnate productivity. In post green revolution
period, the global crop loss due to insect pest was 10.8%. Now days, with the
increases of global temperature insects are going to be hungrier and their
population will increase manifold. These warmer climate will increase the
metabolic rate of insects that make them hungrier and so they will devour
more crops leads to more crop losses. For control of insect pests, the farmers
Page | 3
have adopted higher dose of pesticides that leads to environmental pollution,
adverse effect on natural enemies, resurgence of secondary insect pests and
development of resistance in insect. Along with chemical control, there was
other alternative biotechnological system i.e., Bacillus thuringiensis (Bt). Its
efficacy was threatened by the development of resistance in some insects
such as Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae),
Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and Spodoptera
frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). These problems have
motivated researchers globally to develop novel and environmental friendly
insect control strategies. Hence, the prime focus of modern agriculture has
been to achieve enhanced yields with the existing land and resources for
global food security and agricultural sustainability. Recently, with the
advance of genomics and sequencing of plant genomes have opened new
avenues for their exploitation in crop improvement programs. This led to the
inception of the next level of biotechnological intervention called “Genome
engineering” or “Genome editing”. In the present scenario of constricted
agricultural fields and increased load of insect pests on crop plants, genome
editing will serve as a potential tool to combat insect pests, and hence, it has
been described as the “new breeding technology” [1].
The genome editing, is a type of genetic engineering in which DNA is
inserted, deleted or replaced in the genome of a living organism using
“engineered nucleases” or "molecular scissors” [2]. It is used to precisely and
efficiently edit DNA within a cell. In fact, the genome editing of engineered
nucleases were selected by Nature Methods. Due to the advancement of
research and development in 2011, the genome editing was called as the
“Method of the Year”. Recently, the gene knock down and gene knock out
are the techniques used to prevent gene expression in insects. The main
difference between them is the level of repression. In gene knock down, the
expression of a particular gene of an organism is suppressed (reduced or
silenced). It leads to abortive protein translation and degradation of that
mRNA. It includes siRNA interference and pre-mRNA splicing sites, or
nuclease cleavage sites used for maturation of other functional RNAs
(morpholino oligos). Whereas, in gene knock out, the removal of genes from
DNA of an organism through different genetic techniques. It involved in
complete erasing of the target genes or inactivating them through nonsense
mutations. The gene knockout includes two methods, homologous
recombination and site-specific nucleases or Molecular scissors or
endonucleases. The homologous recombination (HR) involves creating
a DNA construct containing the desired mutation. This method is used to
break double strand DNA lesions, maintains genomic stability by repairing
Page | 4
broken DNA strands and incorporation of correct DNA sequence that are
homologous to the site of damage [3]. The site specific nucleases method is
used to ligating the two cut ends together, insertions or deletions of base
pairs which cause frame shift mutations called as Non homologous End
Joining NHEJ). These mutations can render the gene in which they occur
nonfunctional, thus creating a knockout of that gene. This process is more
efficient than homologous recombination. The four kinds of programmable
endonucleases are currently being used for plant genome editing i.e., Mega
nucleases, Zinc finger nucleases (ZFNs), Transcription activator-like effector
nucleases (TALENs) and Clustered regularly interspaced short palindromic
repeats (CRISPR-Cas9).
2. History and evolution of genome editing
The timeline of genome editing technology starting from 1989, when the
homologous recombination mediated genome editing targeting was first
described in mouse embryonic stem cells. In 1991, the insect genome editing
was first carried out in Drosophila melanogaster. In 1992, the Cre-lox
editing technology was successfully used for site specific recombination in
mice. The target specific DNA sequences, zinc finger protein was first time
discovered in 1998. In 2000, the CRISPR/Cas defense system was identified
first time in prokaryotes. In Xanthomonas bacteria, DNA binding proteins
like TALENs was discovered in 2009. For mammalian cell genome editing,
the CRISPR/Cas 9 system was first time used in 2013 [4].
3. Different types of site-specific nucleases
1. Mega nucleases
The mega nucleases were the first class of sequence-specific nucleases
used in plants and they continue to be deployed to achieve complex genome
modifications. It is also known as Homing endonucleases. It was the first
tool used for double strand break-induced genome manipulation. It occurs
naturally in yeast and in Chlamydomonas. It cut the large portion of the
DNA (14 to 40 base pairs). The DNA binding site overlaps with those for
DNA cleavage sites. The advantage of mega nucleases is their smallest
nuclease comprising only 165 amino acids. The most widespread and best-
known mega nucleases were the proteins in the LAGLIDADG family. These
homing endonucleases enzymes introduce site specific DNA breaks and are
encoded by homing endonucleases genes and flanking DNA into the broken
chromosome [5].
Page | 5
2. Zinc Finger Nuclease (ZFN)
These were first identified as a DNA-binding motif in Transcription
factor TFIIIA from African clawed frog (Xenopus laevis). Zinc finger protein
are small structural motif that is characterized by the coordination of one or
more zinc ions in order to stabilize the fold. That contain multiple finger-like
protrusions that make tandem contacts with their target molecule. The FokI,
naturally found in Flavobacterium okeanokoites is a bacterial type IIS
restriction endonuclease consisting of an N-terminal DNA-binding domain
and a non-specific DNA cleavage domain at the C-terminal. Each ZFNs
composed of two functional domains i.e., DNA-binding and DNA-cleavage
domains derived from the FokI restriction enzyme. Each ZFNs have three
nucleotide recognizes one amino acids. The zinc DNA binding domain
attach to FokI restriction enzyme by linker sequence which targeted at that
specific site on DNA and induced double stranded breaks that lead to both
targeted mutagenesis and targeted gene replacement at remarkably high
frequencies [6].
3. Transcription Activator-Like Effector Nucleases (TALENs)
The TALENs are bacterial proteins that were injected into infected plant
cells via a type III secretion system by pathogens of the genus Xanthomonas.
In 1989, these proteins were first time reported by Ulla Bonas in
Xanthomonas oryzae. These proteins were typically composed of N-terminal
translocation domain, a central repeat domain that mediated binding to the
DNA and a C-terminal transcriptional activation domain. In the cell, these
factors recognize DNA targets in the host genome and activate the
expression of genes necessary for pathogen multiplication and spread. The
N-terminal region was used for transcription secretion whereas central repeat
domain was used for attachment of site-specific sequence DNA. It have
Repeat Variable Diresidues (RVD) that provide specificity for binding with
DNA, present at 12th & 13th position of amino acid in 34 amino acid
sequences that provide restriction for binding on DNA at once. Each
TALENs have one nucleotide recognizes one amino acids. The C- terminal
region contains nuclear localization signals and an acidic activation domain.
As similar to ZFNs, TALENs have two domains i.e., DNA-binding and
DNA-cleavage domains which targeted at that specific site on DNA and
induced double stranded breaks that lead to both targeted mutagenesis and
targeted gene replacement at remarkably high frequencies [7].
Page | 6
4. Clustered regularly interspaced short palindromic repeats
(CRISPR-Cas9)
CRISPR-Cas9 belongs to a family of DNA sequences found in the
genomes of bacteria and archaea. These were first discovered downstream of
the alkaline phosphatase isozyme gene (iap) in Escherichia coli. The
palindromic repeats are separated by short (32 to 36 bp) sequences derived
from the DNA of viruses that have previously infected the cell or its
predecessors. The CRISPR systems have evolved as an adaptive immune
system for Archaea and bacteria and are broadly classified into class I and II
based on the type of effector molecules. They are further grouped into six
distinct types (I-VI) according to the architecture of CRISPR/Cas (CRISPR-
associated) loci and type of Cas proteins with crRNA for CRISPR
interference. Type II CRISPR system derived from Streptococcus pyogenes
is the most widely used genome editing tool that uses a distinct DNA
endonuclease, Cas9 to recognize dsDNA substrates and have been developed
as versatile genome-editing tools for a wide variety of potential applications.
It has two domain DNA-binding which contained Protospacer Adjacent
Motif (PAM), single guide RNA (sgRNA) and DNA-cleavage domain which
had Cas-9 protein that can be targeted to a specific genomic sequence by an
easily engineered 20 base pairs (bps) RNA guide sequence and cut at that
specific site. CRISPR-Cas9 induced double-strand breaks which are subject
to cellular DNA repair processes that lead to both targeted mutagenesis and
targeted gene replacement at remarkably high frequencies.
Table 1: Different Cas proteins and their function
Protein Distribution Process Functions

Spacer DNAse, not sequence specific, can bind RNA;
Cas1 Universal
acquisition present in all types.
Spacer specific to U-rich regions; present in all Types.
Cas2 Universal
acquisition
Type I Target DNA helicase, endonuclease.
Cas3
signature interference
Spacer RecB-like nuclease with exonuclease activity
Cas4 Type I, II
acquisition homologous to RecB.
crRNA RAMP protein, endoribonuclease involved in
Cas5 Type I
expression crRNA biogenesis; part of CASCADE.
Cas6 Type I, III
Cas7 Type I
Cas8 Type I crRNA Large protein with McrA/HNH-nuclease
Page | 7
expression domain and RuvC-like nuclease; part of
CASCADE.
Large multidomain protein with McrA-HNH
Type II Target nuclease domain and RuvC-like nuclease
Cas9
signature interference domain; necessary for interference and target
cleavage.
crRNA HD nuclease domain, palm domain, Zn ribbon;
Type III
Cas10 expression and some homologies with CASCADE elements.
signature
interference
Fig 1: Types of genome editing technologies

Table 2: Comparison among different site-specific nucleases
Typical features Mega nucleases ZFNs TALENs Cas9

Size of encoding
~1 Kb ~1 Kb ~3 Kb ~4.2 Kb
cDNA
Target
recognition
Target DNA- ZFN domains
domain TALE domains sgRNA fused
binding domain fused with
(LAGLIDADG) fused with FokI with Cas9
and nucleases FokI
with homodimers endonuclease nucleases
cleavage endonuclease
or hybrid
endonuclease
Polymeric state Acts as dimers Acts as dimers Acts as dimers Acts as
Page | 8
monomer
Nucleotide length 20 bp
of recognized (additional 3
14 to 40 bp 18 to 36 bp 30 to 40 bp
extended DNA bp PAM
sequences motif)
High (largely
Frequency of
Low High High depends on
targeting sites
PAM motif)
Easy to
deliver (using
Smaller in size,
Delivery of Not feasible to direct delivery
which makes it Easy to deliver
nucleases deliver of sgRNA-
easier to deliver
Cas9
complex)
Table 3: Advantages and limitations of site-specific nucleases
Site specific
Advantages Limitations
nuclease system
• It has smallest nucleases- • Difficult to manipulate the
Meganucleases comprising only 165 amino DNA binding site.
acids size. • It has small recognition site.
• High specificity • Non-modular, difficult and
• Low immunity laborious engineering process
• Difficult to predict cytotoxic
target effects.
• Pair of ZFNs required to target
Zinc Finger specific DNA sequence.
Nuclease (ZFNs) • Some nucleotide triplets do not
have corresponding zinc
finger.
• Site specific insertion of gene
drives.
• It has limited target sites.
• Fusing multiple TALE • 5’ base of TALENs target site
Transcription domains does not affect must be a thymine.
Activator like binding specificity. • Off target effects.
Effector Nucleases • Similar targeting efficiency • TALENs binding is negatively
(TALENs) with less cytotoxic effects. impact by DNA methylation.
• Relatively low in efficiency.
• Easy to generate via simple • Requirement for a PAM
molecular cloning sequence adjacent to target
Cluster Regularly
techniques site.
Interspaced Short
• Higher target efficiency • Higher off target effects.
Palindromic
Repeats (CRISPR)/ • Ability to multiplex for • Binding efficiency impacted
targeting of multiple genes. by chromatin accessibility.
Cas9
• Less time consuming
compared to ZFNs or
Page | 9
TALENs.
• Simultaneous introduction
of double stranded breaks
at multiple sites and that
can be used to edit several
genes at the same time.
Table 4: List of insects genes successfully modified by using the genome editing
technologies
Sr. Genome editing

Insect Target gene Gene function Reference
No. technologies
To develop a new
silkworm strain with a
high degree of
[8]
1. CRISPR/Cas9 Silkworm KO resistance to Bombyx
mori nuclear
polyhedral virus
infection.
It replaced the
silkworm fibroin
heavy chain gene
(FibH) with the major [9]
2. TALENs Silkworm FibH
ampullate spidroin-1
gene (MaSp1) from
the spider Nephila
clavipes
It implicated in
Aedes olfaction and host [10]
3. ZFNs Null gene
aegypti finding capability in
mosquito
Its essential role in the
CRY2
Danaus functioning of the [11]
4 ZFNs (Cryptochrome
plexippus circadian clock in the
gene)
D. plexippus.
It shows anomalous
segmentation and
Spodoptera Abdominal-A [12]
5. CRISPR/Cas9 ectopic pigmentation
litura (Slabd-A) gene
during the larval
stage.
BmBLOS2
(Biogenesis of
It controls the
lysosome-
ZFNs and formation of uric acid [13]
6. B. mori related
TALENs granules in the larval
organelles
epidermis
complex 1,
subunit 2 gene)
[14]
7. TALENs B. mori BmOR1 gene The Odorant receptors
Page | 10
(ORs) for sex
pheromone substances
have been identified
as sex pheromone
receptors in various
moth species.
4. Why CRISPR/Cas9 is advantages over other genome editing

technology in insects????
The CRISPR/Cas9 system is highly versatile, efficient and affordable
tool for targeting chosen sites in the genome. It cleaves a specific sequence
using the Cas9 nuclease coupled with a single guide RNA (sgRNA),
inducing double stranded DNA breaks at target genomic loci of insect that
are repaired either by non-homologous end joining or by homologous
recombination in the presence of homologous donor template DNA. It is not
only limited to deliver it to cells and to select mutation efficiently in the
target cell. This gene editing system has been used to precisely edit genomes
of numerous eukaryotic organisms, including insect orders of Coleoptera [15],
Diptera, Lepidoptera [16] and Hemiptera. Thus, this gene editing technique
facilitates functional genomic studies of model and non-model species and
the development of novel genetics-based pest control approaches.
5. Application of genome editing in entomology
Insects are one of the daunting limitations to agricultural productivity
there is a need to extensive exploration of genome editing based
technologies in agricultural and medicinal insect pest management. Among
the genome editing systems, in particular the CRISPR/Cas9 system have
new opportunities to manage insect pests because of its relative simplicity,
higher target efficiency and less time consuming etc.
Fig 2: Application of genome editing technologies in entomology
Page | 11
5.1 Suppression of pest population
For reducing the pest population and damage of insects in field, the
scientists are developing different genetic pest control strategies. In that,
sterility-based systems such as the Sterile Insect Technique (SIT) reduced
the pest population by using radiation and mutagenic chemicals. The insects
treated with certain x-rays or gamma radiation are unable to produce a
normal number of living progeny. These sterile males or female mated with
normal population leads to interference of reproduction in insects occurs.
However, there is some disadvantages by using this techniques i.e., cost of
production is more, require reliable supply of sterile insects and sterile
insects should not inflict direct damage. The new, precision-guided sterile
insect technique (pgSIT) has developed recently in Drosophila that alters key
genes that control insect sex determination and fertility [17]. In this technique,
the CRISPR to simultaneously disrupt key genes that control female viability
(Fightless) and male fertility in pest species then they produce sterile male
progeny with 100 percent efficiency. The sterile eggs are then shipped to a
pest location virtually anywhere in the world, circumventing the need for a
production facility on-site. Once the eggs are deployed at the pest location,
the newly born sterile males will mate with females in the wild and be
incapable of producing offspring, driving down the population. That novel
twist makes it extremely portable from one species to another species to
suppress populations of mosquitoes or agricultural pests. In the future, pgSIT
may provide an efficient, safe, scalable, and environmentally friendly
alternative next-generation technology for wild population control of
disease-spreading mosquitoes resulting in wide-scale prevention of human
disease transmission.
5.2 Gene drive
It refers to gene editing technology that enhances the probability that a
specific gene will be inherited by an organism’s offspring by bias
inheritance. These has been identified as a potential new biotechnological
strategy to insect pest management, especially for disease vectoring insects
and potentially for agricultural 15 insects and other invasive insect species.
Gene drive technology works by tricking the rules of natural inheritance. In
sexual reproduction, when one set of chromosomes contain a gene drive, it
dominates over the other partner’s genetic makeup and drives the gene flow
to be inherited with greater frequencies than those predicted by Mendelian
inheritance. This “super Mendelian inheritance” allows passage of such
genes from one generation to another even when they reduce the fitness of
the particular organism. Gene drive technology on its own, without the
Page | 12
CRISPR-Cas9 system, is not as effective and vice versa. Gene drive and
CRISPR-Cas9 are basically two separate technologies with the same
objective, which is making gene alterations using gene editing technology to
create populations of organisms that carry a desired/altered gene. For
example in Anopheles gambiae the CRISPR–Cas9-targeted disruption of the
intron 4-exon 5 boundary aimed at blocking the formation of functional
AgdsxF did not affect male development or fertility, whereas females
homozygous for the disrupted allele showed an intersex phenotype and
complete sterility. The knock out of dsx in A. gambiae sex determination,
represent a substantial step toward the development of effective gene drive
vector-control measures that aim to suppress insect vector populations to
levels that do not support malaria population [18].
Fig 2: Schematic representation of gene drive in insects

5.3 Alteration and reduction of pest population
By using this genome editing technologies, scientists employed in a
two-step strategy the alterations of target insects, and their subsequent
release into the natural environment that leads to reduce the pest population
below the ETL level in the field. For managing insect population, different
genome editing in insects was taking place in that disruption of host plant
volatile, mating partner identification and deformation of body segments,
prolegs embryonic lethality and anomalous gonads. In insects, the olfactory
receptors (ORs) are important for the recognition of host plant as well as
Page | 13
mating partner odorant. Orco forms heterodimers with olfactory receptors
(ORs) that act as odor-gated ion channels at the membrane of olfactory
sensory neurons. By using CRISPR/Cas9 technology that target a gene that
could interrupt the chemical communication of this species, the receptor co-
receptor-1. CRISPR/Cas9-mediated Orco knockout caused defects in plant
odor and sex pheromone olfactory detection in homozygous individuals
would impact mate detection of lepidopteran pest (S. littoralis) [19]. The sex
hormone releases from female that will attract the male moth. By knocking
out of this Orco mutant, the male moth unable to detect the female moth,
thus resulting in mating with immature females that subsequently leads to
dumping of sterile eggs. This technique development may lead to the long
term novel pest control strategies in agricultural crops. Similarly, knock out
of the Orco gene in migratory locust through CRISPR/Cas9 severely
impaired electrophysiological and olfactory behavioral responses [20]. The
Orco mutant locusts lost an attraction response to aggregation pheromones
under the crowding conditions. By adoption of this technologies will be a
potential option to keep insect pests away from the crops and prevent insect
damage.
Most of the herbivores insects species are specialist feeding on a small
number of related host plants. These specialist typically display rapid and
efficient mechanisms for detoxification and sequestration of specific toxins
of their phytochemically similar host plants. The multifunctional activities of
cytochrome P450 monooxygenases in phase 1 reactions of insect
detoxification contribute to biochemical defense mechanism against natural
and synthetic toxins in insect from host phytochemicals to synthetic
insecticides. The CYP6AE gene responsible for detoxification of insecticides
in insect. To evade this, resistance developments in insects efforts are being
made to engineer receptors such that resistance management can be
efficacious. The CRISPR/ Cas9 system provides a promising tool for genetic
manipulation of P450 and CYP6AE genes. Knock out of CYP6AE gene
cluster in H. armigers proved the role of these enzymes in detoxification of
various toxic phytochemicals [22]. Another approach, is knocking out of
developmental genes such as abd-A (Abdominal A) gene, a transcriptional
factor involved in downstream regulation of various target genes that are
extensively involved in development. The CRISPR/Cas9 system was used to
edit the sequence of abd-A mutants in S. litura, P. xylostella and S.
frugiperda that produces deleterious phenotypes such as malformed
segments, abnormal prolegs, anomalous gonads and embryonic lethality [21].
This CRISPR/Cas9 system may be employed to convert these potential
heterozygous mutations to homozygous ones. The utilization of this
Page | 14
technology, may be a viable control approach to lepidopteran populations
have developed resistance to multiple classes of insecticides and Bt corn. In
this way, this technology has potential to accelerate the study of resistance
mechanisms and the development of innovative genetics-based control
strategy.
5.4 Modification of plant genes
The genome editing technology in plants for insect pest management has
been less exploited. Most of the polyphagous pests recognize host plants
using the plant’s own volatile blends, gustatory clues, oviposition sites,
visual appearance and their interactions are coevolved. The insect prefer to
lay eggs on host plant based on the availability of food source for their
young ones. Plant volatile blends are a mixture of volatiles, of which only a
few are recognized by insects as clues for host selection and oviposition site
detection. Plants have evolved the defensive strategies depending on the
pest. The plants produces different secondary metabolites like salicylic acid,
jasmonic and ethylene. Alteration of plant volatile blends through genome
editing can be an alternative strategy in pest management. The care should
be taken such that the modification in plants will not lead to deleterious
effects toward beneficial insect population. Visual appearance of plants plays
a prominent role in the ability of insects to recognize and attack host plants.
Alteration in plant pigmentation has been found to modify insect host
preferences. The insect utilize their host plants based upon the concentration
of proximate nutrients, especially proteins and the concentration of
allelochemicals. The plant variability with nutrient factor is important factor
affecting the dietary nature of plant nutrients for insects. This phenomenon
was reported in red leaf tobacco, a transgenic that was developed by the
modification of anthocyanin pathway. The study demonstrated that
overproduction of anthocyanin pigmentation resulted in the red coloration of
leaves in the transgenic tobacco plant. This change in leaf color proved to be
acting as a deterrent to the herbivores, S. litura and H. armigera confirming
the importance of leaf color and appearance on host recognition in insects
[23]
. The deterrence of insects from plants due to change in the secondary
metabolite substance and visual appearance will be possible approach for
genome editing for pest management.
6. Conclusion
Genome editing has great potential in overcoming the limitations of
conventional resistance breeding. Among the genome editing technologies,
CRISPR-Cas9 is showing high revolution due to their facile engineering,
Page | 15
robustness and efficiency in the functional genomics of insects. The
knockout of key regulators in genome editing would serve as a futuristic
approach to combat insect pests. In addition, regulatory and ethical
considerations may limit the wide applications of these technologies.
Keeping in view of past experiences, there is a need to improve the
technology to avoid regulatory hurdles and ensure that its fruits are within
reach for the poor and for subsistence farmers. Genome-editing technologies
are poised to reshape the future of plant agriculture and food security to feed
the world’s burgeoning population.
7. References
1. Belfort M. Tyagi S, Kesiraju K, Saakre M, et al. Genome editing for
resistance to insect pests: An emerging tool for crop improvement. ACS
omega. 2020;5(33):20674-20683.
2. Reid W, O’Brochta DA. Applications of genome editing in insects.
Current Opinion in Insect Science. 2016;13:43-54.
3. Montecillo JAV, Chu LL, Bae H. CRISPR-Cas9 system for plant
genome editing: current approaches and emerging developments.
Agronomy. 2020;10(7):10-33.
4. Randhaw S, Sengar S. The evolution and history of gene editing
technologies. Progress in Molecular Biology and Translational Science.
2021;178:1-62.
5. Belfort M, Bonocora RP. Homing endonucleases: from genetic
anomalies to programmable genomic clippers. Homing Endonucleases.
2014;1:26.
6. Rémy S, Tesson L, Ménoret S, Usal C, Scharenberg AM, Anegon I.
Zinc-finger nucleases: a powerful tool for genetic engineering of
animals. Transgenic Research. 2010;19(3):363-371.
7. Mounadi K, Floriano ML, Garcia-Ruiz H. Principles, applications, and
biosafety of plant genome editing using CRISPR-Cas9. Frontiers in
plant science. 2020;11:56.
8. Chen S, Hou C, Bi H, Wang Y, Xu J, Li M, et al. Transgenic clustered
regularly interspaced short palindromic repeat/Cas9-mediated viral gene
targeting for antiviral therapy of Bombyx mori Nucleo polyhedral virus.
J Virol. 2017;91(8):16-20.
9. Xu J, Dong Q, Yu Y, Niu B, Ji D, Li M, et al. Mass spider silk
production through targeted gene replacement in Bombyx mori. Proc
Natl Acad. Sci USA. 2018;115(35):8757-8762.
Page | 16
10. Liesch LL, Bellani LB, Vosshall. Functional and genetic
characterization of neuropeptide Y-like receptors in Aedes aegypti. Plos
Negl Trop Dis, 2013, 7.
11. Merlin C, Beaver LE, Taylor OR, Wolfe SA, Reppert SM. Efficient
targeted mutagenesis in the monarch butterfly using zinc finger
nucleases. Genome Research. 2013;23:159-158.
12. Bi HL, Xu J, Tan AJ, Huang YP. CRISPR/Cas9-mediated targeted gene
mutagenesis in Spodoptera litura. Insect Sci. 2016;23(3):469-77.
13. Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, et
al. Targeted mutagenesis in the silkworm Bombyx mori using zinc finger
nuclease mRNA injection. Insect biochemistry and molecular biology.
2010;40(10):759-765.
14. Sakurai T. Targeted disruption of a single sex pheromone receptor gene
completely abolishes in vivo pheromone response in the silkmoth.
Scientific reports. 2015;5:1-11.
15. Gilles AF, Schinko JB, Averof M. Efficient CRISPR-mediated gene
targeting and transgene replacement in the beetle Tribolium castaneum.
Development. 2015;142(16):2832-9.
16. Li X, Fan D, Zhang W, Liu G, Zhang L, Zhao L, et al. Outbred genome
sequencing and CRISPR/Cas9 gene editing in butterflies. Nat Commun.
2015;6:8212.
17. New CRISPR-based technology developed to control pests with
precision-guided genetics. Science Daily. University of California-San
Diego, 2019 Jan.
18. Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt Anti, et al. A
CRISPR-Cas9 gene drive targeting double sex causes complete
population suppression in caged Anopheles gambiae mosquitoes. Nature
Biotechnology. 2018;36(11):1062-1066.
19. Koutroumpa FA, Monsempes C, François MC, De Cian A, Royer C, et
al. Heritable genome editing with CRISPR/Cas9 induces anosmia in a
crop pest moth. Scientific Reports. 2016;6(1):1-9.
20. Li Y, Zhang J, Chen D, Yang P, Jiang F, et al. CRISPR/Cas9 in locusts:
successful establishment of an olfactory deficiency line by targeting the
mutagenesis of an odorant receptor co-receptor (Orco). Insect
Biochemistry and Molecular Biology. 2016;79:27-35.
Page | 17
21. Wu K, Shirk PD, Taylor CE, et al. CRISPR/Cas9 mediated knockout of
the abdominal-A homeotic gene in fall armyworm moth (Spodoptera
frugiperda). PLoS ONE. 2018;13(12):1-16.
22. Wang H, Shi Y, Wang L, Liu S, et al. CYP6AE gene cluster knockout
in Helicoverpa armigera reveals role in detoxification of
phytochemicals and insecticides. Nature Communications. 2018;9(1):1-
8.
23. Malone LA, Barraclough EI, Lin-Wang K, Stevenson DE, Allan AC.
Effects of red-leaved transgenic tobacco expressing a MYB transcription
factor on two herbivorous insects, Spodoptera litura and Helicoverpa
armigera. Entomol. Exp. Appl. 2009;133(2):117-127.
Page | 18
Chapter - 2
Remote Sensing Application in Crop Pest
Management
Authors
Tumma Mounika
Research Scholar, Department of Agricultural Entomology,
Uttar Banga Krishi Viswavidyalaya, Pundibari, West Bengal,
India
Tapan Kumar Hath
Professor, Department of Agricultural Entomology, Uttar
Banga Krishi Viswavidyalaya, Pundibari, West Bengal, India
Page | 19
Page | 20
Chapter - 2
Remote Sensing Application in Crop Pest Management
Tumma Mounika and Tapan Kumar Hath
Abstract
Agriculture is the back bone of India. The production of agriculture is
manly affected by biotic factors such as predators, parasites, pathogens and
aboitic factors like temperature, humidity, rainfall etc. Monitoring and
forecasting of the crop pest has been the integrated part of the crop
production system. Remote sensing in recent times became the popular tool
which provides the data for timely management of the potential pest. It aims
in being as a bridge gap between the existing systems securing a regular
information flow about the areas affected by the pests and diseases and other
yield reducing factors on the nationwide basis. Hence recent advancements
in the field of remote sensing provides an ample scope to use the technology
in agriculture for pest monitoring, detecting and timely management with
high precision.
Keywords: Remote sensing, forecasting, monitoring, technology
Introduction
Agriculture is the dominated sector of Indian economy which
contributes 17% of GDP (Vardhan et al., 2022). Nearly 49% of population in
India are involved in Agriculture and allied sectors. The production of
agriculture is significantly affected by some factors i.e., biotic and abiotic
factors. The biotic factors such as crop pests, predators, parasites, pathogens
where as abiotic factors like temperature, humidity, rainfall etc. among them
insect’s pest’s cause’s enormous losses at production and storage level. In
the developed countries, to overcome and manage the problem of pests,
forecasting and monitoring has been an integrated parts of the crop
production system. Several technologies have been developed for monitoring
of pests, yield forecasting, for timely management of potential pests and
early warnings to the crop growers. Among them remote sensing technology
has become popular. This paper gives a basic knowledge regarding the
functioning of remote sensing and its application in agriculture with main
emphasis on pest management.
Page | 21
What is remote sensing?
Remote sensing implies sensing from a distance. It is a non-contact
measurements of radiations reflected and emitted from the agricultural fields
due to the interactions of electromagnetic radiations with soil and plant
material. It is an outgrowth of aerial photography. The Term “Remote
sensing” was first time introduced by Evelyn L. Pruitt of U.S. Office of
Naval Research in the year 1960 (Baumann, 2014).
Remote sensing incorporates new technologies which provide efficient,
complete, accurate and timely information. These new technologies together
with historical photographs and provides the information as a practical
management tool for site specific management of crops. With the launch of
Earth Resource Technology Satellite (ETRS)-I the supply of remote sensing
data has been increased (Aggarawal, 2004). Remote sensing along with other
technologies such as the Global positioning systems (GPS), Geographic
Information Systems (GIS) and Variable Rate Technology (VRT) that
scientist can implements to help the farmers in maximizing the economic
and environmental benefits of area-wide pest management through precision
agriculture (Huang et al., 2008).
Components and functioning of remote sensing
Electromagnetic energy is utilized for the remote sensing.
Electromagnetic energy refers to the energy that moves with the velocity of
light in a harmonic wave pattern. Electromagnetic energy ranges from short
wavelength ultra violet through visible, near infrared and thermal infrared to
longer wavelength active radar and passive microwave systems.
Page | 22
The wave concept explains the propagation of Electromagnetic energy,
but the electromagnetic energies are detectable only when it interacts with
the matter. In this interaction, the emitted radiations or reflected/scattered in
different parts of electromagnetic spectrum are detected, recorded, measured
and analyzed using visual of digital analysis techniques. Using different
kinds of sensors of electromagnetic radiation (EMR), from aerial
photographs and satellites, one can remotely collect data and analyze them to
obtain information about the object or phenomenon sensed. Every object is
having characteristic spectral signatures i.e. reflectance/ scattering or
emittance from objects in different spectral bands which vary depending
upon their structure, nature of the surface and temperature. The analysis is
based on the spectral radiances observed in different bands which facilitates
in the data acquisitions and identification of different objects.
Fig 1: Working of a camera with flash gun (Process of data acquisition)

Types of remotes sensing
Remote sensing has been recognized as a valuable tool for viewing,
analysing, characterizing and making decisions about the environment.
Remote sensing technology has advanced on three fronts (Levin, 1999):
1) From military uses to a variety of environmental analysis
applications that relate to land, ocean and atmosphere issues.
2) From photographic systems to sensors that convert energy from
many parts of the electromagnetic spectrum to electronic signals.
3) From aircrafts to satellite platforms
Conventional remote sensing is of two types:
i) Aerial photography
ii) Satellite imagery
Page | 23
Aerial photography
It is the oldest form of remote sensing. Despite of increasing availability
of more sophisticated imaging systems, aerial photographs remains one of
the most reliable and widely used sources of remote sensed data. The first
aerial photography was taken in the year 1858 before the term remote
sensing came into existence. USDA operationally started using it from
1930’s to record land use and for soil mapping. Here the photographs are
taken from an aircrafts flying at a particular heights (Baumann, 2014), with
the axis of the aerial camera kept vertical, horizontal or oblique. For
environmental survey purpose vertical photography are suitable for obtaining
uniform coverage.
Fig 2: Aerial photography by aircraft

Aerial photography remains a powerful tool to use for both routine and
specialized applications. Two variants of aerial photos are obtained i.e.,
black and white or colour, are the vertical (sees Earth straight downward)
and oblique (sees at various angles, usually less than 45°). Photography
commonly takes place in the visible spectra region but the film sensitive to
the very near infrared or ultra violet allows image taking in those spectral
regions. For the management of biosphere reserve aerial photography type of
remotes sensing is most important. The advantages of aerial photography are
superior resolution, relative simplicity of photography and film processing,
relatively low-cost equipment and providing considerable amount of
information. But the disadvantage is the range of sensitivity confined by the
film emulsion technology to visible and near infrared regions (0.4 to 1μm).
Page | 24
Fig 3: Oblique view of aerial photgraphy
Satellite imagery
The satellite imagery is also called as earth observation imagery, space
borne photography. Here many satellites with remote sensing instruments are
involved in taking the photographs. Satellites can cover the more land space
than planes and monitor the areas on regular basis. With the advancement of
the technology the images became digital in format rather than analogue.
The digital format displays and analyse the images of earth which were
recorded by sensors in several different portions of the electromagnetic
spectrum. Satellite imagery technology made possible to see the things
occurring on the surface of the earth that looking at a normal aerial
photography one could not detect (Baumann, 2009).
Fig 4: Satellite imageries

The satellite scans the earth line by line in many discrete light ranges in
the visible and thermal portions of the spectrum (0.3 to 14μm). A scan line
consists of several measurement values representing the energy reflected or
emitted from the surface area. The values are recorded on the magnetic tapes
which can be directly used in a computer for analysis. A computer can
handle multispectral data simultaneously and make decisions about
identification of surface features using statistical approaches.
Page | 25
Fig 5: Satellite scanning the earth
Application of remote sensing
Remote sensing technology has amply demonstrated its efficacy in
making resources inventories, mapping and monitoring of natural/ physical
resources. This leads further to invaluable information concerning the
characters, distribution, productivity and use of natural resources which is
essential for optimum management of our physical resources. Periodic
inventories of our natural resources both renewable and non-renewable,
enables us to plan the judicious management and utilization.
The major areas of application of remote sensing are
1) Agriculture including crop protection
2) Forestry
3) Mineral resources
4) Water resources
5) Oceans and costal resources
6) Land use including urban land use
7) Environmental monitoring
8) Cartography
Application of remote sensing in crop protection:
Remote sensing is considered as a fast, relatively cost effective and non-
destructive method to study the biophysical and biochemical parameters of
vegetation across the vast areas (Ngie et al., 2014). Remote sensing
(including aerial photography and satellite imagery) helps in obtaining the
baseline information for land use and other spatial planning in areas where
maps are not available and provides input for the modelling of alternative
land use options (i.e., Agriculture or Biological conservation).
Page | 26
Remote sensing offers great promise for the detection of insect pests,
diseases of insect pests, diseases and nematodes which causes damage to the
crop plants since it affords an opportunity to survey large areas rapidly,
which is considered in saving the manpower and expenses. Remote sensing
involves in the responses to a particular conditions or situations that arises
during course of season such as insect pest management. It detects the
emerging pest population in the field and involves in collecting the images
frequently and comparing them with previous images.
Feeding injury by the insects on foliage causes discolouration of the
foliage, geometric distortion of leaves. Depending on what portion of the
plant is been subjected to stress the damage intensity can vary. The
quantitative measurement of the degree of damage can be made and can
possibly be correlated with the density of insect infestation.
Remote sensing of insect pests of fruit trees was pioneered in 1968,
especially in citrus, detecting the insect infestation using presence of sooty
mould. The use of remote sensing in agriculture in India started with the
detection of wilt disease in coconut at Kerala Coast (Dakshinamurty et al.,
1971). But later main emphasis was shifted to crop acreage estimation, crop
condition assessment and yield forecasting using different crop simulation
models (Shin et al., 2009; Basso et al., 2010; 2012).
Some examples are illustrates the importance of remote sensing
programmes in studying the various aspects of insect life which help in
formulating their management programmes.
a) Sooty mould producers
Sooty mould production occurs due to honey dew, a highly
proteinaceous material. The deposition of honey dew on the foliage gives a
glistering effect. This deposition serves as a host of sooty mould fungus
(Capnodium citri). The black hypae spreads across the surface of the foliage
and covers as a thick mat. Reflectance measurement showed that citrus
blackfly, brown soft scale and citrus mealy bug deposits thinnest,
intermediate and thickest sooty moulds on citrus leaves. The remotes sensing
with photography helps in early infestation by three insects (Gausman and
Hart, 1974). Aphids have been detected by using very powerful 10cm radars
upto 1200m above the ground level.
Page | 27
b) Ants
Ant mounds considerably vary in its shape, colour and structure.
Remote sensing helps in detection and separation of species. The harvester
ants, destroys a variety of crop plants in range lands and other turf areas. It
destroys grass in pastures by turning up the soil in mound building activities
and interfere with reseeding of pasture lands. The mounds that makes home
of this harvester ants are flat and very circular. The entrance hole at the
centre provides a dark spot. Aerial colour infrared photographs clearly show
the distinct circular pattern with the dark spot at the centre (Hart et al.,
1971).
Page | 28
c) Crown distortion
The larvae of root borer, Prinus spp. Feeds on the roots of pecan trees;
roots are gridled and sometimes severed by their activities (Payne et al.,
1971). Heavy feeding leads to the death of limbs of pecan tree. Aerial survey
using Kodak Agrochrome Infrared 2443 films produced photographs
showing a finger like appearance where shadows of the infested trees
occurred.
For forcasting and monitoring of the desert locust, Schistocerca
gregaria infestation over large areas of Africa and Asia, RS data is used
which is based on the canopy green biomass of LAI (leaf area index).
d) Colour changes of foliage
European red mite, (Panonychus ulmi) which damages the foliage of
peach trees by sucking juices from the leaves. Reduction of chlorophyll
causes a loss of green colour. When injury becomes serious, the upper leaf
surface develops a bronze or silver appearance. The aerial colour infrared
photograph of a peach orchard with the above-mentioned mite showed that
infected trees had a ragged appearance and were lighter red than the non-
infested trees.
Traditional verses remote sensing methods of agricultural surveys
Remote sensing in agriculture involves extremely complex crops and
soils because of their dynamic and inherent complexity of biological
materials and soils. Remotes sensing offers a numerous advantages over
traditional methods in conducting agricultural and resource surveys.
Advantages
1) Capability of achieving a synoptic view under relatively uniform
lighting conditions.
2) Potential for accelerate.
3) Availability of multispectral data providing a lot of information.
4) Capability of repetitive coverage to depict seasonal and long term
changes, the relative inexpensive cost of monitoring from space.
5) The opportunity of integrating existing survey into updated
monitoring system.
6) The change detection capabilities needed by regulatory programmes
for updating the information on vegetation/terrain conditions.
7) Availability of imagery with minimum distortion, thereby
permitting direct measurement of important agro-physical
parameters.
Page | 29
8) The fact that remotely sensed data provides a permanent record.
Limitations
1) The main limitations in detecting and monitoring pests and plant
diseases are the problems of identification of the real cause of the
spectral variations in plants. Finding out spectral response changes
caused by disease in control conditions and its extra polation to
natural environment is of primary importance. It is also important to
find out the variations in spectral response due to pests and diseases
and other stresses.
2) The other formidable limitations being faced in operationalisation
of remotes sensing technology in India is the non- availability of the
cloud free data of desired data. This problem is particularly acute
for kharif crops.
Conclusion
Remote sensing help in providing the information regarding the crop
condition, detection of pest development in the remote and inaccessible
areas. It aims in being as a bridge gap between the existing systems securing
a regular information flow about the areas affected by the pests and diseases
and other yield reducing factors on the nationwide basis. Forecasting of the
insects pests in agro-ecosystem’s provides an opportunity to inform the
farmers about possible out breaks for preparedness and taking of timely
actions to apply biocontrol agents, mechanical means and pesticides, which
ultimate decrease the cost of cultivation and serves as a tool in precision
farming. Hence recent advancements in the field of remote sensing provides
an ample scope to use the technology in agriculture for pest monitoring,
detecting and timely management with high precision.
Reference
1. Basso B, Cammarano D, Troccoli A, Chen D, Ritchie JT. Long-term
wheat response to nitrogen in a rainfed Mediterranean environment:
Field data and simulation analysis. European Journal of Agronomy.
2010;33:132-138.
2. Basso B, Fiorentino C, Cammarano D, Cafiero G, Dardanelli J. Analysis
of rainfall distribution on spatial and temporal patterns of wheat yield in
Mediterranean environment. European Journal of Agronomy.
2012;41:52-65.
3. Baumann PR. History of remote sensing, Satellite Imagery Part II, 2009.
Available:
Page | 30
http://www.oneonta.edu/faculty/baumanpr/geosat2/RS%20History%20I
I/ RS-History-Part- 2.html (Retrieved 15 April 2015).
4. Baumann PR. History of remote sensing, Aerial Photography Part I,
2014. Available: http://www.oneonta.edu/faculty/baumanpr/geosat2/
RS%20History%20I/ RS- History Part- 1.html (Retrieved on 15 April
2015).
5. Dakshinamurty C, Krishnamurthy B, Summanwar AS, Shanta P,
Pisharoty PR. Remote sensing for coconut wilt. In: Proceedings of 6th
International Symposium on Remote Sensing of Environment.
Environmental Research Institute at Michigan, Ann Arbor, U.S.A.,
1971, 25-29.
6. Gausman HW, Hart WG. Reflectance of four levels of sooty mold
deposits produced from the honey dew of three insects species. J Rio
Grande Valley Hort. Sci. 1974;28:131-136.
7. Hart WG, Ingle SJ, Davis MR, Mangum C, Higgins A, Boling JC. Some
uses of infrared aerial color photography in entomology. Proc. 3 rd Bienn.
Workshop color Aerial Photogr. Plant sci. Gainesvielle, Fl, 1971 March,
99-113.
8. Huang Y, Lan Y, Hoffmann WC. Use of airborne multi-spectral
imagery for area-wide pest management. Agricultural Engineering
International: The CIGR E-journal. Manuscript IT 07 010. 2008
Feb;10:14.
9. Levin N. Fundamentals of remote sensing. 1st Hydrographic data
management course, IMO-International Maritime Academy, Trieste,
Italy Remote Sensing Laboratory, Geography Department, Tel Aviv
University, Israel GIS unit, the Society for the Protection of Nature in
Israel, 1999.
10. Ngie A, Ahmad F, Abutaleb K. Remote sensing potential for
investigation of maize production: Review of literature. South African
Journal of Geomatics. 2014 Aug ;3(2):163-184. Available:
http://www.ajol.info/index. php/sajg/issue/view/11348 (Retrieved 15
April 2015).
11. Payne JA, Hart WG, Davis MR, Jones LS, Weaver DJ, Hortpn BD.
Detection of peach and becan pests and diseases with color infrared
aerial photography. Proc. 3rd Bienn. Workshop color Aerial Photogr.
Plant sci. Gainesvielle, Fl, 1971 March, 216-230.
Page | 31
12. Shin DW, Baigorria GA, Lim YK, Cocke S, La Row TE, O’Brien JJ, et
al., Assessing crop yield simulations with various seasonal climate data.
Science and Technology Infusion Climate Bulletin. NOAA’s National
Weather Service. 7th NOAA Annual Climate Prediction Application
Science Workshop, Norman, OK, 2009 Oct.
Page | 32
Chapter - 3
Management Tactics for Plant Parasitic
Nematodes
Authors
Babita Kumari
Ph.D. Scholar, Department of Nematology, CCS, Haryana
Agricultural University, Hisar, Haryana, India
Sujata
Shweta
Page | 33
Page | 34
Chapter - 3
Management Tactics for Plant Parasitic Nematodes
Babita Kumari, Sujata and Shweta
Abstract
Plant-parasitic nematodes (PPNs) reckoned economically most important
among all the agricultural pests due to their enormous host range, wide spread
occurrence and hidden mode of life cycle. They takes nutrients directly from
the roots and hampers the uptake of food and water which results wilting and
yellowing of plants. Due to their hidden mode of parasitism and root feeding
nature, symptoms caused by PPNs often goes unnoticed. There are several
methods to control nematodes viz., cultural, physical and biocontrol methods.
Cultural methods include crop rotation, fallowing, deep summer ploughing,
use of resistant varieties flooding trap cropping etc. Adoption of single method
to manage nematodes is not satisfactory to keep population of these tiny
organism below ETL (Economic threshold level).
Keywords: Plant-parasitic nematodes, cultural, physical and biocontrol
methods and economic threshold level
Introduction
Plant-parasitic nematodes cause heavy losses in crop production
worldwide. An estimate shows that phytonematodes cause far more damage
each year compared to insect pests. The extent of direct damage by the
nematodes to plants depends on several factors. These includes initial
nematode population, nematode density in soil, nature of parasitism
(ectoparasite or endoparasite, migratory or sedentary), host susceptibility,
cropping pattern, edaphic factors (soil texture, moisture etc.), climatic
conditions, maximum temperature and moisture. The goal of the management
practices is to manage crop losses due to these microscopic organisms and to
get maximum production by least disturbances to microflora and microfauna
of the soil so as to sustain natural balance. Secondly, keep nematode
population below ETL (economic threshold level). The nematode control
methods are:
Page | 35
Cultural control: These approaches are agronomical practices which are
adopted in order to reduce plant-parasitic nematodes problem in crops.
1) Crop rotation: It is universal fact that soil fertility reduces if same
crop is taken on same piece of land in successive manner. This
monoculture leads to extreme exhaustion of a particular essential
nutrients or amassing of organic acids released by monocropping and
most importantly due to continuous provide of favorable food
buildup of plant-parasitic nematodes population. In such conditions,
crop rotation is very helpful. Rotating of susceptible crop with non-
susceptible crop is helpful in reducing the nematode population. Crop
rotation of Tomato with cotton-wheat-cotton resulted in fewer galls
of M. javanica and gave maximum yield (Hashmi and Hasmi, 1990).
Population of M. javanica on tomato crop considerably decreased
when carrot, capsicum and onion crop was included in crop rotation
(Kanwar and Bhatti, 1992). Wheat growing of non-host crop like
gram, mustard, fenugreek and carrot for one year resulted in 50%
decline in H. avenae population after one year and 75% decline after
2 years.
2) Selection of healthy seed material: Plants which are propagated
through vegetative means nematodes can be eliminate by selecting
the disease-free plants for vegetative propagations. The Globodera
rostochiensis pest of potato, Radopholus similis and Pratylenchus
spp. nematodes of banana can be avoid by selecting nematode free
planting materials.
3) Altering the time of planting: Altering the time of planting helps to
reduce nematode damage. When the crops planted in winter time the
soil temperature is low and at that time the nematodes cannot be
active or their life cycle is extended due to low temperature.
4) Fallowing: Leaving the field fallow or without any vegetation for a
specific period, preferably after ploughing helps to expose the
nematodes to direct sunlight and the nematode die due to hunger
without host plant. Population of M. incognita infecting tomato
reduced when infected field was left fallow (Khan et al., 1975).
5) Deep summer ploughing: In month of May-June, when temperature
is high, infested field is ploughed with disc plough and exposed to
sunlight, which leads to increased soil temperature and kills the
nematodes. Three deep summer ploughing each at 10 days interval
in month of June (40-46 oC) at Hisar, Haryana led to 96.5 per cent
reduction in root-knot nematode population (Jain and Bhatti, 1987).
Page | 36
6) Flooding: This practice can be adopted where enormous availability
of water is exists. Under water-logged conditions, anaerobic
condition develops in the soil which kills the nematodes by
asphyxiation. Also, some lethal chemicals like fatty acids, hydrogen
sulphide and ammonia are produced in flooded conditions.
7) Trap cropping: Two crops are grown in the field, out of which one
crop is highly susceptible to the nematode. The nematode attacks the
susceptible crop easily and quickly. The susceptible crop can be
grown prior to main crop and harvested as a fodder or green manure.
Examples: Crotalaria have been used to reduce population of
Meloidogyne spp. other crops are Chrysanthemum and Tagetes.
Marigold crop gave maximum reduction in root-knot nematode
population infecting tomato nursery and increased the germination
rate of seed (Rangaswamy et al., 1999).
8) Antagonistic crops: Crops like marigold, mustard and neem etc.
have chemicals or alkaloids or nematotoxic compounds as root
exudates which don’t allow nematode to penetrate or if invasion
occurs further growth is arrested. In marigold (Tagetes spp.) plants
the α-terthienyl and bithinyl compounds are present all over the plant
from root to shoot tips. These chemicals kill the nematodes.
Table 1: List of plants having active compounds against plant-parasitic nematodes
Sr. Associated active Nematode

Plant species References
No. compounds species
Asparagus Glycoside, Asparagusic Takasugi et al.,
1 -
officinalis acid 1975
Azadirachta Meloidogyne
2 Nimbecidine Lal et al., 2012
indica incognita
3 Brassica nigra Allyl isothiocyanate - Dahlin et al., 2020
2-Phenyl ethyl Globodera Dandurand et
4 Brassica spp.
glucosinolate rostochiensis al., 2017
Meloidogyne
5 Crotalaria Monocrotaline Bratley, 1942
spp., coffeae,
Gloriosa Meloidogyne
6 Colchicine Nidiry et al., 1993
superba incognita
Lanatoside, Linaroside, Meloidogyne
7 Lantana camara Qamar et al., 2005
Carminic acid incognita
Lycopersicum Root-knot
8 Alpha tomatine Elliger et al., 1988
esculentus nematode
Meloidogyne DeViala et al.,
9 Rudbeckia hirta Thiarubrine C
incognita and 1998
Page | 37
Pratylenchus
penetrans
Aspartic acid, Glutamic
acid, Valive, Proline, Meloidogyne
10 Til Tanda et al.,1989
Serine, Glycine and Incognita
Lucine
11 Sorghum Dhurrin - Curto et al., 2012
12 Tagetes erecta Alpha terthienyl, bithienyl D. dipsaci Bakker et al., 1979
9) Removal and destruction of infested plants: Diagnosis of infected

plants and removal is helpful to lessen nematode spread. After crop
harvesting the stubbles of infested plants should be removed from the
main field.
Physical control and mechanical control
It is easy to kill the nematodes in laboratory by means of heat, osmotic
pressure, irradiation etc. These physical management methods may be harmful
to plant or the man working with the treatments. Insects, weed seeds,
nematodes, bacteria and fungi are killed by steam sterilization. In such cases
steam is introduced into the soil through perforated iron pipes buried in the
soil. The soil surface needs to be covered with plastic sheet during this
operation. Transparent sheets of 25 to 30 micron thickness are laid down on
soil in lightly moist condition during summer months. In case of laboratory
and pot culture experiments autoclaves are used for sterilization soil.
Anaerobic soil disinfection: It is also known as biological soil disinfection. In
this technique, organic matter is added to soil and soil is covered by polyether
sheet, upon decomposition these organic matter produces toxic gases which
help in killing nematodes. HWT (Hot water treatment) is commonly used for
controlling nematodes. Irradiation: Cyst of Globodera rostochiensis exposed
to 20,000γ contained only dead eggs and at 40,000 γ exposure, the eggs lost
their contents. Washing process: PPNs are generally spread by soil adhering
to potato tubers, bulbs and other propagating materials. Washing of such
material under tap water helps to avoid the nematodes in spreading to new
planting field.
Table 2: HWT for dramatizing planting material
S. No. Nematode Crop/Plantig material Temperature Time

1. Aphelenchoides besseyi Rice seeds 52oC 10 min
2. Anguina tritici Wheat seeds 54-56 oC 10-15 min
3. Radopholus similis Banana 55 oC 20-25 min
Tylenchulus
4. Grapevine 51 oC 5 min
semipenetrans
Page | 38
5. Meloidogyne javanica Grapevine 50 oC 5 min
6. Ditylenchus dipsaci Narcisus bulb 43.3 oC 4 hrs
7. Ditylenchus dipsaci Gladiolus bulb 50 oC 7 min
8. Ditylenchus dipsaci Onion bulb 43 oC 120 min
9. Radopholus citrophilus Citrus bare root 50 oC 10 min
10. Scutellonema bradys Yam tuber 50-55 oC 40 min
Use of resistant varieties: Several nematode resistant varieties are

available in different agricultural crops.
Table 3: Some important resistant variety against PPNs
S.
Crop Against nematode Resistant variety
No.
1. Barley Heterodera avenae BH- 75, Rajkiran, C-164
Meloidogyne incognita, Black beauty, Pusa purple long,
2. Brinjal
M. javanica Neelkantha
Meloidogyne incognita,
3. Chilli Pusa jwala, Mohini, NP-46 A
M. javanica
4. Citrus Tylenchulus semipenetrans Trifoliate root stock
5. Coffee Pratylenchus coffeae Robusta root stock
6. Cowpea GAU-1, C-152
M. javanica
7. Cotton Meloidogyne incognita Paymaster, Bikaner nerma, Sharda
Meloidogyne incognita, Kishmish, Beli, Cardinal, Early
8. Grapevine
M. javanica Muscat, Loose Perlett
9. Mungbean ML-30, ML-62, ML-80
M. javanica
10. Okra Long green smooth
M. javanica
11. Potato Globodera rostochiensis Kufri swarna
12. Tomato PNR-7, Hisar lalit
M. javanica
13. Tobacco Meloidogyne incognita NC-45
14. Wheat Heterodera avenae Raj MR-1
R-gene
• Hs1pro-1 gene shows resistance against Heterodera schachtii, isolated
from Beta procumbens, a wild species of beet (Cai et al., 1997).
• Cre3 gene isolated from wild wheat Triticum tauschii which shows
reistance to cereal cyst nematode, Heterodera avenae, in wheat
(Triticum aestivum L.), (Lagudah et al., 1997).
Page | 39
• Mi 1.2 gene derived from Lycopersicon peruvianum wild tomato
introduced in Lycopersicon esculentum shows resistance against
Meloidogyne incognita, M. javanica and M. arenaria, but not against
M. hapla (Williamson, 1998; Milligan et al., 1998). This gene also
shows resistance against potato aphid (Macrosiphum euphorbiae)
and the sweet potato whitefly (Bemisia tabaci) (Nombela et al.
2003).
• Gpa2, gene derived from Solanum tuberosum ssp. andigena provides
resistance against Globodera pallida, (Rouppe et al., 1999; Stiekema
et al., 1999).
• Hero gene shows 95% resistance to Globodera rostochiensis and
80% resistance to G. pallida isolated from tomato (Ernst et al. 2002)
and Gro1-4 gene shows resistance to G. rostochiensis pathotype
Ro1(Paal et.al., 2004).
• CaMi gene shows against the root-knot nematode, M. incognita,
which is isolated from Capscicum annum (Chen et al., 2007).
• XiR1gene shows resistance in grape against Xiphinema index
Resistance 1 which is first report for ectoparasitic nematode (Hwang
et al., 2010).
• The Ma gene confers complete-spectrum, heat-stable, and high-level
resistance to RKN, which is derived from the Myrobalan plum
(Prunus cerasifera) (Claverie et al., 2011).
• Major quantitative trait locus gene Rhg4 contributes resistance to
Heterodera glycines (Liu et al., 2012).
Phytoalexins are phenolic compounds which are not present in healthy
plant but are produced upon stimulation of a plant by a pathogen or by a
mechanical or chemical injury. The term phytoalexin is derived from greek
phyto- meaning plant and alexin mean warding off compound. are defined as
"low molecular weight, anti-microbial compounds that are both synthesized
and accumulated in plants after exposure to microorganisms or abiotic agents"
(Paxton, 1980; VanEtten et al., 1994). More toxic to non-pathogen than to the
pathogens. Antimicrobial compounds from plants are broadly classified into
two categories: Phytoanticipins and Phytoalexins (Mansfield, 1999).
Phytoanticipins are low molecular weight, antimicrobial compounds, present
in plants before challenge by the micro-organisms.
Page | 40
Types of phytoalexins
1) Ipomeamarone: It is an abnormal sesquiterpenoids induced in sweet
potato tissue infected with black rot fungus Ceratocystis fimbriata.
It has a striking inhibitory effect on the fungus even in 0.1% conc.
More phytoalexin is produced in resistant variety than in susceptible
one.
2) Pisatin: It has the chromo coumarin ring system and is a phenolic
ether. Production is elicited in lima bean resistant to Pratylenchus
scribneri but not in susceptible snap bean. Both lima bean and snap
bean constitutively produce coumestans at 5-20 ug/g but only lima
bean is induced to produce more after infection.
3) Phaseollin: It is similar to pisatin in chemistry and function. It is
fungicidal at high conc. and fungistatic at low conc. A no. of
compounds such as Phaseolin soflavan and kievitone which are
structurally similar to phaseollin have been identified.
4) Glyceollin: Soybean anti-nematode phytoalexin but also has
antifungal activity at 50-110 ppm. Inoculation of fungal races
resulted in higher conc. In incompatible host cultivar than in
inoculation of fungal races on compatible cultivars. Due to reduced
biodegradation rather than increased biosynthesis.
5) Isocoumarin: Isolated from carrot root tissues. It can also be
produced in response to no. of non-pathogenic micro-organisms.
Chemically related to the pterocarpan phaseollin.
6) Trifolirhizin: It is a new glucoside which has been isolated from
roots of cloves. It structures indicates that it is chemically closely
related to pisatin is has formula C6H12O5.
7) Rishitin: Mullar and Boerger (1940) were first to show that the
potato tuber blight resistance responseded when inoculated with
avirulent race of P. infestans by producing a phytoalexin that
inhibited the development of virulent race. It is a bicyclic non-
sesquiterpene alcohol.
8) Gossypol: Cotton anti-fungal phytoalexin which affects Verticillium
dahliae and M. incognita. It is an ether soluble phenol.
9) Xanthotoxin: Isolated from parsnip root. Inoculation with other non-
pathogens resulted in production of xanthotoxin.
10) Capsidiol: It is a sesquiterpene phytoalexins produced in pepper
fruits inoculate with a non-pathogenic fungus. Produced conc. are
sufficient to inhibit these fungi in vitro.
Page | 41
11) Medicarpin: Alfalfa (Medicago sativa) inoculated with a series of
pathogens have been studied. Antifungal compound was isolated and
identified as medicarpin.
Biological control
There are several methods for the management of plant parasitic
nematodes, but all have limitations also. Among all methods, biological
control has been found to be ecofriendly option for control of nematodes. It
aims to manipulate the parasites, predators and pathogens of nematodes in the
rhizosphere in order to control the plant parasitic nematodes. It automatically
occurs in soil nature without human’s intervention.
Predacious nematodes: Predacious nematodes have specialized open
stoma armed with teeth to catch and swallow the plant parasitic nematodes.
Addition of organic amendments helps to encourage the multiplication of
predacious nematodes such as Mononchus spp. other genera like Diplogaster
spp. and Tripyla spp. are also come under the group of predacious nematodes.
1) Predacious fungi: Most of the predacious fungi come under
Moniliales and Phycomycetes. There are two types of predacious
activities among these fungi.
I. Trapping fungi: The nematode trapping fungi have adhesive
networks and sticky knobs produced by the mycelium to capture the
plant parasitic nematodes.
The nematode trappers are grouped as follows:
a) Sticky hyphae: e.g. Stylopage, Dactylella cinophaga.
b) Sticky branches: The fungal mycelia have short lateral branches and
they anastomose to form loops. The nematode trapped in this loop.
c) Sticky networks: The mycelium curls around and anastomoses with
similar branches. These loops produce complex three-dimensional
structures. The adhesive surface of network helps to hold the
nematode e.g. Arthrobotrys oligospora.
d) Sticky knob: Small spherical or sub spherical lobes are present on
one or two celled lateral hyphae. Only the terminal knob is sticky to
hold the nematodes e.g. Monacrosporium ellipsosporum.
e) Constricting ring: The short hyphal branch curls back on itself and
anastomoses and forming a ring. When the nematode enters the ring
and contact the inner walls of the ring cells bulge inward filling the
lumen of the ring and kills the nematode e.g. M. bembicoidesd and
Dactylaria brachophaga.
Page | 42
f) Non-constricting rings: The trap is formed similar to the
constricting ring. The ring becomes an infective structure and kills
the nematode e.g. Datura candida.
In addition to formation of traps and adhesive secretions, the predacious
fungi may also produce toxin which kill the nematodes.
II. Endozoic fungi: The endozoic fungi usually enter the nematode by
a germ tube that penetrates the cuticle from a sticky spore. The fungal
hyphae ramify throughout the nematode body, absorb the contents
and multiply. The hyphae then emerge from dead nematode.
Catenaria vermicola often attacks sugarcane nematodes.
III. Endophytic fungi: Arbuscular mycorrhizal fungi and Glomus spp.
are two major endophytic fungi which promote plant growth and
indirectly inhibit nematode multiplication.
IV. Parasitic fungi: Purpureocillium lilacinum and Pochonia
chlamydosporia in an effective egg parasite on many nematodes. The
parasitic fungus is particularly effective against Meloidogyne,
Heterodera, Rotylenchulus and Tylenchulus. The fungus attacks the
eggs as they are deposited in groups as a mass. Pochonia
chlamydosporia produces a pneumotaxic compound named
Spironolactone.
Table 4: Commercial product of fungi
Sr. No. Product Fungal spp. Type of fungi Country

1. Royal 300 Arthrobotrys superba Predacious France
2. Royal 350 Arthrobotrys irregular Predacious France
Purpureocillium Egg parasitic
3. Biocon Philippines
lilacinus producing antibiotics
Soyabean root Purpureocillium Egg parasitic
4. China
Bio Protectant lilacinus producing antibiotics
5. Di-tera Myrothecium Producing antibiotics USA
6. Nemaout Fungi - Saudi Arab
2) Bacteria: Recent studies have shown the influence of introduced

microbial antagonist in controlling the nematodes. Several bacteria
are found pathogenic to nematodes. Seed treatment with
Pseudomonas fluorescens has been found to reduce the cyst
nematode, H. cajani in cowpea. The rhizobacteria viz., Bacillus
cereus, Burkholderia cepacia and P. fluorescens were found to be
effective against M. incognita in tomato and banana.
Page | 43
• Bacillus thuringiensis: Delta endotoxins and Beta exotoxin
(thuringiensis). Cry 5B is the most effective crystal toxin found
against nematodes.
• Biocure and Sparsh is the commercial product of Pseudomonas
fluorescens.
• Pasteuria penetrans is first described by Thorne in 1940. It is an
obligate bacterium, difficult for in vitro production. Econem is the
only commercial product developed by Bioscience Inc USA. It
containing spore of the bacteria Pasteuria usage against
Belonolaimus longicaudatus.
Life cycle of Pasteuria penetrans: it has four stages:
a) Endospores attaching to nematodes.
b) Germination (4-10 days).
c) Proliferation inside the nematode pseudocoel.
d) Releasing of mature endospores.
Endospores are the dominant stages of bacterium. At 20 oC P. penetrans
require 120 days to complete the life cycle and minimum temperature is 17
o
C. The highest endospore attachment occurred at pH 9 and decreased at low
pH value. Attachment was higher at pH 7 than pH 4and 9 in tap water and
lowers at pH 7 than pH 4and 9 in distilled water. Pasteuria penetrans require
host nematodes to complete its life cycle. Without host nematode, Pasteuria
penetrans can’t amplify its life cycle.
• Pasteuria nishizawae: Against nematode Heterodera spp.
• Pasteuria penetrans: Against nematode Meloidogyne spp.
• Pasteuria thornei: Against nematode Pratylenchus spp.
• Pasteuria thornei: Against nematode Belonolaimus spp.
Chemical control: Among all the nematode control method, chemical
control method is more reliable, effective and rapid in action and can be used
when and where required but they have comparatively high cost and cause
environmental pollution hazards. Nematicide is defined as a substance or
mixture of substances used for killing, repelling, or otherwise preventing the
plant parasitic nematodes. Kuhn (1881) first tested CS2 (Carbon disulphide)
to control sugar beet cyst nematode (Heterodera schachtii) in Germany but he
could not get encouraging results. Bessey (1911) test CS2 for the control of
root-knot nematodes later formaldehyde, cyanide, quicklime. Mathews (1919)
chloropicrin (test gas) against plant parasitic nematode in England. In 1944,
Page | 44
scientists from California and Florida states of USA reported the efficacy of
EDB, DD paved way for chemical control.
Chemical control
Table 5: Some important nematicides
S. Common
Trade Name Use Dose
No. name
Ethylene Bromofume and 60-120 liter or
1. Injected/dibbed into soil
dibromide Dowfume. 200 kg ai/ha
Soil treatment before
planting, at the time of
Nemagone, planting, post-plant
2. DBCP Fumazone. treatment, effective when 10-60 lit/ha
soil temperature is above
200C, sprinkled or mixed
with irrigation water.
Dibromomethane,
3. DD mixture - 225-280 lit/ha
Dorlone.
Termite/wood
beetle @ 32-64
Kills the rodents, insects, g/m3, fumigation
Methyl fungi and weeds in the soil, of live plant @
4. -
bromide highly dangerous to warm 16-32 g/m3.
blooded animals. Application of
nematode/ insect
@ 4-7 ml/ft2.
16-48 g/m3. It is
Acquinite & Pic Tear gas, non-inflamable, in control of
5. Chloropicrin
fume good penetrating effect. nematode/ insect
in soil.
Systemic nematicides,
6. Fensulfothion Dasanit, Terracur effective against golden -
nematode in Nilgiri hills
Systemic nematicide,
effective on root-knot
7. Fenamiphos Nemacur 40 EC --
nematode and cyst
nematodes, 1-5 % granules
Systemic, effective against
8. Ethoprop Mocap -
juvenile nematode
10% granules, fumigant
9. Phorate Thimet -
action
Vapam, Sistan,
Metham
10. Vitafume and Injected in to soil. 100-200 ml/m2
sodium
Unifume,
It is a systemic, 10%
11. Aldicarb Temik -
granule, 30-35 days remain
Page | 45
residual in plant.
acropetal action
systemic insecticide cum
12. Carbofuran Furadan applied @ 1-2 kg
nematicide
ai/ha
against insects, mites and
13. Methomyl Lannate -
nematodes
40% EC systemic,
14. Oxamyl Vydate effective against foliar -
nematode
The pesticides or chemicals can be classified on their mode of entry, mode

of action and also by their chemical nature.
I) Classification based on mode of entry
1) Stomach poison: The chemicals are applied on foliage and other
parts of plant when ingested by the nematode, it acts on digestive
system and cause death. E.g. Lead Arsenate and Phosphamidon.
2) Contact poison: The toxicant which brings death of the pest species
by means of contact and are directly absorb by the cuticle. e.g.
Methyl Parathion.
3) Fumigant: The toxicant in its gaseous state penetrates the organism
and kill them. CS2, DD and EDB are example for fumigants.
II) Classification based on mode of action
1) Physical poison: The toxicant which brings death of organism by
exerting a physical effect is known as physical poison. The heavy oils
like tar oil leads to asphyxiation and cause death.
2) Protoplasmic poison: A toxicant responsible for precipitation of
protein, especially destruction of cellular protoplasm of intestine
epithelium. e.g. Formaldehyde, Ethylene oxide, Nitro phenols etc.
3) Respiratory poison: A chemicals which blocks the cellular
respiration or inactivate cellular respiratory enzymes are respiratory
poisons. e.g. H2S, DD and EDB.
4) Nerve poison: These chemicals are anti acetylcholinesterase activity
which lead to constant excitation of nerves in the target organism.
Due to this, the organism faces convulsions, tremors, muscle
paralysis and lead to death. e.g. diazinon & aldicarb.
III) Classification based on chemical nature
1) Synthetic inorganic compounds: These compounds are systemic
inorganic salts which acts as stomach poison and kill the target
Page | 46
organism. e.g. Calcium arsenate.
2) Synthetic organic compounds: These group are further classified as
i) Halogenated hydrocarbon: e.g. Chloropicrin, methyl bromide, DD,
EDB and DBCP etc.
ii) Organophosphorus compound: The basic constituents of
organophosphorus compound are carbon, hydrogen and chlorine and
certain compounds may have oxygen and sulphur also. e.g.
Parathion, Dichlorofenothion, Thionazin, Phorate etc.
iii) Carbamates: Carbamate compounds are derivatives of carbamic
acid. e.g. Aldicarb, Carbofuron etc.
iv) Substituted phenols: In this compound the phenol is substituted by
any other group e.g. Binapacryl.
v) Thiocyanates: e.g. Lethane and Thanite
vi) Flourine compound: e.g. Flourine sodium flouroacetate.
vii) Sulphur compounds: e.g. CS2. H2S and Endosulphan.
IV) Natural products
Nicotine, Pyrethrin, Neem cakes. α terthinyl in Marigold, Catechol in
Ergrostris, Mustard, Sesame, Bitter cucumber.
New nematicides
1) Nimitz (granular form) produced by Adama company against root-
knot nematodes in tomato, okra, brijal and chilli.
2) Velum prime (liquid form) produced by Bayr company against root-
knot nematodes in tomato.
Domestic Quarantine is effective against the transportation of potato
tubers from Nilgiri hills (Tamil Nadu) to other part of the country to prevent
infestation of potato cyst nematode Globodera rostochiensis. Under the
schedule of 4,5,6 of Plant Quarantine Order. 2003- restricted plant parts are
listed with their origin country to prevent spread of new or already exiting
nematode. Plant quarantine order, 2003, came into force on the 1st January,
2004.
Nematodes of quarantine importance
1) Aphelenchoides arachidis-Groundnut Testa Nematode.
2) Aphelenchoides fragariae-Foliar nematode.
3) Aphelenchoides besseyi-White tip nematode.
Page | 47
4) Aphelenchoides ritzembosi-Foliar nematode.
5) Bursaphelenchus xylophilus-Pine wood nematode.
6) Cactodera cacti-Cactus cyst nematode.
7) Ditylenchus dipsaci-Stem and bulb nematode.
8) Ditylenchus destructor-Potato tuber nematode.
9) Globodera pallida-Potato cyst nematode.
10) Globodera rostochiensis-Potato cyst nematode.
11) Heterodera goettingiana-Pea cyst nematode.
12) Heterodera glycines-Soybean cyst nematode.
13) Heterodera humuli-Hop cyst nematode.
14) Heterodera schachtii-Sugarbeet cyst nematode.
15) Heterodera tabacum-Tobacco cyst nematode.
16) Radopholus citrophilus-Citrus burrowing nematode.
17) Rhadinaphelenchus cocophilus-coconut red ring nematode.
References
1. Bakker J, Gommers FJ, Nieuwenhuis I, Wynberg H. Photoactivation of
the nematicidal compound alpha-terthienyl from roots of marigolds
(Tagetes species). A possible singlet oxygen role. Journal of Biological
Chemistry. 1979;254(6):1841-1844.
2. Bratley HE. Weed host plants of the nematode found in the three year
tobacco rotation. Proceeding of Soil Science Society of Florida.
1942;4:118-120.
3. Cai D, Kleine M, Kifle S, Harloff H. Positional cloning of a gene for
nematode resistance in sugar beet. Science. 1997;275:832-834.
4. Chen R, Li H, Zhang L, Zhang J, Xiao J, Ye Z. CaMi, a root-knot
nematode resistance gene from hot pepper (Capsium annuum L.) confers
nematode resistance in tomato. Plant cell reports 2007;26(7):895-905.
5. Claverie M, Dirlewanger E, Bosselut N, Van Ghelder C, Voisin R,
Kleinhentz M, et al. The Ma gene for complete-spectrum resistance to
Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal
post-LRR region. Plant Physiology. 2011;156:779-792.
6. Curto G, Dallavalle E, De Nicola GR, Lazzeri L. Evaluation of the
activity of dhurrin and sorghum towards Meloidogyne incognita.
Nematology. 2012;14(6):759-769.
Page | 48
7. Dahlin P, Hallmann J. New insights on the role of allyl isothiocyanate in
controlling the root knot nematode Meloidogyne hapla. Plants.
2020;9(5):603.
8. Dandurand LM, Morra MJ, Zasada IA, Phillips WS, Popova I, Harder C.
Control of Globodera spp. using Brassica juncea seed meal and seed meal
extract. Journal of Nematology. 2017;49(4):437.
9. De Viala SS, Brodie BB, Rodriguez E, Gibson DM. The potential of
thiarubrine C as a nematicidal agent against plant-parasitic nematodes.
Journal of Nematology. 1998;30(2):192.
10. Elliger CA, Waiss AC, Dutton HL, Rose MF. α-Tomatine and resistance
of tomato cultivars toward the nematode, Meloidogyne incognita. Journal
of chemical ecology. 1988;14(4):1253-1259.
11. Ernst K, Kumar A, Kriseleit D, Kloos DU, Phillips MS, Ganal MW. The
broad‐spectrum potato cyst nematode resistance gene (Hero) from tomato
is the only member of a large gene family of NBS‐LRR genes with an
unusual amino acid repeat in the LRR region. The Plant Journal.
2002;31(2):127-136.
12. Hashmi S, Hashmi G. Crop rotation and Meloidogyne javanica in
vegetable production. International Nematology Network Newsletter.
1990;7(1):19-21.
13. Hwang CF, Xu K, Hu R, Zhou R, Riaz S, Walker MA. Cloning and
characterization of XiR1, a locus responsible for Dagger nematode
resistance in grape. Theoretical and applied genetics. 2010;121(4):789-
799.
14. Jain RK, Bhatti DS. Population development of root‐knot nematode
(Meloidogyne javanica) and tomato yield as influenced by summer
ploughings. International Journal of Pest Management. 1987;33(2):122-
124.
15. Kanwar RS, Bhatti DS. Influence of cropping sequence on population of
Meloidogyne javanica and crop yield. Haryana Agricultural University
Journal of Research. 1992;22:40-45.
16. Khan AM, Saxena SK, Siddiqi ZA, Uadhyay RS. Control of nematodes
by crop rotation. Indian Journal of Nematology. 1975;5:214-221.
17. Kuhn J. Die Ergebnisse der Versuche zur Ermittelung der Ursache der
Rübenmüdigkeit und zur Erforschung der Natur der Nematoden.
Schönfeld G., 1881, 3.
Page | 49
18. Lagudah ES, Moullet O, Appels R. Map-based cloning of a gene sequence
encoding a nucleotide-binding domain and a leucine-rich region at the
Cre3 nematode resistance locus of wheat. Genome. 1997;40(5):659-665.
19. Lal B, Rana BP. Efficacy of neem products as seed treatment against
Meloidogyne incognita in Okra. Annals of Plant Protection Sciences.
2012;20(1):268-269.
20. Liu S, Kandoth PK, Warren SD, Yeckel G, Heinz R, Alden J, et al. A
soybean cyst nematode resistance gene points to a new mechanism of
plant resistance to pathogens. Nature. 2012;492(7428):256-260.
21. Mansfield JW. Antimicrobial compounds and resistance: the role of
phytoalexins and antianticipins. In: Mechanisms of Resistance to Plant
Diseases. Slusarenko AJ, Fraser RSS and Van Loon LC (Ed). Kluwer,
Amsterdam, 1999.
22. Mathews DJ. Report of the work of the WB Randall research assistant.
Rep. Exp. Res. Stn., Cheshunt. 1919;5:18-21.
23. Milligan SB, Bodeau J, Yaghoobi J, Kaloshian I, Zabel P, Williamson
VM. The root knot nematode resistance gene Mi from tomato is a member
of the leucine zipper, nucleotide binding, leucine-rich repeat family of
plant genes. The Plant Cell. 1998;10(8):1307-1319.
24. Nidiry ESJ, Khan RM, Reddy PP. In vitro nematicidal activity of
Gloriosa superba seed extract against Meloidogyne incognita.
Nematologia Mediterranea, 1993, 127-128.
25. Nombela G, Williamson VM, Muñiz M. The root-knot nematode
resistance gene Mi-1.2 of tomato is responsible for resistance against the
whitefly Bemisia tabaci. Molecular Plant-Microbe Interactions.
2003;16(7):645-649.
26. Paal J, Henselewski H, Muth J, Meksem K, Menéndez CM, Salamini F,
et al. Molecular cloning of the potato Gro1‐4 gene conferring resistance
to pathotype Ro1 of the root cyst nematode Globodera rostochiensis,
based on a candidate gene approach. The Plant Journal. 2004;38(2):285-
297.
27. Paxton JD. A new working definition of the term “phytoalexin” Plant
Disease. 1980;64:734.
28. Qamar F, Begum S, Raza SM, Wahab A, Siddiqui BS. Nematicidal
natural products from the aerial parts of Lantana camara Linn. Natural
product research. 2005;19(6):609-613.
Page | 50
29. Rangaswamy SD, Parvatha Reddy P, Nanje Gowda DN. Management of
root-knot nematode, Meloidogyne incognita in tomato by intercropping
with marigold and mustard. Pest Mang. Hortic. Ecosyst. 1999;5:118-121.
30. Rouppe Van der Voort JR, Kanyuka K, Van der Vossen E, Bendahmane
A, Mooijman P, Klein-Lankhorst R, et al. Tight physical linkage of the
nematode resistance gene Gpa2 and the virus resistance gene Rx on a
single segment introgressed from the wild species Solanum tuberosum
subsp. Andigena CPC 1673 into cultivated potato. Molecular Plant-
Microbe Interactions. 1999;12(3):197-206.
31. Stiekema WJ, Van der Vossen EAG, Sandbrink H, Peters S, Rouppe van
Der Voort J, Kanyuka K, et al. Two paralogous genes of the Gpa2
resistance locus confer both virus and nematode resistance to potato,
1999.
32. Takasugi M, Yachida Y, Anetai M, Masamune T, Kegasawa K.
Identification of asparagusic acid as a nematicide occurring naturally in
the roots of asparagus. Chemistry Letters. 1975;4(1):43-44.
33. Tanda AS, Atwal AS, Bajaj YPS. In vitro inhibition of root-knot
nematode Meloidogyne incognita by sesame root exudate and its amino
acids. Nematologica. 1989;35(1):115-124.
34. Thorne G. Duboscqia penetrans n. sp. (Sporozoa: Microsporidia,
Nosematidae), a parasite of the nematode Pratylenchus pratensis (de
Man) Filipjev. Proceedings of the Helminthological Society of
Washington. 1940;7:51-53.
35. Van Etten HD, Mansfield JW, Bailey JA, Farmer EE. Two classes of plant
antibiotics: phytoalexins versus "phytoanticipins". The Plant Cell.
1994;6(9):11-91.
36. Williamson VM. Root-knot nematode resistance genes in tomato and
their potential for future use. Annual Review of Phytopathology.
1998;36(1):277-293.
Page | 51
Page | 52
Chapter - 4
Splat: A New Pheromone Dispensing Technology
Authors
Venugopal U
Department of Entomology, University of Agricultural
Science, Bengaluru, Karnataka, India
Neelima K
Department of Entomology, Sam Higginbottom University of
Agricultural Technology & Science, Naini Allahabad,
Uttar Pradesh, India
Nagaraju MC
Manju KL
Page | 53
Page | 54
Chapter - 4
Splat: A New Pheromone Dispensing Technology
Venugopal U, Neelima K, Nagaraju MC and Manju KL
Abstract
Pesticides have received widespread scrutiny for their negative impacts
on non-target organism including human health, thus the identification of
reduced-risk alternatives, for use in these area-wide control programs is of
high priority. In this chapter attempt has been made SPLAT® significantly
decreases environmental impacts and enable commercialization of more
affordable semiochemical based control products. SPLAT® mating disruption
and attract-and-kill formulations have been developed for management of
agricultural and forest pests.
Keywords: SPALT, insect pest, pheromones, traps
Insect pheromones
Semiochemicals, such as sex pheromones, are chemical signals that
insects use to communicate with one another. Sex pheromones are released by
female insects to let males know they are available to mate. The female
releases this chemical signal, which is carried by the wind to males. When a
male becomes aware of the pheromone, he follows the plume upwind to reach
the female and mate with her. The majority of insect pheromone products use
synthetic copies of the female pheromones, which will only attract males.
Female sex pheromones have no effect on females. Pheromones are also
species specific, so there is no effect on any other species other than the target
species (Mafra-Neto et al., 2014).
The use of a good pheromone dispenser plays a key role in achieving
high-performance mating disruption. First, an ideal pheromone dispenser
should remain effective for a prolonged period, not waste active ingredients
are inexpensive to produce, be easy to use in the field, and be nontoxic
Furthermore, pheromone dispensers should be amenable to use at varying
densities and deployment dates according to pest pressure. In addition, it
should achieve the availability of a controlled release device to encourage
growers to adopt mating disruption technology.
Page | 55
ISCA Technologies, Inc. (Riverside, CA U.S.A.) acquired SPLAT®
(Specialized Pheromone and Lure Application Technology) in 2004.
SPLAT® is a chemical controlled-release emulsion technology that has been
used to dispense compounds to control a variety of insect pests. SPLAT®
formulations have been commercialized both domestically and
internationally. Each focusing on one of three semiochemical-based insect
control techniques: Mating disruption, attract-and-kill, and repellents. Each
section provides an introduction to the technique and summary of existing
commercial SPLAT® products for insect control using that technique, and
follows with one or more case studies of new SPLAT® formulations being
developed to control agricultural or forestry pests.
Semiochemical methods of pest control have numerous advantages over
the use of conventional insecticides, both in terms of efficacy and
environmental sustainability. Semiochemicals, such as sex pheromones used
by insects to locate a mate, and host plant kairomones, which serve as
important cues to help insects navigate their environment, are naturally
occurring, often species-specific substances. Artificial introduction of such
substances into a field environment often allows the grower to elicit a specific
response from a specific insect pest, leaving desirable nontarget organisms,
like natural enemies and pollinators, often unaffected. Behavioral
manipulation by means of semiochemical applications has been shown to
involve less risk of the target species developing resistance to the active
ingredient (AI), a common shortcoming among conventional insecticides.
Furthermore, unlike conventional insecticides, which are often applied as
cover sprays over the entire crop, semiochemicals tend to be deployed in
discrete point sources. This reduces the likelihood of non-target organisms
coming into contact with the compound and enables growers to avoid
contamination of food crops. Despite these advantages, it has proven difficult
to develop and commercialize effective, economically-viable semiochemical-
based pest control technologies.
SPLAT® formulations typically become rain fast within 3 hours
following application, and can provide continuous controlled release of
semiochemical AIs for intervals ranging from 2 weeks to 6 months. The rate
of release of the incorporated AIs into the field is determined by the AI
molecules’ rate of diffusion through the SPLAT® matrix, which occurs in
accordance with Fick’s First Law of Diffusion, stating that molecules move
from regions of high concentration to regions of low concentration at a rate
that is directly proportional to their concentration gradient. This rate may be
precisely controlled or adjusted by subtle changes in the formulation’s
Page | 56
characteristics and application method. Because of its controlled-release
capacity, SPLAT® formulations typically use smaller amounts of AIs than
other semiochemical products, reducing application costs and environmental
impact (Mafra-Neto et al., 2014).
The primary obstacle to wide-spread adoption of these technologies is the
high cost of semiochemicals, which are generally more expensive than
conventional pesticides. In many cases, however, it is not the intrinsic cost of
the semiochemical itself that makes the end product economically unfeasible,
but rather the method by which it must be deployed in the field. Past and
current semiochemical formulations have typically been developed as devices,
which by their very nature require manual application. This can lead to
prohibitively high application costs, especially in large and/or heavily infested
fields, requiring high application densities. These costs can be further
compounded by the physical limitations of the active semiochemical (i.e.,
chemical instability/volatility, sensitivity to light or heat), as well as by the
mechanism of behavior manipulation in which it is employed (Mafra-Neto et
al., 2014).
Controlled-release technology
The aqueous component of the SPLAT® emulsion gives the product its
liquid character, allowing it to flow. The non-aqueous component of the
emulsion is the controlled-release device. It comprises the active ingredients
(e.g., semiochemical compounds or pesticides) and the additives that will
protect these and fine-tune their release rates from the dispenser. Upon
application, the aqueous component of SPLAT® evaporates from the
dispenser within 3 hours, leaving the rainfast, nonaqueous component firmly
affixed to the substrate, where it will release the active ingredients until all
available molecules are dispensed (Figure 1). The longevity of the
Fig 1: Following application, the SPLAT® emulsion dries and becomes rainfast
within 3 hours, then releases active ingredients at a controlled rate for 2 weeks to 6
months
Page | 57
dispenser depends on the manner in which the particular SPLAT®
formulation was created, its composition, how it was applied, as well as the
environmental conditions to which it is exposed following application.
SPLAT® products are typically formulated to release semiochemicals for 2
weeks to 6 months. SPLAT® is a “matrix-type” or “monolithic” diffusion-
controlled release device. Diffusion-controlled release devices are ones where
the diffusion of the active ingredient through the device controls its release
rate (Mafra-Neto et al., 2014).
Monolithic dispensers are diffusion-controlled release devices where the
active ingredient is dispersed or dissolved in a matrix. If the active ingredient
is dispersed in the matrix, it must dissociate from the other molecules in its
crystal cell and solubilize into the matrix before release can occur. If it is
dissolved in the matrix, this first step is bypassed. In the majority of cases, we
expect hydrophobic arthropod pheromones to be dissolved in the SPLAT®
matrix when the product is applied. The movement of the active ingredient
dissolved within the matrix occurs by diffusion and follows Fick’s First Law,
which states that molecules move down their concentration gradients at a rate
that is directly proportional to their concentration gradient. Movement of the
molecule within the matrix occurs in one of two ways. If the molecule is very
small compared to the size of the amorphous spaces in the matrix, it diffuses
through the matrix by moving from one such space to another (Faleiro et al.,
2014).
Upon reaching the surface of the matrix, the active ingredient is released
into the environment. Whether the release rate of the active ingredient to the
environment is zero or first order depends on the partition coefficient of the
active ingredient between the matrix and the environment. If the active
ingredient readily partitions to the environment, then its rate of release will be
diffusion-controlled and first order.
SPLAT vs. traditional pheromone dispensers
• Multiple methods of application: Having a wide range of
viscosities and application methods (e.g. applicator sprays, aerial
applicator sprays, caulking gun type tubes, etc.) SPLAT increases
productivity by mechanizing the application of pheromone
dispensing points.
• Easy application for small-scale & large-scale operations: The
amorphous and flowable quality of this highly adaptable product
allows for an easy transition from small scale manual applications to
large-scale mechanical applications.
Page | 58
• Adjustable strategies same amount of AI: A fixed quantity of this
material can be applied differently depending on the pest population
pressure. The application of this matrix can be tailored by the user to
best match the pest distribution and density in the field.
Using a fixed amount of SPLAT per area, one can choose:
1) A high density of small point-sources, thus maximizing the mating
disruption effect (recommended for high pest pressure).
2) A low density of larger point-sources, thus increasing the longevity
of the application (recommended for lower pest population pressure).
• Rain Fast Formulation: Once cured, SPLAT will not wash off of
vegetation.
• Season-long Protection & More: SPLAT can remain effective in
managing pest populations up to a six month duration.
• Mixes with Kairomones and Feeding stimulants: SPLAT can be
mixed with a variety of feeding stimulants or attractants including
liquids, solids and oils to enhance attraction or stimulate feeding.
Fig 2: SPLAT vs. Traditional Pheromone Dispensers
Page | 59
Methods of applications
SPLAT® formulations typically have a paste or cream-like consistency.
SPLAT® is a non-Newtonian, shear-thinning, thixotropic fluid, which means
that SPLAT® viscosity decreases when the emulsion is placed under stress,
such as when it is stirred or pumped, but increases again when the stress is
removed. This property is advantageous in that the less viscous SPLAT® can
easily be manipulated (e.g., stirred or pumped), but quickly thickens upon
application to a surface, aiding in product adhesion. A wide variety of manual
and mechanical applicators can be used to apply SPLAT®. The most basic
SPLAT® applicator can be a stick, spatula, or knife. More advanced manual
applicators include syringes, grease guns and caulking guns. Indeed, SPLAT®
formulations are regularly sold loaded into standard caulking tubes and
applied with off the shelf caulking guns. In addition, numerous mechanical
applicators have been adapted or created specifically to apply SPLAT® with
a variety of motorized vehicles, including tractors, all-terrain vehicles, and
even motorcycles. SPLAT® has also been sprayed from motorized backpack
sprayers and applied aerially (Faleiro et al., 2014).
Type of SPLAT Formulations
SPLAT® formulations have been developed to release a variety of
compounds, including sex pheromones, kairomones, attractants, repellents,
phagostimulants, and insecticides. Several SPLAT® mating disruption and
attract-and-kill formulations are commercially available. SPLAT® repellent
formulations will also soon become available commercially. Development of
novel SPLAT® products is an active area of research. Efficacy trials
conducted in the development of several of the newest SPLAT® mating
disruption attract-and-kill, and repellent formulations.
SPLAT® mating disruption formulations
Mating disruption is an insecticide-free pest management strategy that
uses synthetically produced nature identical pheromones to confuse males and
limit their ability to locate females for mating. By reducing the likelihood of
successful mating through mating disruption, the infestation level decreases.
Mating disruption consists of dispensing a synthetic form of one or more
components of the natural pheromone blend of insect or biologically-
equivalent compounds, where host plant(s) are present. The presence of the
species’ sex pheromones in the environment delays or prevents mating of the
insect, reducing fecundity and subsequent populations. Although using
semiochemicals to disrupt communication among insects, rather than using
these compounds as they are naturally used in insect communication (e.g., to
Page | 60
attract or repel insects), was a relatively revolutionary idea when the first
insect sex pheromones were identified 50 years ago, the use of
semiochemicals for mating disruption was suggested and tested early on by
several scientists. It has proven to be a powerful technique for insect control
and has become the most commonly-used semiochemical-based insect control
method.
Mechanisms of mating disruption
1) Competitive attraction, also referred to as “false trail following”
occurs when males orient and respond to synthetic pheromone
plumes produced by semiochemical dispensers, rather than the
natural plume emitted by a calling female. This mechanism of mating
disruption is population density dependent, and decreases in efficacy
as the population of the pest increases.
2) Camouflage, unlike competitive attraction, is density independent,
and requires that the environment be saturated with synthetic
pheromone to the extent that mate-seeking males are incapable of
homing in on females.
3) Desensitization takes place when males’ responses to the female sex
pheromone are significantly diminished due to over-exposure, and
may occur either through adaptation of the males’ olfactory receptor
neurons, or by habituation of the insects’ central nervous system.
4) Sensory imbalance achieves mating disruption by impeding the
male’s ability to recognize the female sex pheromone within the
treated environment, thereby preventing him from responding to it
(Mafra-Neto et al., 2013).
Season-long mating disruption of Grapholita molesta (Lepidoptera:
Tortricidae) by one machine application of pheromone in wax drops
(SPLAT-OFM)
The current research tested the hypothesis that increasing the size of
dispensed drops above the 0.04 ml average volume achieved by the initial
applicator prototype, as well as, re-formulating the wax for longer release over
time in hot temperatures would maintain and improve longevity.
The specific objectives were to:
1) Modify the mechanical applicator to release larger wax drops.
2) Evaluate an experimental microcrystalline wax formulation of G.
molesta pheromone (SPLAT-OFM) (Stelinski et al., 2007).
Page | 61
Disruption of male orientation
Disruption of male G. molesta orientation to synthetic sex pheromone was
assessed using four pheromone traps deployed per replicate plot. Two traps
were deployed 15 m apart in the central row of plots and two traps were placed
two rows from plot borders. Traps were baited with red septa loaded with 0.1
mg of (Z)-8-dodecenyl acetate:(E)-8-dodecenyl acetate:(Z)-8-dodecen-1-ol in
a 100:6:10 blend. Traps were deployed approximately 3-4 m above-ground in
the upper third of the tree canopy following application of wax. New
pheromone lures were deployed at the onset of each moth generation resulting
in 2 replacements over the course of the season. Moths captured in traps were
counted and removed twice weekly.
Following a single mechanized application of SPLATOFM on April 28th,
98% inhibition of male catch (Fig. 3a) was recorded season long. The mean
(§SE) number of males caught per block per generation was significantly (F’s
= 72.3, 27.4, 41.7; df = 1, 3; P < 0.05) greater in control plots (9.1 § 2.8, 10.9
§ 1.9, 21.3 § 4.1) than in pheromone-treated plots (0.02 § 0.02, 0.02 § 0.02,
0.9 § 0.3) for moth generations 1-3, respectively. Inhibition of moth catch in
traps was >99% for generation 1 and 2 and 96% for the third generation
(Stelinski et al., 2007).
Fig 3: Mean captures of Grapholita molesta males per week throughout the season in
replicated 0.8 ha pheromone-treated and control plots (a)
Disruption of female mating
Disruption of moth mating was also directly assessed by deploying
tethered virgin female moths in plots weekly for the duration of the
experiment. Eight to 15 female moths (2-4-day old) were tethered per plot
during each of 17 deployment dates (10, 18, 24, 30 May; 7, 14, 24, 29 June;
4, 13, 19, 25 July; 03, 10, 17, 24, and 31 August) throughout the season.
Page | 62
Female moths were attached to branches of trees with polyester thread tied to
the base of one wing. They were tethered on 50 cm of thread, which allowed
moths to relocate themselves on branches following placement. Of the 1,016
moths deployed, 72% were recovered. Females were collected from the Weld
approximately 18–20 h following deployments. Dissections were conducted
to determine mating status by removal of the bursa copulatrix and inspection
for the presence of a spermatophore.
Fig 4b: Mean proportion of virgin female Grapholita molesta mating during 18–20
hr of Weld deployment in plots receiving a mechanized application of SPLAT
dispensers of pheromone versus untreated control blocks (b)
Excluding days on which no mated females were recovered from both
pheromone-treated and control plots (21 June and 19 July), the proportion of
female moth mating in control plots averaged 27% over the course of the
season (N = 32–60 per treatment per sampling date) (Fig. 4B). Mating of
tethered virgin female G. molesta was completely disrupted in pheromone
treated blocks season long This formulation could have broad application for
many pest species provided that the pheromonal active ingredients can be
adequately stabilized and protected from degradation and that they are not
phytotoxic so as to damage foliage and/or mark fruit.
Carob Moth, Ectomyelois ceratoniae
Ectomyelois ceratoniae (Lepidoptera: Pyralidae), the carob moth, is a
widely distributed polyphagous pest that infests numerous fruit and nut crops.
SPLAT® EC was formulated for controlled release of (Z,E)-7,9,11-
dodecatrienyl formate, a parapheromone (pheromone mimic) of the major
component of the carob moth pheromone, (Z,E)-9,11,13-tetradecatrienal.
Todd et al. synthesized (Z,E)-7,9,11-dodecatrienyl formate, a more stable
analogue of (Z,E)-9,11,13-tetradecatrienal, and demonstrated that it
Page | 63
effectively mimicked the major component of the carob moth pheromone both
at the cellular and behavioral levels (Mafra-Neto et al., 2014).
Three treatments were tested at the first location: SPLAT® EC, 5%
malathion dust and a non-treated control. Two treatments were evaluated at
the second location: SPLAT® EC and 5% malathion dust. SPLAT® EC was
applied as two 2.5-g dollops per tree, one placed at the top of the tree, where
date bunches are located, and the second placed on the trunk, ca. 1.5 m up
from the ground, for a total of ca. 610 g SPLAT® EC per ha. A single
application of SPLAT® EC was made at the beginning of the trial. Malathion
dust was applied ca. every 2 weeks throughout the trial for a total of 4
applications. The SPLAT® EC and malathion-treated plots were evaluated
with male captures in carob moth parapheromone-baited traps. One
parapheromone-baited trap was placed in the center of each plot and male
moths in each trap were counted weekly. Moth capture data were analyzed by
repeated measures ANOVA on square root-transformed data (Mafra-Neto et
al., 2014).
Table I: Field efficacy of SPLAT® EC for carob moth control in dates. of fruit
infestation at harvest
Male moth captures in the SPLAT® EC-treated plots were lower than in
the malathion-treated plots at both locations. At location 1, the differences in
moth captures in SPLAT® EC- and malathion-treated plots were statistically
significant throughout the trial (Figure 5). At location 2, moth captures in the
SPLAT® EC- and malathion-treated plots were significantly different on all
sampling dates, except November 2, when the difference was nearly
significant. The extremely low numbers of male moths captured in
parapheromone-baited traps in the SPLAT® EC-treated plots demonstrated
that mating disruption was nearly complete from the one-time SPLAT® EC
application through harvest. Whereas moth capture data were collected only
in SPLAT® EC-and malathion-treated plots, fruit infestation data were
collected in all plots, including the non-treated control plots at location 1.
Carob moth infestation at location 1 was equivalent in SPLAT® EC and
malathion-treated plots and significantly lower in these treatments compared
to the non-treated control (Table I).
Page | 64
Fig 5: Field efficacy of SPLAT® EC for carob moth control in dates. Male moth
captures in parapheromone-baited traps at A. location 1
Results at location 2 were similar, with no significant difference in fruit
infestation in the SPLAT® EC- and malathion-treated plots. The results were
economically-important because the cost of the single SPLAT® EC
application (material and application) was equivalent to the total cost for all
malathion dust treatments.
SPLAT® Attract-And-Kill Formulations
The attract-and-kill strategy is also referred to as “lure-and-kill” and
“attracticide”, as well as by other terms (e.g., male annihilation, lure-and-
sterilize, lure-and-infect, bait spray), depending on the type of attract-and-kill
strategy being used. Broadly, attract-and-kill consists of attracting males,
females, or both sexes of a pest species to an insect control agent (e.g.,
insecticide, sterilant, or insect pathogen). Upon contact, the insect is either
killed (immediately or after a delay) or sublethal effects of the control agent
diminish the pest population by reducing the insect’s fertility or ability to
mate. The insect attractant can be a chemical attractant, a visual cue, an
acoustic cue, or a combination of these. Crude baits (e.g., food lures) are also
used in attract-and-kill devices and entire plants (e.g., trap crops) have been
used as attractants for this technique as well. Since SPLAT® is a chemical
controlled-release technology; we will focus on a discussion of attract-and-
kill devices that use chemical attractants.
Unlike mating disruption, which can control insect populations via a
variety of mechanisms, there is only one way that attract-and-kill can achieve
Page | 65
insect control. Pest insects (ideally both sexes) must be lured to a control agent
that exerts its affect on the individual following contact. Furthermore, the
attractant must not only. Unlike mating disruption, however, it is less sensitive
to environmental factors, such as site topography and plot size, and can
successfully be used in situations where mating disruption is likely to fail.
Although attract-and-kill products most often contain insecticides, which
make them less environmentally-friendly and potentially more of a concern to
the public than mating disruption products, they offer several advantages over
conventional insecticides. Another important difference between mating
disruption and attract-and-kill products for manufacturers is that unlike mating
disruption products, these insecticide-containing formulations cannot take
advantage of legislation which has simplified and reduced the cost of EPA
registration of arthropod pheromone-based insect control products that do not
contain insecticides.
Greenhouse and Field evaluation of a new male annihilation technique
(MAT) product, SPLAT-MAT spinosad ME™, for the control of Oriental
fruit flies (Diptera: Tephritidae) in Taiwan
Male annihilation treatments (MAT’s) consisting of a toxicant mixed with
the powerful, male-specific parapheromone attractant methyl eugenol (ME)
form the basis of most control programs for B. dorsalis. Control by MAT
products is achieved though the selective removal of males from a fruit flies
population, thus reducing mating and eventually leading to a population crash.
Eradication of B. dorsalis has been achieved in many locations through the
area-wide application of MAT products containing ME Spinosad, an
insecticide derived from metabolites of the soil bacterium Saccharopolyspora
spinosa, is an effective insecticide with highly favorable mammalian
toxicology and environmental profiles. Spinosad was initially registered in
1997 and is considered a naturally derived product. As a result, certain
formulated products have been approved for use in the organic agriculture by
the Organic Materials Review Institute (OMRI) and numerous other national
and international certification bodies Five treatments with four replications
were compared:
1) SPLAT-Spin-ME (containing 2% spinosad a.i., 50% methyl eugenol,
and 48% SPLAT; a novel MAT product recently introduced by Dow
AgroSciences in partnership with ISCA Technologies.
2) Naled+ME (containing 5% naled and 90% methyl-eugenol,), the
most widely-used MAT product in Taiwan.
Page | 66
3) GF-120 (0.24% a.i. spinosad, Dow AgroSciences, Indianapolis, IN)
4) malathion 50 EC protein bait spray (malathion Probelte®, Dow
AgroSciences, Spain + Buminal®, hydrolyzed proteins 30% p/v, SL,
Bayer, Spain).
5) Untreated control (water): A total of ten, 20 μL droplets of distilled
water were applied onto the inside Teflon surface of a 7 mm-diameter
plastic lid for both greenhouse and field cage experiments (Hsu et al.,
2010).
Fig 6: The naled-ME (prior sticking) and SPLAT-spinosad-ME treatments of the

Teflon surface of the lids that were then put into the trap for these experiments. A. 40
μL of naled+ME placed onto filter papers, at 10 μL each; B. 40 μL of naled+ME
added into a caneite block; C. 50 mg of SPLAT-spinosad-ME; D. 1 gram of SPLAT-
spinosad-ME; E. A caneite block with 8 mL of naled-ME
Greenhouse Attraction and Mortality Bioassays
Attraction bioassays were conducted inside a ventilated greenhouse A
treatment of freshly-prepared SPLATSpin-ME (50 mg/lid) was included at
weathering time-points 0, 1, 2 and 3 weeks for comparison. Each of 5 treated
lids from each treatment/weathering time-point was placed inside of a
perforated plastic fruit fly trap. These traps were then suspended by means of
a wire from the roof of a large, organdy 1 m3 cage. A total of 50 male flies
were then introduced into the cage and supplied with cotton wicks soaked in
a protein/sugar/water solution as a food source. Relative attraction was
assessed as the percentage of the 50 original male fruit flies found within the
perforated plastic trap hanging within the cage after 1 hour of exposure.
Page | 67
Mortality bioassays took place directly following the attraction bioassays
in the same ventilated greenhouse, using the same organdy 1 m3 cage, and
using the same weathered, treated plastic lids. The treated lids were removed
from the perforated plastic traps used in the previous attraction bioassay.
Field Attraction and Mortality bioassays
These field cage trials were similar in most respects to the greenhouse
bioassays described above, except that the weathering period extended to 8
weeks, the field bioassays were run in cages held outdoors, and higher rates
of SPLAT-MAT Spinoad ME (1.0 g/lid) and naled+ME (7.5 g/lid) were used
in these field cage trials relative to the prior greenhouse evaluations. Two
SPLAT-Spin-ME treated lids, two naled+ME treated lids, and a single
untreated water check lid from each treatment/ weathering time-point were
evaluated. For each replicate, a treated, weathered lid was initially presented
to the male fruit flies within a perforated plastic trap suspended from the roof
of a 1 m3 organdy cage (Hsu et al., 2010).
Test cages were maintained outdoors in the shade under ambient Taiwan
weather conditions (Temp. 6-32 °C). A total of 50 male flies were introduced
into the cage and supplied with water and a dry protein/sugar mixture (ratio:
1/4) as a food source. Attraction was measured as the percentage of the 50
original male fruit flies found within the perforated plastic trap hanging within
the cage after the first hour. After the one hour attraction exposure, the plastic
traps and flies were removed from the cages, and the weathered lids (SPLAT-
Spin-ME) or caneite blocks (naled+ME) were suspended directly from the
roof of the cages, thus allowing unimpeded access to the treated lids from all
sides. A total of 50 new male fruit flies were introduced into the organdy cages
and observed for an additional 48 h.
This series of greenhouse and field experiments compared the attraction
and toxicity over time of SPLAT-Spin-ME and other commercially-used
MAT products (naled+ME) and bait spray products (GF-120 bait and
malathion protein bait) in controlling the B. dorsalis populations in Taiwan
under ambient outdoor conditions. In the greenhouse attraction bioassays
SPLAT-Spin-ME and naled+ME were the most attractive treatments for male
B. dorsalis based on the trap captures after a 1 h exposure period. SPLAT-
Spin-ME was numerically but not significantly superior to naled+ME over all
0-3 weeks weathering time-points.
Page | 68
Table 2: Attraction of Bactrocera dorsalis males (based on the capture rate within a
baited trap during a 1 h exposure period) to four MAT and bait spray treatments
versus a water control in a greenhouse cage experiment
Table 3: Mortality of Bactrocera dorsalis males within baited cages to 4 MAT and
bait spray treatments and a water control in a field cage experiment at 24 h and 48 h
grading periods
In the field attraction bioassays the relative attraction of SPLAT-Spin-ME

vs naled+ME was reversed compared to the greenhouse results, with
naled+ME the more attractive and longer-lasting treatment in these field cage
studies. A likely reason for this reversal in measured attraction is the fact that
in the field evaluations the overall applied concentration of ME in the
naled+ME treatment was raised from 37 mg/lid in the greenhouse evaluations
to 7.5 g/lid in the field experiments. The overall applied concentrations of ME
in the SPLAT-Spin-ME treatments were 25 mg/lid in the greenhouse
evaluations and 0.5 g/lid in the field experiments.
In the greenhouse mortality bioassays SPLAT-Spin-ME consistently
resulted in higher levels of B. dorsalis mortality than naled+ME at nearly all
of the 0-3 weeks weathering time-points. This was true for the 24 h and 48 h
grading periods. In the field mortality bioassays there was a reversal in the
relative efficacy of SPLAT-Spin-ME vs. naled+ME relative to the greenhouse
results, with naled+ME now enjoying a relative advantage. Mortality rates for
SPLAT-Spin-ME and naled+ME were uniformly high and generally
equivalent through weathering time-points 0-3 weeks, but by the 4th week
SPLAT-Spin-ME mortality fell off markedly and continued at these lower
Page | 69
levels through weathering time-points 4-8 weeks whereas naled+ME
maintained its effectiveness for the entire 0-8 weeks weathering period.
Repellent SPLAT formulation
An insect repellent is a chemical compound or blend of compounds that
deters insect activity on or near otherwise attractive substrates. Although
repellents have played a key role in the control of hematophagous insects and
a large number of repellent semiochemicals. The vast majority of repellents
are labile semiochemicals that quickly vanish once applied in the field, as
conventional slow-release formulation technologies are often difficult to apply
and/or inefficient in controlling the emission rate of the active ingredient.
Phytophagous insects may have the ability to discriminate between hosts
and nonhosts, as well as between hosts of different quality. For example,
herbivore-induced plant volatiles are important signals for an ovipositing
female, allowing her to judge the quality of the host plant before laying eggs,
which is a crucial decision for the survival and development of her offspring.
Volatiles released from nonhost plants are also important cues that may warn
insects to avoid nonhost or less preferred host plants, or enable them to select
the right habitat and a suitable host plant. For example, nonhost volatiles may
modulate host-selection behaviors of bark beetles by reducing their attraction
to pheromones or host kairomones, or by enhancing the effect of anti-
aggregation pheromones.
SPLATACP Repel
Field tests of SPLAT formulations releasing DMDS were conducted in
Florida in 20-tree blocks of mature sweet oranges, Citrus sinensis var.
“Valencia,” with four replicate blocks per treatment (SPLAT and control).
Trees were 12 years old, planted with 3 × 6 m spacing, and averaged 4 m in
height. Yellow sticky card traps were used to assess population densities of
Diaphorina citri. SPLAT ACP Repel was applied at a rate of 6.2 kg/ha, that
is, approximately 30 g of SPLAT per tree (six 5-g dollops per tree). This test
proved that Diaphorina citri population densities can be significantly reduced
by the application of DMDS to small plots of citrus. A follow-up field trial
was performed with SPLAT ACP Repel, containing varying concentrations of
the DMDS, and several different additives slowed the release rate of the active
ingredient. This resulted in a formulation of SPLAT ACP Repel that provides
5 weeks of repellency toward Diaphorina citri in field conditions.
Diaphorina citri are known to recolonize citrus groves 1–2 weeks after
an application of insecticide, because insecticide residues are relatively short-
lived, and Diaphorina citri adults are highly mobile.119,120 This creates the
Page | 70
need for a high frequency of insecticide sprays. Some citrus growers in Florida
and Brazil have been applying insecticides every 3-4 weeks during the field
season to manage Diaphorina citri. To evaluate whether the application of
SPLAT ACP Repel could help growers reduce the frequency of insecticide
sprays, we designed the following experiment to determine whether
Diaphorina citri recolonization in an area previously treated with insecticide
was slowed by the presence of SPLAT ACP Repel compared to those areas
receiving insecticide sprays alone.
Using a simple, randomized block design with two treatments, (1)
insecticide followed by SPLAT ACP Repel and (2) insecticide only, all
treatment plots received a label rate (40.0 oz./ha) application of the pyrethroid
insecticide After 2 weeks, half of the plots were treated with SPLAT ACP
Repel and the remaining half were left untreated. In untreated plots,
populations of Diaphorina citri began to rebound 6 weeks after treatment,
with populations returning to pretreatment levels by week 7. However, in
those plots that received the SPLAT ACP Repel treatment 2 weeks after the
insecticide spray, Diaphorina citri populations did not return to pretreatment
levels for the entire 10 weeks of study (Figure 7). These data indicate that
recolonization by Diaphorina citri was impeded by SPLAT ACP Repel and
suggest that integration of this product into a Diaphorina citri management
program has the potential to reduce the number of insecticide sprays needed
while suppressing the Diaphorina citri population below critical thresholds
This work indicates that the guava-derived semiochemical, DMDS, is an
effective repellent for Diaphorina citri at concentrations as low as 10% in
SPLAT ACP Repel. When incorporated into SPLAT, the resulting matrix
demonstrated the capacity to extend the release period of this extremely
volatile compound, prolonging the repellent’s efficacy in the field. Field trials
have shown that SPLAT ACP Repel with DMDS alone significantly reduces
Diaphorina citri captures in infested orchards, indicating the potential for this
formulation to reduce HLB infection rates in areas treated with SPLAT ACP
Repel.
Page | 71
Fig 7: Delayed immigration of Diaphorina citri into insecticide-treated plots
following application of SPLAT with dimethyl disulfide (DMDS). In untreated plots,
populations of Diaphorina citri began to rebound 6 weeks after treatment, with
populations becoming equivalent to pretreatment levels by week 7. In plots that
received the SPLAT treatment 2 weeks after the insecticide spray, Diaphorina citri
populations did not return to pretreatment levels for the entire 10 weeks of study 8
SPLAT Verb
An ideal formulation of verbenone for management of Dendroctonus
ponderosae should provide monolithic reservoir-type release at adequate rates
(>50 mg/day) for an adequate period (~3 months in Pinus contorta) allow for
the application of a relatively high density of point sources per unit area (>375
ha); allow for application by manual or mechanized means using conventional
equipment; and be fully biodegradable (within ~12 months), allowing the
formulation to be left in the field. Point sources eventually fall to the ground
and biodegrade without consequence. Most importantly, the release rate of
most active ingredients from SPLAT is surprisingly constant, decreasing
slowly over a period of weeks to months depending on the formulation. The
amorphous and flowable quality of this highly adaptable product allows for
easy transition from small-scale manual applications (single trees) to large-
scale mechanized applications (campgrounds, stands, etc.).
Fieldwork
Protecting Individual Pinus contorta from Dendroctonus ponderosae.
Trees treated with SPLAT Verb and untreated controls (n = 21 SPLAT Verb,
due to limited quantities available; n = 30 untreated control) were confirmed
to be uninfested by Dendroctonus ponderosae prior to treatment. Four large
Page | 72
dollops of SPLAT Verb were applied at approximately 3 m in height on the
tree bole, using a Graco 15:1 automotive-style pneumatic-grease pump with a
5-m hose attached to a spray nozzle. The pump was powered by a portable
gasoline-driven air compressor. A total of 15 trees received approximately 32
g of (−)-verbenone per tree (~533 g of SPLAT Verb) and the remaining 6 trees
received approximately 39 g of verbenone per tree (~650 g of SPLAT Verb).
Treatments were applied in mid-July (13.9 °C-25.6 °C, 33%-65%
relative humidity, winds <5 km/h) approximately 7-10 days after the initiation
of Dendroctonus ponderosae flight in the area. A 10-minute rainfall occurred
immediately after application. All SPLAT Verb-treated and-untreated control
trees were baited with one Dendroctonus ponderosae tree bait positioned at a
northern aspect approximately 2.4 m in height on the tree bole. Untreated
control trees were baited before those treated with SPLAT Verb, an important
caveat but one we feel had little impact given that only limited flight (~3
weeks) occurred before the baiting of SPLAT Verb-treated trees. Baits were
removed from all experimental trees approximately 30 days after baiting, at
which time all trees treated with SPLAT Verb were visually evaluated for
dollop integrity.
Only one SPLAT Verb-treated tree showed signs of Dendroctonus
ponderosae attack, whereas 83.3% of untreated control trees suffered mass
attack (boring dust and/or pitch tubes encircling the tree bole) by
Dendroctonus ponderosae at levels high enough to suggest that mortality was
imminent. Evaluations of tree mortality in the following year indicated that
SPLAT Verb provided 100% tree protection, whereas only 6.7% of the
untreated control trees survived (Table 3 & 4).
Table 3: Effectiveness of SPLATVerb for Protecting Individual Neighboring Pinus
contorta from Dendroctonus ponderosae Attack, Bridger-Teton National Forest
Table 4: Effectiveness of Verbenone Treatments for Protecting Small Stands of

Pinus contorta in Montpelier Ranger District, Caribou-Targhee National Forest
Page | 73
Over several decades, substantial research has focused on the use of
antiaggregants, primarily verbenone but also other inhibitory compounds, 15
to disrupt the responses of Dendroctonus ponderosae to attractant-baited traps
and attractant-baited and attractant-unbaited trees in hopes of developing
tactics to reduce levels of tree mortality attributed to Dendroctonus
ponderosae.
Conclusion
SPLAT® is unique among commercial semiochemical dispensers in
providing a matrix that is capable of dispensing a wide variety of compounds
and can be applied using a virtually unlimited number of manual and
mechanical techniques. The versatility of SPLAT® makes it adaptable for use
in any semiochemical based insect control program, regardless of
semiochemical, crop, or plot size. SPLAT® mating disruption and attract-and-
kill formulations has been developed for important agricultural and forestry
pests both domestically and internationally. Several of the existing SPLAT®
mating disruption formulations have also been certified for use in organic crop
production. Repellent formulations are currently being developed for
important agriculture and forestry pests, as well as attractant formulations for
beneficial insects. ISCA will continue to work with collaborators to test the
limits of the SPLAT® matrix for dispensing insect behavior-modifying
compounds in a variety of environments as it participates in research to create
new semiochemical-based insect control tools.
References
1. Faleiro JR, Al-Shawaf AM, Al-Dandan AM, Al-Odhayb A, Al-Rudayni
A, Abdallah AB, et al., Controlled Release Products for Managing Insect
Pests. Outlooks on Pest Management. 2016;86:15-21.
2. Heuskin S, Verheggen FJ, Eric Haubruge, Wathelet JP, Lognay G. The
use of semiochemical slow-release devices in integrated pest management
strategies. Biotechnol. Agron. Soc. Environ. 2011;15:459-470.
3. Hsu JC, Liu PF, Hertlein M, Mau RF, Feng HT. Greenhouse and field
evaluation of a new male annihilation technique (MAT) product, SPLAT-
MAT Spinosad ME, for the control of Oriental fruit flies (Diptera:
Tephritidae) in Taiwan. Form. Entomol. 2010;30:87-101.
4. Mafra-Neto A, Christopher J, Fettig A, Munson S, Stelinski L. Use of
Repellents Formulated in Specialized Pheromone and Lure Application
Technology for Effective Insect Pest Management. American Chemical
society. 2013;16:209-311.
Page | 74
5. Mafra-Neto A, Frederique ML, Christopher JF, Munson AS, Stelinski LL,
Vargas RI. Manipulation of insect behavior with special pheromone and
lure application technology (SPLAT®). In: Pest management with natural
products, American Chemical Society, ACS Symposium series,
Washington, 2014, 32-58.
6. Stelinski LL, Miller JR, Ledebuhr R, Siegert P, Gut LJ. Season-long
mating disruption of Grapholita molesta (Lepidoptera: Tortricidae) by
one machine application of pheromone in wax drops (SPLAT-OFM). J
Pest. Sci. 2007;80:109-117.
Page | 75
Page | 76
Chapter - 5
Biocontrol Agent: A Promising Tool towards
Sustainable Agriculture
Authors
Ushma Joshi
Department of Microbiology Faculty of Sciences, Ganpat
University, Mehsana, Gujarat, India
Mrunali Patel
Department of Biotechnology, Faculty of Sciences, Ganpat
Nehal Rami
Department of Microbiology, Faculty of Sciences, Ganpat
Dhvani Patel
Department of Microbiology, Faculty of Sciences, Ganpat
Kruti Dave
Department of Biotechnology, Faculty of Sciences, Ganpat
Page | 77
Page | 78
Chapter - 5
Biocontrol Agent: A Promising Tool towards Sustainable
Agriculture
Ushma Joshi, Mrunali Patel, Nehal Rami, Dhvani Patel and Kruti Dave
Abstract
Biocontrol can be defined as the control of the type of living organisms
with the application of another. The use of Biocontrol agents are also called
biological control and are mainly used for the reduction of pest population
and produce yields that are free of any pests. The biocontrol methods are a
long-term method and most importantly, a self-sustaining one that helps in
the control of invasive species of plants. Nowadays, Plant and Microbes
based biological control is considered to be a rapidly developing natural
phenomenon in research area with implications for plant yield and food
production. They may help to maintain the quality of crops, food and feeds
and reduce the undesirable usage of chemical pesticides and toxic chemicals.
The present study includes the current progress of biological control by
using molecular docking and understanding about the mode of mechanism.
Keywords: Biotic stress, abiotic stress, biocontrol, molecular docking
1. Introduction
1.1 Role of agriculture in India
India has achieved remarkable place in the sector of Agriculture from
past 30 years; this has become possible because of number of families of
farmers and scientists working in the field of agriculture. This tremendous
growth sector is flourishing and contributing Indian agriculture with help of
research and analysis of crop, government policies for crop production.
Trading and export of agricultural products is a key factor for an economical
growth of any region; specifically developing Nation. Export of such
agricultural products can effectively balance the economical investment
done by any country like India for petroleum products, capital goods and any
other product necessary for sustained growth. Agriculture plays very
important role in the Indian economy and over 70% of the rural household
depends on agriculture, which contributes sixty per cent to the total
population andabout seventeen per cent to the total GDP [1].
Page | 79
Fig 1: Role of Agriculture in Indian economy
1.2 Problem in agriculture sector
Plant crop during their growth life cycle faces many challenges which
cause stress during their growth period. These factor causing plant growth
are majorly categorized into biotic stress and Abiotic stress.
1.2.1 Abiotic stress
A variety of environmental factors like Alkality, drought, salinity,
temperature, pH, nutrient deficiency, high light are the major abiotic factors
that affects the plant growth and its metabolic activity. Abiotic stress affects
plant via various mechanisms like change in the salt concentration results in
decrease of vacuolar and cytosolic volumes, when amount of available free
water decreases in soil it causes drought condition in plant system and
affects plant growth by disturbing biochemical reactions. Concentration of
certain metals like Cu, Zn, Ni, Ag, Mg plays very important role as their
unavaibility can hinder plant growth as they play role as cofactor in various
enzymatic reactions and their excessive amount can have some toxic effect.
Difference in temperature can have adverse effect on crop yield; low
temperature can have some freezing effect on cytosol of temperate crop and
increase in temperature leads to disturbance in plant metabolic activity [2].
1.2.2 Biotic stress
On a global scale, plant diseases have drastic impact on social,
economical and ecological impact. Potato late blight and chestnut blight are
Page | 80
the best example in the context of plant diseases which has led to human
migration and massive human death [3].
In India during past few years agriculture crop production has achieved
285-300 million MT because of the green revolution technology, but at the
same time this total yield is getting affected by certain biotic and abiotic
stress. In India approximately 15-20% yield loss in major crops has been
estimated because of biotic and abiotic factors [4].
Biotic factor responsible for creating stress in plant growth includes
weed, insects, nematode pest and pathogenic microorganism like pathogenic
bacteria, viruses and fungi. Vivid variety of pest and insects including plant
hoppers, whitefly, mealybug, are highly responsible for decrease in crop
yield. Nematodes are one of the potent parasites infecting crop yield, they
have become foremost threat to the total yield of ornamental and vegetable
crops, oilseed crops and pulses of the country. Meloidogyne enterolobii is a
nematode pest causing severe effect on Pomegranate and Guava crops;
oilseed crops and pulses has been reported to be damaged by Rotylenchulus
spp. and Pratylenchus spp. Microorganisms like viral, bacterial and fungal
pathogens can be transmitted in variety of plant crops species via insect
vectors, water, soil, air and water and they can habituate and pass on from
generation to generation for long period of time. After entering inside the
plant host they can have a variety of impact on plant survival, growth and
reproduction.
Fungal diseases are severe threat to the primarily important crops of
India including rice, wheat and maize. A huge yield loss in different crops
has been reported for the same. Psedomonas sp., Xanthomonas sp., Erwinia
sp., Ralstonia sp., Agrobactrium sp., like bacteria has been reported to cause
disease in plants, over 200 plant pathogenic bacteria [5].
Table 1: List of plant pathogens along with disease they are causing and host plant
Plant Crop Plant pathogen Plant Disease

Rice Helminthosporium oryzae Destruction of Rice
Wheat Puccinia graminis Stem rust
Rice Pyricularia oryzae Blast of Rice
Rice Rhizoctonia solani Sheath blight
Stripe/yellow rust
Wheat andother cereals Puccinia striiformis
pathogen
Fusarium graminearum Gibrella Stalk and ear rot
Maize Aspergillus flavus Aspergillus ear
Cercospora zeae-maydis Grey Leaf spots
Page | 81
Cotton Xanthomonas citri Bacterial blight
Fusarium oxysporium Fusarium Wilt
Goundnut rossete Virus
Groundnut Groundnut rossete Disease
Groundnut rossete assistor Virus
Ralstonia solanacearum Brown Rot
Potato
Phytophthora infestans Late blight
Phytophthora infestans Late blight
Tomato
Yellow leaf curl virus Yellow leaf curl
Tomato is the second most cultivated vegetable crop in India, it is a

good source of antioxidant compounds, anti-cancerous compounds and
Vitamin C. Crop yield of tomato is highly getting affected by biotic stress.
Psedomonas syringae, Perenospora parasitica, Xanthomonas campestris are
the bacterial pathogen which cause disease in tomato plant. Fungal pathogen
Fusarium oxysporum and pathogenic viruses like Tomato mosaic virus,
Cucumber mosaic virus and Turnip mosaic virus causes different kind of
disease in Tomato plant which results in to loss of crop yield. Tomato
spotted wilt virus are responsible viruses for causing disease in tomato leaf
and stem drying respectively. Yellow curl disease highly affects plant by
developing interveinal chlorosis on lower leaves and bronzing of leaves [6].
Groundnut or Peanut was initially cultivated in West India, Brazil,
Maxico, Bolivia, Peru; worldwide it is harvested in to 19 million hectares,
Asia is the top most producer of groundnut and India is the largest producer
in the world. However, yield of Groundnut is getting affected because of
various factors including pest and disease [7].
1.3 Solution available and problems with available solution
Chemical pesticides and fertilizers and fungicide are considered is a
must needed part of recent agriculture sector and crop protection as they
attacks plant pathogens, insects and pests. Because of this high impact, their
demand is increasing day by day. Though they increases plant yield their
consent and excessive use has resulted in severe physical, chemical and
biological changes in soil ecosystem. A long run farming with application of
chemical insecticides, fertilizers and pesticides highly affects on quality of
organic matter, pH, soil structure and nutrient cycling [8]. This kind of
problems generated by agricultural activities has become a severe threat
globally. Excessive use of chemicals along with affecting agroecosystem,
they are affecting ground water, surface water, and air which ultimately
affects human health and environment. Some harmful chemicals which is
used as pesticides also disturbing food chain. Along with affecting
environment this chemicals have severe adverse effect on plant system [9].
Page | 82
Table 2: List of Fungicide, Herbicide and insecticide and their adverse effect on plant
[9]
Fungicide
Fungicide/Pesticides Chemical family Adverse effect on plant
Mefenoxam Amide Affects RNA synthesis
Metalaxyl Amide Inhibits RNA synthesis
Chloramphenicol Antibiotic Inhibits peptidyl transferase enzyme
Validamycin Antibiotic Inibits enzyme trehalase
Carbendazim Benzimidazole Inhibits turbulin formation
Affects respiration and production of
Clorothalonil Organoclorin
energy
Herbicide
Diflufecnican Amide Inhibition of caratenoid biosynthesis
Dinoterb Dinitophenol Disturb photosynthesis
Inhibitors of photosynthesis at photosystem
Atrazine Traazine
II site A
Aurora 40 WG Sulfonyurea Inhibition of acetolacte synthase
Insecticide
Endosulfan Organophosphate Altefration of enzymes
Cypermetherin Pyrethoids Interfere with sodium channels
Affects respiration process and production
Methomyl Carbamates
of energy
Novaluron Benzoylphenylurea Chitin synthesis inhibitor
2. Biological agents and their importance

Biological control of plant pathogens has become an integral component
of pest management in light of the environmental and health isuues
attributed to fungicides in agriculture. Biological control agents (BCAs)
have a fascinating role in enhancing global plant health. Microbes, animals,
and plants are all included in BCAs. The significant disparity between
groups reflects a variation in the approach to improving plant health.
Bacteria, viruses fungus and protozoa belong to the first group, while mites,
spinders, nematodes and insects belong to the second group. Trees and a
variety of flora make up the third group.
The BCAs maintain the pest within acceptable limits. The ability of
these three groups towards plants can generally be determined using direcy
approach, an indirect way, or a combination of both methods. The direct
method is more interesting since it can be used to control plant infections,
weeds and pests (plant enemies) by directly targeting them. The indirect
Page | 83
strategy is less successful and primarily works by managing or directing
the growth of plant enemies. Among the several BCAs, microbes have high
efficacy and are simple to create [10].
Weeds can be controlled using plant-suppressive microorganisms that
inhibit the growth of grass weeds. The effect of bacterial variety on plant
health and growth may be seen in these microbes. Because of their transient
nature, this category of bacteria is sometimes disregarded; however,
research into plant-suppressive bacteria has resulted in advances in crop
management methods and has shown promise for biological weed
control of various weed species. Because they are active colonisers of the
roots and residue, these bacteria are good biological control agents. They
serve as a direct delivery system for the insecticide they manufacture
naturally. More importantly, these bacteria illustrate the vast amount of
genetic material found in soil that biotechnology might use [11].
2.1 Plant based phytochemicals, their importance and application
Plant produces secondary metabolites as a defence against insects,
pathogenic bacteria and harsh climatic circumstances. In that, flavonoids,
phenolic compounds, carotenoids, phytosterols, terpenoids, alkaloids, tannins
and saponins are primary groups. Antibiotic resistance is one of the most
critical public health issues. This issue arises due to pathogenic
microorganism adaptation to antimicrobials used in various settings,
including food animals, crop production, medicine and disinfectants in
farms, homes and hospitals. The microorganisms have evolved resistance to
all known antibiotics, resulting in a considerable economic cost associated
with multi drug resistant bacteria [12].
The plants had investigated as sources for identifying effective and
different antimicrobials to acquire different antimicrobial agents with unique
mechanisms of action. Plants are significant source of antimicrobial
compounds with the majority of them being effective against yeasts, insects,
bacteria, other plants and fungus [13]. Phytochemicals inhibit peptidoglycan
formation, harm microbial membrane structures, change the hydrophobicity
of bacterial membrane surfaces and regulate quorum-sensing (QS).
2.1.1 Alkaloid compounds
Alkaloids are nitrogen-containing heterocyclic chemicals commonly
found in plants. Theyare one of the most significant natural artefacts due to
their enormous number, complexity and structural diversity. Alkaloids have
been found to have antibacterial properties after extensive research.
Page | 84
Alkaloids restrict bacterial growth by inhibiting protein synthesis and
nucleic acid, damaging cell wall and cell membrane, modifying the bacterial
cell membrane permeability, inhibiting efflux pumps, inhibiting bacterial
metabolism [14].
2.1.1.1 Inhibition of the bacterial nucleic acid and protein synthesis
DNA and RNA are the nucleic acids found in bacteria. Generally RNA
molecules serve as a messenger molecules that ensuring appropriate protein
synthesis, whereas DNA molecules store, transfer and copy the genetic
information. As a result, DNA/RNA molecule damage or DNA replication
inhibition limits the expression of virulence genes, which impacts
microorganism characteristics, reproduction and growth. Filamentous
temperature sensitive protein Z (FtsZ) is involved in developing
diaphragms after that it creates ring structure at division region, which
regulates the bacterial cell division process. As a result, FtsZ is a target for
developing novel antimicrobials [15].
In E. coli, the FtsZ protein is required for cell cycle. It helps to construct
the diaphragm and develop ring structure at division region to keep the
bacterial cell division method under control.
Sanguinarinerine causes filamentation in gram positive and gram
negative bacteria and inhibits cytokinesis, limiting bacterial cell division. In
E. coli, it causes cyto-kinetic Z rings development to be disrupted. In
addition, sanguinarinerine affects the assembly of pure FtsZ and lowers
protofilament bundling in vitro. Matrine also prevents the protein synthesis
involved in development of cell also division in E. coli and S. aureus,
preventing bacteria from dividing and growing. Matrine attaches to protein
in cell, producing aggregates, which causes cytoplasm to disintegrate, killing
the bacterium [16].
2.1.1.2 Effect on bacterial cell membrane permeability-Damage of cell
membrane and its cell wall
Bacterial cell membrane comprises phospholipid bilayers and proteins
and is elastic and semipermeable. It functions cell identification and electron
transport and provides a generally controlled inner environment for bacterial
life activities. The capacity to build biofilms gives bacteria a selection
advantage for surviving in hostile environments [17]. Impairment to bacterial
cell membrane compromises protective barrier of cell, allowing a high
number of molecules to escape. In addition, the defence role of cell wall is
vanished and transport of cell membrane and data transfer capabilities are
hampered. The leakage of intracellular electrolyte accelerates the
Page | 85
conductivity of the culture medium into medium. As a result, changes in the
conductivity of culture supernatant promotes in the permeability of the
bacterial cell membrane. Normally, alkaline phosphatase (AKP) is present
in-between bacterial cell wall and cell membrane and the permeability of the
bacterial cell wall increases, further, AKP would break the cells. The
integrity of bacterial cell wall can be inferred from AKP activity
measurements [18].
2.1.1.3 Inhibition of efflux pumps
Drug efflux pumps are transmembrane protein complexes seen in
bacteria. When the efflux pump system completes well timed exclusion of
membrane permeable antibacterial agents, then the pathogenic bacterial cell
avoids contact with the antibacterail agent in the membrane, reducing the
bactericidal impact of antibacterial agent and incresing pathogen resistance
through the membrane [19]. Many studies have found that E. coli biofilms
have stronger drug resistance than planktonic cells, which has been
linked to enhanced efflux pump gene expression in biofilms, which could
cause to an increse in the division density or activity of cell membrane efflux
pumps. While inhibiting efflux pump protein expession can lead to drug
resistance in biofilm forming strains.
2.1.1.4 Inhibition of bacterial metabolism
Berberine can impact Streptococcus pyogenes (Group A streptococcus,
GAS) carbohydrate metabolism via enhancing carbohydrate transformation
and absorption while decreasing carbohydrate consumption. It also
stimulated the conversion path of further compounds to monosaccharides
and their derivatives, which boosted carbohydrate conversion andabsorption.
Further, Berberine also increased the activity of the ATP-binding cassette
transporter and phosphotransferase systems elaborate in glucose absorption.
Many different process in glycolysis, pyrimidine metabolism, purine
metabolism, fructose and mannose metabolism and fatty acid biosynthesis
were repressed in the berberine treatment groups to lower carbohydrate
consumption [20]. The redox and energy substance metabolisms enhance
imbalanced due to the disruption of carbohydrate metabolism. These
findings revealed that berberine stimulated excessive ROS in GAS, which
inhibited the bacteria.
Page | 86
Fig 2: Antibacterial mechanism of natural alkaloids
2.1.2 Phenolic compound

Antioxidant, anti-allergic, anti-inflammatory, anticancer,
antihypertensive and antibacterial activities are all polyphenols. Polyphenols
are split into two groups based on their chemical structure: non- flavonoids
and flavonoids. Flavonoids are group of structurally varied chemicals. The
link between structure and antibacterial action in chalcones has been widely
investigated among flavonoids. The chalcone molecule has an open
heterocclic ring, which when closed transforms into flavanone. According
to the current findings, the chalcone skeleton offers an appealingtemplate for
chemical changes that could improve antibacterial potential [21].
2.1.2.1 Membrane disruption by phenolic compounds
Membrane disruption contributes to the antibacterial activity of
maximum plant phenolics, which has been mechanically examined in both
gram positive and gram negative bacteria. More unusual tests have been
carried out, such as measuring the likelihood of a hydrophilic water drop to
spread or bead while in contact with a bacterial lawn to quantify cell surface
hydrophobicity [22]. This test focused on the effects of Ferulic and Gallic
acids, which increased surface hydrophobicity of Gram-negative and Gram-
positive bacteria. However, this amount of surface hydrophobicity could be
related to direct or indirect interactions through internal components that can
impact cell wall chemistry.
Individual binding assays or model membrane insertion assays can
assuredly evaluate straight interactions between specific components once a
Page | 87
membrane effect exists [23]. The straight interaction of epigallocatechin
gallate and epicatechin gallate along with membranes has been tested using
model membranes [24]. However, the wide range of bacterial membrane
composition would almost certainly necessitate individual testing for each
organism of interest. Elementary membrane integtrity analysis with added
exogenous magnesium is another distinctive experiment that may be useful
in measuring direct membrane binding. This analysis demostrated a
magnesium dependence similar to ethylene diamine tetra acetic acid (EDTA)
regulating membrane disrupting activity of gallic acid [25]. The magnesium
ions are recognised to stabilize gram negative bacterial membrane. This
simple analysis could be used to analyze direct magnesium-mediated
membrane interactions and other essential magnesium-dependent cell
activities.
2.1.2.2 Synergistic antibacterial activity
Membrane-disrupting chemicals, such as phenolics, have been linked to
various synergistic effects, including allowing intracellular poisons to be
more accessible and faster to access their targets [26]. Moreover, strains that
use efflux pumps to remove toxins can be targeted by inhibiting the efflux
pumps with one chemical, using an intracellular toxins as another component
for synergistic death [27]. The expression of efflux pumps has also been found
to be downregulated. Other modes of action may have synergistic effects,
increasing the effectiveness of existing antibacterial agents, particularly in
addressing drug resistance processes.
2.1.3 Tannin compound
Tannins are polyphenolic chemicals found in plants that bind to
proteins, amino acids, and alkaloids, causing them to precipitate. They are
antibacterial biomolecules that are well- known. Natural products are being
studied as anti-infective medicines, and the structures of tannins with
antifungal, antiviral and antibacterial properties have been discovered.
Tannin can penetrate through the cell wall and into the interior membrane,
interfering with cell metabolism and ultimately killing it. Tannin has a fast
action on Gram-positive bacteria. However, it takes longer in Gram-negative
bacteria with a bilayered membrane. It is an essential distinction because
various bacteria flourish in specific microbial environments. Tannins
inhibit the fungal enzymes, cellulases and pectinases, and which are required
for the fungal digestion of plant material [28].
2.1.4 Terpenoid compound
Essential oils are made up of a complex blend of chemicals, ranging in
concentration from 20 to 60 [29]. Terpenes, the significant ingredients of
Page | 88
essential oils, are generated and released from specific plant tissues via the
isoprenoid pathway. They are made up of isoprene units, which are used to
classify them: two isoprene units make monoterpenes, three units make
sesquiterpenes, four units make diterpenes, six units make triterpenes, and
eight units make carotenoids. Alcohols (linalool, geraniol, carveol,
citronellol, terpineol, menthol, borneol, and bisabolol), aldehydes (citral and
citronellal), phenols (thymol and carvacrol), ketones (carvone and
camphor), ethers (eucalyptol), and hydrocarbons (cymene, pinene, limonene
and phellandrene) groups.
There have been some mechanisms proposed. The rupturing of the cell
membrane and alterations in the ion channels (Na+, K+, Ca2+, or Cl) in
the cell membrane, for example,may enhance permeability and result in the
release of critical intracellular constituents and inhibition of target enzymes
[30]
. A recent study using scanning electron microscopy revealed that
thymol's fundamental mode of action is membrane disruption, implying that
it might be used instead of synthetic antibiotics to treat S. Typhimurium.
Hydroxyl groups, such as thymol, terpineol, eugenol and carvacrol are
highly reactive and establish hydrogen bonds with target enzymes active
sites, rendering the inactive, resulting in cell membrane malfunction or
rupture. For example, Thymol is molecule found in many essential oils that
inhibits gram positive and gram negative bacteria such as Bacillus subtilis,
E. coli, Klebsiella pneumoniae, and Staphylococcus aureus [31].
3. Microbes based
3.1 Indirect biocontrol

The indirect methods involve targeting plant pathogens without killing
them directly but decrease inhibitions in the growth of this community. The
plant-beneficial microbial group deploys several tools including competition
for space and nutrients, producing secondary compounds and antibiotics,
inducing plant defenses, and enhancing tolerance and plant growth. The
biological control agents from plant groups with plant pathogens competed
Page | 89
on nutrients such as carbon and iron. Several microbial species of biocontrol
agents such as bacteria and fungi can produce secondary compounds and
antibiotics inhibiting the growth of the plant enemies. Pseudomonas
fluorescens WR-1 produced volatile organic compounds, which caused a
reduction in growth and virulence of plant pathogen Ralstonia solanacearum.
Several useful microbes can induce systemic resistance and defense in the
plant against the plant enemies, i.e., both plant pathogens and pests [32].
3.2 Direct biocontrol
The direct effect is a suitable method for a reduction in the numbering
of plant enemies. The ability to impact plant enemies directly through fungus
parasitism is an example. A group of bacteria are considered natural
enemies of plant pathogens and are found abundantly in the soil. These
bacteria utilize a variety of techniques to accomplish their objectives,
including the production of toxins, antibiotics, volatile organic compounds
(VOCs), and enzymes, as well as competing for resources and inducing
systemic resistance in plants. Microbacterium nematophilum, Pseudomonas
spp., Bacillus thuringiensis, Pasteuria penetrans and Serratia marcescens
were found among the microorganisms that have been deadly against
nematodes from various species [33].
Pseudomonas: Heterotrophic bacterium, a gram-positive that can
induce plant protection against plant pathogens including cucumber mosaic
virus, bacteria like Xanthomonas campestris pv. malvacearum, and fungi like
F. oxysporum f. sp. cubense tropical race 4 Mohammed et Pseudomonas
putida developed nematicidal substances which including cyclo (L-ProeL-
Leu), that caused the individual of plant-parasitic nematode M. incognita to
die as well as the rate of egg hatching to reduce [34].
Bacillus thuringiensis: Bacillus thuringiensis releases Cry toxins and is
a Gram-positive bacterium. During the spore germination process with this
poison, it forms a parasporal body called crystal. It's an insecticidal protein
that's found in nature. D-endotoxins, commonly referred to as Cry or Cyt
proteins, make up the protein crystal. The d-endotoxins are active against
lepidopteran insects such as butterflies, beetles, mosquitoes, ants, and
mites, as well as other invertebrates such as nematodes, flatworms, and
protozoans [35].
3.2.1 Hydrolytic enzymes
Chitinases, in addition to other cell wall dissolving enzymes like 1,3-
glucanase, are commonly produced by plants as a defence strategy.
Chitinases, which are an essential biocontrol mechanism in the rhizosphere,
Page | 90
can also be produced by microbes [36]. Their role in the rhizosphere
suggests that microbial chitinase activity in the phyllosphere may be
studied further. The B. subtilis J9 strain has been shown to protect
strawberry plants from B. cinerea in field settings and to produce
extracellular chitinase and protease [37].
We recently discovered that lactobacilli that produce bifunctional
enzymes with chitinase/peptidoglycan hydrolase activity can limit fungus
hyphae development in vitro [38]. Lactobacilli are not common phyllosphere
residents, and their survival rate is frequently poor. However, they have been
demonstrated to dominate the endosphere of Origanum vulgare plants and to
be inversely connected with disease symptoms of cucumber leaf spot, likely
caused by P. syringae pv. Lachrymans [39].
Microbes can trigger the development of chitinases in the host plant,
which is a frequent defence mechanism in plants, in addition to the creation
of hydrolytic enzymes by the biocontrol agents themselves. In the section
"Indirect Interactions", we go over how a pathogen can be inhibited by
activating a defence response in the host.
3.2.2 Quorum Sensing and Quenching
If a specific concentration threshold of signalling molecules is exceeded,
quorum sensing systems cause bacteria to change their activity. Non-
pathogenic bacteria, interestingly, use the same signaling molecules as
pathogens and, as a result, can contribute to disease progression or
inhibition, depending on how they interfere. The bacteria that cause olive
tree knot disease, Pseudomonas agglomerans, Erwinia tolerance, and
Pseudomonas savastanoi, all had an AHL-signal-based quorum-sensing
system in common. Symbionts, or opportunistic pathogens, in this case,
benefit from the niche produced by the pathogen's illness development and
so engage in its communication system. E. toletana and P. agglomerans
worsened the infection in the olive trees in this study by working with the
pathogen [40].
Other P. agglomerans strains, on the other hand, showed biocontrol
activity in tomato plants against the pathogen P. syringae pv. Tomato [41], but
it is unclear whether quorum sensing is involved. Actual biocontrol agents'
ability to have this effect on both target and non-target diseases has yet to be
determined.
3.2.3 Competition for Nutrients and Space
The Niche-Overlap Index (NOI) is a metric for determining how similar
two microorganisms' carbon source profiles are [42]. Wilson and Lindow
Page | 91
(1994) estimated the NOI by dividing the total number of carbon sources
used by both strains by the total number of carbon sources used by one
strain. The ability of the epiphytic bacteria Pseudomonas fluorescens, P.
agglomerans, Stenotrophomonas maltophilia and Methylobacterium
organophilum to coexist with the pathogen P. syringae on the phyllosphere
of beans was found to be inversely related to their NOI (Phaeseolus
vulgaris).
The ability to inhibit disease caused by P. syringae pv. tomato was
associated with the NOI of 36 non-pathogenic phyllosphere bacteria in
another investigation [43]. These findings back with theory that "antagonism
owing to competition between strains would rise proportionally to the
overlap of their ecological niches". This notion was based on field research
in which ice nucleation-deficient P. syringae mutants successfully
antagonised the wild-type P. syringae strain in field circumstances when the
mutant was treated two days before the wild-type strain.
The mutants were able to outcompete the wild-type strain under these
conditions, and there was a reduction in frost harm to the plants. The
mutants, on the other hand, had the advantage of being the first to occupy the
ecological niche. Priority effects play a significant role in microbial
competition and the formation of phyllosphere communities as a result, some
biocontrol agents work better as a preventative strategy than as a therapy.
3.2.4 Siderophores
However, for the biocontrol agent P. syringae pv. syringae 22d/93,
siderophore production is an essential adaptation factor, since the population
number of a siderophore-negative mutant was 2 orders of magnitude lower
than that of the wild-type 10 days after inoculation. Furthermore, in
numerous systems, siderophores have been implicated in induced systemic
resistance (ISR) (see section "Induced Systemic Responses"). It's not
impossible that the injury in Wensing et al. (2010)'s experiment induced ISR
via host jasmonic acid (JA) and ethylene-mediated pathways (see "Plant
Hormones" section). The presence of an extra siderophore-induced ISR was
no longer required after wounding, and the strain had no biocontrol function
in the wounded plants.
In addition to direct interactions, biocontrol chemicals can indirectly
inhibit infections by modifying the immune system or hormone levels of the
plant. Indirect interactions between microbes and plants that defend the plant
from pathogen infection are explored here.
Page | 92
Biological control agents (BCAs) have benefits over chemical pesticides
in that they are less environmentally damaging. Chemical pesticide residue
has the adverse effect on the environment. Depending on the type of
chemical pesticide and its beneficial effects on plant enemies, that is still a
synthetic chemical, and excessive chemical residue can damage the
environment for a long time, require a significant financial investment to
remove and restorea clean ecology complimentary of carcinogenic as well as
other chemicals. Plant enemies do indeed produce chaos and destruction and
devastate crop fields, resulting in starvation and the demise of the country, as
well as migration to other locations.
4. Fungi as bio-control agents
The use of fungal endophytes as a technique of raising yields and
protecting plants from harm is becoming more common in organic farming
systems. Endophytic fungus of several species has been highlighted as
possible indirect biocontrol agents in large-scale agricultural applications. As
a result, using biotechnology to improve crops by inoculating plants with
modified fungal strains would lessen toxicity to humans, livestock, and the
environment. The genes of fungal endophytes might be genetically altered
by removing harmful genes or adding new helpful ones. Endophytes might
therefore be utilised as surrogate hosts to genetically modify crops [44].
In this regard, the development of biopesticides has developed with
various functionalities, offering a promising potential for indirect usage at a
cheap cost, making it appropriate for agriculture. Biopesticides based on
fungi take things to the next level. Many of these items include parasite
fungus, which grow within the body of an insect and feed on its internal
tissue until it dies (and sometimes beyond that). As seen by the growing
number of commercial treatments available or in development, fungi are
increasingly being used to manage invertebrate pests and illnesses. Plant
productivity, animal and human health, and food production are all affected
by fungal biological management, which is an attractive and fast increasing
study subject. Fungi are particularly appealing as biocontrol agents because
of their widespread distribution, high degree of host specificity, host death,
persistence, dispersion effectiveness, and simplicity of culture and laboratory
maintenance. Fungal biological control agents (BCAs) research,
development and eventual commercialization continue to face a variety of
challenges, ranging from clarifying vital basic biology information to socio-
economic aspects. Although significant progress has been achieved in
several areas, it is critical to integrate and share this new results [45].
Page | 93
Soil microorganisms like nitrogen-fixing symbiotic bacteria and
mycorrhizal fungi, which aid in mineral nutrition, play an important part in
plant development and crop success. Nonetheless, further study is needed to
determine the entire scope of plant associated microorganisms and the
capacity to switch conventional fertilisers. Scientists already completed the
significant development in understanding the composition and behaviour of
rhizospheric microbiomes in recent years. Today, novel research
investigations are being done to improve the utilisation of microbes in weed
control, soil recovery, fertility enhancement, and even antibiotic production.
Plants may also help microbes grow in a complimentary fashion; after
all, it is a symbiotic connection. The release of organic compounds into the
rhizosphere by plants is a major component in shaping the population of a
microbiome. As with plants, soil microorganisms' growth and development
are influenced by a variety of environmental conditions such as the amount
of obtainable nutrients, pH, temperature, humidity and aeration degree. As a
result, the mix of plants and bacteria in the soil has an impact on the life
forms that live there. In the literature, three strategies have been proposed to
clarify how microbial action hinders with plant growth: altering plant
hormone signalling, repelling or outperforming other disease producing
pathogens, and boosting soil nutrient diversification. As a result,
microorganisms contribute to the long-term viability of agro-ecosystems, not
only by lowering agricultural's ecological effect, but also by providing high-
performance fertilisers. Entomopathogenic fungi are mostly utilised in
agricultural regions to control arthropods, although their usage is limited
owing to environmental factors. Its effect on arthropod hosts has been
researched for hundreds of years, but their additional roles have remained
mostly unknown. Nonetheless, recent literature has shown a vast scenario in
which entomo-pathogens can performance an important role in nature by
encouraging plant development, functioning as endophytes and opponents to
plant diseases, and colonising the rhizosphere. Biocontrol treatments for
insects and ticks usually have a direct influence on arthropods, but they
have little influence on parasite growth in the future. In this regard, the
development of biopesticides has developed with various functionalities,
offering a promising potential for indirect usage at a cheap cost, making it
appropriate for agriculture [46].
4.1.1 Trichoderma
Trichoderma is a genus of rhizocompetent filamentous fungal strains
that serve as saprophytic fungi and mycoparasites in a wide range of
environments and are commonly utilised as biological control agents.
Page | 94
Trichoderma species generate a lot of conidia, which are generally
yellowish or green, ovoid and uninucleated. Conidiophores resemble
pyramidal shrubs in that they are branching. The genus is perhaps the utmost
researched for its benefits on plant disease control. The capacity to utilise
a wide range of compounds as carbon and nitrogen sources, as well as
resistance to inhibitors produced by other microorganisms and tolerance to
various types of fungicides, distinguishes members of this species.
Furthermore, their rapid growth, production of chlamydospores, and high
ability to degrade structural and nonstructural carbohydrates make them
suitable for biological control, as these ecological characteristics allow
them to compete for, colonise, and protect important pathogen entry points
in host plants such as roots, lesions, and senescent tissue. Trichoderma spp.
have the ability to antagonise, parasitize, or even kill other fungi, which has
led to the use of several species in the field for biological control of
phytopathogenic fungi. This fungus has demonstrated a high opportunity to
boost numerous benefits to plants, which include defence against
phytopathogens, advancement of growing plants via processes such as
phytohormone production, improved nutritional provision in the soil thru
the recycling nutrients, and thus enhanced crop productivity, as well as
the production of enzymes andmetabolites for industrial applications.
The interaction between Trichoderma and plants occurs in the root area,
where the growth promoting process occurs, which is connected to the
production of plant hormones, vitamins, or the transformation of resources
into useable forms. Trichoderma can act as plant growth biostimulants by
releasing phytohormones that promote root development. They may
colonise the root system, the surfaces, and the interior of plant
tissues without causing sickness. The exchange of bioactive chemicals
between the plant and the fungus in this contact zone, which creates a
chemical interaction zone, is how they communicate. The fungus'
metabolites thicken plant cell walls and cause the synthesis of phenolic
compounds, which restrict the fungus from spreading beyond the infection
site and causing harm to the plant [48].
4.1.2 Clonostachys
Clonostachys corda (Ascomycota, class Sordariomycetes, order
Hypocreales, family Bionectriaceae) and Clonostachys rosea (LINK) are
fungi which live in soil or are associated with decaying plant tissue. This
species produces light-hued colonies, which usually gain plenty of pink,
yellow, or green, due to the aggregation of coloured conidial masses or the
formation of exudates in the culture media. This fungus features
Page | 95
morphologically appressed branches and phialides, as well as dimorphic
hyaline hyphae and conidiophores that proliferate throughout the colony. C.
rosea is commonly used, especially in agriculture. These mycoparasitic
fungi can parasitize other fungal species and function as biological control
agents for phytopathogenic fungi-caused root illnesses. C. rosea has been
demonstrated to be effective in suppressing B. cinerea, which produces grey
mould in a variety of crops, in both controlled and field trials. C. rosea
isolates are capable of epiphytic and endophytic colonisation once applied to
the aerial section of plants. Nonetheless, after a few days, the surviving
biomass in leaf tissues drops substantially, necessitating weekly
reapplications to successfully manage this disease in the field. Plant tissues
are rapidly colonised by C. rosea when they become senescent, owing to a
large number of enzyme- coding genes involved in the breakdown of
plant carbohydrates in this species' genome, which is equivalent to that
observed in phytopathogenic species. The ability to create bioactive
chemicals of secondary metabolisms, such as peptaibols, bisorbicillinoids,
and glisoprenins, has also been connected to Clonostachys antagonistic
potential against other fungi. Furthermore, significant amounts of hazardous
compounds generated by other fungi, including as mycotoxins and oxidising
agents, as well as fungicides routinely employed in plant disease treatment,
are tolerated. C. rosea also uses chitinolytic enzymes in its interactions with
other fungus [49].
4.1.3 Fusarium
It may be used to manage insects and weed pathogens by infecting them
with a variety of illnesses. Fusarium moniliforme is used to control
jimsonweed (Datura stramonium), ‘Fusarium arthrosporioide against
broomrapes (Orobanche spp.)’, ‘Fusarium lateritium against giant ragweed
(Ambrosia trifida)’, ‘Fusarium oxysporum f. sp. strigae against Striga
hermonthica’, and ‘Fusarium sp. against Parthenium hysterophorus’. Adding
cellulase to medium improves the penetration of the weed disease Fusarium,
increasing its pathogenicity. Fusarium produces phytotoxins such as
‘trichothecenes (T2-toxin and others), naphthazarins, fusaric acid, related
pyridine derivatives and fumonisins’ as a pathogenicity factor.
4.1.4 Saccharomyces cerevisiae
S. cerevisiae is a kind of yeast. In diverse hosts, yeast produces
nonspecific defensive systems. S. cerevisiae resistance is connected to the
occurrence of certain carbohydrates and glycoproteins in the cell wall.
Page | 96
4.2 Fungi mediated bio-control mechanisms
‘Biological control’ is the outcome of a range of organism-to-organism
connections. Newly, investigators have concentrated on defining the
mechanisms at work in distinct experimental settings. The existence and
action of other organisms that comes into touch with inevitably worsen
pathogens. Many of antagonism's bio-control mechanisms take place
through a variety of interactions that are closely associated with the amount
of inter-species contact and the interactions type. ‘Hyper-parasitism’ by plant
pathogen obligatory parasites would be the utmost kind of direct antagonism
attributed to no other organism's activity.
4.2.1 Direct antagonism
Hyper-parasitism is the death of a pathogen caused by direct antagonism
or lysis by another microorganism, and mycoparasites are fungi that
parasitize other fungi. Cook (1993) divided the encounters into three
categories: unilateral hostility, mutual antagonism, and no antagonism [50].
Ampelomyce squisqualis is a naturally occurring deuteromycetehyper-
parasite that infects conidiophores, cleistothecia and powdery mildew
hyphae to produce pycnidia (fruiting bodies). The mildew development of
colony is slowed and subsequently killed by hyperparasitism. For more than
50 years, A. quisqualis is the focus of several studies on biological
management of powdery mildews. Weindling identified and published in
1932 that Trichoder malignorum (T. viride) parasitizes Rhizoctoniasolani
hyphae and suggested that inoculating citrus plants with Trichoderma spores
might help reduce damping off. Trichoderma species' mycoparasitic
competence against a variety of economically significant plant infections
enables for the development of biocontrol. Microbial predation is more
widespread and pathogen agnostic as compare to hyperparasitism that
resulting in less predictable disease management.
4.2.2 Antibiosis
Antibiosis is the process by which antagonist fungi secrete
antimicrobial chemicals in order to inhibit pathogenic fungus in the
surrounding area of their development region. Most fungi are able to secret
certain antibiotic-active chemicals and secondary metabolites, which are
typically linked to certain stages of morphological differentiation and the
active growth phase. Some fungal secondary metabolites have the capability
to amend plant development and metabolism, whereas others appear to target
particular fungal processes like sporulation and hyphal extension. As a
result, the expression of secondary metabolites in some fungi, particularly
Page | 97
those utilised in agriculture, may occur at a predictable period throughout
their typical life cycle. In another experiment, hyphal penetration was
missing in spite of close interaction between Trichodermaspp. Hyphae and
Fusariummonil iforme/Aspergillus flavuson co-culturing, indicating that
‘mycoparasitism’ was not the single source of the detected inhibitory effects.
As a result, metabolites generated by ‘Trichoderma spp.’ were thought to be
likely antibiosis components.
The metabolites are ‘linear’, ‘amphipathic polypeptides’ known as
‘peptaibols’ and ‘peptaibiotics’ and their physico-chemical and biological
features, which comprise lipidmembrane disruption, antimicrobial action and
plant resistance induction, are also described. When nutrients are scarce,
certain biocontrol organisms engage in predatory behaviour. However, under
normal growth conditions, such activity is rarely seen. Some Trichoderma
species, for example, release a variety of enzymes that attack fungi's cell
walls. Peptaibols and their relatives, ‘peptaibiotics’, are amphipathic linear
polypeptides. These compounds, which are made up of 5 to 20 amino acids
and are typically made in micro heterogeneous mixtures, have better
antimicrobial activity against gram-positive bacteria and fungi, and theywork
in conjunction with cellwall-degrading enzymes (CWDEs) to inhibit fungal
pathogen growth and generate pathogen resistance in plants. Non-ribosomal
biosynthesis produces peptaibols and peptaibiotics with unique amino acid
composition. Peptide synthetases are huge multifunctional enzymes that
build these compounds from a variety of precursor which can be N-
methylated, acylated or reduced via the multiple carrier thio-template
method. 'Peptaibols and peptaibiotics' have distinct physicochemical and
biological properties, such asthe ability to puncture bilayer lipid membranes.
4.2.3 Competition
Because ‘starvation’ is the greatest prevalent cause of mortality for
microorganisms, struggle for limited resources leads in fungal phyto-
pathogens being biologically controlled. Celar (2003) led a study on the
most common nutrient types seen in phytopathogenic and antagonistic fungi.
It has been noted that biocontrol grounded on competition for
uncommon but necessary micronutrients, such as ‘Iron’, which is highly
restricted in the rhizosphere, is possible, depending on soil pH. Iron is found
in very oxidised and aired soil in the ferric state that is insoluble in water
(pH 7.4) and has a concentration as low as 10-18 M. This concentration is
insufficient to allow microorganism development, which requires
concentrations of about 10-6 M. To live in such an environment, organisms
Page | 98
have been discovered to release siderophores, which are iron-binding
ligands with a high affinity for sequestering iron from the micro-
environment. To mobilise ambient iron, most filamentous fungi ingest iron
as an important mineral for viability and under iron deficiency, and most
fungi excrete low- molecular-weight ferric-iron specific chelators, known as
siderophores. Iron is then retrieved from the ferri-siderophore complexes via
specialised uptake processes. Siderophore biosynthesis is adversely
controlled by carbon supply in Aspergillus fumigates and Aspergillus
nidulans. The development of Ustilagomaydis is influenced by gene products
associated to iron uptake. Some Trichoderma Biocontrol agents create
extremely effective siderophores that chelate iron and prevent other fungi
from growing. As a result, the biocontrol efficiency of Pythium by
Trichoderma is influenced by soil composition and iron availability. T.
harzianum T35 also suppresses Fusarium oxysporum by competing for
rhizosphere colonisation and nutrients, with biocontrol becoming more
effective as nutrient concentrations fall. Competition has proven to be crucial
in the biocontrol of phytopathogens like Botrytis cinerea, the most common
pathogen. The commensal bacteria' higher iron absorption efficiency is
suggested to be a significant element in their capacity to aggressively
colonise plant roots and help in the displacement of harmful organisms from
potential infection sites. As a result, the given cases demonstrated the
importance of nutrient competition for fungal development as a bio-control
agent.
5. In Silico approach for biological agent
Protein-protein, enzyme-substrate, protein-nucleic acid, drug-protein,
and drug-nucleic acid interactions all play important roles in many essential
biological processes, including signal transduction, transport, cell regulation,
gene expression control, enzyme inhibition, antibody-antigen recognition
and even multi-domain protein assembly. These interactions frequently
result in the development of stable protein-protein or protein-ligand
complexes, which are required for the proteins to carry out their biological
tasks [51]. Understanding the binding mechanism and affinity between
interacting molecules necessitates an understanding of protein tertiary
structure. However, obtaining complicated structures using experimental
methods such as X-ray crystallography or NMR is frequently challenging
and costly. As a result, docking computing is regarded as a crucial tool for
comprehending protein-protein or protein-ligand interactions [52]. As the
number of three-dimensional protein structures determined by experimental
method grows, structure databases such as Protein Data Bank (PDB) and
Page | 99
Worldwide Protein Data Bank (wwPDB) have over 188085 protein
structures, Many of which play crucial roles in key metabolic processes and
might be used as therapeutic targets and particular databases containing the
structures of binary complexes, as well as information on their binding
affinities, become accessible, such as in PDBBIND [53], PLD [68], AffinDB
[54]
and BindDB [55], Molecular docking methods are improving, and they are
becoming more important than ever [56]. Molecular docking is a commonly
used computer simulation approach for predicting the shape of a receptor-
ligand complex, in which the receptor is often a protein or a nucleic acid
molecule and the ligand is usually a small molecule or another protein.
(Figure 1)
Fig 3: Elements in molecular docking

5.1 Experimental docking procedures
There are several excellent reviews of molecular docking methods
available, as well as several studies evaluating the efficacy of various
molecular docking tools [57]. Following that, we'll go over the four-step
procedure used to perform molecular docking in this study.
5.1.1 Target selection
In an ideal world, the target structure would be established
experimentally using X-ray crystallography or nuclear magnetic resonance,
both of which may be obtained from the PDB; nonetheless, docking has
proven to be more successful than homology models or threading. The
model must be of high quality. Validation tools such as Molprobity can be
used to test it [58]. After choosing a model, it must be prepared by removing
water molecules from the cavity, stabilizing charges, filling in missing
residues, and producing side chains, all according to the specifications
supplied. The receptor should be physiologically active and in a stable state
at this time.
5.1.2 Ligand selection and preparation
The sort of docking ligands used will be determined by the objective. It
may be retrieved from variety chemical databases, such as ZINC or
Page | 100
PubChem, or drawn using the Chemsketch programme [59]. Filters are
frequently used to minimize the amount of molecules that must be docked.
Net charge, molecular weight, polar surface area, solubility, commercial
availability, similarity thresholds, pharmacophores, synthetic accessibility,
and absorption, distribution, metabolism, excretion, and toxicological
qualities are all examples of attributes.
5.1.3 Docking
The ligand gets docked onto the receptor and the interactions are
verified in this last stage. The scoring function calculates a score based on
the best ligand chosen.
5.1.4 Evaluating docking results
The root-mean-square deviation (RMSD) between the experimentally
observed heavy-atom locations of the ligands and the one(s) anticipated by
the algorithm is commonly used to assess the efficacy of docking algorithms
in predicting ligand binding poses. The system's flexibility presents a
significant difficulty in the hunt for the best pose. The amount of degrees of
freedom used in the conformational search is a key factor in determining the
effectiveness of the search. When the RMSD is less than 2, it is regarded to
be an excellent performance.
Fig 4: Steps of Docking
Page | 101
5.1.2 Docking software description
There are a plethora of methods for assessing and rationalising ligand-
protein or protein- protein interactions, and the number is growing all the
time. In docking techniques, speed and precision are critical qualities for
achieving good outcomes. Several algorithms have similar approaches with
unique expansions aimed at achieving a quick technique with the highest
possible accuracy. AutoDock, DOCK, FlexX, GOLD [60], ICM [61], ADAM
[62]
, DARWIN [63], DIVALI [64], and DockVision [65] are the most popular
docking programmes.
6. Conclusions
Plant infections, pests, and weeds are only a few of the species in the
environment that may devastate the community of plant enemies. In reality,
one collection's relationship with another is linked. This approach can give a
collection of biological elements that can be usedto effectively manage plant
enemies. This category includes microbes, animals, and plants, all of which
are capable of controlling plant enemies. Some biocontrol agents may be
successful in eradicating the population of plant adversaries at a percent
below an economic threshold, whereas other biological factors have not
shown effectiveness in maintaining the plant adversaries in the same way.
As a result, integrated pest management must incorporate biological
factors as well as other management tactics such as chemicals, physical, and
other. A virus has strong efficiency against insects and some status against
plant viruses, fungi, and bacteria, which is unusual. In addition, bacteria,
fungus, nematodes, spiders, insects, and plants have been discovered to
have substantial promise in managing a variety of plant enemies rather
than chemical pesticides. The biological control approach, on the other
hand, takes a long time to establish that it is effective against plant enemies.
At the same time, they provide a long-term and cost-effective solution for
keeping the environment clean and avoiding the detrimental impacts of
residue from synthetic chemicals used in pesticide manufacturing. Finally,
numerous biological control agents, whether microbial, animal, or plant, can
be recognised and exploited in the production of biopesticides and
biofertilizers in the environment around plant enemies.
References
1. Goyal SK, Pandey RN, Singh JP. India ’ s Agricultural Exports Growth
and Instability, 1993.
2. Ramakrishna A, Ravishankar GA. and esio science notdistribute and
esio science notdistribute. 2011;6(11):1720-1731. Doi:
10.4161/psb.6.11.17613.
Page | 102
3. Vela C, Castroverde CDM, He SY. Review Plant-Pathogen Warfare
under Changing Climate Conditions, no. Figure 1, 2018, 619-634. Doi:
10.1016/j.cub.2018.03.054.
4. Kumar J, Jain SK, Sivalingam PN, Dixit A, Mallikarjuna J, Ghosh PK.
Biotic stresses of agricultural crops in India: re-visiting national status
and mitigation strategies. 2021;120(2):17-18.
5. Shennan C. Biotic interactions, ecological knowledge and agriculture,
2007-2008 Aug, 717-739. Doi: 10.1098/rstb.2007.2180.
6. Hirota T, Natsuaki T, Murai T. Yellowing disease of tomato caused by
Tomato chlorosis virus newly recognized in Japan, 2010, 168-171, Doi:
10.1007/s10327-010-0219-4.
7. Taylor P, Mishra A, Gohel VR, Valand GB, Patel JG, Shukla DD.
International Journal of Pest Management Peanut stripe virus disease of
groundnut-a review, 2015 May, 37-41, Doi:
10.1080/09670879309371793.
8. R. Article, “Stem Rot Disease of Groundnut (Arachis hypogaea L.)
Induced by Sclerotium Rolfsii and Its Management”.
9. Divya J, Belagali SL. Effect of Chemical Fertilizers on Physico-
Chemical Characteristics of Agricultural Soil Samples. 2012;6:181-187.
10. Riah Z, Saleem M, Lett EC, Trinsoutrot-gattin I. Riah et al., 2014. Doi:
10.1007/s10311-014-0458-2.
11. Andrés JA. Role of Pseudomonas aurantiaca in Crop Improvement,
2011 June.
12. Mcewen SA, Fedorka-Cray PJ. Antimicrobial Use and Resistance in
Animals. 2002;34(3):93-106.
13. Abreu AC, Borges A, Simões LC, Saavedra MJ. Antibacterial Activity
of Phenyl Isothiocyanate on Escherichia coli and Staphylococcus
aureus, 2013, 756-761.
14. Zhang Q, Lyu Y, Huang J, Zhang X. Antibacterial activity and
mechanism of sanguinarine against Providencia rettgeri in vitro, 2020.
Doi: 10.7717/peerj.9543.
15. Clifford T, Howatson G, West DJ, Stevenson EJ. The Potential Benefits
of Red Beetroot Supplementation in Health and Disease”, 2015, 2801-
2822. Doi: 10.3390/nu7042801.
Page | 103
16. Souza CRM, Bezerra WP, Souto JT. Marine Alkaloids with Anti-
Inflammatory Activity: Current Knowledge and Future Perspectives,
2020.
17. Mabhiza D, Chitemerere T, Mukanganyama S. Antibacterial Properties
of Alkaloid Extracts from Callistemon citrinus and Vernonia adoensis
against Staphylococcus aureus and Pseudomonas aeruginosa, 2016.
18. Hara S, Yamakawa M. Moricin, a Novel Type of Antibacterial Peptide
Isolated from the Silkworm, Bombyx mori*, J Biol. Chem.
1995;270(50):29923-29927. doi: 10.1074/jbc.270.50.29923.
19. Yan Y, Li X, Zhang C, Lv L, Gao B, Li M. Research Progress on
Antibacterial Activities and Mechanisms of Natural Alkaloids: A
Review”, 2021.
20. Ito A, Taniuchi A, May T, Kawata K, Okabe S. Increased Antibiotic
Resistance of Escherichia coli in Mature Biofilms †. 2009;75(12):4093-
4100. Doi: 10.1128/AEM.02949-08.
21. Pommier Y, Leo E, Zhang H, Marchand C. Review DNA
Topoisomerases and Their Poisoning by Anticancer and Antibacterial
Drugs, Chem. Biol. 2010;17(5):421-433. Doi:
10.1016/j.chembiol.2010.04.012.
22. Liu XL, Xu YJ, Go ML. Functionalized chalcones with basic
functionalities have antibacterial activity against drug sensitive
Staphylococcus aureus, 2008, 43. doi: 10.1016/j.ejmech.2007.10.007.
23. Ferreira C, Saavedra MJ, Simo M. Antibacterial Activity and Mode of
Action of Ferulic, 2013, 00(0). Doi: 10.1089/mdr.2012.0244.
24. Wu T, et al., Biochimica et Biophysica Acta A structure-activity
relationship study of fl avonoids as inhibitors of E. coli by membrane
interaction effect, BBA-Biomembr. 2013;1828(11):2751-2756. Doi:
10.1016/j.bbamem.2013.07.029.
25. Taylor P, Kumazawa S, Kajiya K, Naito A, Saitô H, Tuzi S. Direct
Evidence of Interaction of a Green Tea Polyphenol, Epigallocatechin
Gallate, with Lipid Bilayers by Solid-state Nuclear Magnetic Resonance
Direct Evidence of Interaction of a Green Tea Polyphenol,
Epigallocatechin Gallate, with Lipid Bilayers, no. October, 2014, 37-41.
Doi: 10.1271/bbb.68.1743.
26. Nohynek LJ et al., Berry Phenolics : Antimicrobial Properties and
Mechanisms of Action Against Severe Human Pathogens Berry
Page | 104
Phenolics: Antimicrobial Properties and Mechanisms of Action Against
Severe Human Pathogens, 2009-2013 June, 37-41.
27. Jeon B. Synergistic anti-Campylobacter jejuni activity of
fluoroquinolone and macrolide antibiotics with phenolic compounds.
2015 Oct;6:1-9. Doi: 10.3389/fmicb.2015.01129.
28. Prasch S, Bucar F. Plant derived inhibitors of bacterial efflux pumps: an
update, Phytochem. Rev. 2015 July. Doi: 10.1007/s11101-015-9436-y.
29. Kurhekar JV. TANNINS-antimicrobial Chemical, 2017 July.
30. Senthil-Kumar M, Ryu C, Kang L, Kirankumar S. Plant Physiology.
2016;158(4):1789-1802. Doi: 10.1104/pp.lll.189217.
31. Chouhan S, Sharma K, Guleria S. Antimicrobial Activity of Some
Essential Oils-Present Status and Future Perspectives, 2017, 1-21. Doi:
10.3390/medicines4030058.
32. Ouattara B, Simard RE, Holley RA, Piette GJ. Antibacterial activity of
selected fatty acids and essential oils against six meat spoilage
organisms. 1997;37:155-162.
33. Chauhan AK, Kang SC. Thymol disrupts the membrane integrity of
Salmonella ser. typhimurium in vitro and recovers infected macrophages
from oxidative stress in an ex vivo model, Res. Microbiol.
2014;165(7):559-565. doi: 10.1016/j.resmic.2014.07.001.
34. Nawaz H, Suleman S, Khan A, Rani M, Uddin V, Iqbal M.
Phytomedicine Biotransformation of monoterpenoids and their
antimicrobial activities, Eur. J Integr. Med. 2014;1(12):1597-1626. doi:
10.1016/j.phymed.2014.05.011.
35. Mukherjee PK, Horwitz BA, Herrera-Estrella A, Schmoll M, Kenerley
CM. Trichoderma Research in the Genome Era. Doi: 10.1146/annurev-
phyto-082712-102353.
36. Tawfeeq Al-Ani LK, et al., Biological Control Agents and Their
Importance for the Plant Health. Elsevier Inc., 2020.
37. Zhai Y, et al., Cyclo(L-Pro–L-Leu) of pseudomonas putida MCCC
1A00316 isolated from antarctic soil: Identification and characterization
of activity against meloidogyne incognita, Molecules, 2019, 24(4). doi:
10.3390/molecules24040768.
38. Soares Figueiredo C, Nunes Lemes AR, Sebastião I, Desidério JA.
Synergism of the Bacillus thuringiensis Cry1, Cry2 and Vip3 Proteins in
Page | 105
Spodoptera frugiperda Control, Appl. Biochem. Biotechnol.
2019;188(3):798-809. Doi: 10.1007/s12010-019-02952-z.
39. Phani V, Somvanshi VS, Shukla RN, Davies KG, Rao U. A
transcriptomic snapshot of early molecular communication between
Pasteuria penetrans and Meloidogyne incognita, BMC Genomics.
2018;19(1):1-15. doi: 10.1186/s12864-018-5230-8.
40. Veliz EA, Martínez-Hidalgo P, Hirsch AM. UCLA Chitinase-producing
bacteria and their role in biocontrol, 2017. Doi:
10.3934/microbiol.2017.3.689.
41. Badiaa E, Abdeljabbar H, Mohamed RH, Abdellatif B, Najla SZ. In vivo
and in vitro evaluation of antifungal activities from a halotolerant
Bacillus subtilis strain J9, African J Microbiol. Res. 2012;6(19):4073-
4083. Doi: 10.5897/ajmr11.403.
42. Legein M, et al., Modes of Action of Microbial Biocontrol in the
Phyllosphere, Front. Microbiol., 2020 July, 11. Doi:
10.3389/fmicb.2020.01619.
43. De Mastro G, Gobbetti M, Francesca N. Dynamic and Assembly of
Epiphyte and Endophyte Lactic Acid Bacteria During the Life Cycle of
Origanum vulgare L. 2018 June;9:1-16. doi:
10.3389/fmicb.2018.01372.
44. Filiatrault MJ. The olive knot disease as a model to study the role of
interspecies bacterial communities in plant. June 2015;6:1-12. doi:
10.3389/fpls.2015.00434.
45. Morella NM, Weng FCH, Joubert PM, Jessica C, Lindow S, Koskella B.
Successive passaging of a plant-associated microbiome reveals robust
habitat and host genotype-dependent selection, Proc. Natl. Acad. Sci.
U.S.A. 2020;117(2):1148-1159. Doi: 10.1073/pnas.1908600116.
46. Halperin I, Ma B, Wolfson H, Nussinov R. Principles of Docking: An
Overview of Search Algorithms and a Guide to Scoring Functions. 2002
Jan;443:409-443. Doi: 10.1002/prot.10115.
47. Pyoverdine F, Remus-emsermann MNP. “crossm”. 2020;86(8):1-12.
48. Biological F, Agents C. Fungal biological control agents, 2000, 186-
191.
49. Adesemoye AO, Kloepper JW. Plant-Microbes Interactions in Enhanced
Fertilizer-Use Efficiency Plant-Microbes Interactions in Enhanced,
2009. Doi: 10.1007/s00253-009-2196-0.
Page | 106
50. Meena K, Salim H. Evaluation of biocontrol potential of Arthrobotrys
oligospora against Meloidogyne graminicola and Rhizoctonia so...,
1965. Doi: 10.1016/j.biocontrol.2011.10.006.
51. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M, Pathology P.
Trichoderma Species-Opportunistic, Avirulent Plant Symbionts, 2004
Jan, 2. Doi: 10.1038/nrmicro797.
52. Moreira M, Ferreira D, Abreu LM, Rodrigues-Filho E, Pfenning LH,
Donnell KO. Diversity of Clonostachys species assessed by molecular
phylogenetics and MALDI-TOF mass spectrometry, 2014, 8.
53. Hahn SM, et al., Taxol in Combination with Doxorubicin or Etoposide
Possible Antagonism in vitro, 2705-2711.
54. Huang S, Zou X. Advances and Challenges in Protein-Ligand Docking,
2010, 3016-3034. Doi: 10.3390/ijms11083016.
55. Yang C, Falkner J. The PDBbind database : methodologies and updates
Related papers,” no. Part I.
56. Puvanendrampillai D, Mitchell JBO. Protein Ligand Database (PLD):
additional understanding of the nature and specificity of protein-ligand
complexes. 2003;19(14):1856-1857. Doi:
10.1093/bioinformatics/btg243.
57. Block P, Sotriffer CA, Dramburg I, Klebe G. AffinDB: a freely
accessible database of affinities for protein-ligand complexes from the
PDB. 2006;34:522-526. doi: 10.1093/nar/gkj039.
58. Liu T, Lin Y, Wen X, Jorissen RN, Gilson MK. BindingDB: a web-
accessible database of experimentally determined protein-ligand binding
affinities. 2006-2007 Dec;35:198-201. Doi: 10.1093/nar/gkl999.
59. Dias R, Filgueira W, Jr DA. Molecular Docking Algorithms, 2009-2014
June. Doi: 10.2174/138945008786949432.
60. Abagyan R, Totrov M. High-throughput docking for lead generation
Potential of Mean Force, 2001, 375-382.
61. Chen VB, et al., Feature articles Macromolecular structure
determination using X-rays, neutrons and electrons: recent
developments in Phenix, 2019, 861-877. Doi:
10.1107/S2059798319011471.
62. Jones G, Willett P, Glen RC, Leach AR, Taylor R, Uk KBR.
Development and Validation of a Genetic Algorithm for Flexible
Docking, 1997.
Page | 107
63. Abagyan R, Totrov M, Kuznetsov D. New Method for Protein Modeling
and Design: Applications to Docking and Structure Prediction from the
Distorted Native Conformation. 1994;15(5):488-506.
64. “jmbi.1994.1656.pdf.”.
65. Taylor JS, Burnett RM. DARWIN: A Program for Docking Flexible
Molecules. 2000 May;191:173-191.
66. Clark KP. Flexible Ligand Docking without Parameter Adjustment
across Four Ligand-Receptor Complexes, 1995, 16(10).
67. Hart TN, Read RJ. A Multiple-Start Monte Carlo Docking Method
Plim: The Protein Ligand Interaction Modeler Protein Modeling by
Homology Using Distance Information, 1992, 10.
Page | 108
Chapter - 6
Recent Scenario of Termite Infestation and Their
Management
Authors
Vijay Kumar
Research Scholar, Department of Entomology, ANDUA&T,
Kumarganj, Ayodhya, Uttar Pradesh, India
Umesh Chandra
Assistant Professor, Department of Entomology, ANDUA&T,
Pradip Kumar Patel
Arvind Kumar
Vishnoo Omar
Page | 109
Page | 110
Chapter - 6
Recent Scenario of Termite Infestation and Their
Management
Vijay Kumar, Umesh Chandra, Pradip Kumar Patel, Arvind Kumar and Vishnoo Omar
Abstract
Termites are eusocial insects that belong to the Blattodea order and are
classified as either infraorder Isoptera or epifamily Termitoidae. Termites are
a eusocial insect species. Termites are known as the "silent destroyer" because
they may live and thrive in your home or yard without causing any visible
damage. Termites eat cellulose-based plant components in their entirety.
Termites are detritivorous, meaning they eat dead plants and trees, as well as
the dead components of living trees, such as wood and soil wood. Termites
account for 10% of animal biomass in the tropics and subtropics of the world,
and this proportion jumps to 95% when just soil insects are considered.
Termite natural activities improve soil pH, organic carbon content, water
content, and porosity for aeration. The termite colony or society, is a well-
organized and well-connected unit. There is a caste system in place, with
labour division depending on the structure, function and behaviour of colony
members. The reproductive, soldier, and worker castes are the most important
in the colony. Soldiers and employees are either male or female and are sterile.
Uses of several methods to management of termite.
Keywords: Termite, biology, recent scenario, management
Introduction
Termites are eusocial insects that belong to the Blattodea order and are
classified as either infraorder Isoptera or epifamily Termitoidae (along with
cockroaches). Termites were once thought to be a separate order from
cockroaches, but recent evolutionary studies have revealed that they evolved
from cockroaches and are the sister group of wood-eating cockroaches of the
genus Cryptocercus. Separation occurred during the Jurassic or Triassic
periods, according to prior estimates. The first fossil remains date from the
Early Cretaceous, and they are assumed to have originated in the Late Jurassic.
Currently, 3,106 species have been identified, with a few hundred more to be
discovered.
Page | 111
Formosan subterranean termite
(Coptotermes formosanus) Soldiers (red-coloured heads)
Workers (pale-coloured heads)
Termites, sometimes known as white ants, are a widespread insect
nuisance belonging to the Isoptera order. They are prolific and widespread
throughout the world's tropical and sub-tropical climates. They live in big
groups, primarily in underground nests, and are well-known for their
depredations. They construct little clay mounds or passages that are visible
above ground.
Termites are a eusocial insect species. Termites are known as the "silent
destroyer" because they may live and thrive in your home or yard without
causing any visible damage. Termites eat cellulose-based plant components in
their entirety. Termites are detritivorous, meaning they eat dead plants and
trees, as well as the dead components of living trees, such as wood and soil
wood. The mouth of a termite is capable of breaking woody material into small
bits. This capacity is what creates concern in human residences, as termite
workers' eating habits can cause significant property damage. Termites can
gnaw on the foundations of houses, furniture, shelves, and even books.
Termites harm field crops, ornamental plants, cash crops, and forest trees. As
nature's scavengers, they transform stables, limbs, and plant debris to humus.
Termites account for 10% of animal biomass in the tropics and subtropics
of the world and this proportion jumps to 95% when just soil insects are
considered. Termite natural activities improve soil pH, organic carbon
content, water content, and porosity for aeration, resulting in favourable
conditions for primary production in the tropics and subtropics, where they
are the lead decomposers and boost soil formation. They are, however, unique
among insects in that they can degrade cellulose from living, dead, and rotting
wood, as well as other plant materials. They are a major insect pest to humans,
Page | 112
posing a serious threat to a wide range of agricultural crops, constructions,
especially wooden materials. Termites have attracted the attention of science
since the seventeenth century due to their massive negative impacts on the
artificial earth. Because of their natural food source and habitat, termites come
into contact with humans. The fight between man and termite results in
significant damage to structures and facilities, as cellulose inside the wood and
woody tissue are their primary food sources. Termite damage not only causes
interior damage but also external damage. In the tropics and subtropics of the
planet, termites account for 10% of animal biomass, and this figure jumps to
95% when only soil insects are considered. Termite natural activities aid in
soil preparation by increasing pH, organic carbon content, water content, and
porosity. They are, however, unique among insects in that they can degrade
cellulose from living, dead and rotting wood, as well as other plant materials.
They are a major insect pest to humans, posing a serious threat to a wide range
of agricultural crops, constructions, especially wooden materials domestic
supplies, but it also includes the storage of commercial wood/wooden objects
crops and farmhouses, and in certain cases, the complete residence and/or
construction composed of wood resources. Subterranean termites wreak havoc
on man-built structures and agricultural crops; termites cause over 95 percent
of structural damage, and they've been identified in practically every structure,
including those that aren't made of wood. Termites have a lot of negative
consequences on the man-made planet.
The International Symposium on Termites in the Humid Tropics' was
held in New Delhi under the auspices of UNESCO and the Indian Zoological
Survey. Termites' destructive influence was also discussed and dramatised in
the conference proceedings, along with other elements of termites. During this
time, scientists and researchers in India were working on a variety of termite
issues, including damage and long-term control strategies. Despite the fact that
termite research in India has primarily focused on the forestry and wood
science sectors, numerous writers have documented the destructive elements
of termites in structures and constructions. Many wood-destroying termites
were listed in Sen-Sarma et alpaper.'s 'Wood Destroying Termites of India,'
which was released in 1975 by the Forest Research Institute.
Distribution and diversity
Except for Antarctica, termites can be found on every continent. Termite
species diversity is modest in North America and Europe (10 species in
Europe and 50 in North America), but it is high in South America, where there
are over 400 species. 1,000 of the 3,000 termite species now recognised can
be found in Africa, where mounds are common in some areas. In the northern
Page | 113
Kruger National Park alone, there are over 1.1 million active termite mounds.
There are 435 species of termites in Asia, the majority of which are found in
China. Termite species in China are only found south of the Yangtze River in
moderate tropical and subtropical climates. All ecological categories of
termites (dampwood, drywood, and subterranean) are native to Australia, with
over 360 species recognised. Termites account for a disproportionate share of
the world's insect biomass due to their strong sociality and abundance.
Termites and ants make only around 1% of all insect species, but they account
for more than half of all insect biomass.
Diversity of Isoptera by continent
North South
Continent Asia Africa Europe Australia
America America
Estimated number of
435 1,000 50 400 10 360
species
Termites do not live in cool or cold environments due to their delicate

cuticles. Termites are classified into three ecological groups: dampwood,
drywood and underground. Dampwood termites can only be found in
coniferous woods, while drywood termites can only be found in hardwood
forests; subterranean termites can be found in a wide range of environments.
The West Indian drywood termite (Cryptotermes brevis), which is an invasive
species in Australia, is one of the drywood termite species.
Termites are typically small, ranging in length from 4 to 15 millimetres
(316 to 916 in). The queens of the species Macrotermes bellicosus are the
largest of all extant termites, measuring up to 10 centimetres (4 in) in length.
The extinct Gyatermes styriensis, a huge termite with a wingspan of 76
millimetres (3 in) and a body length of 25 millimetres, thrived in Austria
during the Miocene.
Different species of termite
Termites are widely regarded as the world's most devastating insect pests.
Each year, these pests do extensive damage to buildings and structures,
resulting in significant financial losses.
There are 220 different species of termites in India that cause problems to
properties:
 Coptotermes gestroi
 Coptotermes heimi
 Heterotermes indicola
Page | 114
 Schedorhinotermes spp.
 Odontotermes spp.
 Psammotermes rajathanicus
 Macrotermes gilvus
 Microcerotermes spp.
 Nasutitermes sp.
Behaviour and ecology
Diet
Termites are detritivores, meaning they eat dead plants at any stage of
decay. They also help the environment by recycling waste materials like dead
wood, faeces and plants. Many animals ingest cellulose and have a specialised
midgut that breaks it down. Termites are a large source (11%) of atmospheric
methane, one of the most potent greenhouse gases, which is created through
the breakdown of cellulose. Termites rely on symbiotic protozoa
(metamonads) and other bacteria in their intestines to digest the cellulose for
them, allowing them to absorb the end products for their own consumption.
Many species identified in the termite stomach microbial ecosystem are found
nowhere else on Earth. Termites are born without these symbionts in their
stomachs and must create them after being fed a termite culture.
Trichonympha, for example, rely on symbiotic bacteria lodged on their
surfaces to manufacture part of the digestive enzymes they require. Higher
termites, particularly those in the Termitidae family, can produce cellulase
enzymes on their own, but they rely on bacteria for the majority of their
cellulase needs. In Termitidae, the flagellates have vanished. Spirochetes
living in termite stomachs have been discovered to be capable of converting
air nitrogen into a form that the insect can use. The relationship between the
termite digestive tract and the microbial endosymbionts is currently poorly
understood by scientists; nonetheless, all termite species feed the other
members of the colony with substances produced from the digestion of plant
material, either through the mouth or from the anus. The gut microbiota of
termites and cockroaches is thought to be derived from their dictyopteran
ancestors based on closely comparable bacterial species.
Predators
Termites are eaten by many different predators. Hodotermes
mossambicus, a single termite species, was identified in the stomach contents
of 65 birds and 19 mammals. Termites are consumed by arthropods such as
ants, centipedes, cockroaches, crickets, dragonflies, scorpions, and spiders, as
Page | 115
well as reptiles like lizards and amphibians like frogs and toads. Two spiders
in the Ammoxenidae family are specialist termite predators. Ants are the most
effective predators against termites. Termite predators are found in several ant
genera. Megaponera, for example, is a termite-eating (termitophagous) genus
that engages in raiding operations that might last several hours. Paltothyreus
tarsatus is another termite raider, with each individual stacking as many
termites as possible in its mandibles before returning home, while also
recruiting extra nestmates.
Parasites, pathogens and viruses
Because termites are normally well protected in their mounds, they are
less likely to be attacked by parasites than bees, wasps, or ants. Termites,
however, are parasitized by a variety of parasites. Dipteran flies, Pyemotes
mites, and a variety of nematode parasites are among them. The order
Rhabditida contains the majority of nematode parasites; others include
Mermis, Diplogaster aerivora and Harteria gallinarum. A colony may
migrate to a different area if it is at danger of being attacked by parasites.
Certain fungi, such as Aspergillus nomius and Metarhizium anisopliae, are
severe hazards to termite colonies because they are not host-specific and can
infect wide areas of the colony; transmission is mainly through direct physical
contact. The termite immune system is known to be weakened by M.
anisopliae. A. nomius infection occurs only when a colony is under extreme
stress. There are over 34 fungus species that live as parasites on termites'
exoskeletons, many of which are host-specific and only cause indirect harm to
their hosts.
Locomotion and Foraging
Termite foraging behaviour is determined by the type of termite. For
example, some species eat the wood buildings they live in, while others gather
food around the nest. Most workers are rarely seen in the open and do not feed
exposed; instead, they use sheeting and runways to keep predators at bay.
Workers who find food sources deposit a phagostimulant pheromone that
attracts other workers, and workers who find food sources recruit more
nestmates by depositing a phagostimulant pheromone that draws other
workers.
Competition
When two colonies compete, they will always act agonistically against
one other, resulting in battles. These battles can result in death on both sides
as well as the gain and loss of territory in some situations. According to
studies, when termites meet in foraging regions, some of them will
Page | 116
purposefully block channels to prevent other termites from entering. The
discovery of dead termites from other colonies in exploratory tunnels isolates
the area, necessitating the construction of new tunnels.
Communication
Because most termites are blind, they rely on chemical, mechanical and
pheromonal cues to communicate. Foraging, identifying reproductives, nest
construction, recognition of nestmates, nuptial flight, locating and fighting
enemies, and nest defence are just a few of the activities that these kinds of
communication are utilised for. Antenna communication is the most prevalent
method of communication. Contact pheromones (which are transferred while
workers are engaged in trophallaxis or grooming) as well as alarm, trail, and
sex pheromones are all known.
Defence
To defend a colony, termites use alarm communication. When the nest
has been broken or is being attacked by enemies or potential infections, alarm
pheromones might be emitted. Through vibrational signals generated by
infected nest mates, termites always avoid nest mates infected with
Metarhizium anisopliae spores. Other defence mechanisms include jerking
violently and secreting secretions from the frontal gland, as well as defecating
faeces containing warning pheromones.
Relationship with other organisms
A fungus has been shown to imitate termite eggs, allowing it to dodge its
natural predators. The eggs are rarely killed by these minute brown balls,
known as "termite balls" and in some circumstances, the workers tend to them.
This fungus imitates these eggs by manufacturing glucosidases, a cellulose-
digesting enzyme. Trichomoniasis beetles and certain termite species
belonging to the Reticulitermes genus have a unique imitating behaviour.
Beetles and termites share the same cuticle hydrocarbons and even
biosynthesize them. The beetles are able to blend in with the termite colonies
thanks to their chemical mimicry. Austrospirachtha mimetes can imitate a
termite worker thanks to its evolved appendages on the physogastric abdomen.
Page | 117
Rhizanthella gardneri is the only orchid known to be pollinated by termites
Caste of termite
The termite colony, or society, is a well-organized and well-connected
unit. There is a caste system in place, with labour division depending on the
structure, function, and behaviour of colony members. The reproductive,
soldier, and worker castes are the most important in the colony. Soldiers and
employees are either male or female and are sterile.
A new termite colony is usually established through the dispersal of

winged adults (alates), which develop in a mature colony during specific
seasons of the year. After moulting into winged adults, alates spend many days
or weeks in specific chambers along the nest's edge. High atmospheric
Page | 118
humidity, in combination with temperature, climatic, and seasonal parameters
that vary by species, is usually related with alate emergence and flight. Some
species may only emerge once a year, whereas others may have multiple
flights in a row.
Prior to the alates' emergence, workers prepare tunnels to the surface,
open exit holes, and sometimes build launching platforms. Soldiers defend the
exit holes during emergence, not only to keep adversaries out, but also to keep
alates from returning to the nest. The alates, which ordinarily avoid light,
become attracted to it at the time of emerging and fly out of the nest. They are
poor flyers and, unless carried by the wind, will crash into the original colony
within a few hundred metres. The flight, also known as a nuptial or mating
flight, is nothing more than dispersal. After the flight, mating takes place.
Swarming from many colonies occurs in a given location at the same time and
can be closely synchronised in areas separated by hundreds of kilometres. An
advantage of synchronization might be intercolony mating.
Reproductives: Possess compound eyes and are more or less brown due
to their sclerotized cuticle. Developing reproductives have wing buds, wings
or wing stumps. Reproductives can be further divided into:
Alates, both sexes' juvenile winged reproductives. Approximately 100 to
1000 alates leave the colony for mating and colonising flights on a regular
basis. After mating, a pair chooses an appropriate location, such as a rotting
scar on a tree, to start a new colony.
De-alates, alates who shed their wings after the colonising flight and
transform into queens and kings in the process. Only a few eggs are deposited
and raised by a female de-alate at first. As the colony expands in size,
additional workers become available to assist the young queen in caring for
the brood. After three to five years, the number of individuals in a pest species'
colony has grown to the point where it can become harmful.
Page | 119
Queen and king, who are the colony's primary reproductive individuals.
Once the queen has a large number of workers to assist her, her only job is to
create a large number of kids. A huge queen can lay over 1000 eggs in a single
day. A queen's life span can be as long as 50 years.
Neotenics-As the queen's productivity declines, they help her lay eggs.
One of the neotenics takes the queen's position when she dies or deteriorates.
That is why the removal of a queen from her colony does not inevitably imply
the colony's demise.
Workers: Males and females who are sterile, wingless, and blind.
Because their cuticle is unpigmented and unhardened, the animals must be
kept in a dark, wet environment. Workers construct the nest and galleries, as
well as bringing in food, caring for the brood, and feeding reproductives and
troops. The life expectancy of a worker is one to two years.
Soldiers: are infertile, wingless and blind males and females with an
unpigmented, unsclerotized cuticle, similar to workers. Soldiers use powerful
jaws and/or spew a white sticky repellant from an orifice on their head to
defend their colony from invaders. Soldiers are unable to feed themselves and
must rely on others to do so. The number of soldiers is typically substantially
lower than the number of labourers. Depending on the species, soldiers might
be mandibulate or nasute. As a result, soldiers can be used to identify different
termite species. Soldiers have a lifespan of one to two years.
Relationship with the environment
Environmental changes, including those caused by human involvement,
can have a significant impact on termite populations. A Brazilian study used
65 x 2 m transects to sample the termite assemblages of three Caatinga sites
under varied levels of anthropogenic disturbance in the semi-arid region of
northeastern Brazil. The three sites had a total of 26 species of termites, with
196 encounters recorded in the transects. The termite assemblages differed
Page | 120
significantly between sites, with a clear decrease in both variety and
abundance as disturbance increased, which was linked to decreased tree
density and soil cover, as well as the degree of trampling by cattle and goats.
The wood-feeders were the feeding group that was the most badly impacted.
Nests
The inanimate and animate portions of a termite colony can be divided
into two categories. The animate component is the colony's entire population
of termites, whereas the inanimate component is the structure itself, which is
built by the termites. Subterranean (totally underground), epigeal (protruding
above the soil level) and arboreal (above the soil surface) are the three basic
types of nests (built above ground, but always connected to the ground via
shelter tubes). Epigeal nests (mounds) are built of soil and mud that protrude
from the earth with ground contact. A nest serves a variety of purposes,
including providing a safe dwelling environment and protection from
predators. Rather than multipurpose nests and mounds, most termites build
underground colonies. Termites now, like termites millions of years before,
build their nests in wooden constructions such as logs, stumps, and the dead
sections of trees. To build their nests, termites primarily use faeces, which
have many desirable properties as a construction material. Other building
components include partially digested plant material, which is utilised in
carton nests (arboreal nests made of faeces and wood), as well as earth, which
is used in subterranean nest and mound construction. Because many nests in
tropical forests are underground, not all nests are visible. Apicotermitinae
species are good examples of underground nest builders because they only live
in tunnels. Other termites live in wood, and as they eat the wood, they build
tunnels. The termites' soft bodies are protected by nests and mounds from
desiccation, light, diseases and parasites, as well as predators. Because carton
nests are particularly vulnerable to invading predators, the residents employ
counter-attack methods.
Page | 121
Mounds
If a nest protrudes from the earth's surface, it is classified as a mound. A
mound provides termites with the same level of protection as a nest, but it is
more effective. Because of their clay-rich construction, mounds in locations
with torrential and constant rainfall are at risk of erosion. Those made of carton
can give rain protection and can even survive heavy precipitation. In the event
of a breach, some portions of mounds are employed as strong points.
Cubitermes colonies, for example, construct tiny tunnels that serve as strong
points since their diameter is too small for soldiers to obstruct. The queen and
king are housed in a highly secure room known as the "queen's cell," which
serves as a last line of defence.
Macrotermes species form gigantic mounds, which are possibly the most
complex constructions in the insect world. These mounds are among the
world's largest, with chimneys, pinnacles, and ridges reaching a height of 8 to
9 metres (26 to 29 feet). Amitermes meridionalis, another termite species, can
construct nests that are 3 to 4 metres (9 to 13 feet) high and 2.5 metres (8 feet)
broad. The world's tallest mound, measuring 12.8 metres (42 feet), was
discovered in the Democratic Republic of the Congo.
Termite mound in Queensland, Australia
Page | 122
Termite mound in Namibia
Termites in a mound, Analamazoatra Reserve, Madagascar

Relationship with humans
As pests
Many termite species can cause severe damage to unprotected buildings
and other wooden structures due to their wood-eating behaviour. Termites
play an important role as decomposers of wood and vegetative material and
where structures and landscapes with structural wood components, cellulose-
derived structural materials and ornamental vegetation provide termites with
a consistent source of food and moisture, conflict with humans arises.
Termite damage on external structure

As food
Humans eat 43 termite species, and livestock eat them as well. The protein
from termites can assist enhance the human diet, which is especially essential
in impoverished nations where malnutrition is frequent. Termites are ingested
Page | 123
in many parts of the world, but this practise has only recently gained popularity
in affluent countries.
Termites are eaten by people in a variety of cultures all over the world.
Alates are a major component of aboriginal populations' diets in many parts
of Africa. Collecting or cultivating insects is done in a variety of ways by
different groups, with soldiers from various species being collected at times.
Queens are considered a delicacy, despite their difficulty in obtaining them.
Termite alates are nutrient-dense, with appropriate fat and protein levels. They
have a nut-like flavour after cooking and are regarded as tasty.
These flying alates were collected as they came out of their nests in the ground
during the early days of the rainy season
In agriculture
Termites can be serious agricultural pests, especially in East Africa and
North Asia, where crop losses can be significant (3–100 percent in Africa).
Water infiltration has substantially improved thanks to termite tunnels in the
soil, which allows precipitation to soak in deeper, reducing runoff and thereby
soil erosion through bioturbation. Termite infestations in South America can
cause significant damage to cultivated plants such as eucalyptus, upland rice,
and sugarcane, including attacks on leaves, roots, and woody tissue. Cassava,
coffee, cotton, fruit trees, maize, peanuts, soybeans, and vegetables are all
susceptible to termites.
Termite infestation in Rice and Wheat crops
Page | 124
Termite attack on Maize field
Termite attack on constructions in various countries with economic
estimation of damage
Country with reference Economic extent of termite damage in structures
within parenthesis (annually)
Worldwide A sum of $40 billion
India Sum of 280 million Indian Rupees
China Sum of 1700-2000 million Renminbi (Chinese currency)
Malaysia Up to $ 8-12 million during 2000-2003
Philippines Millions of American $
Taiwan Over $ 3-4 million
Japan About $ 1 billion
Australia More than 100 million Australian $
United States of America $ 1.5 billion in southern parts of USA
In science and technology

The termite gut has sparked a slew of research projects aiming at
replacing fossil fuels with more environmentally friendly, renewable energy
sources. Termites are powerful bioreactors that can produce two litres of
hydrogen from a single sheet of paper. Within the termite hindgut, around 200
species of microorganisms thrive, releasing hydrogen that has been trapped
inside the wood and plants that they eat. The work of unidentified enzymes in
the termite gut breaks down lignocellulose polymers into sugars, which are
then converted to hydrogen. The complex mounds that termites make have
spurred the creation of autonomous robots capable of constructing elaborate
structures without human aid. Termites utilise advanced technology to
regulate the temperature of their mounds.
Page | 125
In culture
The East gate Centre is a shopping mall and office building in central
Harare, Zimbabwe, designed by Mick Pearce, who utilized passive cooling
inspired by native termites. It was the first significant structure to use termite-
inspired cooling techniques, and it drew worldwide attention. The Learning
Resource Center at the Catholic University of Eastern Africa and Melbourne's
Council House 2 building are two such examples.
Management of termite
Get rid of moisture: Moisture is one of the key factors that attracts bugs
into your home, including termites. Termites can be kept at bay by removing
excess moisture from your home. A dehumidifier might aid you if you live in
a humid area. During the summer, you can turn on the air conditioner
intermittently throughout the day to keep the house cool and remove extra
moisture from the air.
Fix leaks: Keep an eye out for any leaks or signs of degradation in your
home. Termites love termite-infested roofs and moisture-soaked walls. Repair
and seal leaks as soon as possible, and inspect your home on a regular basis,
especially in basements and dark places. These areas are frequently
disregarded, yet they are the first to attract pests. A leak in the basement is
particularly appealing because it is closer to the ground, making it easier for
termites to attack.
Declutter your house: Whether you have hired professionals to treat
your home for termites or bed bugs, it is critical to clear your home. Empty
papers, cardboards, old magazines, and newspapers should be given extra
attention since they provide an ideal environment for pests like termites to
thrive. If termites have infested one of your rooms, don't move anything from
that room, including furniture, to other parts of your house that aren't infested
with termites.
Maintain a distance between soil and wood: If you have a garden, make
sure that the soil and the wood are separated. Most experts agree that a spacing
of at least eighteen inches is necessary. Termites will be less likely to attack
your home's foundation and furniture if you do this. To establish a physical
barrier for termites, use stones or cement to divide soil from the wooden area,
especially in patios, gardens, and other areas.
Use borate on wood before priming or painting: Borate is a termite
repellant that is widely used. Prior to priming and painting, you can spray
borate on the wood. It absorbs into the wood, preventing termites from biting
Page | 126
and gnawing on it. After the termite spray has cured, prime and paint it as
usual, then use it to build window frames, doors, and furniture. This borate
spray is long-lasting enough to keep termites at bay.
Place infected items in the sun: If termites are causing damage to a piece
of furniture, expose it to direct sunshine for at least three days. Summer is a
good time to use this termite protection approach because termites can't endure
the heat. By leaving infected furniture in the sun, the heat will kill the termites
and dry out the furniture, reducing the risk of re-infestation. Before putting the
furniture back into the house, it's a good idea to dust it completely and treat it
with a termite repellent. You can do the same thing with furniture that isn't
likely to be harmed by termites to ensure that it remains termite-free.
Management of termite in field crops
 Cow manure that hasn't decomposed should be avoided.
 Flood irrigation should be used and water should not be allowed to
stagnate in the field.
 Plant residue cleaning, collection and burning.
 Using phenyl 1 percent in irrigation water to deter termites from the
farm bed.
 Pentachlorophenol is used as a termite repellant.
 Termite control with the fungus Metarhizium anisopliae (trade name
Bio Blast).
 Prior to sowing the seed treatment with chlorpyriphos 20EC @
400ml in 5 litres of water per quintal of seeds should be done, and
the seeds should be dried in sheds.
 During field irrigation, Chlorpyriphos 20 EC @ 2-3 litre was utilised.
 Seed treatment with Imidacloprid at 70 percent WS (Gaucho) and 48
percent FS.
 Use of fumigation for the control of termite as methyl bromide and
chloropicrin
Different technology used for termite control:
Barrier technology
 Physical barrier: Fine material particles, such as sand and crushed
volcanic cinders, were found to have the ability to act as a barrier
against the subterranean termite Reticulitermes hesperus in this
technique. To operate as an effective barrier, Logan and his coworker
determined that the particle size should be between 1.7 and 2.4 mm.
Page | 127
 Chemical barriers: can be divided into two categories: repellant and
non-repellant. Non-repellent chemicals used against termites include
Cloranthraniliprole, Imidacloprid, Chlorfenapyr and Fipronil.
Chlorpyriphos, Bifenthrin, Permenthrin and other repellent
chemicals.
Bait technology
Baiting is a promising technology of termite management in building
construction. Baiting technique is almost proved against lower termites
including Reticulitermes and Coptotermes. Bait toxicants were divided bait
toxicants into three groups viz.
i) The metabolic inhibitors.
ii) Biological control agents.
iii) Insect growth regulators (IGRs).
Borate technology
 Borate refers to the compounds containing boron and oxygen, usually
naturally exists in the form of sodium and calcium borate. Indian has
recommended use of borate compounds for wood preservation in the
form of BCCA (Borate CCA) and ACZB (Ammonical Copper Zinc
Borate).
 Gel treatment: gel is a newer formulation in insecticides, but in Indian
context Gel formulations of termiticides.
References
1. Alves RRN. Edible and medicinal termites: a global overview. Journal of
Ethnobiology and Ethnomedicine. 2015;11(1):29.
2. Claybourne Anna. A colony of ants and other insect groups. Chicago, Ill.:
Heinemann Library, 2013, 38.
3. Culliney TW, Grace JK. Prospects for the biological control of
subterranean termites (Isoptera: Rhinotermitidae), with special reference
to Coptotermes formosanus. Bulletin of Entomological Research.
2000;90(1):9-21.
4. Chouvenc T, Efstathion CA, Elliott ML, Su NY. Resource competition
between two fungal parasites in subterranean termites. Die
Naturwissenschaften. 2012;99(11):949-58.
5. Costa-Leonardo AM, Haifig I. Pheromones and exocrine glands in
Isoptera. Vitamins and Hormones. 2010;83:521-549.
Page | 128
6. Costa-Leonardo AM, Haifig I. Termite communication during different
behavioral activities in Biocommunication of Animals. Springer
Netherlands, 2013, 161-190.
7. Freymann BP, Buitenwerf R, Desouza O, Olff. The importance of
termites (Isoptera) for the recycling of herbivore dung in tropical
ecosystems: a review. European Journal of Entomology.
2008;105(2):165-173.
8. Higashi Masahiko, Yamamura Norio, Abe Takuya, Burns Thomas P.
(1991-10-22). Why don't all termite species have a sterile worker caste?.
Proceedings of the Royal Society B: Biological Sciences.
2008;246(1315):25-29.
9. Heidecker JL, Leuthold RH. The organisation of collective foraging in
the harvester termite Hodotermes mossambicus (Isoptera). Behavioral
Ecology and Sociobiology. 1984;14(3):195-202.
10. Hölldobler B, Wilson EO. The Ants. Cambridge, Massachusetts: Belknap
Press of Harvard University Press, 1990, 559-566.
11. Jost C, Haifig I, De Camargo-Dietrich CRR, Costa-Leonardo AM. A
comparative tunnelling network approach to assess interspecific
competition effects in termites. Insectes Sociaux. 2012;59(3):369-379.
12. Kok OB, Hewitt PH. Bird and mammal predators of the harvester termite
Hodotermes mossambicus (Hagen) in semi-arid regions of South Africa.
South African Journal of Science. 1990;86(1):34-37.
13. Korb J. Termites, hemimetabolous diploid white ants?. Frontiers in
Zoology. 2008;5(1):15.
14. Mitchell JD. Termites as pests of crops, forestry, rangeland and structures
in Southern Africa and their control. Sociobiology. 2002;40(1):47-69.
15. Machida M, Kitade O, Miura T, Matsumoto T. Nitrogen recycling
through proctodeal trophallaxis in the Japanese damp-wood termite
Hodotermopsis japonica (Isoptera, Termopsidae). Insectes Sociaux.
2001;48(1):52-56.
16. Meyer VW, Braack LEO, Biggs HC, Ebersohn C. Distribution and
density of termite mounds in the northern Kruger National Park, with
specific reference to those constructed by Macrotermes Holmgren
(Isoptera: Termitidae). African Entomology. 1999;7(1):123-130.
17. Matsuura K. Termite-egg mimicry by a sclerotium-forming fungus.
Proceedings of the Royal Society B: Biological Sciences.
2006;273(1591):1203-1209.
Page | 129
18. Matsuura K, Yashiro T, Shimizu K, Tatsumi S, Tamura T. Cuckoo fungus
mimics termite eggs by producing the cellulose-digesting enzyme β-
glucosidase. Current Biology. 2009;19(1):30-36.
19. Nyakupfuka A. Global Delicacies: Discover Missing Links from Ancient
Hawaiian Teachings to Clean the Plaque of your Soul and Reach Your
Higher Self. Bloomington, Indiana: Balboa Press, 2013, 40-41.
20. Noirot C, Darlington JPEC. Termite Nests: Architecture, Regulation and
Defence in Termites: Evolution, Sociality, Symbioses, Ecology Springer,
2000, 121-139.
21. Perna A, Jost C, Couturier E, Valverde S, Douady S, Theraulaz G. "The
structure of gallery networks in the nests of termite Cubitermes spp.
revealed by X-ray tomography. Die Naturwissenschaften.
2008;95(9):877-884.
22. Reinhard J, Kaib M. Trail communication during foraging and
recruitment in the subterranean termite Reticulitermes santonensis De
Feytaud (Isoptera, Rhinotermitidae). Journal of Insect Behavior.
2001;14(2):157-171.
23. Rosengaus RB, Traniello JFA, Chen T, Brown JJ, Karp RD. Immunity in
a social insect. Naturwissenschaften. 1999;86(12):588-591.
24. Su NY, Scheffrahn RH. Termites as Pests of Buildings in Termites:
Evolution, Sociality, Symbioses, Ecology. Springer Netherlands, 2000,
437-453.
25. Slaytor M. Cellulose digestion in termites and cockroaches: What role do
symbionts play? Comparative Biochemistry and Physiology B.
1992;103(4):775-784.
26. Simpson SJ, Sword GA, Lo N. Polyphenism in Insects. Current Biology.
2011;21(18):738-749.
27. Tokuda G, Watanabe H, Matsumoto T, Noda H. Cellulose digestion in
the wood-eating higher termite, Nasutitermes takasagoensis (Shiraki):
distribution of cellulases and properties of endo-beta-1,4-glucanase.
Zoological Science. 1997;14(1):83-93.
28. Traniello JFA, Leuthold RH. Behavior and Ecology of Foraging in
Termites. Springer Netherlands, 2000, 141-168.
29. Toledo Lima J, Costa-Leonardo AM. Subterranean termites (Isoptera:
Rhinotermitidae): Exploitation of equivalent food resources with
different forms of placement. Insect Science. 2012;19(3):412-418.
Page | 130
30. "Termite Power". DOE Joint Genome Institute. United States Department
of Energy. 14 August 2006. Archived from the original on 22 September
2006. Retrieved 11 September 2015.
31. "Termites Green Architecture in the Tropics". The Architect.
Architectural Association of Kenya. Archived from the original on 22
March 2016. Retrieved 17 October 2015.
32. Vasconcellos Alexandre, Bandeira Adelmar G, Moura Flávia Maria S,
Araújo Virgínia Farias P, Gusmão Maria Avany B, Reginaldo,
Constantino. Termite assemblages in three habitats under different
disturbance regimes in the semi-arid Caatinga of NE Brazil. Journal of
Arid Environments. Elsevier. 2010 Feb;74(2):298-302.
Page | 131
Page | 132
Chapter - 7
Role of Transgenic Plants in Pest Management
Authors
Ajit Pandey
Banda University of Agriculture and Technology, Banda,
Aditya Patel
Sardar Vallabhbhai Patel University of Agriculture &
Technology, Meerut, Uttar Pradesh, India
Rishi Nath Pandey
Arvind Kumar
Page | 133
Page | 134
Chapter - 7
Role of Transgenic Plants in Pest Management
Ajit Pandey, Aditya Patel, Rishi Nath Pandey and Arvind Kumar
Abstract
Genetically modified plants can tackle many of the world's food security
and malnutrition problems and contributing to the conservation and
protection of the environment by improving productivity and relying on
chemical pesticides and herbicides. They are formed by Agrobacterium-
mediated mutations and other direct DNA transfer techniques. Many
transgenes that are resistant to pests, diseases and herbicides have been
introduced into crop plants from variety of plants and bacterial systems. In
the most cases, the genes expressed in GM plants pass on to offspring
without harming the receiving plant. Furthermore, under field conditions,
GM plants have high level of pest resistance. Genetically modified plants
include corn, wheat, soybeans, tomatoes, potatoes, cotton, rice, etc. Among
these herbicide-tolerant and pest-tolerant cotton varieties, Bacillus
thuringiensis (Bt) gene carriers are commercially grown maize and soybeans.
The development of insect-resistant varieties by incorporating one or more
specific genes is becoming an important factor in reproduction. The usage of
endotoxin genes like as Bt and plant-derived genes “proteinase inhibitors” at
specific levels will open up new possibilities for pest control. Transgenic
methods should be included into the whole system approach to pest
management and environmental friendliness.
Keywords: Malnutrition, environmental protection, agrobacterium,
transgenic, management
Introduction
A quote derived from famous war epic, The Art of War, narrating
Chinese military strategist Sun Tzu (“Master Sun”) rules of warfare: “If you
know the enemy and know yourself, you need not fear the result of a
hundred battles. If you know yourself but not the enemy, for every victory
gained you will also suffer a defeat”. (Singh et al., 2020).
Page | 135
Transgenic plants are those who’s DNA has been altered through the use
of genetic engineering. Transgenic refers to an organism that has a transgene
that was introduced through technology (rather than breeding) means.
Transgenesis means the process of creating transgenic organisms.
Transgenesis
Transgenesis is the process of inserting a gene (referred to as a
transgene) from one organism into another. The goal is for the transgenic
organism to express the gene and demonstrate a new property or attribute.
The fact that all living organisms have the same genetic code makes this
possible.
Transgenic crops
Transgenic plants are plants that have been genetically modified to
contain DNA from another species (if the DNA is endogenous to the plant
species, the plants are called cisgenic plants; otherwise, the word
"transgenic" will be used to refer to all genetically modified plants). Plants
that are transgenic can have genes that provide resistance to insects, diseases,
herbicides, or abiotic challenges, as well as genes that enhance nutritional
value, storability, or flavour (Nap et al. 2003). Genetic engineering can
achieve these features considerably more efficiently than conventional
breeding programmes. Insect-resistant transgenic crops can help farmers
save money by reducing pest management and labour expenses, as well as
increasing yields due to reduced pest damage (Kalaitzandonakes 1999). The
cultivation of genetically engineered crops has expanded dramatically since
their first commercialization in the mid-1990s, from 2 million hectares in
1996 to 148 million hectares in 2010(James, 2010). At the moment, all
commercially present insect-resistant transgenic crops are intended for direct
pest resistance, and they all contain genes for Bacillus thuringiensis (Bt)
toxins that harm certain herbivores (Aronson and Shai, 2001; Chen et al.
2008). Plants have two types of herbivorous insect resistance mechanisms:
direct and indirect. Herbivores are affected directly by plant features that are
either physical (e.g. thorns) or chemical (e.g. poisons or digestibility
reducers). Indirect resistance increases the effectiveness of herbivore natural
enemies, such as the emission of volatile herbivore-induced secondary plant
compounds that attract the natural enemies (Karban and Baldwin, 1997;
Dicke and Baldwin, 2010). In 2017, India has the world's fifth largest
planted area of genetically modified (GM) crops, at 11.4 million hectares
(mh). However, unlike other large growers, its whole GM crop acreage is
dedicated to cotton, which incorporates genes from the Bacillus thuringiensis
or Bt soil bacterium that code for resistance to Heliothis bollworm insect
Page | 136
pests. The United States has the most transgenic crop land, with 75 million
hectares. The International Service for the Acquisition of Agri-Biotech
Applications (ISAAA) states that the 75 million hectares of GM land
included 34.05 million hectares of soyabean, 33.84 million hectares of maize
(corn), 4.58 million hectares of cotton, 1.22 million hectares of alfalfa, 0.876
million hectares of canola, 0.458 million hectares of sugar-beet, 3,000
hectares of potato and around 1,000 hectares each of apples, squash and
papaya.
According to the latest ISAAA research, "Global Status of

Commercialized Biotech/GM Crops in 2017", farmers throughout the world
planted 189.8 million hectares of transgenic crops last year. This compares
to 1.7 million hectares in 1996, the first year they were commercially
cultivated. The total planted area increased rapidly in the first decade of this
century, but has slowed in the subsequent five years.
According to the research from the global crop biotech advocacy
organisation, soyabean (94.1 mh) had the largest percentage of the world's
total 189.8 mh GM crop area in 2017, followed by maize (59.7 mh), cotton
(24.1 mh), canola (10.2 mh), alfalfa (1.2 mh) and sugar-beet (1.2 mh) (0.50
mh). Insect resistance and tolerance to glyphosate herbicide application were
among the GM features introduced into these crops via the introduction of
alien genes into host plants.
Development of transgenic crops

In the laboratory, genetically modified plants are developed by changing
the genetic make-up of a plant, typically by adding one or more genes from
Page | 137
its genome. The new transgenic DNA is aimed at the nucleus of the plant
cell. The biolistic method (particle gun method) or the Agrobacterium
tumefaciens mediated transformation method is used to create genetically
modified plants.
Corn and rice crops are the most commonly used the "gene gun"
method, also known as micro-projectile bombardment. Cannons are used to
transmit DNA combined with micro gold or tungsten particles into plant
tissue or single plant cells under increased pressure (H. S. Chawla, 2000).
Rapid cells penetrate both the cell wall and the membranes. DNA is
separated from the enclosed metal inside the nucleus and incorporated into
the plant gene. This approach has worked for a range of crops, particularly
monocots like wheat and maize, which have struggled to transform with
Agrobacterium tumefaciens (Shrawat et al. 2006). This is a safe and secure
technique. The main disadvantage of this method is that it may result in
considerable damage to living tissue. The "Agrobacterium" approach is the
next step in the evolution of genetically engineered plants. It makes use of
soil-dwelling bacteria called Agrobacterium tumefaciens. With the help of
DNA fragmentation, it infects plant cells. Plant-derived DNA fragments
attach to its chromosome by tumor-inducing plasmid (Ti plasmid). Plasmid
Ti is a large circular DNA molecule that replicates without the help of the
bacterial chromosome (H. S. Chawla, 2000). This plasmid is important
because it contains sections of transmitted DNA (t DNA) that the researcher
can insert into the gene. Transferred to plant cell by "floral dip". Immersion
of flowering plants in the solution of Agrobacterium containing the desired
gene, followed by collection of GM seeds from the plant (H.S. Chawla,
2000). This method is advantageous because it is a natural transfer and
therefore a more acceptable strategy. Furthermore, "Agrobacterium" has the
ability to swiftly transport large DNA fragments. One of the most serious
disadvantages of Agrobacterium is that it may not infect all key food crops
(H.S. Chawla, 2000). This method is particularly effective for
dicotyledonous plants such as potatoes, tomatoes and tobacco plants.
Page | 138
Fig 1: Agrobacterium mediated transformation
Because of well-developed transformation techniques, easy propagation,
and well-studied genomes, tobacco and Arabidopsis thaliana are the major
GM crops in the research (Koornef et al. 2010). Other plant species are used
as specimens for them. Transgenic plants have also been used in
bioremediation of contaminated soil. Organic pollutants such as mercury,
selenium and polychlorinated biphenyls (PCBs) were removed from the soil
using genetically modified plants containing genes for bacterial enzymes
(Meagher 2000).
Success story of transgenic crops
Bt and Biopesticides
Bacillus thuringiensis or Bt, is a gram-positive bacterium found
naturally in soil all over the world. Bacteriologists have known for decades
Page | 139
that certain strains of Bt kill specific insects and that the toxic ingredient
responsible for the insects' mortality is a protein. When certain insects
consume the bacterium or the protein produced by the bacterium (the protein
is known as d-endotoxin), the operation of their digestive systems is
interrupted, eventually leading to death. Sudden death occurs when the dose
is too high. The Bt protein is not toxic to humans, birds, or fish, nor does it
harm beneficial insects. Mammals, including humans, lack d-endotoxin
receptors in their stomachs, and all Bt proteins examined thus far disintegrate
within 20 seconds in the presence of mammalian digestive juices. Although
Bt is not efficient against all insects, some Bt strains are beneficial against
specific species.
The major families of insects that respond to Bt are:
Lepidoptera (caterpillars e.g. European corn borer or cotton bollworm).
Coleoptera (beetles e.g. Colorado potato beetles).
Diptera (flies and mosquitoes).
Page | 140
Bt: Endotoxins and Their genes
Initially, Bt toxins were classified into 14 distinct groups and 4 classes
(Höfte and Whiteley classification (Höfte and Whiteley, 1989) based on their
host range.
These are:-
 CryI (active against Lepidoptera [“Cry” stands for “crystalline”
reflecting the crystalline appearance of the d-endotoxin; “Cry” is
used to denote the protein whereas “cry” represent the respective
gene]).
 CryII (Lepidoptera and Diptera).
 CryIII (Coleoptera).
 CryIV (Diptera).
Cry Origin Major Target
Common names
protein (Bt sub-species) Insects Order
Silk worm, Tobacco horn worm,
CryIA(a) Kurstaki L
European corn borer
Tobacco horn worm, Cabbage worm,
CryIA(b) Berlineri L&D
Mosquito
Tobacco budworm, Cabbage lopper,
CryIA(c) Kurstaki L
Cotton bollworm
CryIA(d) Aizawai L Several Lepidoptera
CryIA(e) Alesti L Tobacco budworm
CryIB Thuringiensis L Cabbage worm
CryIB(c) Morrisoni L Several Lepidoptera
CryIC Entomocidus L&D Cotton leaf worm, Mosquito
CryIC(b) Galleriae L Beet army worm
Beet army worm, Tobacco horn
CryID Aizawai L
worm
CryIE Kenyae L Cotton leaf worm
CryIE(b) Aizawai L Several Lepidoptera
European corn borer, Beet army
CryIF Aizawai L
worm
CryIG Galleriae L Greater wax moth
CryIIA Kurstaki L&D Gypsy moth, Mosquito
Gypsy moth,
CryIIB Kurstaki L
Cabbage lopper, Tobacco horn worm
CryIIC Shanghai L Tobacco horn worm, Gypsy moth
CryIIIA San diego C Colorado potato beetle
Page | 141
CryIIIA(a) Tenebrionis C Colorado potato beetle
CryIIIB Tolworthi C Colorado potato beetle
CryIIIC N/a C Spotted cucumber beetle
CryIIID Kurstaki C N/a
CryIVA Israelensis D Mosquito (Aedes and Culex)
CryIVB Israelensis D Mosquito (Aedes)
CryIVC N/a D Mosquito (Culex)
CryIVD N/a D Mosquito (Aedes and Culex)
European corn borer, Spotted
CryV N/a L&C
cucumber beetle
CryIX Galleriae L Greater wax moth
Source: (Rajamohan and Dean 1995 and Crickmore et al. 1996)
Refugia
Crop pest management efforts in an agro-ecosystem can be boosted by
altering habitat (refugia). Refugia is a type of microhabitat that provides
spatial and/or temporal refuge for pest natural enemies like as predators and
parasitoids, as well as biotic interaction components in ecosystems such as
pollinators or pollinating insects (Perba et al. 2019). Several strategies for
managing Bt toxin resistance in transgenic crops have been presented.
These are:
a) Transgenic plant mixtures, mosaics or rotations.
b) Time-or tissue-specific toxin expression.
c) Low doses of toxin in association with natural enemies.
d) Co-expression of multiple cry genes.
e) High expression (dosage) with refugia of non-Bt plants.
The last technique is the one that is now suggested (Frutos et al. 1999),
and it is based on three assumptions: i.e.:
1) Resistance will be transmitted as a functionally recessive trait, with
heterozygous progeny being killed at the same rate as homozygous
susceptible larvae, restricting the propagation of resistance alleles in
the population.
2) Insects with Bt resistance will be relatively rare in comparison to
susceptible individuals, and they will nearly always mate with
susceptible wild-type insects, producing heterozygous progeny.
3) That resistant individuals may be at a considerable competitive
disadvantage as a result of the fitness cost associated with
harbouring the resistance allele.
Page | 142
The spatial and temporal position of the refugia in relation to the Bt crop
has not been thoroughly researched, while current EPA regulations in the
United States require refugia to be planted within 0.6 km of Bt corn fields
(EPA, 2001). The influence of the spatial arrangement of the refugia on the
rate of resistance increase should be closely tied to the migration of
susceptible insects within the field from surrounding subpopulations.
Simulation model
Bt plant or a non-Bt plant and was set out in four spatial patterns:
1) Refugia as a border.
2) Refugia in the central part of the field.
3) Refugia equidistant, from the center of the field.
4) Random.
Border Central
Equidistant Random
In computer models, spatial patterns of refugia were used. The
percentage of refugia equals 20%. Refugia (non-Bt plant patches) are
indicated in red and Bt transgenic plant patches are indicated in white (Cerda
et al. 2004).
Page | 143
Protease inhibitors
Protease inhibitors (PIs) are tiny proteins found primarily in storage
tissues such as tubers and seeds, but sometimes in the aerial parts. Protease
inhibitors are found all over the plant world and perform critical roles in
defence against herbivores and pathogens. They form complexes with
proteases, limiting proteolytic action, and operate as store proteins in plant
storage organs. Hilder et al. revealed direct evidence of PIs' protective effect
against insect herbivores. Since then, PIs have been used to create a huge
variety of transgenic plants. There are four types of protease inhibitors:
serine, cysteine, aspartic, and metallo-protease inhibitors. The most
numerous are serine PIs, which are found in the seeds, leaves, and tubers of
various Fabaceae, Poaceae, and Solanaceae families. Furthermore,
mechanical injury, insect chewing, and microbial infection all significantly
increased the level of PIs in both local and remote tissues. (Jagdish et al.
2020).
Lectin
Lectins are nonimmune glycoproteins that identify and bind
carbohydrates. These proteins can be found in a wide range of organisms
(viruses, bacteria, fungi, seaweed, animals and plants). This review is
primarily centred on knowledge about plant lectins, which have been
discovered to be crucial new biological control agents. A large number of
studies have indicated lectin toxicity in insects of several orders, including
Lepidoptera, Coleoptera, and Hemiptera. Lectins are rapidly gaining
popularity as insecticidal agents against sap-sucking insects such as aphids
and leaf and plant hoppers, with no effect on human metabolism.
Plant lectins are classified into four groups based on their number of
domains and features.
 Merolectins are lectins that possess a single carbohydrate-binding
domain. As a result, the merolectins lack of agglutinating activity.
 Hololectins contain two or multivalent carbohydrate-binding sites.
 Chimerolectins possess a carbohydrate-binding domain and an
additional domain that confers other biological activities.
 Superlectins are lectins with two or multivalent carbohydrate
domains that are able to recognize structurally unrelated sugars.
Page | 144
Source: Reyes-Montano et al., 2018
Advantages of transgenic plants
To date, GM technology has been employed to develop a wide range of
crop species. Food is becoming increasingly scarce as the world's population
grows. Improved production output, decreased transportation costs, and
enhanced nutritional content are among advantages of genetically altered
foods. Developments leading to commercially produced crops in countries
such as the United States and Canada have focused on providing resistance
to pests, pests or viruses as well as enhancing herbicide resistance. While
these traits benefited farmers, consumers have had difficulty identifying any
other benefit. In some cases, a cheaper price is possible due to decreased
costs and increased ease of production (Flack-zepeda et al. 1999). A variety
of GM crops for malnutrition are scheduled to be released for cultivation in
the next five to ten years (Davis kevin, 2007).
Herbicide resistant plants
Plants that can survive herbicides are known as herbicide resistant
plants. Many broad-spectrum herbicides contain glyphosate as an active
ingredient. The aro A gene was inserted into a glyphosate EPSP synthetase
from Salmonella typhimurium and E. coli to produce glyphosate-resistant
Page | 145
transgenic tomato, potato, tobacco, cotton, and other plants. The mutant ALS
(acetolactate synthetase) gene from Arabidopsis is used to make
sulphonylurea-resistant tobacco plants. The QB protein of Photosystem II is
injected from mutant amaranth hybrids into tobacco and other crops to
produce atrazine-resistant transgenic plants.
Insect resistant plants
Bacillus thuringiensis is a pathogenic bacterium that infects a wide
variety of insect pests. Its lethal effect is caused by the protein toxin it
produces. The toxin gene is delivered directly into the plant's gene using
recombinant DNA techniques, where it is produced and protects the plant
from insect pests.
Virus resistant plants
By injecting viral coat proteins into tobacco and tomato plants, TMV
resistance can be achieved. There are other transgenic plants that are
resistant to viruses.
a) Potato virus resistant potato plants.
b) RSV resistant rice.
c) YMV resistant black gram.
d) YMV resistant green gram etc.
Pest resistant plants
If transgenic plants gain resistance to a certain pest, there is definitely an
advantage to farmers. Since 1996, papaya resistant to the papaya ring spot
virus has been commercialised and farmed in Hawaii (Gonsalves, 1998).
Reduced pesticide use can also help the environment. Genetically modified
crops with bactericidal genes from Bacillus thuringiensis have dramatically
reduced the amount of pesticides used on cotton in the United States. Pest
and disease-causing organism populations, on the other hand, adapt quickly
and develop pesticides resistant.
Nutritional benefits
Vitamin A deficiency causes 500,000 children to become partially or
completely blind each year (Conway, 1999). For the majority of the world's
population, milling rice is the staple food. Conventional breeding efforts
have failed to produce crops with high vitamin A concentrations.
Researchers introduced three genes into rice, two from daffodils and one
from a bacterium. The transgenic rice seed is yellow in colour and generates
more beta-carotene, a precursor of vitamin A. (Ye, X., Al-Babili et al. 2000).
Page | 146
Rice that is yellow or golden in colour could be an effective therapy for
vitamin A deficiency in tropical newborns.
Use of marginalized land
Because of excessive salinity and alkalinity, a vast landmass, both
coastal and terrestrial, has been marginalised over the world. A salt tolerance
gene has been found, cloned, and transferred to other plants from mangrove
forests (Avicenna marina). Genetically modified plants have been found to
tolerate high concentrations of salt. Salt-tolerant genetically modified maize
plants were created using the Gut D gene from Escherichia coli. Such genes
could be used to build farming systems for neglected territories (MS
Swaminathan, Personal Communication, 2000). Transgenic tomatoes that
grew well in salty soils were developed by researchers at the University of
California Davis campus. The transgene was a highly expressed
sodium/proton antiport pump that trapped excess sodium in leaf cell
vacuoles. The fruit contained no sodium accumulation.
Reduced environmental impact
Scarcity of water and inefficient use have become global issues. Soils
that have been treated to extensive tillage (ploughing) for weed control and
seed bed preparation are prone to erosion and have lost a large amount of
water. Low-tillage methods have been employed in traditional communities
for many years. Crops that thrive in such conditions must be developed,
including root disease resistance and herbicides that can be used instead of
tillage (Cook et al. 2000).
Therapeutic proteins from transgenic plants
Proteins of therapeutic value, such as those employed in the treatment
and diagnosis of human diseases, can be created in plants utilising
recombinant DNA technology. When these transgenic plants are scaled up to
fields, they produce industrial quantities of proteins. Molecular farming,
often known as pharming, is a branch of research that combines molecular
biotechnology and agriculture. Proteins produced in transgenic plants for
medicinal purposes fall into three categories:
a) Antibodies.
b) Proteins.
c) Vaccines.
Transgenic plants have been shown to produce antibodies against dental
caries, rheumatoid arthritis, cholera, E. coli diarrhoea, malaria, some
Page | 147
malignancies, HIV, rhinovirus, influenza, hepatitis B virus, and herpes
simplex virus. Plants such as potatoes and bananas have developed vaccines
against infectious illnesses of the gastrointestinal tract (Thanavala et al.,
1995). Cereal grains would be an additional viable target. An anti-cancer
antibody that detects cells from lung, breast, and colon cancer has recently
been discovered in rice and wheat seed, and could be valuable in future
detection and therapy (Stöger et al. 2000).
Other benefits of transgenic plants
An important advantage is that genetically modified foods can withstand
long distance transport. GM crops are grown green and are allowed to
mature while shipping, giving them a longer shelf life. Even after an
extended shipping and storage period, the product will reach its destination
without being damage.
According to the manufacturers of these GM crops, the use of these
seeds has a variety of benefits, including increased yield and lower cost.
They proposed GM foods as the second "green revolution" in the world with
billions of people starving for food.
Disadvantages of transgenic crops
Transgenic crops have been a source of controversy for a long time.
There have been numerous issues highlighted, which can be divided into two
categories.
 Concerns about the potential impact of genetically modified
materials on human health. Transgenic crops, for example, have
been associated to allergies in some persons, while it is unclear if
transgenic crops are the origin of these reactions. Furthermore,
antibiotic resistance genes put into these crops have been related to
antibiotic resistance, resulting in superbugs that cannot be
eliminated with antibiotics (Losey et al. 1999). The public is wary
of swallowing DNA that has come from another source, such as a
virus or bacteria.
 Concerns have been raised about whether transgenic crops harm the
environment. One example is pollen from transgenic corn, which
has the ability to kill Monarch butterfly larvae. It has been
demonstrated that hybrid corn pollen contains a bacterial poison
that is distributed by wind across a distance of 60 metres. Corn
pollen is deposited on other plants around corn fields in this region,
where it might be consumed by non-target creatures such as the
monarch butterfly (Losey et al. 1999).
Page | 148
However, the following are potential issues of concern for plant protein
production.
1) Plant protein glycans and other plant antigens can cause allergic
responses.
2) Contamination of plants and products by mycotoxins, pesticides,
herbicides and endogenous metabolites.
3) Regulatory uncertainty, particularly for proteins requiring human
medicine approval (Doran, 2000).
Regulation of transgenic plants
In the United States, the coordinated framework for biotechnology
controls the regulation of genetically modified species, including plants. It is
the Government's position that this is untrue and that we agree that the
World Health Organization's protocols are adequate to ensure that any
potentially harmful effects on GM foods can be detected.
The three Agencies involved in this are:
 The USDA Animal and Plant Health Inspection Service (APHIS)
Biotechnology Regulatory Services (BRS) programme is
responsible for regulating the introduction (importation, interstate
movement and field release) of genetically engineered (GE)
organisms that may pose a plant pest risk, according to USDA
Animal and Plant Health Inspection Service. APHIS protects both
agriculture and the environment by ensuring that biotechnology is
developed and used safely. Through a solid regulatory framework,
BRS enables the safe and limited introduction of new GE plants
with rigorous safeguards, preventing the unintended release of any
GE material. The Environmental Protection Agency (EPA) assesses
potential environmental consequences, particularly for pesticide-
producing genes.
 DHHS, FDA-Food and Drug Administration-assesses the risk to
human health, if the plant is intended for human consumption.
Should we use transgenic crops?
In order to produce an environmentally friendly and harmless crop, the
perceived advantages and disadvantages of GM crops must be married
together. This is well known to growers and researchers of GM crops. To
date, however, there is very little evidence to back up the claim. More
research is needed to determine the true safety of these plants, as well as if
Page | 149
they are healthy for the environment and the people who consume them in
the long run. Most people agree that the potential benefit of developing crops
that produce more and cheaper food for the human population is that genetic
modification technology is making valuable progress.
The future
Although genetically modified crops have the potential to alleviate
global food shortages, their practicality for production remains under doubt.
The increased cultivation of GM crops to end hunger has unintended
consequences for the environment and human health. The debate continues,
and the future of genetically modified crops is uncertain. From 1994 to 2002,
the commercial success of transgenic crops shown that utilising transgenic
crops for commercial production on a farmer's property can yield significant
benefits. Some of the main benefits are as follows:
i) Reduced damage caused by insects and viruses and reduced use of
pesticides.
ii) Post-harvest losses due to improved shelf life and marketing
convenience (tomato) due to storage pest resistance.
iii) Improved nutritional value (canola oil).
iv) More effective hybrid seed production.
Because insecticide and pesticide use will be decreased, the above will
not only contribute to a more sustainable food security system, but also to a
safer environment. This will necessitate the seed business responding to the
changing scenario by providing farmers with seed of these superior crops.
Creating countries will be responsible for developing methods and
commercialising transgenic crops. In the future, transgenic crops will be
employed for features including food processing, pharmaceuticals (including
edible vaccinations), and specialised chemicals, in addition to better
agronomic traits. A transgenic rubber tree has also been developed, which
will be used for a number of applications. As a result, transgenic crops have
a promising future. In 2000, the market for these crops is predicted to reach 3
billion dollars, and in 2005, it is expected to reach $6 billion dollars. These
objectives will be met via concerted efforts in both developed and
developing countries. The general people and farmers will have to adjust to
the new situation. To achieve the benefits of these GM crops, which will be
produced in large numbers in the current decade (2000-2010), both the
public and business sectors will have to play a considerable role.
Page | 150
Conclusion
Researchers believe that in the future, GM crops will be able to deliver
vaccines and medications, making treatment more accessible to people in
developing countries. Traditional medications are more difficult to transport
and preserve than food-based medicines. Many people's lives have been and
will continue to be impacted by transgenic plant developments. Transgenic
plants can now be used to manufacture and inject human antibodies. Genetic
engineering is well-known in the production of biopharmaceuticals such
erythropoietin, which is used to treat anaemia, and insulin, which is used to
treat diabetes. Future generations of GM plants will be engineered to endure
harsh environments while also improving nutrient content, pharmaceutical
production and bioenergy and biofuel production.
Reference
1. Shrawat H Lorz. Agrobacterium-mediated transformation of cereals: a
promising approach crossing barriers Plant Biotechnol Journal.
2006;4(6):575-603.
2. Aronson AI, Shai Y. Why bacillus thuringiensis insecticidal toxins are
so effective: unique features of their mode of action. Fems microbiology
letters. 2001;195:1-8.
3. Cerda H, Wright DJ. Modeling the spatial and temporal location of
refugia to manage resistance in bt transgenic crops. Agriculture,
ecosystems & environment. 2004;102(2):163-174.
4. Chawla H. Introduction to plant biotechnology. CRC Press, 2011.
5. Chen M, Zhao JZ, Shelton AM, Cao J, Earle ED. Impact of single-gene
and dual-gene bt broccoli on the herbivore pieris rapae (lepidoptera:
pieridae) and its pupal endoparasitoid pteromalus puparum
(hymenoptera: pteromalidae). Transgenic research. 2008;17:545-555.
6. Conway G. The doubly green revolution: food for all in the twenty-first
century. Cornell University Press, 2019.
7. Conway G, Toenniessen G. Feeding the world in the twenty-first
century. Nature. 1999;402(6761):C55-C58.
8. Cook RJ. Agricultural biotechnology and the poor: proceedings of an
international conference, Washington, DC, 21-22 October 1999.
Science-based risk assessment for the approval and use of plants in
agricultural and other environments, 2000.
9. Gonsalves D. Annu Rev Phytopathol. 1998;36:415-437.
Page | 151
10. Davies KM. Genetic modification of plant metabolism for human health
benefits. Mutation Research/Fundamental and Molecular Mechanisms
of Mutagenesis. 2007;622(1-2):122-137.
11. Dicke M, Baldwin IT. the evolutionary context for herbivore-induced
plant volatiles: beyond the 'cry for help'. Trends in plant science.
2010;15:167-175.
12. Doran PM. Foreign protein production in plant tissue cultures. Current
Opinion in Biotechnology. 2000;11(2):199-204.
13. Epa. United states environment protection agency bt. Cotton refuge
requirements for the 2001 growing season, 2001-2002 June.
Http://www.epa.gov/pesticides/biopesticides/otherdocs/ bt cotton refuge
2001.htm.
14. Falck-Zepeda JB, Traxler G, Nelson RG. Rent creation and distribution
from the first three years of planting Bt cotton. Ithaka, NY: ISAAA,
1999.
15. Ferber D. Risks and benefits: GM crops in the cross hairs. Science.
1999;286(5445):1662-1666.
16. Frutos R, Rang C, Royer M. Managing insect resistance to plants
producing bacillus thuringiensis toxins. Crit. Rev. Biotechnol.
1999;19:227-276.
17. Conway G. Biotechnology, food & drought Proceedings of the World
Commission on Water, 1999 Nov.
18. Höfte H, Whiteley HR. Insecticidal crystal proteins in bacillus
thuringiensis. Micorbiology review. 1989;53:242-255.
19. Jagdish M, Koundal KR. Constitutive expression of protease inhibitor
gene isolated from black gram (Vigna mungo L.) Confers resistance to
spodoptera litura in transgenic tobacco plants, 2020.
20. James C. Global status of transgenic crops in 1997. Isaaa briefs no.
1997;5:31.
21. James C. Global status of commercialized biotech/gm crops:
International service for the acquisition of agribiotech applications
(isaaa), Ithaca, New York, USA, 2010.
22. James C. ISAAA brief 43, global status of commercialized biotech/gm
crops: ISAAA briefs. Ithaca, New York, 2011.
Page | 152
23. James C. Global review of the field testing and commercialization of the
transgenic plants: 1986 to 1995” ISAAA. (international services for the
acquisition of agri-biotech applications, 1996.
24. Kalaitzandonakes N. A farm level perspective on agrobiotechnology:
how much value and for whom? Agbioforum. 1999;2:61-64.
25. Karban R, Baldwin IT. Induced responses to herbivory. The university
of Chicago Press, Chicago, Illinois, USA, 1997.
26. Koornneef M, Meinke D. The development of Arabidopsis as a model
plant. The Plant Journal. 2010;61(6):909-921.
27. Losey JE, Rayor LS, Carter ME. Transgenic pollen harms monarch
larvae. Nature. 1999;399(6733):214-214.
28. Meagher RB. Phytoremediation of toxic elemental and organic
pollutants. Current opinion in plant biology. 2000;3(2):153-162.
29. Nap JP, Metz PLJ, Escaler M, Conner AJ. The release of genetically
modified crops into the environment-part i. Overview of current status
and regulations. Plant journal. 2003;33:1-18.
30. Purba E. Insect management with refugia plant in upland rice (Oryza
sativa L.). In IoP conference series: earth and environmental science.
IoP publishing. 2019 May; 260(1):012-138.
31. Rani SJ, Usha R. Transgenic plants: types, benefits, public concerns and
future. Journal of pharmacy research. 2013;6(8):879-883.
32. Reyes-Montaño EA, Vega-Castro NA. Plant lectins with insecticidal
and insectistatic activities. Insecticides-agriculture and toxicology
[Prieiga Per Internetą 2018-11-03]. Intech, 2018, 17-41.
33. Singh S, Singh A, Kumar S, Mittal P, Singh IK. Protease inhibitors:
recent advancement in its usage as a potential biocontrol agent for insect
pest management. Insect science. 2020;27(2):186-201.
34. Stöger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, et al.
Cereal crops as viable production and storage systems for
pharmaceutical scFv antibodies. Plant molecular biology.
2000;42(4):583-590.
35. Thanavala YANG, Yang YF, Lyons PMHS, Mason HS, Arntzen C.
Immunogenicity of transgenic plant-derived hepatitis B surface antigen.
Proceedings of the National Academy of Sciences. 1995;92(8):3358-
3361.
Page | 153
36. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, et al.
Engineering the provitamin A (β-carotene) biosynthetic pathway into
(carotenoid-free) rice endosperm. Science. 2000;287(5451):303-305.
Page | 154
Chapter - 8
Role of Nano-Pesticides, Nano-Fertilizers and
Nano Bio-Sensors in Agriculture
Authors
Vijay Kumar
Department of Entomology, SKRAU, Bikaner, Rajasthan, India
Lalita Verma
Department of Agronomy, Rajasthan Agricultural Research
Institute, SKNAU, Jobner, Jaipur, Rajasthan, India
Page | 155
Page | 156
Chapter - 8
Role of Nano-Pesticides, Nano-Fertilizers and Nano Bio-
Sensors in Agriculture
Vijay Kumar and Lalita Verma
Abstract
Presently, nanomaterial-based inputs exhibit fascinating prospects over
traditional sensors. Nanotechnology have marked advantages such as
enhanced detection sensitivity/specificity and possess great potential for its
applications in different fields including environmental and bioprocess
control, quality control of food, agriculture, bio defence, and, particularly,
medical applications. But here we are concerned with the role of nano
biosensor in agriculture and agro-products.
Some of the potential applications of nano biosensors are listed below:
 As Diagnostic Tool for Soil Quality and Disease Assessment
 As an Agent to Promote Sustainable Agriculture
 As a Device to Detect Contaminants and Other Molecule
 As Tool for Effective Detection of DNA and Protein
Keywords: Biopesticides, biofertilizers, bio-sensors, nano technology
Introduction
Nano-pesticides
 Nano-pesticides are defined as any formulation that includes
elements of nm size range and/or claims novel properties associated
with these small size range.
 Nano-pesticides are plant protection chemicals, in which either the
active ingredient or the carrier molecule is developed through
nanotechnology.
 The major aim in the development of nano-pesticides is to lessen
the environmental hazards of a pesticide active ingredient through
improving the efficacy of a chemical.
Page | 157
 The size of a nanoparticle generally ranges 1-100 nanometer
and a nanometer is one billionth of a meter. When the size gets this
small, particles reach a very large surface area and thus more
volume of pesticides get contact with the pests.
Formulations of nano-pesticides
 Nano-pesticides are formulated according to their intended purpose
as formulations improving solubility, slow release of active
ingredients, preventdegradation etc.
Some foremost nano-formulations are:
 Nano-emulsions: In this formulation active ingredient of the
chemical is dispersed as nanosized droplets in water, with
surfactant molecules confinedat the pesticide-water interface.
 Nano-suspension: Nano-suspensions, also termed as nano-
dispersions, are formulated by dispersing the pesticide as solid
nanosized particles in aqueous media.
 Polymer based nano-particles: Polymer-based pesticide
nanocarriers are majorly deployed in the slow and controlled
release of active ingredients to the target site.
 Nano-encapsulation: This confines the hydrophobic or hydrophilic
activeingredient, surrounded by a polymer coating or membrane.
 Nanospheres: These are homogeneous vesicular structures, in
which the bioactive ingredient is uniformly dispersed throughout
the polymer matrix.
 Nanogels: These are also known also hydrogel nanoparticles.
These are formulated by cross linking of polymeric particles having
hydrophilic groups.
 Nano-fibres: Nano-fibres are developed through electrospinning,
thermalinduced phase separation.
Advantages of nano-pesticides
 Improved solubility of active ingredients.
 Better stability of formulation.
 Slow release of active ingredient.
 Improved mobility.
 Higher surface area.
 Uniform leaf coverage.
 Improve pesticide utilization.
Page | 158
 Nano-formulations improve adhesion of droplets to plant surface.
 Eco-friendly approach.
Nano fertilizers
 Nano-fertilizers are nutrient carriers of Nano-dimensions capable
of holding bountiful of nutrient ions due to their high surface area
and release it slowly and steadily that commensurate with crop
demand.
 Nutrient use efficiencies of conventional fertilizers hardly exceed
30-35%, 18-20% and 35-40% for N, P and K respectively. The
data remain constant for the pastseveral decade.
 Nano particles have extensive surface area and capable of holding
abundance of nutrients and release it slowly and steadily such that it
facilitates uptake of nutrients matching the crop requirement
without any associated ill effects of customized fertilizer inputs.
Nano-sensors
 Nano-sensors are any biological, chemical or surgical sensory
points used to convey information about nanoparticles to the
macroscopic world.
 Different types include.
 Sensors using semi-conductor nanowire detection elements.
 Semi-conducting carbon nano tubes.
 Carbon nanotubes and nanowires detect bacteria and viruses.
 Nano cantilevers.
Nano bio-sensors
 Nanobiosensor is a modified version of a biosensor which may be
defined as a compact analytical device/unit incorporating a
biological or biologically derived sensitized element linked to a
physico-chemical transducer.
 Nanosensors with immobilized bioreceptor probes that are selective
for target analyte molecules are called nanobiosensors.
 A nanobiosensor is usually built on the nanoscale to obtain process
and analyze the data at the level of atomic scale.
 Their applications include detection of analytes like urea, glucose,
pesticides etc., monitoring of metabolites and detection of various
microorganisms/pathogens.
Page | 159
Characteristics of ideal biosensor
 Highly specific for the purpose of the analyses i.e. a sensor must be
able to distinguish between analyte and any ‘other’ material.
 Stable under normal storage conditions.
 Specific interaction between analytes should be independent of any
physical parameters such as stirring, pH and temperature.
 Reaction time should be minimal.
 The responses obtained should be accurate, precise, reproducible
and linear over the useful analytical range and also be free from
electrical noise.
 The nanobiosensor must be tiny, biocompatible, nontoxic and non-
antigenic.
 Should be cheap, portable and capable of being used by semi-
skilled operators.
Types of Nanobiosensors
 Mechanical nanobiosensors: Nanoscale mechanical forces
between biomolecules provide an exciting ground to measure the
biomolecular interaction. This helps in the development of minute,
sensitive and label free biosensors
 Optical nanobiosensors: Optical biosensors are based on the
arrangement of optics where beam of light is circulated in a closed
path and the change is recorded in resonant frequency when the
analyte binds to the resonator
 Nanowire biosensors: Nanowire biosensor is a hybrid of two
molecules that are extremely sensitive to outside signals: single
stranded DNA, (serving as the ‘detector’) and a carbon nanotube,
(serving as the transmitter). The surface properties of nanowires can
be easily modified using chemical or biological molecular ligands,
which make them analyte independent, this transduces the chemical
binding event on their surface into a change in conductance of the
nanowire with extreme sensitivity, real time and quantitative
fashion.
 Ion channel switch biosensor technologies: The Ion Channel
Switch (ICS) is based on a synthetic self-assembling membrane that
acts as a biological switch for detecting the signals i.e. the presence
of specific molecules by triggering an electrical current
Page | 160
 Electronic nanobiosensors: Electronic nanobiosensors work by
electronically detecting the binding of a target DNA that actually
forms a bridge between two electrically separated wires on a
microchip
 Viral nanobiosensors: Virus particles are essentially biological
nanoparticles. Herpes simplex virus (HSV) and adenovirus have
been used to trigger the assembly of magnetic nanobeads as a
nanosensor for clinically relevant viruses
 Nanoshell biosensors: Positioning gold nanoshells are used in a
rapid immunoassay for detecting analytes within complex
biological media without any sample preparation
 PEBBLE nanobiosensors: Probes Encapsulated by Biologically
Localized Embedding (PEBBLE) nanobiosensors consist of sensor
molecules entrapped in a chemically inert matrix by a
microemulsion polymerization process that produces spherical
sensors in the size range of 20 to 200 nm. These nanosensors are
capable of monitoring real-time inter- and intra-cellular imaging of
ions and molecules.
Nano technology
 Agriculture has always been the backbone of the developing
countries.
 Nanotechnology is now emerging and fast growing field of science
which is being exploited over a wide range of scientific disciplines
including Agriculture.
 A smarter way for sustainable agriculture appears to be
nanotechnology.
Potential applications of nanotechnology in agriculture
 Increase the productivity using nanopesticides and nanofertilizers.
 Improve the soil quality using nanozeolites and hydrogels.
 Stimulate crop growth using nanomaterials.
 Provide smart monitoring using nanosensors by wireless
communication devises.
Nanotechnology in tillage
 Mechanical tillage practices improve soil structure and increase
porosity leading to better distribution of soil aggregates and
eventually modify the physical properties of soil.
Page | 161
 Nanomaterials usage increase soil pH and soil structure
 It also reduces mobility, availability and toxicity of heavy metals
besides reducing soil erosion
 Nanoparticles in soil reduce cohesion and internal friction besides
reducing the shear strength of the soil. Reduction in adhesion of soil
particles allows easycrushing of lumps with less energy
Nanotechnology in seed science
 Seed is nature’s nano-gift to man. It is self-perpetuating biological
entity that is able to survive in harsh environment on its own.
 Nanotechnology can be used to harness the full potential of seed.
 Seed production is a tedious process especially in wind pollinated
crops. Detecting pollen load that will cause contamination is a sure
method to ensuregenetic purity.
 Pollen flight is determined by air temperature, humidity, wind
velocity and pollenproduction of the crop.
 Use of nanobiosensors specific to contaminating pollen can help
alert the possiblecontamination and thus reduces contamination.
 The same method can also be used to prevent pollen from
genetically, modifiedcrop from contaminating field crops.
 Novel genes are being incorporated into /seeds and sold in the
market.
 Tracking of sold seeds could be done with the help of nanobarcodes
that are encodable, machine-readable, durable and sub-micron sized
taggants.
 Disease spread through seeds and many times stored seeds are
killed by pathogens.
 Nano-coating of seeds using elemental forms of Zn, Mn, Pa, Pt, Au,
Ag will not only protect seeds but used in far less quantities than
done today.
Nanotechnology in water use
 Water purification using nanotechnology exploits nanoscopic

materials such as carbon nanotubes and alumina filters for
nanofiltration.
 It utilizes the existence of nanoscopic pores in zeolite filtration
membranes, nanocatalysts and magnetic nanoparticles.
Page | 162
 Carbon nanotube membranes and Nanofibrous alumina filters can
remove almost all kinds of water contaminants including turbidity,
oil bacteria, viruses and organic contaminants.
Nanotechnology in fertilizers
 Fertilizers have played a pivotal role in enhancing the food grain
production in India.
 Despite the resounding success in grain yield, it has been observed
that yields of many crops have begun to stagnate as a consequence
of imbalanced fertilization and decline in organic matter content of
soils.
 Excessive use of nitrogenous fertilizer affects the groundwater and
also causes eutrophication in aquatic ecosystems.
 A disturbing fact is that the fertilizer use efficiency is 20-50 per cent
for nitrogen and 10-25 per cent for phosphorus.
 Elimination of eutrophication and drinking water with possible
buildup of nutrients in soil is possible only by a doping nano
fertilizers an emerging alternative to conventional fertilizers.
 Additionally, nano-technology has improved nutrient use efficiency
and minimize costs of environmental protection.
 The Nano-composites being contemplated to supply all the nutrients
in right proportions through the “Smart” delivery systems also
needs to be examined closely.
 Currently, the nutrient use efficiency is low due to the loss of 50-
70% of the nitrogen supplied in conventional fertilizers.
 Encapsulation of fertilizers within a nanoparticle is one of these
new facilities which are done in three ways.
a) The nutrient can be encapsulated inside nanoporous materials.
b) Coated with thin polymer film.
c) Delivered as particle or emulsions of nanoscales dimensions.
Nanotechnology in plant protection
 Persistence of pesticides in the initial stage of crop growth helps in
bringing down the pest population below the economic threshold
level and to have an effective control for a longer period. Hence, the
use of active ingredients in the applied surface remains one of the
most cost-effective and versatile means of controlling insect pests.
Page | 163
 To protect the active ingredient from the adverse environmental
conditions and to promote persistence, a nanotechnology approach,
namely “nano-encapsulation” can be used to improve the
insecticidal value.
 Nanoencapsulation comprises nano-sized particles of the active
ingredients being sealed by a thin-walled sac or shell (protective
coating).
 Nanoencapsulation of insecticides, fungicides or nematicides will
help in producing a formulation which offers effective control of
pests while preventing accumulation of residues in soil.
 In order to protect the active ingredient from degradation and to
increase persistence, a nanotechnology approach of “controlled
release of the active ingredient” may be used to improve
effectiveness of the formulation that may greatly decrease amount
of pesticide input and associated environmental hazards.
 Nano-pesticides will reduce the rate of application because the
quantity of product actually being effective is at least 10-15 times
smaller than that applied with classical formulations, hence a much
smaller than the normal amount could be required to have much
better and prolonged management.
Nanotechnology in weed management
 Multi-species approach with single herbicide in the cropped
environment resultedin poor control and herbicide resistance.
 Continuous exposure of plant community having mild
susceptibility to herbicide in one season and different herbicide in
other season develops resistance in due course and become
uncontrollable through chemicals.
 Developing a target specific herbicide molecule encapsulated with
nanoparticle is aimed at specific receptor in the roots of target
weeds, which enter into roots system and translocated to parts that
inhibit glycolysis of food reserve in the root system. This will make
the specific weed plant to starve for food and gets killed.
 In rainfed areas, application of herbicides with insufficient soil
moisture may lead to loss as vapour so controlled release of
encapsulated herbicides is expected to take care of the competing
weeds with crops.
 Now a days, adjuvants for herbicide application are currently
available that claim to include nanomaterials.
Page | 164
 Excessive use of herbicides leave residue in the soil and cause
damage to the succeeding crops. continuous use of single herbicide
leads to evolution of herbicide resistant weed species and shift in
weed flora.
 For example, Atrazine, an s-triazine-ring herbicide, is used
globally for the control of pre-and post-emergence broadleaf and
grassy weeds, which has high persistence (half-life-125 days) and
mobility in some types of soils. Residual problems due to the
application of atrazine herbicide pose a threat towards widespread
use of herbicide and limit the choice of crops in rotation.
Conclusion
On overall basis, it may concluded that with the help of nano-technology
(nano pesticides, nano-fertilizers and bio-sensors) minimize the amount and
cost of agricultural inputs and increase the use efficiency.
References
1. Abbas SS, Haneef M, Lohani M, Tabassum H, Khan AF. Nanomaterials
used as a plants growth enhancer: an update. Int. J Pharm. Sci. Rev. Res.
2016;5:17-23.
2. Androvitsaneas P, Young AB, Schneider C, Maier S, Kamp M, Höfling
S, et al. Charged quantum dot micropillar system for deterministic light-
matter interactions. Phys. Rev. 2016;B93:241-409.
10.1103/physrevb.93.241409
3. Anton N, Vandamme TF. The universality of low-energy nano-
emulsification. Int. J Pharm. 2009;377:142-147.
10.1016/j.ijpharm.2009.05.014.
4. Anton N, Vandamme TF. Nano-emulsions and micro-emulsions:
clarifications of the critical differences. Pharm. Res. 2011;28:978-985.
10.1007/s11095-010-0309-1.
5. Asua JM. Miniemulsion polymerization. Prog. Polym. Sci.
2002;27:1283-1346. 10.1016/S0079-6700(02)00010-2.
6. Aziz N, Faraz M, Pandey R, Sakir M, Fatma T, Varma A, et al. Facile
algae-derived route to biogenic silver nanoparticles: synthesis,
antibacterial and photocatalytic properties. Langmuir. 2015;31:11605-
11612. 10.1021/acs.langmuir.5b03081.
7. Aziz N, Pandey R, Barman I, Prasad R. Leveraging the attributes of
Mucor hiemalis-derived silver nanoparticles for a synergistic broad-
Page | 165
spectrum antimicrobial platform. Front. Microbiol. 2016;7:19-84.
10.3389/fmicb.2016.01984
8. Bakalova R, Zhelev Z, Ohba H, Ishikawa M, Baba Y. Quantum dots as
photosensitizers? Nat. Biotechnol. 2004;22:1360-1361.
10.1038/nbt1104-1360.
9. Berekaa MM. Nanotechnology in food industry; advances in food
processing, packaging and food Safety. Int. J Curr. Microbiol. App.
Sci. 2015;4:345-357.
10. Bhattacharyya A, Duraisamy P, Govindarajan M, Buhroo AA, Prasad R.
Nano-biofungicides: emerging trend in insect pest control, in Advances
and Applications through Fungal Nanobiotechnology ed. Prasad R.
(Cham: Springer International Publishing), 2016, 307-319. 10.1007/978-
3-319-42990-8_15.
11. Khond VW, Kriplani VM. Effect of nanofluid additives on
performances and emissions of emulsified diesel and biodiesel fueled
stationary CI engine: a comprehensive review. Renew. Sustain. Energy
Rev. 2016;59:1338-1348. 10.1016/j.rser.2016.01.051.
12. Khota LR, Sankarana S, Majaa JM, Ehsania R, Schuster EW.
Applications of nanomaterials in agricultural production and crop
protection: a review. Crop Prot. 2012;35:64-70.
10.1016/j.cropro.2012.01.007.
13. Konstantatos G, Sargent EH. Solution-processed quantum dot
photodetectors. Proc. IEEE. 2009;97:1666-1683.
10.1109/JPROC.2009.2025612’
14. Kovochich M, Xia T, Xu J, Yeh JI, Nel AE. Principles and procedures
to assess nanoparticles. Environ. Sci. Technol. 2005;39:1250-1256.
15. Kumar S, Bhanjana G, Sharma A, Sidhu MC, Dilbaghi N. Synthesis,
characterization and on field evaluation of pesticide loaded sodium
alginate nanoparticles. Carbohydr. Polym. 2014;101:1061-1067.
10.1016/j.carbpol.2013.10.025.
16. Llop J, Estrela-Lopis I, Ziolo RF, González A, Fleddermann J, Dorn M,
et al. Uptake, biological fate and toxicity of metal oxide nanoparticles.
Part. Part. Syst. Charact. 2014;31:24-35. 10.1002/ppsc.201300323.
Page | 166

Splat Book

Uploaded by

Copyright:

Available Formats

Splat Book

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Splat Book

Uploaded by

Copyright:

Available Formats

Recent Trends in

Insect Pest Management

Chief Editor: Dr. S.V.S. Raju

The author/publisher has attempted to trace and acknowledge the materials

Chapters Page No.

2. Remote Sensing Application in Crop Pest Management 19-32

3. Management Tactics for Plant Parasitic Nematodes 33-51

4. Splat: A New Pheromone Dispensing Technology 53-75

5. Biocontrol Agent: A Promising Tool towards Sustainable

6. Recent Scenario of Termite Infestation and Their Management 109-131

7. Role of Transgenic Plants in Pest Management 133-154

8. Role of Nano-Pesticides, Nano-Fertilizers and Nano Bio-

Protein Distribution Process Functions

Fig 1: Types of genome editing technologies

Typical features Mega nucleases ZFNs TALENs Cas9

Table 3: Advantages and limitations of site-specific nucleases

Sr. Genome editing

4. Why CRISPR/Cas9 is advantages over other genome editing

Fig 2: Application of genome editing technologies in entomology

Fig 2: Schematic representation of gene drive in insects

Fig 1: Working of a camera with flash gun (Process of data acquisition)

Fig 2: Aerial photography by aircraft

Fig 4: Satellite imageries

Sr. Associated active Nematode

9) Removal and destruction of infested plants: Diagnosis of infected

S. No. Nematode Crop/Plantig material Temperature Time

Use of resistant varieties: Several nematode resistant varieties are

Sr. No. Product Fungal spp. Type of fungi Country

2) Bacteria: Recent studies have shown the influence of introduced

The pesticides or chemicals can be classified on their mode of entry, mode

Fig 2: SPLAT vs. Traditional Pheromone Dispensers

Fig 6: The naled-ME (prior sticking) and SPLAT-spinosad-ME treatments of the

In the field attraction bioassays the relative attraction of SPLAT-Spin-ME

Table 4: Effectiveness of Verbenone Treatments for Protecting Small Stands of

Plant Crop Plant pathogen Plant Disease

Tomato is the second most cultivated vegetable crop in India, it is a

2. Biological agents and their importance

2.1.2 Phenolic compound

3.1 Indirect biocontrol

Fig 3: Elements in molecular docking

Fig 4: Steps of Docking

Termites do not live in cool or cold environments due to their delicate

A new termite colony is usually established through the dispersal of

Termite mound in Queensland, Australia

Termites in a mound, Analamazoatra Reserve, Madagascar

Termite damage on external structure

Termite infestation in Rice and Wheat crops

In science and technology

According to the latest ISAAA research, "Global Status of

Development of transgenic crops

 Water purification using nanotechnology exploits nanoscopic

You might also like