Biodiversity and Biotechnology of Ectomycorrhizae PDF

Soil Biology
Volume 25
Series Editor
Ajit Varma, Amity Institute of Microbial Technology,
Amity University Uttar Pradesh, Noida, UP, India
For further volumes:
http://www.springer.com/series/5138
.
Mahendra Rai l Ajit Varma
Editors
Diversity and Biotechnology

of Ectomycorrhizae
Editors
Mahendra Rai, Ph.D. Prof. Dr. Ajit Varma
Professor and Head Director General
Department of Biotechnology Amity Institute of Microbial Technology
SGB Amravati University Amity University Uttar Pradesh
Amravati 444 602, Maharashtra & Vice Chairman
India Amity Science, Technology & Innovation
mkrai123@rediffmail.com; Foundation
pmkrai@hotmail.com Block A, Amity Campus, Sector 125
Noida, UP 201303
India
ajitvarma@aihmr.amity.edu
ISSN 1613-3382
ISBN 978-3-642-15195-8 e-ISBN 978-3-642-15196-5
DOI 10.1007/978-3-642-15196-5
Springer Heidelberg Dordrecht London New York
# Springer-Verlag Berlin Heidelberg 2011
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,
reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication
or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,
1965, in its current version, and permission for use must always be obtained from Springer. Violations
are liable to prosecution under the German Copyright Law.
The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws
and regulations and therefore free for general use.
Cover design: SPi Publisher Services
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)

Dedicated to my revered teacher Professor R. C. Rajak, Ex-head and Dean,
Department of Bio-Sciences, RD University, Jabalpur, Madhya Pradesh, India
– Mahendra Rai
.
Preface
Roots – the hidden half of the plants – are important organs for absorption of water
and nutrients from the soil. Roots release many carbon compounds as exudate.
These compounds provide support to develop a symbiotic relationship with soil-
borne fungi, often called as mycorrhiza. The fungal partner (mycobiont) provides
the plant with improved access to water and nutrients in the soil due to a profusely
branched hyphal network that spreads from the root surface and extends far into the
soil. The plant, in return, supplies carbohydrate for fungal growth and survival. The
mycorrhizae in general and ectomycorrhizae (ECM) in particular are more benefi-
cial to the plants growing in nutrient-poor soil. Ectomycorrhizal plants are resistant
to soil-borne diseases and often tolerate drought stress. In fact, the ECM is respon-
sible for the succession of ecosystems.
There is enormous diversity of ectomycorrhizal fungi in a forest. The ECM can
be used as indicators of quality and also for the development of forest ecosystem.
ECM could be applied for reforestation as they accelerate the plant growth by
supplying water and nutrients. Interestingly, without ECM, healthy woodland
community cannot be maintained. Moreover, some ectomycorrhizal fungi produce
edible sporocarps (fruiting bodies), which are eaten by the people and thus impor-
tant for the food industry.
For better performance of the plants, it is necessary to inoculate them at seedling
stage by ECM to make their life safe. ECM plays a multifunctional role during
symbiosis with higher plants. These fungi have diverse roles as bioremediators,
bioprotectors, biofertilizers, and stress indicators. They are the true “mycoindica-
tors” of the forest ecosystem. There are many metal chelating molecules produced
by ectomycorrhizal fungi, which have remarkable biotechnological significance.
Furthermore, ECM secretes important secondary metabolites.
Molecular approaches are very important for the identification and differentia-
tion of the fungi forming symbiosis with higher plants. Molecular tools are also
important to understand how the genes are expressed during symbiosis with higher
plants. The ectomycorrhizal fungi can be transformed by using Agrobacterium
tumefaciens.
vii
viii Preface
The main goal of this book is to provide information to the readers regarding
diversity and applications of ectomycorrhizae and the use of modern biotechnologi-
cal tools in understanding and transforming them.
The volume is divided into three parts, viz. (1) diversity, morphology, and
applications, (2) biotechnological aspects of ectomycorrhizal fungi, and (3) func-
tions and interactions. The whole book has been made user-friendly and worth
reading.
Part I includes three chapters, out of which the first chapter explains how
ectomycorrhizal inoculation benefits the members of family diptercarpaceae. The
second chapter discusses the status of ectomycorrhizal fungi in South America,
while the third chapter deals with inocula and the techniques of inoculation into the
host plants.
Part II incorporates the molecular approaches in the systematics of ECM, gene
expression during symbiosis, Agrobacterium-mediated gene transfer, biotechno-
logical process, and signaling in ECM symbiosis and RNA-silencing.
In Part III, ectomycoremediation, functions of ECM when challenged with
heavy metals, scale issues concerning the role of ECM in functioning of ecosystems
as indicators of stress in forests, effect of pesticides on ECM, their secondary
metabolites, carbon and nitrogen interactions, interaction of Cantharellus with
Dendrocalamus, and edible ectomycorrhizal fungi have been included.
This volume would be of utmost importance to students, researchers, and
teachers of botany, mycology, microbiology, forestry, and biotechnology. The
readers should find the book full of information and reader friendly.
In planning this volume, invitations for contributions were extended to leading
international authorities working with ectomycorrhizae. The editors would like to
express sincere appreciation to each contributor for his/her work and for their
patience and attention to detail during the entire production process. We sincerely
hope that these eminent contributors will encourage us in the future as well, in the
greatest interest of the academia.
We are extremely grateful to the staff members of Springer Heidelberg, espe-
cially Hanna G. Hensler-Fritton, Editorial Director Life Sciences/Biomedicine
Europe II, Dieter Czeschlik (now retired), and Jutta Lindenborn for their continued
interest, critical evaluation, constructive criticism, and support. We wish to
acknowledge the help and support given to us by our students, faculty colleagues,
and family members for their constant encouragement.
Amravati, Maharashtra, India Mahendra Rai

Noida, Uttar Pradesh, India Ajit Varma
Contents
Part I Diversity, Morphology and Applications
1 The Importance of Ectomycorrhizas for the Growth

of Dipterocarps and the Efficacy of Ectomycorrhizal
Inoculation Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Francis Q. Brearley
2 The Ectomycorrhizal Symbiosis in South America: Morphology,

Colonization, and Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Alejandra G. Becerra and Marcelo R. Zak
3 Ectomycorrhizal Inoculum and Inoculation Techniques . . . . . . . . . . . . . . 43

Ivan Repáč
Part II Biotechnological Aspect of ECM
4 Systematics and Ecology of Tropical Ectomycorrhizal Fungi

Using Molecular Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Rivière-Dobigny Taiana
5 The Molecular Ectomycorrhizal Fungus Essence in Association:

A Review of Differentially Expressed Fungal Genes During
Symbiosis Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Bartolomeu Acioli-Santos, Helder Elı́sio E. Vieira, Cláudia E.P. Lima,
and Leonor C. Maia
6 Agrobacterium tumefaciens-Mediated Transformation

of Ectomycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Minna J. Kemppainen, Maria C. Alvarez Crespo, and Alejandro G. Pardo
ix
x Contents
7 Biotechnological Processes Used in Controlled Ectomycorrhizal

Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Par Duponnois Robin, Bâ Amadou, Mousain Daniel, Galiana Antoine,
Baudoin Ezékiel, Dreyfus Bernard, and Prin Yves
8 Signaling in Ectomycorrhizal Symbiosis Establishment . . . . . . . . . . . . . . 157

Paul Baptista, Rui Manuel Tavares, and Teresa Lino-Neto
9 RNA Silencing in Ectomycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Minna J. Kemppainen and Alejandro G. Pardo
Part III Functions and Interactions
10 Ectomycoremediation: An Eco-Friendly Technique

for the Remediation of Polluted Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Heike Bücking
11 Metal Elements and the Diversity and Function

of Ectomycorrhizal Communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Alexander Urban
12 A Conceptual Framework for Up-Scaling Ecological Processes

and Application to Ectomycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Virgil Iordache, Erika Kothe, Aurora Neagoe, and Felicia Gherghel
13 Mycobioindication of Stress in Forest Ecosystems . . . . . . . . . . . . . . . . . . . 301

Hojka Kraigher and Samar Al Sayegh Petkovšek
14 Effects of Pesticides on the Growth of Ectomycorrhizal Fungi

and Ectomycorrhiza Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Miguel Marin
15 Metal-Chelating Agents from Ectomycorrhizal Fungi

and Their Biotechnological Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Ángela Machuca
16 Ectomycorrhiza and Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . 371

Hanna Dahm and Patrycja Golińska
17 C:N Interactions and the Cost:Benefit Balance

in Ectomycorrhizae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Ana Corrêa and Maria-Amélia Martins-Loução
Contents xi
18 Ectomycorrhizal Interaction Between Cantharellus

and Dendrocalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Rohit Sharma and Ram C. Rajak
19 Edible Ectomycorrhizal Fungi: Cultivation, Conservation,

and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Alka Karwa, Ajit Varma, and Mahendra Rai
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
.
Contributors
Acioli-Santos, Bartolomeu Departamento de Micologia, Universidade Federal

de Pernambuco, Av. Prof. Nelson Chaves, s/n, Cidade Universitária, Recife PE,
50670-420, Brasil, bartacioli@cpqam.fiocruz.br, bartacioli@yahoo.com
Al Sayegh Petkovšek, Samar ERICo Velenje, Ecological Research & Industrial

Cooperation, Koroška 58, 3320, Velenje, Slovenia, samar.petkovsek@erico.si
Alvarez Crespo, Maria C. Laboratorio de Micologı́a Molecular, Departamento

de Ciencia y Tecnologı́a, Universidad Nacional de Quilmes, y Consejo Nacional de
Investigaciones Cientı́ficas y Tecnicas (CONICET). Roque Sáenz Peña 352,
(B1876BXD) Bernal, Provincia de Buenos Aires, Argentina, apardo@unq.edu.ar
Amadou, Bâ IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire

des Symbioses Tropicales et Méditerranéennes (LSTM), TA A-82/J, Campus
International de Baillarguet, Montpellier, Cedex 5, France
Antoine, Galiana IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Labor-

atoire des Symbioses Tropicales et Méditerranéennes (LSTM), TA A-82/J, Campus
Baptista, Paula CIMO / Escola Superior Agrária, Instituto Politécnico de

Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança,
Portugal, pbaptista@ipb.pt
Becerra, Alejandra G. Instituto Multidisciplinario de Biologı́a Vegetal

(CONICET), Cátedra de Diversidad Vegetal I, Facultad de Ciencias Exactas,
Fı́sicas y Naturales, Universidad Nacional de Córdoba, C.C. 495, 5000 Córdoba,
República Argentina, abecerra@efn.uncor.edu
xiii
xiv Contributors
Bernard, Dreyfus IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2,

Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), TA A-82/J,
Campus International de Baillarguet, Montpellier, Cedex 5, France
Brearley, Francis Q. Department of Environmental and Geographical Sciences,

Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK, f.q.
brearley@mmu.ac.uk
Bücking, Heike Biology and Microbiology Department, South Dakota State

University, Brookings 57007, SD, USA, heike.bucking@sdstate.edu
Corrêa, Ana Departamento de Microbiologı́a del Suelo y Sistemas Simbióticos,

Estación Experimental del Zaidı́n, CSIC, Profesor Albareda 1, 18008 Granada,
Spain, ambcorrea@gmail.com
Dahm, Hanna Department of Microbiology, Institute of General and Molecular

Biology, Faculty of Biology and Earth Sciences, University of Nicolaus Coperni-
cus, 87-100 Toruń, Gagarina 9, Poland, dahm@biol.uni.torun.pl
Daniel, Mousain INRA. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2,

Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM). TA A-82/J,
Campus International de Baillarguet, Montpellier, Cedex 5, France
Ezékiel, Baudoin IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Labor-

atoire des Symbioses Tropicales et Méditerranéennes (LSTM), TA A-82/J, Campus
Gheghel, Felicia UMR 1136 Interactions Arbres/Microorganismes, INRA

54280, Champenoux, France
Golińska, Patrycja Department of Microbiology, Institute of General and

Molecular Biology, Faculty of Biology and Earth Sciences, University of Nicolaus
Copernicus, 87-100 Toruń, Gagarina 9, Poland, plgolinska@umk.pl
Iordache, Virgil Department of Systems Ecology, University of Bucharest,

Bucharest, Romania, virgil.iordache@g.unibuc.ro
Karwa, Alka Department of Biotechnology, SGB Amravati University,

Amravati 444 602, Maharashtra, India, alkajaju@rediffmail.com
Kemppainen, Minna J. Laboratorio de Micologı́a Molecular, Departamento de

Ciencia y Tecnologı́a, Universidad Nacional de Quilmes, y Consejo Nacional de
Investigaciones Cientı́ficas y Tecnicas (CONICET). Roque Sáenz Peña 352,
(B1876BXD) Bernal, Provincia de Buenos Aires, Argentina, apardo@unq.edu.ar
Contributors xv
Kothe, Erika Microbial Phytopathology, Institute of Microbiology, Friedrich

Schiller University, Jena, Germany
Kraigher, Hojka Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana,

Slovenia, hojka.kraigher@gozdis.si
Lima, Cláudia E.P. Departamento de Micologia, Universidade Federal de

Pernambuco, Av. Prof. Nelson Chaves, s/n, Cidade Universitária, Recife, PE
50670-420, Brasil
Lino-Neto, Teresa BioFIG, Departamento de Biologia, Universidade do Minho,

Campus de Gualtar, 4710-057 Braga, Portugal, tlneto@bio.uminho.pt
Machuca, Ángela Department of Plant Science and Technology, Universidad

de Concepción, Campus Los Ángeles, J.A.Coloma, 0201 Los Ángeles, Chile,
angmachu@udec.cl
Maia, Leonor C. Departamento de Micologia, Universidade Federal de Pernam-

buco, Av. Prof. Nelson Chaves, s/n, Cidade Universitária, Recife, PE 50670-420,
Brasil
Marin, Miguel Department of Physiology, Genetics and Microbiology, University

of Alicante, Carretera San Vicente del Raspeig s/n, 03690 San Vicente del Raspeig
(Alicante), Spain, mmarinz@ua.es
Martins-Loução, Maria-Amélia Faculdade de Ciências, Centro de Biologia

Ambiental, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Neagoe, Aurora Research Center for Ecological Services (CESEC), University

of Bucharest, Bucharest, Romania
Pardo, Alejandro G. Laboratorio de Micologı́a Molecular, Departamento de

Ciencia y Tecnologı́a, Universidad Nacional de Quilmes, y Consejo Nacional de
Investigaciones Cientı́ficas y Tecnicas (CONICET), Roque Sáenz Peña 352,
(B1876BXD) Bernal Provincia de Buenos Aires, Argentina, apardo@unq.edu.ar
Rai, Mahendra Department of Biotechnology, SGB Amravati University,

Amravati 444 602, Maharashtra, India, mkrai123@rediffmail.com, pmkrai@
hotmail.com
Rajak, Ram C. SGHG Center for Biotechnology, Priydarshini Society, Dumna

Road, Jabalpur Madhya Pradesh, India, rcmykes@yahoo.com
Repáč, Ivan Forestry Faculty, Department of Silviculture, Technical Univer-

sity in Zvolen, T.G. Masaryka 24, 96053 Zvolen, Slovak Republic, repac@vsld.
tuzvo.sk
xvi Contributors
Robin, Par Duponnois IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2,

Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), TA A-82/J,
Campus International de Baillarguet, Montpellier, Cedex 5, France, Robin.
Duponnois@ird.fr
Sharma, Rohit Microbial Culture Collection, Agarkar Reserch Institute, Pune,

411 007, Maharashtra, India, rsmushrooms@yahoo.co.uk
Taiana, Rivière-Dobigny IRD, Avenue de Maradi, 11416 Niamey Niger,

taianariviere@yahoo.co.uk
Tavares, Rui Manuel BioFIG, Departamento de Biologia, Universidade do

Minho, Campus de Gualtar, 4710-057 Braga, Portugal, tavares@bio.uminho.pt
Urban, Alexander Department of Systematic and Evolutionary Botany, Univer-

sity of Vienna, Rennweg 14, 1030 Vienna, Austria, alexander.urban@univie.ac.at
Varma, Ajit Amity Institute of Microbial Technology, Amity University, Noida

Uttar Pradesh, India, ajitvarma@aihmr.amity.edu
Vieira, Helder Elı́sio E. Departamento de Micologia, Universidade Federal de

Pernambuco, Av. Prof. Nelson Chaves, s/n, Cidade Universitária, Recife PE
50670-420, Brasil
Yves, Prin IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire

des Symbioses Tropicales et Méditerranéennes (LSTM). TA A-82/J, Campus
Zak, Marcelo R. Cátedra de Recursos Naturales y Gestión Ambiental, Departa-

mento de Geografı́a, Facultad de Filosofı́a y Humanidades, Universidad Nacional
de Córdoba, C.C. 495, 5000 Córdoba, República Argentina, marcelzak@gmail.com
.
Part I
Diversity, Morphology and Applications
.
Chapter 1
The Importance of Ectomycorrhizas for the
Growth of Dipterocarps and the Efficacy
of Ectomycorrhizal Inoculation Schemes
Francis Q. Brearley
1.1 Introduction
The Dipterocarpaceae is the most important tree family in the tropical forests of
South-east Asia as they are the ecosystem dominants, especially in lowland forests
of this region, where they often contribute up to a quarter of all stems and half of the
above-ground biomass (Proctor et al. 1983; Davies et al. 2003; Brearley et al.
2004). They also play major role in timber production from this area as they are
favoured for their fast growth rates, tall cylindrical boles and high quality wood.
In addition, they are a source of non-timber forest products such as oils and
resins (Shiva and Jantan 1998). Given the continued exploitation and degradation
of lowland tropical forest habitats worldwide, there is interest in reforestation
programmes to maintain forest cover and to provide a sustained supply of wood
products. Many of these programmes have focussed on fast-growing exotic tree
species but, to provide the highest quality timber, reforestation efforts now need to
focus on the Dipterocarpaceae.
The majority of trees in tropical forests form arbuscular mycorrhizas but it was
first noted by Singh in 1966 that dipterocarps, like many temperate forest trees,
formed ectomycorrhizas (EcMs). Since then, many subsequent studies have exam-
ined the roots of numerous dipterocarps and found them to be colonised by EcM
fungi (Alexander and H€ ogberg 1986; Chalermpongse 1987; Smits 1992; Lee 1998;
Hoang and Tuan 2008). There are also minor reports of arbuscular mycorrhizal
(Shamsuddin 1979; Ibrahim et al. 1995; Tawaraya et al. 2003) and ectendomy-
corrhizal (Tupas and Sajise 1976) fungi in dipterocarp roots, but more details
are needed on this potential dual colonisation before anything more concrete can
be written.
In this chapter, I have (1) given a brief overview of mycorrhizal symbioses in
dipterocarps, I now plan to (2) note which are the important fungal species involved
F.Q. Brearley
School of Science and the Environment, Manchester Metropolitan University, Chester Street,
Manchester M1 5GD, UK
e-mail: f.q.brearley@mmu.ac.uk
M. Rai and A. Varma (eds.), Diversity and Biotechnology of Ectomycorrhizae, 3

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_1,
4 F.Q. Brearley
in the EcM symbiosis and then (3) focus on the role of this symbiosis in improving
plant nutrition, growth and performance, and complete (4) with a special focus on
how we might critically use this knowledge to determine if we need to apply EcM
inoculation in reforestation programmes.
1.2 Fungal Species Associated with Dipterocarps
We know, from fruit body surveys, that the most speciose groups of fungi found in
dipterocarp-dominated forests are the families of Amanitaceae, Boletaceae and
Russulaceae (Watling and Lee 1995, 1998, 2007; Watling et al. 1998, 2002; Lee
et al. 2002, 2003), and these appear to form a reasonable proportion of the EcM root
tips belowground (Lee et al. 1997; Ingleby et al. 1998). However, it is known that
above-ground and below-ground views of the EcM community rarely show close
concordance (Gardes and Bruns 1996; Yamada and Katsuya 2001), and it has
recently been noted that fungi with cryptic fruiting bodies such as members of
the Thelephoraceae, which appear to have been overlooked in fruiting body
surveys, are also important EcM formers. For example, Ingleby et al. (2000)
found a Thelephoraceae species to be common on seedlings planted in soil from
Vietnamese forest and I have found two Thelephoraceae species common on
nursery-grown seedlings in Malaysia (Brearley et al. 2003, 2007; Brearley 2006).
Furthermore, sequences from this group dominated the molecular studies on
dipterocarp EcM communities conducted by Sirikantaramas et al. (2003) and
Yuwa-Amornpitak et al. (2006). As an example, half of the sequences generated
by Sirikantaramas et al. (2003) were from this family. In this regard, the use of
molecular identification techniques has shown us that the EcM community
in dipterocarp-dominated forests is more similar to many boreo-temperate EcM
communities (e.g. Richard et al. 2005; Ishida et al. 2007; Morris et al. 2008) than
originally thought. As molecular work continues on the EcMs communities of
dipterocarps, it will reveal a much clearer below-ground picture of the dominant
species involved in this symbiosis.
1.3 Nutrient Relationships
There are numerous experiments showing that EcMs can improve plant nutrient
uptake (Smith and Read 2008) and, in the Dipterocarpaceae, this has also been
shown to be the case. For example, Lee and Lim (1989) correlated percentage EcM
colonisation (% EcM) with foliar phosphorus (P) in dipterocarp seedlings they
studied. Seedlings from a site with lower available soil P showed a positive
correlation between foliar P and % EcM suggesting that EcMs enhanced uptake
of P at this site. Hadi and Santoso (1988, 1989) presented data suggesting inocula-
tion with EcM fungi increased foliar concentrations of nitrogen (N), P, potassium
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 5
(K), calcium (Ca) and magnesium (Mg) of five dipterocarp species (although it is
not entirely clear if their data are for nutrient concentrations or total nutrient content
in the seedlings). The first experiments to show an unequivocal increase in foliar
nutrients in response to EcM colonisation were those of Lee and Alexander (1994)
and Yazid et al. (1994) who clearly showed increased P concentrations in response
to EcM colonisation in two species of Hopea studied. Additionally, in the experi-
ments of Lee and Alexander (1994), whilst there were no suggestions of EcM
colonisation increasing shoot N, K or Mg concentrations, there was some indication
of improved Ca nutrition. These differential responses may be due to the experi-
mental conditions used but may also represent a certain degree of inter-specific
difference in fungal benefits to the host plant or may even be due to an EcM
diversity effect on nutrient uptake (Baxter and Dighton 2005).
Improved mineral nutrition has also been shown under field conditions where
reduction of EcM colonisation (by Mancozeb fungicide) led to reduced foliar N and
P in both Hopea nervosa and Parashorea tomentella in addition to reduced Ca and
Mg in Hopea nervosa (Brearley 2003). I have also shown, through stable isotope
analysis, that EcM dipterocarps can also obtain more N from organic material
(Brearley et al. 2003) with a positive correlation found between % EcM colonisa-
tion and uptake of organic N.
An inoculation experiment that purports to show increases in nutrient uptake of
Shorea seminis when inoculated with two EcM species is that of Turjaman et al.
(2006). Total shoot N and P contents were indeed greater in the EcM inoculated
seedlings but, surprisingly, on a dry-weight basis, inoculation actually led to a
decrease in concentrations of these elements in the shoot (up to 55% in the most
extreme case). Smits (1983) also considers that dipterocarp EcMs may be providing
thiamin to plants although, in the absence of a strong presentation of data, we
should discount this hypothesis for now.
Increased nutrient concentrations within plants are generally likely to lead to
improved growth, and the role of foliar nutrients in determining seedling perfor-
mance is also important during out-planting as it has been shown that higher levels
of foliar N allow dipterocarp seedlings to better avoid photodamage when trans-
ferred to high irradiance conditions and to allow more rapid acclimation to these
conditions (Bungard et al. 2000).
1.4 Inoculation Experiments
Many of the inoculation experiments reported are in the grey literature and have a
number of shortcomings. The most serious of these is that they are poorly reported
and do not provide sufficient detail for the experiments to be evaluated fully nor
repeated by other researchers. Pseudoreplication or the lack of statistical analyses in
many cases also makes evaluation of the results problematic. In this chapter, I will
focus on papers which, I feel, have mostly overcome these shortcomings or are
otherwise noteworthy.
6 F.Q. Brearley
The first reported experiment attempting to inoculate dipterocarp seedlings

with EcM fungi and determine seedling responses was that of Hadi and Santoso
(1988). They inoculated species of Boletus, Russula and Scleroderma using
pieces of chopped fruiting body on the roots of five dipterocarp seedling species.
A shortcoming of this experiment was that the roots were not examined to deter-
mine the extent of EcM formation by any contaminant fungi. Nevertheless, after
6 months growth, inoculation appeared to at least double seedling height in all
fungus/seedling combinations. Furthermore, their approach to inoculation is rarely
used as it is difficult to control the inoculum viability and potential supplied to the
roots with this method.
Initial experiments, using chopped root inoculum (Lee and Alexander 1994),
found that EcM colonisation increased plant dry weights in Hopea odorata and
Hopea helferi. This increase was generally greatest in the absence of additional
nutrients supplied to the soil. However, the problem with experiments using root
inoculum is that the species of fungi on the inoculant roots cannot be determined
and therefore controlled experiments using defined EcM species are needed. In
Malaysia, inoculation experiments have focussed on strains of Pisolithus species
and a member of the Thelephorales (FP160: Lee et al. 2008). In Indonesia, the use
of Scleroderma for inoculation appears more popular although the range of species
being used has increased recently (Turjaman et al. 2007).
1.4.1 Inoculation with Single Species
1.4.1.1 Malaysian Inoculation Experiments
In Malaysia, exotic Pisolithus isolates from Brazil (Pt 441 originally from under
Eucalyptus citriodora) and Thailand (Pt msn) were effective at forming EcMs
on four dipterocarp seedling species (although there was a certain degree of host-
fungal compatibility with one or other Pisolithus isolate having a greater % EcM on
each of the seedling species; Lee et al. 1995). Using Chilvers et al.’s (1986)
cardboard inoculum technique, Yazid et al. (1994) showed that Pt 441 formed a
high percentage of functional EcM colonisation (c. 80%) on Hopea odorata and
Hopea helferi and that this increased the growth (dry weights increased by 7.3 and
3.6 times, respectively) and foliar P concentrations (by at least 40%) after 9 months
growth. Similar results in terms of the growth of Hopea odorata were seen
(although with a lesser growth response) when coconut husk:vermiculite inoculum
was added in a ratio of 1:4 to a sterilised soil:sand mix (Yazid et al. 1996). In
contrast, problems were found when trying to inoculate a local species: Pisolithus
aurantioscabrosus (Lee et al. 1995). Why this is, is not clear but may simply
reflect the ability of Pisolithus tinctorius to form EcMs with a wide host range
(Martin et al. 2002), whereas Pisolithus aurantioscabrosus has only been reported
to be associated with Shorea parvifolia and Shorea macroptera to date (Watling
et al. 1995a, b; Martin et al. 2002).
Initial tests of successful inoculation between Hopea odorata and Hebeloma

crustulinifome (Lapeyrie et al. 1993) were reported, but growth responses were not
shown and this exotic European strain does not appear to be used any more. Recent
work has focussed on Thelephoraceae FP160 (Lee et al. 2008), which significantly
increased stem height, root length and biomass of Hopea odorata after 6 months
growth in the nursery by 30%, 62% and 40%, respectively (Lee et al. 2008).
It currently appears very difficult to bring further tropical dipterocarp-associated
EcM species into culture (S.S. Lee, pers. comm.), and the species that are being
used form a very limited subset of those available. The best approach here would be
a wide-ranging screening using a variety of fungal structures, media and growth
conditions although, of course, this will be very labour intensive with potentially
little reward. Perhaps, the floating culture technique of Sangtiean and Schmidt
(2002) may help South-east Asian researchers to culture some of the later stage
EcM species found in these forests. This technique allowed Sangtiean and Schmidt
(2002) to carry out culture experiments on Amanita, Lactarius and Russula species,
which are common in South-east Asian forests (see above).
1.4.1.2 Indonesian Inoculation Experiments
In Indonesia, the use of Scleroderma species (and especially Scleroderma colum-

nare) appears to be favoured, probably from the initial work of Ogawa (1993,
2006) in the early 1990s. Sadly, much of this early work is difficult to evaluate as
it is not clearly reported (e.g. Supriyanto et al. 1993; Kikuchi 1997) but, more
recently, a number of much better controlled inoculation experiments have been
conducted by Turjaman et al. (2005, 2006, 2007). They showed that the growth of
Shorea pinanga was improved by the addition of spore tablets of Pisolithus
tinctorius (aka Pisolithus arhizus) and Scleroderma columnare species. Both
fungal species improved the growth of Shorea pinanga (150% increase in dry
weight after 7 months) although there was some EcM colonisation of the controls.
Survival rates (86–87%) were also much higher than the control (16%), which is
an equally important factor to take into consideration when planning reforestation
schemes. In a follow-up experiment (Turjaman et al. 2006), tabletted spore
inoculum was compared with alginated bead mycelial inoculum of Pisolithus
tinctorius and Scleroderma columnare. Percentage EcM colonisation was higher
(61–65%) when seedlings were inoculated with spores than with mycelium
(35–37%), and there was, again, at least a doubling of dry weight after 7 months
growth. In the most recently reported experiment (Turjaman et al. 2007), inocula-
tion of four fungal species on the roots of Shorea balangeran increased seedling
growth. Whilst we might expect the use of Boletus sp., Scleroderma sp. and
Strobilomyes sp. to increase seedling growth as these are known to be EcM
forming fungi, the use of Calvatia sp. also increased seedling growth, which
was unexpected as this is not thought to be an EcM forming genus (Rinaldi
et al. 2008).
8 F.Q. Brearley
1.4.2 Other Inoculation Methods
1.4.2.1 Mycorrhizal Tablets
Where sterile facilities are not available to cultivate species aseptically, researchers
have used “mycorrhizal tablets”. In this case, spores or mycelium are mixed with a
carrier (clay or alginate beads) and applied to seedlings’ rooting zones to allow
hyphal contact and subsequent EcM formation. The first record of this in the South-
east Asia region appears to be that of Ogawa (1993). Species used for this method
are often those such as Scleroderma species, which have the advantage that
their spores can be collected much more easily from their enclosed fruit bodies
than many other gilled or pored fruit bodied species. Clay tablets at 1:100 (crushed
fruit bodies:clay) were used in the experiments by Turjaman et al. (2005, 2006), and
these showed increased growth of Shorea pinanga and Shorea selanica when
inoculated as compared with uninoculated controls. However, in such experiments,
there is a need to confirm that the tablets do not contain additional nutrients that
might improve seedling growth in the absence of a mycorrhizal effect.
1.4.2.2 Mother Tree Inoculation
Other inoculation methods include inoculation from a colonised mother tree in the
nursery – in other words, simply letting newly germinated seedlings’ roots contact
hyphae already radiating out in the soil around established, colonised trees. This
method was first used by Roeleffs (1930, in Nara et al. 1999) to inoculate seedlings
of Pinus species. The technique, also known as inoculation beds, is a low-tech
method that allows EcM inoculation before planting-out but it can be rather
haphazard in terms of the speed and reliability of inoculation (see Kikuchi 1997).
For example, Ogawa (2006) shows a diagram of the spread of Scleroderma colum-
nare fruiting bodies through a nursery containing seedlings of Shorea leprosula and
Shorea academica to be between 1 and 2 m per year.
1.4.3 Production of Inoculum
In order to produce inoculum rapidly, conditions for the optimum growth of fungi in
culture should be evaluated. For example, Patahayah et al. (2003a) showed that the
most rapid growth of Pisolithus albus (aka Pt Gemas) was obtained at 25 C when
grown on Oddoux medium but at 30 C when grown on MMN or Pachlewski’s
medium (Patahayah et al. 2003b). We have also shown that this species grows
best when N is supplied in an organic form (BSA in the experiments conducted;
Brearley et al. 2005). Thelephoreaceae FP160 shows best growth at 25 C
(Patahayah et al. 2003b) but has minimal preferences for N source (Brearley
et al. 2005). In terms of efficient spread of EcM inoculum in the nursery, Nara et al.
(1999) considered that seedlings are often maintained under sub-optimal conditions
in potted soil with a high clay content and hence poor aeration, thereby slowing
growth of fungal hyphae. They found that, by using a growth medium with particles
of 2–4 mm diameter, the optimum growth of EcM mycelium (Th1 on Shorea
roxburghii) was obtained. It is also important to consider the longevity of the
different forms of inoculum. For example, Fakuara and Wilarso (1993) showed
that mycorrhizal tablets remained effective up to 4 months in storage (this was the
longest period tested). More experiments are clearly needed in this area, with longer
test periods, to gain a better idea of spore longevity.
1.4.4 Field Experiments
There is now a need to determine how well inoculated seedlings and their symbiotic
EcM species survive in the wild when seedlings are out-planted. This is important
as, if considerable effort is being put into inoculation programmes, this is simply
being wasted if seedlings or their inoculant fungal species are dying unnecessarily.
Furthermore, in terms of reforestation schemes, growth is not necessarily the most
important parameter, seedling survival is arguably equally as important.
Chang et al. (1994, 1995) showed that the species of Pisolithus in the Malaysian
inoculation experiments noted above did not remain competitive when colonised
seedlings of Shorea glauca were planted into the field; indeed Pisolithus had mostly
disappeared from the roots after 6 months suggesting that they are either early stage
fungi, or are poorly adapted to the biotic or abiotic environments of the Malaysian
forest soils. Using Thelephoraceae FP160, Lee et al. (2008) found it to remain
competitive on the roots of seedlings (Hopea odorata and Shorea leprosula) for up
to 23 months after out-planting in a sandy tin mine tailings site (after this time
contaminant EcM fungi had only colonised up to 15% of the root tips of less than
half the seedlings). However, the improved growth of Hopea odorata seen in
the nursery due to inoculation with this fungus (see above) was not seen in the
field (by measurement of root collar diameter) and improved growth of Shorea
leprosula was only seen for up to 3 months following out-planting. Under field
conditions, I found that the reduction in EcM colonisation by fungicide addition
to the roots of two species (Hopea nervosa and Parashorea tomentella) did not
lead to changes in seedling growth but that foliar nutrient concentrations were
reduced (Brearley 2003). In field experiments in a degraded peat swamp forest in
Kalimantan, Turjaman et al. (2007) showed that a spore suspension of Boletus sp.
and Scleroderma sp. applied to the seedling rooting zone led to increased growth of
Shorea balangeran but application of Calvatia sp. and Strobilomyces sp. did not.
For the two fungal species that were beneficial, it took around 8 months for growth
improvements to be seen, perhaps due to the very wet conditions at the start of the
experiment (Turjaman et al. 2007). However, it is difficult to determine if the
species applied were those that maintained improved seedling growth as the roots
10 F.Q. Brearley
at the end of this 40-month experiment were not examined to determine which EcM
fungi were present – it would have been a notable improvement to the study design
to do this. The study of Tata et al. (2009) did report this examination at the end of
their experiments with Shorea selanica and Shorea lamellata, which were inocu-
lated with spore tablets of Scleroderma columnare and planted in natural forests or
rubber agroforests in Sumatra. Their results were complex but did not show
consistent increases in growth, performance or survival of the two dipterocarp
species over a 2-year period. It is notable that, among the 19 EcM types they
identified using PCR-RFLP at the end of the experiment, none of them were
Scleroderma species indicating that the inoculated fungus did not remain competi-
tive on the roots for more than this length of time.
There is clearly a need to further evaluate the growth and survival of inoculated
seedlings in the field as positive responses to EcM inoculation in the ecologically
simplified, and somewhat benign, nursery environment are unlikely to be represen-
tative of that found at out-planting sites. There is an argument to be made to use
indigenous species for inoculation schemes as they are anticipated to be the most
effective, but we may also need to consider the potential impact of biological
invasions if using exotic fungal species (Vellinga et al., 2009).
1.5 Under What Conditions Will EcM Inoculation

Be Beneficial?
Whilst the body of this chapter thus far has outlined how inoculation with EcM fungi
may improve dipterocarp seedling growth, and the various methods to do so, it is
certainly worth considering whether inoculation should indeed be conducted at all. In
the first paper on dipterocarp EcMs, Singh (1966) noted that “mycorrhizal infection
should not be taken as the ‘cure of all ills’ in the establishment of trees in all sorts of
habitats”, and this warning still stands, more than 40 years later. I now pose three key
questions for consideration before starting to plan inoculation schemes.
It is often considered that there is a need to inoculate seedlings prior to out-
planting but, in fact, in most cases inoculation will occur naturally, and inoculation
schemes may not yield any major benefits for seedling growth or survival (although
we cannot be confident that the same species, or most beneficial species, of EcM
fungi will be formed on each seedlings’ roots every time). The first key question is,
therefore, will inoculation be of benefit to the seedlings? The major benefits of
inoculation are knowing that a seedling, at out-planting, is mycorrhizal with a
known species of fungus which is functionally beneficial, and thus it will not
need to wait to form EcMs with an unknown group of fungi present in the soil
which may or may not promote seedling growth; this gives it something of an initial
advantage over any out-planted non-mycorrhizal seedlings. However, the main
benefits of inoculation are more likely to be shown under poor soil conditions, as
I outline below.
1.5.1 Successful Inoculation Schemes
For inoculation schemes to be successful, a series of well-defined and consistently

repeatable techniques is needed. In other words, a pure culture of inoculum is
needed to allow a regular supply, and currently there are very few fungal species
being maintained in pure culture in the South-east Asian region. Access to a
laboratory with sterile facilities is needed which may be problematic for a number
of sites. In the absence of this, the production of mycorrhizal tablets may be useful
although vagaries of fungal fruit body production and genetic variation between
individual genets will remain unaccounted for. Infrequent production of dipterocarp
seeds can make regular production of planting stock difficult although production
of cuttings from a selected number of dipterocarp species now appears fairly routine
(Moura-Costa and Lundoh 1994; Itoh et al. 2003; Haji Ahmad 2006). It must also
be shown that the inoculant fungus has the ability to improve seedling growth or
survival over that of non-inoculated seedlings under field conditions. It appears
much easier to culture species such as Pisolithus or Scleroderma but it must be
remembered that these species are not necessarily those which are most beneficial
to seedling growth or, indeed, are found commonly on dipterocarp seedling roots.
1.5.2 When and Where to Inoculate?
It is often suggested that inoculation may be beneficial for seedlings planted

following logging operations. However, in most cases after logging, there are still
a number of smaller dipterocarp trees which will have EcM fungi associated with
them and, as long as the light conditions are not detrimental to seedling growth, this
should allow the rapid formation of EcMs on seedlings by hyphae, sclerotia and
spores already present in the soil (Lee et al. 1996; Ingleby et al. 1998). There is little
evidence so far that selective logging seriously impoverishes the fungal flora
(Watling et al. 1998) although there is an indication of a loss of the some of the
rarer EcM species in logged forest (Lee et al. 1996). Out-planted dipterocarp
seedlings are almost certain to become colonised within a short period of time as
long as they have below-ground access to roots and mycelium radiating from adult
trees (Lee 1991; Alexander et al. 1992; Lee and Alexander 1996; Lee et al. 1996).
The second key question is, therefore, is inoculation beneficial under all situations?
If not, which situations or conditions are most likely to be improved by inoculating
seedlings prior to out-planting?
Inoculation is considered more likely to be of benefit when seedlings are planted
in areas where suitable EcM inoculum is not available. This may include severely
degraded areas such as mine tailings (Lee et al. 2008), burnt areas (Akema et al.
2009), degraded peatlands (Turjaman et al. 2007) and areas previously used for
agriculture (Ingleby et al. 2000). For example, Ingleby et al. (2000) found that the
inoculation potential of soils which had been under agriculture for over 20 years
12 F.Q. Brearley
was essentially absent when compared with an undisturbed forest or plantation in

Vietnam. The work of Turjaman et al. (2007) in degraded peat swamp forest is also
of relevance here as they showed improved growth of inoculated dipterocarp
seedlings when out-planted in a degraded area.
The final key question is, is simply adding colonised soil appropriate as inocu-
lum? In many cases, local soil from the vicinity of dipterocarp trees may be equally
as effective as any inoculation schemes although these EcMs are not necessarily the
best species to promote seedling growth and there is no way to control which fungal
species successfully colonise the seedlings’ roots. Smits (1992) outlines a simple
method by which large numbers of seedlings can be inoculated by soil colonised by
EcM hyphae and spores. He advocates the use of soil collected from beneath an
adult tree of the same species, but this is based on his weak assertions (Smits 1983,
1985) of a high degree of host specificity. I suggest it is equally likely that host-
specific pathogens will be present (Packer and Clay 2000) and therefore suggest a
general soil inoculum but ensuring that it is collected in the vicinity of dipterocarps.
In the absence of any other schemes, the inclusion of forest soil should be seen as
the minimum to ensure early EcM colonisation of dipterocarp seedlings.
1.6 Conclusions
Whilst EcMs are often thought to be essential for the successful growth of diptero-
carp seedlings, there is surprisingly little evidence confirming this assertion under
natural conditions. In nursery experiments, mycorrhizal inoculation has regularly
been shown to increase seedling growth and nutrient concentrations, but when
similar experiments have been conducted in the field, the results are much more
equivocal with inoculation often showing minimal improvements in growth if
seedlings are planted in natural forests (e.g. Tata et al. 2009). If inoculated seedlings
are planted in degraded soils, the improvement in growth is often more marked
although these improvements may not be maintained if the inoculated fungus does
not remain competively dominant on the seedlings’ roots. I therefore suggest that
researchers and forest restorationists carefully consider whether EcM inoculation is
of benefit in the areas they plan to re-plant.
Acknowledgements I thank Dr. Lee Su See and Dr. Robin Sen for helpful thoughts and com-
ments on the manuscript. My work on dipterocarp ectomycorrhizas was supported by the British
Ecological Society Overseas Research Programme.
References
Akema T, Nurhiftisni I, Suciatmih, Simbolon H (2009) The impact of the 1998 forest fire on
ectomycorrhizae of dipterocarp trees and their recovery in tropical rain forests of East
Kalimantan, Indonesia. JARQ 42:137–142
Alexander IJ, H€ogberg P (1986) Ectomycorrhizas of tropical angiospermous trees. New Phytol
102:541–549
Alexander IJ, Ahmad N, Lee SS (1992) The role of mycorrhizas in the regeneration of some
Malaysian forest trees. In: Marshall AG, Swaine MD (eds) Tropical rain forest: disturbance and
recovery. The Royal Society, London, UK, pp 357–367
Baxter JW, Dighton J (2005) Diversity–functioning relationships in ectomycorrhizal fungal com-
munities. In: Dighton J, White JF Jr, Oudemans P (eds) The fungal community: its organization
and role in the ecosystem, 3rd edn. CRC Press, Boca Raton, Florida, USA, pp 383–398
Brearley FQ (2003) The role of ectomycorrhizas in the regeneration of dipterocarp seedlings. PhD
Thesis, University of Sheffield, UK
Brearley FQ (2006) Differences in the growth and ectomycorrhizal community of Dryobalanops
lanceolata (Dipterocarpaceae) seedlings grown in ultramafic and non-ultramafic soils. Soil
Biol Biochem 38:3407–3410
Brearley FQ, Press MC, Scholes JD (2003) Nutrients obtained from leaf litter can improve the
growth of dipterocarp seedlings. New Phytol 160:101–110
Brearley FQ, Prajadinata S, Kidd PS, Proctor J, Suriantata (2004) Structure and floristics of an old
secondary rain forest in Central Kalimantan, Indonesia, and a comparison with adjacent
primary forest. For Ecol Manage 195:385–397
Brearley FQ, Scholes JD, Lee SS (2005) Nitrogen nutrition and isotopic discrimination in tropical
ectomycorrhizal fungi. Res Microbiol 156:184–190
Brearley FQ, Scholes JD, Press MC, Palfner G (2007) How does light and phosphorus fertilisation
affect the growth and ectomycorrhizal community of two contrasting dipterocarp species?
Plant Ecol 192:237–249
Bungard RA, Press MC, Scholes JD (2000) The influence of nitrogen on rain forest dipterocarp
seedlings exposed to a large increase in irradiance. Plant Cell Environ 23:1183–1194
Chalermpongse A (1987) Mycorrhizal survey of dry-deciduo us and semi-evergreen dipterocarp
forest ecosystems in Thailand. In: Kostermans AJCH (ed) Proceedings of the third round table
conference on dipterocarps. UNESCO Regional Office for Science and Technology, Jakarta,
Indonesia, pp 81–103
Chang YS, Lapeyrie FF, Lee SS (1994) The survival and competitiveness of Pisolithus tinctorius
on outplanted seedlings of Shorea glauca in Malaysia. In: Khoo KC, Appanah S (eds) Pro-
ceedings of the fifth round table conference on dipterocarps. Forest Research Institute of
Malaysia, Kepong, Malaysia, pp 165–169
Chang YS, Lee SS, Lapeyrie FF, Yazid SM (1995) The competitiveness of two strains of
Pisolithus tinctorius on seedlings of three species of dipterocarps under nursery and field
conditions: preliminary results. In: Wickneswari R, Yahya AZ, Shariff AHM, Haji Ahmad D,
Khoo KC, Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the international workshop of
BIO-REFOR, Kangar, 1994. BIO-REFOR, IUFRO-SPDC, Tokyo, Japan & FRIM, Kepong,
Malaysia, pp 208–212
Chilvers GA, Douglas PA, Lapeyrie FF (1986) A paper-sandwich technique for rapid synthesis of
ectomycorrhizas. New Phytol 103:397–402
Davies SJ, Nur Supardi MN, LaFrankie JV Jr, Ashton PS (2003) The trees of Pasoh Forest: stand
structure and floristic composition of the 50-ha forest research plot. In: Okuda T, Manokaran N,
Matsumoto Y, Niiyama K, Thomas SC, Ashton PS (eds) Pasoh: ecology of a lowland rain
forest in Southeast Asia. Springer-Verlag, Tokyo, Japan, pp 35–50
Fakuara Y, Wilarso S (1993) Effect of mycorrhizal tablet storage on seedling growth of Shorea
pinanga Scheff. In: Anon. (ed) BIO-REFOR: proceedings of Tsukuba-workshop.
BIO-REFOR, IUFRO-SPDC, Tsukuba Science City, Japan, pp 174–179
Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata
forest: above and below-ground views. Can J Bot 74:1572–1583
Hadi S, Santoso E (1988) Effect of Russula spp., Scleroderma sp. and Boletus sp. on the
mycorrhizal development and growth of five dipterocarp species. In: Mohinder Singh M (ed)
Agricultural and biological research priorities in Asia, Proceedings of the IFS symposium of
14 F.Q. Brearley
science Asia 87. International Foundation for Science & Malaysian Scientific Association,
Kuala Lumpur, Malaysia, pp 183–185
Hadi S, Santoso E (1989) Accumulation of macronutrients by five dipterocarp species inoculated
with different species of mycorrhizal fungi. In: Mahadevan A, Raman N, Natarajan K (eds)
Mycorrhizae for Green Asia: proceedings of the first Asian conference on mycorrhizae. Centre
for Advanced Studies on Botany, University of Madras, India, pp 139–141
Haji Ahmad D (2006) Vegetative propagation of dipterocarp species by stem cuttings using a very
simple technique. In: Suzuki K, Ishii K, Sakurai S, Sasaki S (eds) Plantation technology in
tropical forest science. Springer-Verlag, Tokyo, Japan, pp 69–77
Hoang PND, Tuan DLA (2008) Investigating the ectomycorrhizal appearance of seedlings in the
Tan Phu forest enterprise’s nursery, Dong Nai Province. J Sci Technol Dev 11(1):96–100
Ibrahim Z, Mahat MN, Lee SS (1995) Response of Hopea odorata seedlings to biological
soil conditioners. In: Wickneswari R, Yahya AZ, Shariff AHM, Haji Ahmad D, Khoo KC,
Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the international workshop of BIO-REFOR,
Kangar, 1994. BIO-REFOR, IUFRO-SPDC, Tokyo, Japan & FRIM, Kepong, Malaysia,
pp 179–182
Ingleby K, Munro RC, Noor M, Mason PA, Clearwater MJ (1998) Ectomycorrhizal populations
and growth of Shorea parvifolia (Dipterocarpaceae) seedlings regenerating under three
different forest canopies following logging. For Ecol Manage 111:171–179
Ingleby K, Thuy LTT, Phong NT, Mason PA (2000) Ectomycorrhizal inoculum potential of soils
from forest restoration sites in South Vietnam. J Trop For Sci 12:418–422
Ishida TA, Nara K, Hogetsu T (2007) Host effects on ectomycorrhizal fungal communities: insight
from eight host species in mixed conifer-broadleaf forests. New Phytol 174:430–440
Itoh A, Yamakura T, Tan S, Kendawang JJ, Lee HS (2003) Effects of resource plant size on
rooting of Dryobalanops lanceolata cuttings. J For Res 8:117–121
Kikuchi J (1997) Ectomycorrhiza formation of dipterocarp seedlings. In: Sangwanit U, Thaiutsa B,
Puangchit L, Thammincha S, Ishii K, Sakurai S, Suzuki K (eds) Proceedings of the fifth
international workshop of BIO-REFOR, Bangkok, 1996. BIO-REFOR, IUFRO-SPDC,
Tokyo, Japan, pp 49–52
Lapeyrie FF, Lee SS, Yazid SM (1993) Controlled ectomycorrhizal inoculation of Hopea odorata
(Dipterocarpaceae) cuttings with Hebeloma crustuliniforme. In: Anon. (ed) BIO-REFOR:
proceedings of Tsukuba-workshop. BIO-REFOR, IUFRO-SPDC Tsukuba Science City,
Japan, pp 189–190
Lee SS (1991) Some views on dipterocarp mycorrhiza research in Malaysia. In: Anon. (ed)
BIO-REFOR: proceedings of pre-workshop. BIO-REFOR, IUFRO-SPDC, Bogor, Indonesia,
pp 66–70
Lee SS (1998) Root symbiosis and nutrition. In: Appanah S, Turnbull JM (eds) A review of
dipterocarps: taxonomy, ecology and silviculture. Center for International Forestry Research,
Bogor, Indonesia, pp 99–114
Lee SS, Alexander IJ (1994) The response of seedlings of two dipterocarp species to nutrient
additions and ectomycorrhizal infection. Plant Soil 163:299–306
Lee SS, Alexander IJ (1996) The dynamics of ectomycorrhizal infection of Shorea leprosula
seedlings in Malaysian rain forests. New Phytol 132:297–305
Lee SS, Lim KL (1989) Mycorrhizal infection and foliar phosphorus content of seedlings of three
dipterocarp species grown in selectively logged forest and a forest plantation. Plant Soil
117:237–241
Lee SS, Lapeyrie FF, Yazid SM (1995) Techniques for controlled ectomycorrhizal inoculation
of dipterocarp seedlings and cuttings. In: Supriyanto, Kartana JT (eds) Proceedings of the
second symposium on biology and biotechnology of mycorrhizae and third Asian conference
on mycorrhizae (ACOM III). BIOTROP Special Publication 56, SEAMEO BIOTROP, Bogor,
Indonesia, pp 217–221
Lee SS, Alexander IJ, Moura-Costa PH, Yap SW (1996) Mycorrhizal infection of dipterocarp
seedlings in logged and undisturbed forests. In: Appanah S, Khoo KC (eds) Proceedings of the
fifth round table conference on dipterocarps. Forest Research Institute of Malaysia, Kepong,
Malaysia, pp 157–164
Lee SS, Alexander IJ, Watling R (1997) Ectomycorrhizas and putative ectomycorrhizal fungi of
Shorea leprosula Miq. (Dipterocarpaceae). Mycorrhiza 7:63–81
Lee SS, Watling R, Noraini Sikin Y (2002) Ectomycorrhizal basidiomata fruiting in lowland rain
forests of peninsular Malaysia. Bois Fôr Trop 274(4):33–43
Lee SS, Watling R, Turnbull E (2003) Diversity of putative ectomycorrhizal fungi in Pasoh Forest
Reserve. In: Okuda T, Manokaran N, Matsumoto Y, Niiyama K, Thomas SC, Ashton PS (eds)
Pasoh: ecology of a lowland rain forest in Southeast Asia. Springer-Verlag, Tokyo, Japan,
pp 149–159
Lee SS, Patahayah M, Chong WS, Lapeyrie FF (2008) Successful ectomycorrhizal inoculation of
two dipterocarp species with a locally isolated fungus in Peninsular Malaysia. J Trop For Sci
20:237–247
Martin F, Dı́ez J, Dell B, Delaruelle C (2002) Phylogeography of the ectomycorrhizal
Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences. New Phytol
153:345–357
Morris MH, Smith ME, Rizzo DM, Rejmánek M, Bledsoe CS (2008) Contrasting ectomycorrhizal
fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland.
New Phytol 178:167–176
Moura-Costa PH, Lundoh L (1994) A method for vegetative propagation of Dryobalanops
lanceolata (Dipterocarpaceae) by cuttings. J Trop For Sci 6:533–541
Nara K, Kawahara M, Okamura K, Sakurai K, Hogetsu T (1999) Prospects and problems
pertaining to the application of ectomycorrhizal fungi to dipterocarp seedlings in tropical
nurseries. In: Anon. (ed) Proceedings of the international symposium “Can Biological Produc-
tion Harmonize with Environment?”. University of Tokyo, Japan, pp 151–154
Ogawa M (1993) Inoculation method of Scleroderma columnare to dipterocarp seedlings. In:
Anon. (ed) BIO-REFOR: proceedings of Tsukuba-workshop. BIO-REFOR, IUFRO-SPDC,
Tsukuba Science City, Japan, pp 185–188
Ogawa M (2006) Inoculation methods of Scleroderma columnare onto dipterocarps. In: Suzuki K,
Ishii K, Sakurai S, Sasaki S (eds) Plantation technology in tropical forest science. Springer-
Verlag, Tokyo, Japan, pp 185–197
Packer A, Clay K (2000) Soil pathogens and spatial patterns of seedling mortality in a temperate
tree. Nature 404:478–481
Patahayah M, Cynthia PC, Lee SS (2003a) Optimizing growth conditions for ectomycorhizal
inoculum production of the Malaysian strain of Pisolithus tinctorius. Tropical forestry research
in the new millenium: international conference on forestry and forest products research 2001,
pp 551–552
Patahayah M, Brearley FQ, Lee SS (2003b) Responses of three ectomycorrhizal fungi to different
temperatures and media in vitro. Poster presentation at conference on forestry and forest
products research 2003, 6–8 October 2003, Kuala Lumpur, Malaysia
Proctor J, Anderson JM, Chai P, Vallack HW (1983) Ecological studies in four contrasting
lowland rain forests in Gunung Mulu National Park, Sarawak. I. Forest environment, structure
and floristics. J Ecol 71:237–260
Richard F, Millot S, Gardes M, Selosse M-A (2005) Diversity and specificity of ectomycorrhizal
fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex.
New Phytol 166:1011–1023
Rinaldi AC, Comandini O, Kuyper TW (2008) Ectomycorrhizal fungal diversity: separating the
wheat from the chaff. Fungal Div 33:1–45
Roeleffs JW (1930) Over kunstmatige verjonging van Pinus merkusii Jungh. et de Vr. en Pinus
khasya Royle. Tectona 23:874–907
Sangtiean T, Schmidt S (2002) Growth of subtropical ECM fungi with different nitrogen sources
using a new floating culture technique. Mycol Res 106:74–85
16 F.Q. Brearley
Shamsuddin MN (1979) Mycorrhizas of tropical forest trees. In: Furtado JI (ed) Abstracts:
fifth international symposium of tropical ecology. University of Malaya, Kuala Lumpur,
Malaysia, p 173
Shiva MP, Jantan I (1998) Non-timber forest products from dipterocarps. In: Appanah S, Turnbull
JM (eds) A review of dipterocarps: taxonomy, ecology and silviculture. Center for Interna-
tional Forestry Research, Bogor, Indonesia, pp 187–197
Singh KG (1966) Ectotrophic mycorrhiza in equatorial rain forests. Malay For 29:13–18
Sirikantaramas S, Sugioka N, Lee SS, Mohamed LA, Lee HS, Szmidt AE, Yamazaki T (2003)
Molecular identification of ectomycorrhizal fungi associated with Dipterocarpaceae. Tropics
13:69–77
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic Press, London, UK
Smits WTM (1983) Dipterocarps and mycorrhiza – an ecological adaptation and a factor in forest
regeneration. Flora Males Bull 36:3926–3937
Smits WTM (1985) Specificity of dipterocarp mycorrhiza. In: Molina R (ed) Proceedings of the
6th North American conference on mycorrhizae. Forest Research Laboratory, Corvallis,
Oregon, USA, p 364
Smits WTM (1992) Mycorrhizal studies in dipterocarp forests in Indonesia. In: Read DJ,
Lewis DH, Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. CAB International,
Wallingford, UK, pp 283–292
Supriyanto, Setiawan I, Omon RM (1994) Effect of Scleroderma sp. on the growth of Shorea
mecistopteryx Ridl. seedlings. In: Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the
International Workshop of BIO-REFOR, Yogjakarta, 1993. BIO-REFOR, IUFRO-SPDC,
Tokyo, Japan, pp 186–188
Tata HL, van Noordwijk M, Summerbell R, Werger MJA (2009) Limited response to nursery-
stage mycorrhiza inoculation of Shorea seedlings planted in rubber agroforest in Jambi,
Indonesia. New For 39:51–74
Tawaraya K, Takaya Z, Turjaman M, Tuah SJ, Limin SH, Tamai Y, Cha JY, Wagatsuma T,
Osaki M (2003) Arbuscular mycorrhizal colonization of tree species grown in peat swamp
forests of Central Kalimantan, Indonesia. For Ecol Manage 182:381–386
Tupas GL, Sajise PE (1976) Mycorrhizal associations in some savanna and reforestation trees.
Kalikasan 5:235–240
Turjaman M, Tamai Y, Segah H, Limin SH, Cha JY, Osaki M, Tawaraya K (2005) Inoculation
with the ectomycorrhizal fungi Pisolithus arhizus and Scleroderma sp. improves early growth
of Shorea pinanga nursery seedlings. New For 30:67–73
Turjaman M, Tamai Y, Segah H, Limin SH, Osaki M, Tawaraya K (2006) Increase in early growth
and nutrient uptake of Shorea seminis inoculated with two ectomycorrhizal fungi. J Trop For
Sci 18:243–249
Turjaman M, Saito H, Santoso E, Susanto A, Gaman S, Limin SH, Shibuya M, Takahashi K,
Tamai Y, Osaki M, Tawaraya K (2007) Effect of ectomycorrhizal fungi inoculated on
Shorea balangeran under field conditions in peat-swamp forests. In: Rieley JO, Banks CJ,
Radjagukguk B (eds) Proceedings of the international symposium and workshop on tropical
Peatland, Yogyakarta, 27–29 Aug 2007. CARBOPEAT, University of Leicester, UK,
pp 143–148
Vellinga EC, Wolfe BE, Pringle A (2009) Global patterns of ectomycorrhizal introductions. New
Phytol 181:960–973
Watling R, Lee SS (1995) Ectomycorrhizal fungi associated with members of the Dipterocarpa-
ceae in Peninsular Malaysia – I. J Trop For Sci 7:657–669
ceae in Peninsular Malaysia – II. J Trop For Sci 10:421–430
Watling R, Lee SS (2007) Mycorrhizal mycodiversity in Malaysia. In: Jones EBG, Hyde KD,
Vikineswary S (eds) Malaysian fungal diversity. Mushroom Research Centre, University
of Malaya & Ministry of Natural Resources and Environment, Kuala Lumpur, Malaysia,
pp 201–219
Watling R, Taylor AFS, Lee SS, Sims K, Alexander IJ (1995a) A rainforest Pisolithus;
its taxonomy and ecology. Nova Hedwig 61:417–429
Watling R, Taylor AFS, Lee SS, Sims K, Alexander IJ (1995b) Pisolithus aurantioscabrosus.
In: Agerer R (ed) Colour atlas of ectomycorrhizae. Einhorn-Verlag, Schw€abisch Gm€und,
Germany, plate 85
Watling R, Lee SS, Turnbull E (1998) Putative ectomycorrhizal fungi of Pasoh Forest Reserve,
Negri Sembilan, Malaysia. In: Lee SS, Dan YM, Gauld ID, Bishop J (eds) Conservation,
management and development of forest resources. Forest Research Institute of Malaysia,
Kepong, Malaysia, pp 96–104
Watling R, Lee SS, Turnbull E (2002) The occurrence and distribution of putative ectomycorrhizal
basidiomycetes in a regenerating south-east Asian rainforest. In: Watling R, Frankland JC,
Ainsworth AM, Isaac S, Robinson CH (eds) Tropical mycology, vol 1, Macromycetes. CAB
International, Wallingford, UK, pp 25–43
Yamada A, Katsuya K (2001) The disparity between the number of ectomycorrhizal fungi and
those producing fruit bodies in a Pinus densiflora stand. Mycol Res 105:957–965
Yazid SM, Lee SS, Lapeyrie FF (1994) Growth stimulation of Hopea spp. (Dipterocarpaceae)
seedlings following mycorrhizal inoculation with an exotic strain of Pisolithus tinctorius.
For Ecol Manage 67:339–343
Yazid SM, Lee SS, Lapeyrie FF (1996) Mycorrhizal inoculation of Hopea odorata (Dipterocar-
paceae) in the nursery. J Trop For Sci 9:276–278
Yuwa-Amornpitak T, Vichitsoonthonkul T, Tanticharoen M, Cheevadhanarak S, Ratchadawong S
(2006) Diversity of ectomycorrhizal fungi on Dipterocarpaceae in Thailand. J Biol Sci
6:1059–1064
.
Chapter 2
The Ectomycorrhizal Symbiosis in South
America: Morphology, Colonization,
and Diversity
Alejandra G. Becerra and Marcelo R. Zak
2.1 Introduction
About 95% of the world’s living species of vascular plants belong to families that
are characteristically mycorrhizal (Trappe 1977). The symbiotic root–fungus asso-
ciations result from the coevolution between plants and fungi, which determined
mycorrhizae to be the norm in terrestrial plant nutrition, not the exception (Trappe
1977, 1987, Brundrett and Cairney 2002).
Among the seven types of mycorrhizae widely described (arbuscular, arbutoid,
ectendo, ecto, ericoid, monotropoid, and orchidaceous), both arbuscular mycorrhi-
zae (AM) and ectomycorrhizae (ECM) are the most abundant and widespread in
forest communities (Allen et al. 2003; Smith and Read 2008).
Forest communities cover approximately 33% of the world’s land surface
(Rumney 1968) being ECM the most frequent and widespread mycorrhizal type
in forests and woodlands of cool temperate and boreal latitudes. Forests character-
ized by the dominance of ECM woody species would have extended both through-
out the hemispheres and upwards in mountain areas at the expense of AM
woodlands (Malloch et al. 1980). On the other hand, even though various tropical
and subtropical trees throughout the world also form ECM (Moyersoen et al. 1998a, b,
A.G. Becerra (*)

Instituto Multidisciplinario de Biologı́a Vegetal (CONICET), Universidad Nacional de Córdoba,
C.C. 495, 5000 Córdoba, República Argentina
and
Cátedra de Diversidad Vegetal I, Facultad de Ciescias Exactas, Fı́sicas y Naturales, Universidad
Nacional de Córdoba, C.C. 495, 5000 Córdoba, República Argentina
e-mail: abecerra@efn.uncor.edu
M.R. Zak
Instituto Multidisciplinario de Biologı́a Vegetal (CONICET), Universidad Nacional de Córdoba,
C.C. 495, 5000 Córdoba, República Argentina
and
Cátedra de Recursos Naturales y Gestión Ambiental, Departamento de Geografı́a, Universidad
Nacional de Córdoba, C.C. 495, 5000 Córdoba, República Argentina
e-mail: marcelzak@gmail.com

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_2,
20 A.G. Becerra and M.R. Zak
2001; Pérez-Moreno 1998; Founoune et al. 2002; Onguene and Kuyper 2002), most
of them form AM (Alexander 1989). This AM are obligate symbionts belonging to
the Glomeromycota phylum (Sch€ ußler et al. 2001), which have been found in trees,
shrubs, and herbs in all tropical regions (Janos 1980, 1985; Béreau et al. 1997;
Smith and Read 2008).
According to Singer and Morello (1960), ECM forests in South America con-
tinue the important ectomycorrhizal complex of Central America into Colombia,
where Quercus sp. (Fagaceae) appears. There is also a strip formed by Alnus
acuminata Kunth (Betulaceae) extended from Venezuela to Argentina, while the
largest and most important ectomycorrhizal area, the Nothofagus (Fagaceae) region
extends from 33 to 50 S. Beyond these naturally occurring forests, human activity
made possible the existence of many ECM forests of plant species naturally found
in the Northern Hemisphere, such as Pinus sp., Eucalyptus sp., Populus sp., Salix
sp., Larix sp., Cedrus sp., Betula sp., and Quercus sp. (Singer and Morello 1960)
with their ECM fungi of European and North American origin.
On this basis, the general aim of the present chapter is to review the ECM
symbiosis in South America with particular emphasis on its anatomical character-
istics. This will provide an organized structure essential for the better under-
standing of the plant–fungus mutualism in ectomycorrhizal associations in South
America, so adding new tools for the management and conservations of threatened
ecosystems.
2.2 ECM Studies in South America
The tradition on ECM research in South America is important. The great diversity
of available studies covers descriptions of mycorrhizal status, ECM fungi, inocula-
tions, colonization, and morphological characterization. Unfortunately, and due in
part to their abundance but often also to their inaccessibility, it is not possible to aim
at describing the whole array of studies carried throughout the continent. Therefore,
this chapter pretends to summarize the wide range of families and genera of
Angiospermae and Gymnospermae forests growing in South America in which
ectomycorrhizal studies have been carried.
Most ECM fungi can associate with various host plants, while the opposite is
also true, being it possible for a single host to form ECM with a number of different
fungi (Moyersoen 1993). Most studies analyzed (Table 2.1) present lists of ecto-
mycorrhizal fungi (most of which belong to the Basidiomycota phylum) and
taxonomic descriptions in exotic plants such as Pinus (P. radiata, P. elliottii),
Eucalyptus (E. citriodora, E. dunii, E. robusta), and Quercus. Among endemic
plants, most studies have been carried out in Nothofagus (N. obliqua, N. dombeyi,
N. alpina) forests. Besides, some of the associations found in these forests
are established with fungi species from exotic plantations (P. radiata en Chile)
(Garrido 1986).
Table 2.1 Description of 103 studies on ectomycorrhizal status in South America
Family Species Publication with countrya and ECM characteristicsb indication
Native plants
Betulaceae Alnus acuminata 1(A;2) 2(A;2) 3(A;1) 4(A;1) 5(A;1) 6(A;1) 7(A;1,4) 8(A;1,3,4) 9(A;1,3,4) 10(A;2)
Caesalpiniaceae Dicymbe corymbosa 11(FG;2,4) 12(FG;2) 13(FG;4) 14(FG;2)
D. altsonii 11(FG;2,4) 12(FG;2) 13 (FG; 4) 15(FG;1,2)
D. jenmanii 11(FG;4)
Dicymbe sp. 14(FG;2)
Dipterocarpaceae Pakaraimaea dipterocarpacea 17(V;1) 18(V;2)
Fabaceae Acacia bonaerensis 19(U;4)
Aldinia heterophylla 20(B;2)
A. insignis 11(G;4) 13(G;4)
A. kunhardtiana 21(V;1,4)
A. latifolia 21(V;1,4)
Calliandra selloi 19(U;4)
C. tweedii 19(U;4)
Gleditsia amorphoides 19(U;4)
Lonchocarpus nitidus 19(U;4)
2 The Ectomycorrhizal Symbiosis in South America
Prosopis spp. 19(U;4)

Fagaceae Nothofagus alpina 22(C;1) 23(C;1) 24(C;3) 25(C;2) 26(C;2) 27(C;1,4) 28(C;4)
N. alessandrii 29(C;3)
N. antarctica 25(C;2) 26(C;2) 30(C;4) 31(C;4) 32(A;4)
N. betuloides 23(C;1) 25(C;2)
N. dombeyi 23(C;1) 24(C;3) 25(C;2) 26(C;2) 28(C;4) 30(C;4) 31(C;4) 32(A;4) 34(A;1) 35(C;1)
36(C;2)
N. glauca 25(C;2) 38(C;3)
N. leonii 25(C;2)
N. nervosa 32(A;4)
N. nitida 25(C;2) 39(C;1)
N. obliqua 23(C;1) 25(C;2) 26(C;2) 30(C;4) 32(A;4) 37(C;4) 38(C;3)
N. obliqua var. macrocarpa 25(C;2)
N. pumilio
21
(continued )
Table 2.1 (continued)
22

23(C;1) 25(C;2) 26(C;2) 32(A;4) 33(C;1) 40(C;1) 41(C;1) 42(C;1) 43(C;1)
44(C;2,4)
Nothofagus spp. 1(C;2) 25(C;2) 26(C;2) 45(A-C;2)
Gnetaceae Gnetum sp. 46(B;4) 47(B;4)
Melastomataceae Graffenrieda emarginata 48(E;1) 49(E;4)
Nyctaginaceae Neea altisima 48(B;4)
N. divaricata 50(E;2,4)
N. laetivirens 52(B;4)
N. obovata, N. robusta 21(V;1,4)
N. tristis 53(FG;4)
Neea sp.1, Neea sp. 2 49(E;4) 51(E;1)
Neea sp. 3, Neea sp. 5 50(E;2,4)
Neea sp. 46(B;4) 47(B;4) 54(P;4)
Guapira sancarlosiana 21(V;1,4) 49(E;4)
Guapira sp. 51(E;1)
Polygonaceae Coccoloba excelsa 21(V;1,4)
C. latifolia 53(FG;4)
C. moliis 53(FG;4)
C. rosea 55(B;1)
Coccoloba sp. 53(FG;4)
Salicaceae Salix humboldtiana 1(A;2) 56(A;1,2) 57(A;4)
Sapotaceae Glycoxylon inphyllum 47(B;4)
Introduced plants
Betulaceae Betula pendula 25(C;2) 58(A;1)
Betula sp. 59(A;2)
Myrtaceae Eucalyptus camaldulensis 60(B;4) 61(B;3)
E. citriodora 61(B;3) 62(B;2) 63(B;2) 64(B;2,3)
E. cloeziana 61(B;3)
E. dunnii 62(B;2) 65(B;2) 66(B;3) 67(B;3) 68(B;3) 69(B;3) 71(B;3) 72(B;3)
E. globulus 25(C;2)
E. grandiis 60(B;4) 61(B;3) 63(B;2) 64(B;2,3) 73(B;2) 74(B;2) 75(B;3) 76(B;3)
A.G. Becerra and M.R. Zak
E. microcorys 63(B;2) 73(B;2)
E. robusta 62(B;2) 64(B;2,3)73(B;2)
E. rostrata 77(A;3)
E. tereticomis 63(B;2)
E. saligna 63(B;2)
E. urophylla 61(B;3) 75(B;3) 76(B;3)
E. viminalis 63(B;2) 77(A;3) 78(A;1,2)
Eucaliptus spp. 25(C;2) 79(Co;2) 80(B;2)
Pinaceae Cedrus atlántica 58(A;1)
C. deodara 81(A;1,2)
Cedrus sp. 59(A;2)
Larix decidua 25(C;2) 59(A;2)
Picea abies 25(C;2)
Pinus caribaea var. hondurensis 82(B;3)
P. contorta 25(C;2)
P. elliotti 58(A;1) 59(A;2) 62(B;2) 83(A;1,3)
P. halepensis 59(A;2) 84(A;3)
P. patula 25(C;2) 59(A;2) 79(Co;2)
P. pinaster 25(C;2) 85(A;3) 84(A;3)

P. pinea 25(A;3) 84(A;3)
P. ponderosa 85(A;2,3,4) 86(A;2) 87(A;2) 88(A;1,2) 89(A;3)
P. radiata 25(C;2) 59(A;2) 79(Co;2) 90(C;2) 91(C;2)
P. sylvestris 25(C;2)
P. taeda 59(A;2) 62(B;2) 65(B;2) 68(B;3) 84(A;3) 92(B;3) 93(B;3) 94(A;3)
P. thunbergii 84(A;3)
Pinus sp. 25(C;2)
Pinus spp. 1(A;2) 95(U-A;2) 96(B;2)
Pseudotsuga menziesii 25(C;2) 86(A;2) 88(A;1,2) 97(A;2,3,4) 98(A;2)
Tsuga heterophylla 25(C;2)
Fagaceae Quercus humboldtii 99(Co;2) 100(Co;2) 101(Co;2) 102(Co;2) 103(Co;2)
Q. robur 25(C;2)
Quercus sp. 59(A;2)
Quercus spp. 79(Co;2)
23
(continued )
24

Salicaceae Populus nigra 25(C;2)
Populus spp. 25(C;2)
Salix viminalis 25(C;2)
Salix spp. 25(C;2)
Each line within the table represents a studied species, with the indication of the Family to which it belongs and all publications (numbered – all respective
citations are found below under References) in which it has been described. Following the paper number is an indication of the characteristics of the study
carried (country and ECM characteristics)
a
Country: A Argentina; B Brazil; C Chile; Co Colombia; E Ecuador; FG French Guyana; P Perú; U Uruguay; V Venezuela
b
ECM characteristics studied: 1: anatomical characteristics, 2: list of ECM fungi, 3: ECM inoculations, 4: mycorrhizal status or colonization
References: (1) Singer (1953), (2) Singer and Morello (1960), (3) Becerra (2002), (4) Becerra et al. (2002), (5) Becerra et al. (2005a), (6) Becerra et al.
(2005b), (7) Becerra et al. (2005c), (8) Becerra et al. (2009a), (9) Nouhra et al. (2003), (10) Nouhra et al. (2005), (11) Henkel et al. (2002), (12) Henkel (1999),
(13) McGuire et al. (2008), (14) Fulgenzi et al. (2007), (15) Henkel et al. (2000), (16) Matheny et al. (2003), (17) Moyersoen (2006), (18) Moyersoen (2008),
(19) Frioni et al. (1999), (20) Singer et al. (1983), (21) Moyersoen (1993), (22) Palfner (1997), (23) Palfner (2001), (24) Godoy and Palfner (1997),
(25) Garrido (1986), (26) Valenzuela et al. (1999), (27) Palfner et al. (2008), (28) Castillo et al. (2006), (29) Flores et al. (1997), (30) Carrillo et al. (1992),
(31) Godoy et al. (1994), (32) Diehl et al. (2008), (33) Palfner (2002a), (34) Beenken (2001), (35) Palfner (2002c), (36) Singer (1971), (37) Castillo et al.
(2006), (38) Pérez et al. (2007), (39) Palfner (2002b), (40) Palfner and Godoy (1996a), (41) Palfner and Godoy (1996b), (42) Palfner (1996), (43) Palfner
(2002d); (44) Villegas et al. (2007); (45) Wrigth (1988); (46) St John (1980), (47) Singer and Araujo (1979), (48) Haug et al. (2004), (49) Kottke and Haug
(2004), (50) Lunt and Hedger (1996), (51) Haug et al. (2005), (52) Janos (1980), (53) Béreau et al. (1997), (54) Alexander and H€ ogberg (1986), (55) Landim de
Souza (2003), (56) Becerra et al. (2009b), (57) Fracchia et al. (2009), (58) Nouhra (1997), (59) Nouhra (1999), (60) Pagano and Scotti (2008), (61) Dos Santos
et al. (2001), (62) Giachini et al. (2000), (63) Yokomizo (1981), (64) Schwan (1984), (65) Giachini et al. (2004), (66) Oliveira et al. (1997), (67) Lupatini et al.
(2008), (68) Rocci (2006), (69) Voigt et al. (2000), (70) Borges de Souza et al. (2004), (71) Borges de Souza et al. (2008), (72) Pinto Bonnassis (2007),
(73) Marx (1977), (74) Hentz de Mello et al. (2006), (75) Arruda Bacchi and Krugner (1988), (76) Liparini Pereira et al. (2005), (77) Halbinger et al. (1986),
(78) Brandán de Weth (2006), (79) Guzmán and Varela (1978), (80) Barros et al. (1978), (81) Daniele et al. (2005), (82) Gross et al. (2004), (83) Nouhra and
Becerra (2001), (84) Tackacs (1961), (85) Barroetaveña and Rajchenberg (2003a), (86) Barroetaveña et al. (2007), (87) Barroetaveña et al. (2005),
(88) Barroetaveña (2004), (89) Martı́nez et al. (2007), (90) Garrido (1983), (91) Garrido (1986), (92) Paloschi de Oliveira et al. (2006), (93) Rocci (2006),
(94) Tackacs (1964), (95) Singer (1968), (96) Putzke and Pereira (1994), (97) Barroetaveña and Rajchenberg (2003b), (98) Barroetaveña et al. (2006),
(99) Singer (1963), (100) Halling (1989), (101) Franco Molano and Uribe Calle (2000), (102) López-Quintero et al. (2007), (103) Vasco-Palacio et al. (2005)
2 The Ectomycorrhizal Symbiosis in South America 25
In South America, most studies on mycorrhizal status and ECM colonization

deal with the Fagaceae, Fabaceae, Nyctaginaceae, and Polygonaceae families
(Table 2.1). For the Nothofagus spp. forests of Chile, a colonization of 1.8–4.8%
and 19.6% has been reported by Carrillo et al. (1992) and Palfner et al. (2008),
respectively, while for the Nothofagus spp. forests of Argentina, it is of 79%
(Diehl et al. 2008). Meanwhile, for some genus of the Fabaceae family, Frioni
et al. (1999) reported a colonization that ranges between 17 and 36%, while
Moyersoen (1993) reported a 65% colonization for Aldinia kunhardtiana.
Most studies carried out for the Nyctaginaceae and Polygonaceae families refer
to the status of their species in terms of presence/absence of ECM (Singer and
Araujo 1979, St John 1980, Janos 1980, Béreau et al. 1997). In the particular case
of Neea obovata, N. robusta, and Guapira sancarlosiana (Nyctaginaceae), coloni-
zation was 100%, 7%, and 96%, respectively, while it was of 56% in Coccoloba
excelsa (Polygonaceae) (Moyersoen 1993).
Inoculation technologies appear as an alternative to the use of fertilizers since
they reduce both the costs of production and environmental contamination
(Garbaye 1990). ECM inoculations in South America have been carried almost
exclusively in introduced plants (Eucalyptus spp., Pinus spp., and Pseudostuga
menziesii), with the exception of Nothofagus spp. and Alnus acuminata. For this,
various techniques have been applied: spores from sporocarps mainly belonging to
the Basidiomycota phylum (Alpova diplophloeus, Laccaria laccata, Rhizopogon
roseolus, Suillus granulatus, among others); ECM culture micelia (micelia, airlift
bioreactors) in Eucalyptus spp. and Pinus spp.; and natural soil (potential inoculum)
for Pinus spp., Pseudotsuga menziesii, Eucalyptus dunii, E. citriodora, Nothofagus
alpina, N. dombeyi, and Alnus acuminata. All three techniques have been used for
introduced trees, while only spores and natural soil have been used for native
species.
2.3 Morphological and Anatomical Features of the ECM

in South America
The morphological and anatomical description of mycorrhizae and the identifi-

cation of their fungal partners are prerequisites for recognizing mycorrhizal biodi-
versity in ecosystems (Agerer 1991; Miller et al. 1991). Meanwhile, the occurrence
of natural ECM associations in the indigenous vegetation types from South
America has been virtually unexplored.
Table 2.2 summarizes the volume of ECM anatomotypes described in South
America for both native and introduced plants. Four anatomical complexes for
recognition of fungal relationships were used: (a) structure of outer mantle layers
in plan view, (b) structure of rhizomorphs, (c) shape of cystidia, and (d) features
of emanating hyphae (Agerer 1999, 2006). Besides, the cross section of mantle
area (e) was also considered, which showed useful for comparing the relative
26
Table 2.2 Anatomical characterization of ECM in native and introduced plants in South American forests
Ectomicorrhiza/host Mantle layers (Plect.: Rhizomorphs Cystidia Emanating hyphae Cross
treea/countryb/ plectenchymatous; Pseud: (color, branching, clamps, section
publicationc pseudoparenchymatous) septa, anastomoses) (mm)
Naucoria escharoides/ Plect. with parallel arranged Uniform-loose hyphal Lackingd Hyaline abundant, 39–56
Aa/A/1 hyphae (Mantle type B) bundles branched, with clamps;
simple anastomoses
Cortinarius helodes/ Plect. loosely interwoven (type B) Uniform-loose Lacking Hyaline abundant, 130–220
Aa/A/2 branched, clampled,
anastomoses open
Gyrodon monticola/ Plect. with globular cells (type F) Boletoid Spherical to clavate Hyaline and brown 24–57
Aa/A/2 abundant, branched,
with clamps
Russula Pseud. with angular cells (type L) Lacking Lacking Colorless, few, clamps 40–60
alnijorullensis/Aa/ lacking
A/3
Cortinarius Plect. with irregularly arranged Uniform-loose Lacking Colorless abundant, clamps 62–120
tucumanensis/Aa/ hyphae (type B) present, anastomoses
A/3 open
Lactarius aff. Pseud. with angular cells (type Lacking Lacking Colorless to 11–30
omphaliformis/Aa/ P-Q) membranaceously
A/3 yellowish, clamps
lacking
Tometella cf. Plect. with hyphal net arrangement Undifferentiated Lacking Yellowish to brownish, 23–36
sublilacina/Aa/A/4 (type A) branched, with clamps
Tometella cf. stuposa/ Pseud., with angular and roundish Not foundd Bottle-shaped with a Membranaceously 45–65
Aa/A/4 cells (type L) straight to bent neck brownish, branched with
clamps
Tometella cf. ellisii/ Plect. with irregularly arranged Not found Lacking Colorless, branched with 70–105
Aa/A/4 hyphae (type B) clamps
Not found Lacking 44–58
Lactarius Pseud. with epidermoid cells Colorless to
omphaliiformis/Aa/ bearing a hyphal net (type Q) membranaceously
A/4 yellowish, without
clamps
Russula sp. /Aa/A/4 Plect. irregularly arranged hyphae Not found Lacking Membranaceously 25–75
(type B) yellowish, branched
without clamps
Unidentified A/No/V/5 Plect. NRd NR Present 75–86
Unidentified B/No/V/5 Compactly mantle NR NR NR 31–49
Unidentified/Nr/V/5 Compactly mantle NR NR NR 33–46
Russula puiggarii/ Plect., loosely with gelatinous NR Not observed Whitish to light yellowish 30 thick
Nsp.1/E/6 matrix
Lactarius sp./Nsp.1/ Plect. hyphae of larger and Undifferentiated Not observed Abundant white, septa 30 thick
E/6 irregular diameter without clamps
Tomentella/ Plect. loosely, hyphae net-like NR NR Brown, septa without 40 thick
Thelephora sp.1/ arranged clamps
Nsp.1/E/6
Tomentella/ Plect., hyphae with clamp NR NR Reddish 40 thick
Thelephora sp.2/
Nsp.1/E/6
Ascomycete/Nsp.1/E/6 Plect., hyphae arranged star-like NR NR Black, simple pores with 30 thick
Woronin bodies
Thelephora/ Plect. loosely NR NR Brown with clamps NR
Tomentella/Nsp.2/E/6
Unidentified/Gs/V/5 Compactly mantle NR NR NR 21–36
Thelephora/ Plect. loosely arrangement NR NR Colorless with clamps NR
Tomentella/Gsp./E/6
Unidentified/Ce/V/5 Compactly mantle NR NR NR, Brown mycorrhizae 40–48
Unidentified/Cr/B/7 Plect. NR NR Dark Brown, abundant 40–65
Unidentified/Ah/V/5 Plect. NR NR Abundant 33–60
Clavulinaceae 1/Pd/ NR NR NR NR NR
V/8
27
(continued )
28

Clavulinaceae 2/Pd/ Plect. on top of a Pseud. Not found Lacking White, non ramified, thick NR
V/8 and rough wall
Sebacina sp./Pd/V/8 Plect. (net on top of a Present (not described) NR Yellowish NR
plectenchyma)
Cortinarius sp./Pd/V/8 Plect. Frequent, hairy, ramified Lacking Whitish, with clamps NR
Inocybe 1/Pd/V/8 NR NR NR NR NR
Inocybe 2/Pd/V/8 Plect. covered by a loose net With smooth margin; clamp Lacking Whitish NR
connections
Amanita sp./Pd/V/8 NR NR NR NR NR
Unidentified/Pd/V/8 Plect. (net on top a plect.) Lacking Elongated White, simple septa NR
Unidentified 2/Pd/V/8 Plect. or pseud. Thelephoroid rhizomorphs Lacking Brown often ornamented, NR
simple septa
Nothofagirhiza Plect., loosely, interwoven hyphae Lacking Lacking Pinkish to dark red, very 10–20
vinicolor/Np/C/9 (type B, E) abundant, with clamps
Russula fuegiana/Np/ Plect. loose net of thin hyphae Not frequent, agglutinated Cylindrical to gradually Not found 13–30
C/10 (type C, D) hyphae tapering with apical
knobs
Thaxterogaster Plect., loosely, interwoven hyphae Numerous undifferentiated Lacking Frequent, branched 13–27
albocanus/Np/C/11 (type B)
Austropaxillus Plect. (type A, B) Rather frequent, highly Cistidia-like hyphal Rather frequent 15–25
boletinoides/Np/ differentiated ends of variable
C/12 shape
Gautieria inapire/Np/ Plect., loosely interwoven (type B) Unconspicuous, simple or Rarely with Gelatinous, cell walls with 1–25
C/15 slightly differentiated acanthocystidia short protuberances
Nothofagirizha Plect. (type B), mantle surface Rhizomorphs-like strands Lacking Light brown, verrucose, 20–30
reticulosa/Np/C/16 often parallely blunded by or simple septa mostly with
anastomoses and short clamps, anastomoses
ramifications also closed by a clamp
Cenococcum Plect. (type G) formed by very Lacking Lacking Numerous black, bristle- NR
geophilum/Np/ densely arranged, star-like like
C/16 pattern
Russula nothofaginea/ Pseud., covered by hyphal net Not found Lacking Colorless infrequent 15–20
Nd/A/19 embedded in a distinct
gelatinous matrix (type Q)
Cortinarius Plect. (type B) Abundant, with attached Lacking Colorless-hyaline hyphae, 30–60
magellanicus/Nd/ spherical sclerotia with clamps
C/14
Stephanopus Plect. (type B) Abundant, blue-bruising Lacking Abundant, with clamps 30–50
stropharoides/Nd/ when squeezed
C/16
Nothofagiriza Pseud. (type K), without clamps, Without Lacking Very few short 20–45
vesiculosa/Nd/ thick-walled
C/16
Cortinarius Plect. (type B) Differentiated Lacking With clamps, open 20–30
austrosalor/Nn/ anastomoses
C/13
Austropaxillus Plect. (type B) Dark brown, pigmented Scattered Clavate Clamped 15–25
boletinoides f. hyphae
olivascens/Nb/
C/16
Boletus loyo/Nob/C/16 Plect. (type B) Scattered, highly Occasionally clavate, Scarse, without clamps 36–45
differentiated slightly thickened
hyphal ends
Boletus putidus/Nob/ Plect. (type A/B) Highly differentiated, Poorly differentiated or Hyphae without clamps 15–25
C/16 thicker, margin with weakly clavate
inflated hyphal elements cystidia or cystidia-
like hyphal ends
(continued )
29
30

Nothofagirhiza Plect. (type D) Not found Three types: slightly Rare, clamps lacking 15–22
tricystidiis/Nob/ tapering with an
C/16 enlarged barrel-
shaped; awl-shaped
or worm-shaped with
enlarged basal cell;
ampullate with an
apical knob
Descolea antarctica/ Plect. with awl-shaped cystidia Not found Lateral, tapering, knob- Infrequent, with clamps 15–20
Na/C/17 (type D) bearing outgrowth
Hymenoscyphoid Compact plect. Lacking Lacking Black hyphae, without NR
(probably clamps
Rhizoscyphus
ericae)/Na/C/18
Inocyboid/Na/C/18 Loose plect. Lacking Lacking Abundant, with clamps, NR
inocyboid
Russuloid/Na/C/18 Plect. Lacking Russuloid gloeocystidia Lacking NR
Thelephoroid/Na/C/18 Compact plect. Whitish or lacking Thelephoroid Abundant, with clamps NR
Tomentelloid/Na/C/18 Pseud. or plect. Dark brown or lacking Present or lacking Scarce or lacking, present NR
or lacking clamp
connections
Rhizoscyphus ericae/ No hyphal mantle; a superficial Lacking Lacking Lacking NR
Gm/E/20 Hartig net (fingerlike branched
hyphae)
Lactarius panuoides/ Mantle densely tomentose to NR Occasionally similar Abundant, with septa NR
Da/G/21 substrigose mass hyphae forming
capitate cystidia
Russula campinensis/ Well developed mantle; coarse net NR NR No evident NR
Da/G/21 of irregularly shaped
Inocybe sp./Sh/A/22 Plect. irregularly arranged hyphae Lacking Lacking Hyaline clampled 19–35
(type A)
Tometella sp./Sh/A/22 Plect. irregularly arranged hyphae Lacking Lacking Hyaline clampled, not 25–31
(type A) branched
Anatomotype III/Sh/ Plect., hyphae without clamps Lacking Lacking Lacking 7–16
A/22
Anatomotype IV/Sh/ Plect. Lacking Lacking Scarse, with clamps 12–16
A/22
Anatomotype V/Sh/ Plect. Lacking Lacking Scarse, without clamps 12–20
A/22
Anatomotype VI/Sh/ Plect. Lacking Lacking Few light to brown, with 12–15
A/22 clamps
Anatomotype VII/Sh/ Plect. Lacking Lacking Few hyaline, without 12–30
A/22 clamps
Leccinum scabrum/Bp/ Plect. (type A) Boletoid, abundant Lacking Rough abundant, clamps NR
A/23 lacking
Suillus granulatus/Pe/ Plect. with brownish drops of Boletoid, abundant Lacking Abundant, clamps lacking NR
A/23 pigments
Amphinema byssoides/ Plect. Lacking NR Abundant yellowish, NR

Pp/A/24 clampled, with spines
Rhizopogon roseolus/ Plect. with a ring-like hyphal Branched white, abundant Lacking Lacking NR
Pp/A/24 pattern hyaline crystals
Rhizopogon aff. Plect. with yellow-brown crystals Abundant, branched, white Lacking Lacking NR
ellenae/Pp/A/24 to pink, with hyaline or
yellow crystals
Rhizopogon inmature Plect. with squarrosely hyphae Thin branched, with crystals Lacking Abundant NR
type/Pp/A/24 with crystals
Rhizopogon coralloid Plect. with hyaline crystals Abundant, branched, with Lacking NR NR
type/Pp/A/24 crystals
Rhizopogon brownish Plect. with abundant crystals Lacking NR Lacking NR
type 42/Pp/A/24
Tuber type/Pp/A/24 Pseud. with irregular cells Lacking Awl-shaped, bristle like NR NR
31
(continued )
32

Brownish coralloid Plect., hyphae with septa Lacking Lacking Lacking NR
anatomotype/Pp/
A/24
Whitish anatomotype/ Plect. with a ring-like arrangement Lacking Lacking With septa NR
Pp/A/24
Dichotomous Plect. Lacking Lacking Clamped NR
yellowish
antatomotype/Pp/
A/24
Brownish Plect., hyphae with septa Lacking NR Lacking NR
antatomotype/Pp/
A/25
Xerocomus Plect. (type A) Boletoid, abundant Lacking Smooth abundant, clamps 40
chrysenteron/Ca/ lacking
A/23
Amanita muscaria/Cd/ Plect. (type A) Undifferentiated, abundant Lacking Smooth, with clamps 32–57
A/25
Tuber type 1/Pm/A/24 Pseud. Lacking Awl-shaped, bristle like Abundant NR
Rhizopogon type 1/ Plect. White branched NR Hyaline, with septa NR
Pm/A/24
White coralloid NR White branched NR NR NR
antatomotype/Pm/
A/24
Brownish Plect. Lacking NR Abundant NR
anatomotype/Pm/
A/24
Each line within the table represents a studied ectomicorrhiza, with an indication of its host trees, country for which it has been described and citation
(numbered – all respective citations are found below under References), followed by a detailed description of the anatomical characteristics of the referred
ECM (structure of outer mantle layers and of rhizomorphs, shape of cystidia, features of emanating hyphae and cross section)
a
Host Tree: Aa Alnus acuminata; No Neea obovata; Nr Neea robusta; N sp. 1 Neea sp. 1; N sp. 2 Neea sp. 2; Gs Guapira sancarlosiana; G sp. Guapira sp.; Ce
Coccoloba excelsa; Cr Coccoloba rosea; Ah Aldinia kunhartiana; Pd Pakaraimaea dipterocarpacea; Np Nothofagus pumilio; Nd Nothofagus dombeyi; Nn
Nothofagus nitida; Nb Nothofagus betuloides; Nob Nothofagus obliqua; Na Nothofagus alpina; Ge Graffenrieda emarginata; Da Dicymbe altsonii; Sh Salix
humboldtiana; Bp Betula pendula; Pe Pinus elliottii; Pp Pinus ponderosa; Ca Cedrus atlantica; Cd Cedrus deodara; Pm Pseudostuga menziessi
b
Country: A: Argentina; B: Brazil; C: Chile; E: Ecuador; G: French Guyana; V: Venezuela
c
References: (1) Becerra et al. (2002), (2) Becerra et al. (2005a), (3) Becerra et al. (2005b), (4) Pritsch et al. (2010), (5) Moyersoen (1993), (6) Haug et al.
(2005), (7) Landim de Souza (2003), (8) Moyersoen (2006), (9) Palfner and Godoy (1996a), (10) Palfner and Godoy (1996b), (11) Palfner (1996), (12) Palfner
(2002a), (13) Palfner (2002b), (14) Palfner (2002c), (15) Palfner (2002d), (16) Palfner (2001), (17) Palfner (1997), (18) Palfner et al. (2008), (19) Beenken
(2001), (20) Haug et al. (2004), (21) Henkel et al. (2000), (22) Becerra et al. (2009b), (23) Nouhra (1997), (24) Barroetaveña (2004), (25) Daniele et al. (2005)
d
Lacking: the author makes its inexistence explicit, NR (not recorded): the author makes no mention to either its presence or absence, Not found: the author
describes it as not found
33
importance of the fungal partner in different plants for different ecosystems,

climates, and soils (Landim de Souza 2003). Table 2.2 does not consider morpho-
logical descriptions of seedlings grown under greenhouse conditions since, as
Agerer (2006) states, descriptions of ECM that are exclusively based on synthe-
sized material often do not provide features occurring in nature.
Out of the 85 ECM anatomotypes described in the different studies (every ECM
not identified to the species level was considered to be a different ECM), 35 were
identified up to their species, 20 to their genus, 5 to supraspecific groups, 1 to its
phylum, 4 were unidentified ECM morphotypes published under a binomial name
(Agerer 1986, 1987–2002, 1994, 1999), and 20 were unidentified ECM anatomo-
types lacking a name.
The associated fungi recorded in the 85 ECM descriptions were mostly Basi-
diomycota (55) and Ascomycota (6), although in 29 descriptions, the fungal
division could not be determined.
Morphotypes belonging to the Helotiaceae family (Ascomycota) showed plec-
tenchymatous mantles or, exceptionally, no mantle, as seen in Graffenrieda emar-
ginata from Ecuador (Haug et al. 2004). Cenococcum, the most cosmopolitan
morphotype, showed a black plectenchymatous mantle forming a characteristically
star-like pattern and frequently numerous black hyphae. Morphotypes belonging to
the Tuberaceae family showed a pseudoparenchymatous mantle with irregular cells
and awl-shaped cystidia.
Fungal families in the Basidiomycota were represented in ECM descriptions
as follows: Amanitaceae (2), Atheliaceae (1), Bolbitiaceae (1), Boletaceae (4),
Clavulinaceae (2), Cortinariaceae (11), Ramariaceae (1), Rhizopogonaceae (6),
Russulaceae (10), Paxillaceae (3), Sebacinaceae (1), Suillaceae (1), Thelephoraceae
(8), plus four supraspecific groups: Inocyboid (1), Russuloid (1), Thelephoroid (1),
and Tomentelloid (1).
Boletoid rhizomorphs were present in most Boletaceae, Paxillaceae, Rhizopo-
gonaceae, and Suillaceae ECM of Boletales families. In general, these morphotypes
showed plectenchymatous mantles frequently with ring-like patterns and the pres-
ence of crystals.
Among the Russulaceae, and as Agerer (2006) states, russuloid rhizomorphs are
combined with plectenchymatous mantles. Meanwhile, Russulaceae species with
pseudoparenchymatous mantles do not form rhizomorphs at all.
ECM belonging to the Cortinariaceae family was characterized by an extrama-
trical organized mycelia and undifferentiated or slightly differentiated rhizo-
morphs, while ECM of the Thelephoraceae family was characterized by a
heterogeneous mantle type assemblage (Agerer 2006). In this ECM, anatomotypes
plectenchymatous and pseudoparenchymatous mantles were found. Different from
Agerer (2006), the morphotype associated with Amanita muscaria (Amanitaceae)
showed a plectenchymatous mantle, while the Sebacina sp. morphotype (Sebaci-
naceae) presented rhizomorphs.
The morphotypes of Amphinema byssoides (Atheliaceae), Descolea anarctica
(Bolbitiaceae), and Gautieria inapire (Ramariaceae) show similar characteristics
than those described by Agerer 2006.
2.4 Conclusion
Although more than 90% of terrestrial plants are associated with mycorrhizal fungi,
and two-thirds of them are AM, ECM tree species are also notorious in South
American forests. This review summarizes the ectomycorrhizal studies carried out
in the neotropical ecozone and provides information about ECM fungi and their
anatomical characteristics.
The analyzed studies reveal that most fungal symbionts are Basidiomycota, and
that most studies on mycorrhizal status and ECM colonization deal with the
Fagaceae, Fabaceae, Nyctaginaceae, and Polygonaceae families. Meanwhile,
the three inoculum techniques (spores from sporocarps, culture mycelia, and natural
soil) have been used for introduced trees (Eucalyptus spp., Pinus spp., and
Pseudostuga menziesii), whereas only spores and natural soil have been used for
native species (Nothofagus spp. and Alnus acuminata). The associated fungi
recorded in the 85 ECM anatomotypes reviewed were mostly Basidiomycota and
a few Ascomycota, mostly found on native species (77%).
An important byproduct of this review is the realization of the existence of many
gaps in the existing knowledge of ECM in South America. This suggests
that mycorrhizologists should focus on little known/studied geographic areas,
ecosystems, host trees, and fungal groups to reveal those aspects of the ECM
symbiosis in South America, which may have practical applications in, for
example, afforestation and environmental management programs. This knowledge
may also be an important contribution to the conservation community, in a time
when firsthand knowledge for urgent decisions is required.
Acknowledgments This work was supported by Conicet, Agencia de Promoción Cientı́fica y

Tecnológica (PICT 438-2006), Secyt, and the National University of Córdoba. The authors wish to
thank all other researchers whom through their continuous efforts contributed with the studies on
which this review was based.
References
Agerer R (1986) Studies on ectomycorrhizae II. Introducing remarks on characterization and

identification. Mycotaxon 26:473–492
Agerer R (1987–2002) Colour atlas of ectomycorrhizae. Einhorn-Verlag, Munich
Agerer R (1991) Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK (eds)
Techniques for the study of mycorrhiza, vol 23, Methods in microbiology. Academic, London,
pp 25–73
Agerer R (1994) Index of unidentified ectomycorrhizae. III. Names and identifications published
in 1992. Mycorrhiza 4:183–184
Agerer R (1999) Anatomical characteristics of identified ectomycorrhizas: an attempt towards a
natural classification. In: Varma A, Hock B (eds) Mycorrhiza: structure, function, molecular
biology and biotechnology, 2nd edn. Springer-Verlag, Berlin, pp 633–682
Agerer R (2006) Fungal relationships and structural identity of their ectomycorrhizae. Mycol
Progress 5:67–107
Alexander IJ (1989) Mycorrhizas in tropical forests. In: Proctor J (ed) Mineral nutrients in tropical
forest and savanna ecosystems. Blackwell Scientific Publications, Oxford, pp 169–188
Alexander IJ, H€ogberg P (1986) Ectomycorrhizas of tropical Angiospermous trees. New Phytol
102:541–549
Allen MF, Swenson W, Querejeta JI, Egerton-Warburton LM, Treseder KK (2003) Ecology of
mycorrhizae: a conceptual framework for complex interactions among plants and fungi. Annu
Rev Phytopathol 41:217–303
Arruda Bacchi LM, Krugner TL (1988) Desenvolvimento ectomicorrı́zico em mudas de Eucalyptus
grandis e Eucalyptus urophylla inoculadas com Pisolithus tinctorius em um vivero comercial.
IPEF 40:21–25
Barroetaveña C (2004) Micorrizas y micorrización controlada en plantaciones y viveros forestales
de pino ponderosa (Pinus ponderosa) y pino oregón (Pseudotsuga menziesii) en la región
Andino Patagónica. Doctoral thesis, Comahue National University, 225p
Barroetaveña C, Rajchenberg M (2003a) Las micorrizas la producción de plántulas de Pinus
ponderosa Dougl. ex Laws. en la patagonia Argentina. Bosque 24:17–23
Barroetaveña C, Rajchenberg M (2003b) Las micorrizas la producción de plántulas de Pseudos-
tuga menziesii (Mirb.) Franco en la patagonia Argentina. Bosque 24:3–15
Barroetaveña C, Rajchenberg M, Cazares E (2005) Mycorrhizal fungi in Pinus ponderosa intro-
duced in central Patagonia (Argentina). Nova Hedwig 80:453–464
Barroetaveña C, Cázares E, Rajchenberg M (2006) Mycorrhizal fungi of Pseudotsuga menziesii,
an introduced tree species in Central Patagonia (Argentina). Nova Hedwig 83:53–66
Barroetaveña C, Cázares E, Rajchenberg M (2007) Ectomycorrhizal fungi associated with
ponderosa pine and Douglas-fir: a comparison of species richness in native western North
American forests and Patagonian plantations from Argentina. Mycorrhiza 17:355–373
Barros HF, Brandi RM, Reis MS (1978) Micorriza em eucalipto. Revista Arvore 2:130–140
Becerra AG (2002) Influencia de los suelos Ustorthentes sobre las ectomicorrizas y
endomicorrizas en Alnus acuminata H.B.K. Master thesis, Facultad de Agronomı́a, Buenos
Aires University, 190p
Becerra A, Daniele G, Domı́nguez L, Nouhra E, Horton T (2002) Ectomycorrhizae between Alnus
acuminata H.B.K. and Naucoria escharoides (Fr.:Fr.) Kummer from Argentina. Mycorrhiza
12:61–66
Becerra A, Nouhra E, Daniele G, Domı́nguez L, McKay D (2005a) Ectomycorrhizas of Cortinarius
helodes and Gyrodon monticola with Alnus acuminata from Argentina. Mycorrhiza 15:7–15
Becerra A, Beenken L, Pritsch K, Daniele G, Schloter M, Agerer R (2005b) Anatomical
and molecular characterization of Lactarius aff. omphaliformis, Russula alnijorullensis and
Cortinarius tucumanensis ectomycorrhizae on Alnus acuminata. Mycologia 97:1047–1057
Becerra A, Pritsch K, Arrigo N, Palma M, Bartoloni N (2005c) Ectomycorrhizal colonization of
Alnus acuminata Kunth in northwestern Argentina in relation to season and soil parameters.
Ann For Sci 62:325–332
Becerra A, Menoyo E, Lett I, Li CL (2009a) Alnus acuminata in dual symbiosis with Frankia and
two different ectomycorrhizal fungi (Alpova austroalnicola and Alpova diplophloeus) growing
in soilless growth medium. Symbiosis 47:85–92
Becerra A, Nouhra E, Silva M, McKay D (2009b) Ectomycorrhizae, arbuscular mycorrhizae and
dark septate fungi on Salix humboldtiana Willd. from Central Argentina: a first assessment in
two riparian populations. Mycoscience 50:343–352
Beenken L (2001) Russula nothofaginea Sing. þ Nothofagus dombeyi (Mirbel) Oersted. Descr
Ectomycorrhizae 5:175–179
Béreau M, Gazel M, Garbaye J (1997) Les symbioses mycorhiziennes des arbres de la Forêt
tropicale humide de Guyane francaise. Can J Bot 75:711–716
Borges de Souza LA, Silva Filho GN, Lopes de Oliveira V (2004) Eficiência de fungos ectomi-
corrı́zicos na absorção de fósforo e na promoção do crescimento de eucalipto. Pesq Agropec
Bras 39:349–355
Borges de Souza LA, Pinto Bonnassis PA, Silva Filho GN, Lopes de Oliveira V (2008) New
isolates of ectomycorrhizal fungi and the growth of eucalypt. Pesq Agropec Bras 43:235–241
Brandán de Weth CI (2006) Estudio de micorrizas en zonas disturbadas, no disturbadas y en
recuperación, en el Parque Biológico Sierra de San Javier (PSSJ). National University of
Tucumán, Tucumán, Argentina
Brundrett MC, Cairney JWG (2002) Ectomycorrhizas in plant communities. In: Sivasithamparam
K, Dixon KW, Barret RL (eds) Microorganisms in plant conservation and biodiversity. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp 105–150
Carrillo R, Godoy R, Peredo H (1992) Simbiosis micorrı́cica em comunidades boscosas del Valle
Central en el Sur de Chile. Bosque 13:57–67
Castillo CG, Borie F, Godoy R, Rubio R, Sieverding E (2006) Diversity of mycorrhizal plant
species and arbuscular mycorrhizal fungi in evergreen Forest, deciduous Forest and grassland
ecosystems of Southern Chile. J App Bot Food Qual 80:40–47
Daniele G, Becerra A, Crespo E (2005) Amanita muscaria (Basidiomycota) y su asociación
micorrı́cica con Cedrus deodara (Pinaceae) en las sierras de Córdoba, Argentina. Bol Soc
Arg Bot 40:45–49
Diehl P, Mazzarino MJ, Fontenla S (2008) Plant limiting nutrients in Andean-Patagonian woody
species: effects of interannual rainfall variation, soil fertility and mycorrhizal infection. For
Ecol Manag 255:2973–2980
dos Santos VL, Muchovej RM, Borges AC, Neves JCL, Kasuya MCM (2001) Vesicular-
Arbuscular/Ectomycorrhiza succession in seedlings of Eucalyptus spp. Braz J Microbiol
32:81–86
Flores R, Godoy R, Palfner G (1997) Morfo-anatomı́a de la ectomicorriza Cenococcum geophilum
Fr. en Nothofagus alessandrii Esp. Gayana Bot 54:157–162
Founoune H, Duponnois R, Bâ AM, El Bouami F (2002) Influence of the dual arbuscular
endomycorrhizal/ectomycorrhizal symbiosis on the growth of Acacia holosericea (A. Cunn.
ex G. Don) in glasshouse conditions. Ann For Sci 59:93–98
Fracchia S, Aranda A, Gopar A, Silvani V, Fernandez L, Godeas A (2009) Mycorrhizal status
of plant species in the Chaco Serrano Woodland from central Argentina. Mycorrhiza
19:205–214
Franco Molano AE, Uribe Calle E (2000) Hongos Agaricales y Boletales de Colombia. Biota
Colombiana 1:25–43
Frioni L, Minasian H, Volfovicz R (1999) Arbuscular mycorrhizae and ectomycorrhizae in native
tree legumes in Uruguay. For Ecol Manag 115:41–47
Fulgenzi TD, Henkel TW, Halling RE (2007) Tylopilus orsonianus sp. nov. and Tylopilus eximius
from Guyana. Mycologia 99:622–627
Garbaye J (1990) Utilisation des mycorhizes en sylviculture. In: Strullu DG (ed) Les mycorhizes
des arbres et plantes cultivées. Lavoisier, Paris, pp 197–250
Garrido N (1983) Las Boletaceae en plantaciones de Pinus radiata D. Don en Chile (Fungi,
Basidiomycetes). Bol Soc Biol 54:77–88
Garrido N (1986) Survey of ectomycorrhizal fungi associated with exotic forest trees in Chile.
Nova Hedwig 43:423–442
Giachini AJ, Oliveira VL, Castellano MA, Trappe JM (2000) Ectomycorrhizal fungi in Eucalyptus
and Pinus plantations in southern Brazil. Mycologia 92:1166–1177
Giachini AJ, Souza LAB, Oliveira VL (2004) Species richness and seasonal abundance of
ectomycorrhizal fungi in plantations of Eucalyptus dunnii and Pinus taeda in Southern Brazil.
Mycorrhiza 14:375–381
Godoy R, Palfner G (1997) Ectomicorrizas en Nothofagus alpina (P. et E.) Oerst y N. dombeyi
(Mirb.) Oerst. del sur de chile. Bol Micol 12:55–61
Godoy R, Romero R, Carrillo R (1994) Estatus micotrófico de la flora vascular en bosques de
conı́feras nativas del sur de Chile. Rev Chil Hist Nat 67:209–220
Gross E, Thomazini Casagrande LI, Caetano FH (2004) Ultrastructural study of ectomycorrhizas
on Pinus caribaea Morelet. var. hondurensis Barr. & Golf. seedlings. Acta Bot Bras 18:1–7
Guzmán G, Varela L (1978) Los Hongos de Colombia III. Observaciones sobre los hongos,
lı́quenes y mixomicetos de Colombia. Caldasia 12:309–338
Halbinger RE, Frontera GM, Correa OS, Gauna FE (1986) Inoculación artificial de hongos
micorrı́ticos de los géneros Boletus y Scleroderma de especies de plantas del género Euca-
liptus. Rev Agr Micologı́a 9:3–9
Halling RE (1989) A synopsis of Colombian Boletes. Mycotaxon 34:93–113
Haug I, Lempe J, Homeier J, Weiß M, Setaro S, Oberwinkler F, Kottke I (2004) Graffenrieda
emarginata (Melastomataceae) forms mycorrhizas with Glomeromycota and with a member of
the Hymenoscyphus ericae aggregate in the organic soil of a neotropical mountain rain forest.
Can J Bot 82:340–356
Haug I, Weis M, Homeier J, Oberwinkler F, Kottke I (2005) Russulaceae and Thelephoraceae
form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical
mountain rain forest of southern Ecuador. New Phytol 165:923–936
Henkel TW (1999) New taxa and distribution records for Tylopilus from Dicymbe forests of
Guyana. Mycologia 91:655–665
Henkel TW, Aime MC, Miller SL (2000) Systematics of pleurotoid Russulaceae from Guyana and
Japan, with notes on their ectomycorrhizal status. Mycologia 92:1119–1132
Henkel TW, Terborgh J, Vilgalys RJ (2002) Ectomycorrhizal fungi and their leguminous hosts in
the Pakaraima Mountains of Guyana. Mycol Res 106:515–531
Hentz de Mello A, Antoniolli ZI, Kaminski J, Lorensi Souza E, Lopes Oliveira V (2006) Fungos
arbusculares e ectomicorrı́zicos em áreas de Eucalipto e de campo nativo em solo arenoso.
Ciência Florestal 16:293–301
Janos DP (1980) Mycorrhizae influences tropical succession. Tropical succession 12:56–64
Janos DP (1985) Mycorrhizal fungi: agents or symptoms of tropical community composition. In:
Molina R (ed) Proceedings of the 6th North American conference on mycorrhizae. Oregon
State University, Corvallis, pp 98–103
Kottke I, Haug I (2004) The significance of mycorrhizal diversity of trees in the tropical mountain
forest of southern Ecuador. Lyonia 7:49–56
Landim de Souza MF (2003) Brazilian Atlantic rainforest remnants and Mycorrhizal symbiosis –
implications for reforestation. A case study in Sergipe, Northeast Brazil. PhD. Universit€at
Bremen, 193pp
Liparini Pereira O, Dutra Costa M, Chaer Borges A, Fernandes Araújo E, Megumi Kasuya MC
(2005) Compatibility and ectomycorrhiza formation among Pisolithus isolates and Eucalyptus
spp. R Bras Ci Solo 29:337–344
López-Quintero C, Vasco-Palacios AM, Franco-Molano AE (2007) Macrohongos de un Bosque
de Roble, Quercus humboldtii Bonpl., en la Vereda Contrafuerte, Municipio de Andes
(Colombia). In: Reserva natural Regional Cuchilla Jardı́n Támesis Antioquia, Una mirada a
su biodiversidad. Corporación Ambiental, Medellı́n, pp 21–34
Lunt PH, Hedger JN (1996) A survey of mycorrhizal infection of trees in the Terra Firme
rainforest, Cuyabeno, Ecuador. Mycologist 10:161–165
Lupatini M, Bonnassis PAP, Steffen RB, Oliveira VL, Antoniolli ZI (2008) Mycorrhizal morpho-
typing and molecular characterization of Chondrogaster angustisporus Giachini, Castellano,
Trappe & Oliveira, an ectomycorrhizal fungus from Eucalyptus. Mycorrhiza 18:437–442
Malloch DW, Pirozynski KA, Raven PH (1980) Ecological and evolutionary significance of
mycorrhizal symbioses in vascular plants (A review). PNAS USA 77:2113–2118
Martı́nez DB, Barroetaveña C, Rajchenberg M (2007) Influencia del régimen de fertilización y del
momento de inoculación en la micorrización de Pinus ponderosa en la etapa de vivero. Bosque
28:226–233
Marx DH (1977) The host range and world distribution of the ectomycorrhizal fungus Pisolithus
tinctorius. Can J Microbiol 23:217–223
Matheny PB, Aime MC, Henkel TW (2003) New species of Inocybe from Dicymbe forests of
Guyana. Mycol Res 107:495–505
McGuire KL, Henkel TW, Granzow de la Cerda I, Villa G, Edmund F, Andrew C (2008) Dual
mycorrhizal colonization of forest-dominating tropical trees and the mycorrhizal status of non-
dominant tree and liana species. Mycorrhiza 18:217–222
Miller SL, Koo CD, Molina R (1991) Characterization of red alder ectomycorrhizae: a preface to
monitoring belowground eco1ogica1 responses. Can J Bot 69:516–531
Moyersoen B (1993) Ectomicorrizas y micorrizas vesiculo-arbusculares en Caatinga Amazonica
del Sur de Venezuela. Scientia Guianae 3:1–82
Moyersoen B (2006) Pakaraimaea dipterocarpacea is ectomycorrhizal, indicating an ancient
Gondwanaland origin for the ectomycorrhizal habit in Dipterocarpaceae. New Phytol
172:753–762
Moyersoen B (2008) Biodiversity and specificity of ectomycorrhizal fungi associated with
Pakaraimaea dipterocarpacea in Guayana, Venezuela. Mycorrhizas in Tropical Forests,
Workshop UTPL, 10p
Moyersoen B, Fitter AH, Alexander IJ (1998a) Spatial distribution of ectomycorrhizas and
arbuscular mycorrhizas in Korup National Park rain forest, Cameroon, in relation to edaphic
parameters. New Phytol 139:311–320
Moyersoen B, Alexander IJ, Fitter AH (1998b) Phosphorus nutrition of ectomycorrhizal and
arbuscular mycorrhizal tree seedlings from a lowland tropical rainforest in Korup National
Park, Cameroon. J Trop Ecol 14:47–61
Moyersoen B, Becker P, Alexander IJ (2001) Are ectomycorrhizas more abundant than arbuscular
mycorrhizas in tropical heath forests? New Phytol 150:591–599
Nouhra E (1997) Boletáceas (Hymenomycetes) de la provincia de Córdoba y sus relaciones
micorrı́cicas con especies arbóreas de la flora autóctona e introducida. PhD thesis, National
University of Córdoba, 115pp
Nouhra E (1999) Novedades em Boletáceas Del centro de la Argentina. Kurtziana 27:403–423
Nouhra E, Becerra A (2001) Sı́ntesis micorrı́cica de Suillus granulatus (Eumycota) y plantines de
Pinus elliotti (Pinaceae). Bol Soc Arg Bot 36:209–215
Nouhra E, Domı́nguez L, Becerra A, Mangeaud A (2003) Colonización micorrı́cica y actinorrı́cica
en plantines de Alnus acuminata y Alnus rubra (Betulaceae). Bol Soc Arg Bot 38:199–206
Nouhra E, Domı́nguez L, Becerra A, Trappe J (2005) Morphology, molecular analysis and
ecological aspects of the South American hypogeous fungus; Alpova austroalnicola sp. nov.
Mycologia 97:598–604
Oliveira VL, Schmidt VDB, Bellei MM (1997) Patterns of arbuscular- and ectomycorrhizal
colonization of Eucalyptus dunnii in southern Brazil. Ann Sci For 54:473–481
Onguene NA, Kuyper TW (2002) Importance of the ectomycorrhizal network for seedling
survival and ectomycorrhiza formation in rain forests of south Cameroon. Mycorrhiza
12:13–17
Pagano MC, Scotti MR (2008) Arbuscular and ectomycorrhizal colonization of two Eucalyptus
species in semiarid Brazil. Mycoscience 49:379–384
Palfner G (1996) Thaxterogaster albocanus Horak & Moser þ Nothofagus pumilio Poepp. et
Endl.) Krasser. Descr Ectomycorrhizae 1:155–160
Palfner G (1997) Descolea antarctica Singer þ Nothofagus alpina (Poepp. et Endl.) Oerst. Descr
Ectomycorrhizae 2:7–12
Palfner G (2001) Taxonomische studien an ektomykorrhizen aus den Nothofagus-W€aldern Mit-
tels€udchiles. Bibliotheca Mycologica. Band 190. J. Cramer. Berlin, Alemania, 243pp
Palfner G (2002a) Austropaxillus boletinoides. In: Agerer R (ed) Colour atlas of ectomycorrhizae.
Einhorn Verlag, Schw€abisch Gm€ und, plate 151
Palfner G (2002b) Cortinarius austrosalor. In: Agerer R (ed) Colour atlas of ectomycorrhizae.
Palfner G (2002c) Cortinarius magellanicus. In: Agerer R (ed) Colour atlas of ectomycorrhizae.
Palfner G (2002d) Gautieria inapire. In Agerer R (ed) Colour atlas of ectomycorrhizae. Einhorn
Verlag, Schw€abisch Gm€ und, plate 154
Palfner G, Godoy R (1996a) “Notofagirhiza vinicolor”þ Nothofagus pumilio (Poepp. et Endl.)

Krasser. Descr Ectomycorrhizae 1:65–70
Palfner G, Godoy R (1996b) Russula fuegiana þ Nothofagus pumilo (Poepp. Et Endl.) Krasser.
Descr Ectomycorrhizae 1:131–136
Palfner G, Canseco MI, Casanova-Katny A (2008) Post-fire seedlings of Nothofagus alpina in
Southern Chile show strong dominance of a single ectomycorrhizal fungus and a vertical shift
in root architecture. Plant Soil 313:237–250
Paloschi de Oliveira L, Rossi MJ, Furigo Júnior A, Silva Filho GN, Lopes de Oliveira V (2006)
Viability and infectivity of an ectomycorrhizal inoculums produced in an airlift bioreactor and
immobilized in calcium alginate. Braz J Microbiol 37:251–255
Pérez F, Palfner G, Brunel N, Santelices R (2007) Synthesis and establishment of Tuber mela-
nosporum Vitt. ectomycorrhizae on two Nothofagus species in Chile. Mycorrhiza 17:627–632
Pérez-Moreno J (1998) La ectomicorriza una simbiosis mutualista en el sostenimiento de Gaia, el
planeta viviente. In: Ferrera-Cerrato R, Pérez-Moreno J (eds) Manejo de agroecosistemas
sostenibles. Veracruzana University, México, pp 93–120
Pinto Bonnassis PA (2007) Caracterização de isolados fúngicos ectomicorrı́zicos na promoção do
crescimento e na colonização radicular de Eucalyptus dunnii Maiden. Master thesis, Santa
Catarina Federal University, 73p
Pritsch K, Becerre A, Põlme S, Tedersoo L, Schloter M, Agerer R (2010) Descrioption and
identification of Alnus awminate ectomycorrhizae from argentinean older stands. Mycologia
(in press)
Putzke JML, Pereira AB (1994) O fungos da familia Boletaceae conhecidos no Rio Grande do Sul,
Brasil (Fungi, Basidiomycotina). Caderno de Pesquisa Sér Bot 6:75–100
Rocci MJ (2006) Tecnologia para produção de inoculantes de fungos ectomicorrı́zicos utilizando
cultivo submerso em biorreator airlift. PhD thesis, Santa Catarina Federal University, 188p
Rumney GR (1968) Climatology and the world’s climate. The Macmillan Company, New York,
643pp
Sch€ußler A, Schwarzott D, Walker C (2001) A new fungal phylum, the Glomeromycota: phy-
logeny and evolution. Mycol Res 105:1413–1421
Schwan KR (1984) Caracterização, incidência e ecologia de micorrizas em viveiro e florestas de
Eucalyptus spp., na Região de Viçosa, Minas Gerais. Master thesis, Viçosa, Universidade
Federal de Vicosa, 55p
Singer R (1953) Four years of mycological work in southern South America. Mycologia
45:865–891
Singer R (1963) Oak mycorrhiza fungi in Colombia. Mycopathol et Mycol Applicata 20:239–252
Singer R (1968) Sand-dune inhabiting fungi of the South Atlantic coast from Uruguay to Bahı́a
Blanca. Mycopathol Mycol Applicata 34:129–143
Singer R (1971) Forest mycology and forest communities in South America. II. Mycorrhiza
sociology and fungus succession in the Nothofagus dombeyi-Austrocedrus chilensis woods
of Patagonia. In: Hacskaylo E (ed) Mycorrhizae, proceedings of the first North American
conference on mycorrhizae. Government Printing Office, Washington, DC, pp 204–215
Singer R, Araujo I (1979) Litter decomposition and ectomycorrhiza in Amazonian forest. I. A
comparison of litter decomposing and ectomycorrhizal basidiomycetes in latosol-terra-firme
forest and white podzol campinarana. Acta Amazonica 9:25–41
Singer R, Morello JH (1960) Ectotrophic tree mycorrhiza and forest communities. Ecology
41:549–551
Singer R, Araujo I, Ivory MH (1983) The ectotrophically mycorrhizal fungi of the neotropical
lowlands, especially Central Amazonia. Nova Hedwig Beihefte 77:1–352
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 2nd edn. Academic Press, New York, 787p
St John TV (1980) A survey of mycorrhizal infection in an Amazonian rain forest. Acta
Amazonica 10:527–533
Tackacs EA (1961) Inoculación de especies de pinos con hongos formadores de micorizas.
Silvicultura 15:5–17
Tackacs EA (1964) Inoculación artificial de pinos de regiones subtropicales con hongos forma-
dores de micorrizas. I. Formación de micorrizas ectotróficas en Pinus taeda L. con Sclero-
derma vulgare Horn. Idia-Suplemento Forestal 12:41–44
Trappe JM (1977) Selection of fungi for ectomycorrhizal inoculation in nurseries. Ann Rev
Phytolaphol 15:203–222
Trappe JM (1987) Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an
evolutionary standpoint. In: Safir GR (ed) Ecophysiology of VA mycorrhizal plants. CRC
Press, Boca Raton, FL, pp 5–25
Valenzuela E, Moreno G, Garnica S, Godoy R, Ramirez C (1999) Mycosociology in native forests
of Nothofagus of the X Region of Chile, diversity and ecological role. Mycotaxon 72:217–226
Vasco Palacio AM, Franco Molano AE, López Quintero CA, Boekhaut T (2005) Macromicetes
(Ascomycota, Basidiomycota) de la región del medio Caquetá, departamentos de Caquetá y
Amazonas (Colombia). Biota Colombiana 6:127–140
Villegas JN, Carrillo RF, Núñez PM, Rodrı́guez M (2007) Caracterización micorrizogénica de un
bosque de Nothofagus pumilio (Poepp. et Endl.) Krasser en el Parque Nacional Villarrica,
Cordillera de los Andes, IX Región, Chile. Biodiversidad y conservación de bosques nativos.
International congress on development, environment and natural resources: multi-level and
multi-scale sustainability, vol 3. Bolivia, pp 1448–1455
Voigt EL, Lopes de Oliveira V, Randi AM (2000) Mycorrhizal colonization and phenolic
compounds accumulation on roots of Eucalyptus dunii Maiden inoculated with ectomycor-
rhizal fungi. Pesq Agropec Bras 35:1905–1910
Wrigth JE (1988) Interrelaciones entre Macromycetes (Fungi) y Nothofagus. Acad Nac Cs
Exactas, Fı́sicas y Naturales. Monografı́a 4:132–155
Yokomizo NKS (1981) Associacáo ectomicorrı́cica de Pisolithus tinctorius (Pers.) Coker e Couch
com espécies de Eucalyptus L. Héritier. Piracicaba Escola Superior de Agricultura “Luis de
Queiróz”. Master thesis, 54p
.
Chapter 3
Ectomycorrhizal Inoculum and Inoculation
Techniques
Ivan Repáč
3.1 Introduction
ECM symbiosis plays an important role in physiology, ecology, resistance, produc-

tion, and other aspects of life of a single trees, populations, and ecosystems of
ectotrophic forest tree species. Among the various ways of improving the early
growth and survival of forest plantations, controlled mycorrhization by inoculating
seedlings with selected fungal strains is an energy-efficient and environment-
friendly alternative to fertilization or soil tilling (Kropp and Langlois 1990; Marx
1991; Garbaye and Churin 1997). Trees planted particularly in soils deficient in
ECM fungi (e.g., mine spoils, polluted areas, agricultural, and other treeless lands)
and even in routine reforestation sites may benefit greatly from ECM inoculation
(Marx 1991; Castellano 1996; Querejeta et al. 1998; Duñabeitia et al. 2004; Núñez
et al. 2006), although the positive effect of inoculation is not consistent (Castellano
1996; Corrêa et al. 2006; Menkis et al. 2007; Baum et al. 2009). Several types of
natural and laboratory-produced inocula and several application techniques have
been used for seedling inoculation. Forest soil, litter, humus, or excised ectomycor-
rhizae were substituted by fungal spores and mycelial inoculum (Marx and Bryan
1975; Le Tacon et al. 1983; Duponnois and Garbaye 1991; Parladé et al. 1999).
Vegetative inoculum appears to be the most appropriate and effective method of
inoculation (Marx and Kenney 1982; Mortier et al. 1988; Garbaye and Churin 1997;
Rincón et al. 2007). Artificial mycorhization with vegetative inoculum consists of
several steps – selection of ECM fungi, isolation and maintenance of fungi in pure
cultures, preparation of fungal inoculum, and inoculation of seedlings.
A large body of literature exists on ECM inoculum preparation and application
in a research scale; a comprehensive review of the literature and of all aspects of
this topic is impossible. Although there have been previous studies on inoculum
preparation and inoculation techniques, this chapter attempts to bring together a
I. Repáč
Department of Silviculture, Technical University in Zvolen, Forestry Faculty, T.G. Masaryka 24,
SK-96053, Zvolen, Slovak Republic
e-mail: repac@vsld.tuzvo.sk

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_3,
44 I. Repáč
brief review of the major inoculum types and methods of their application devel-
oped and to offer a list of selected references as a source of other literature and
detailed description of a particular material, procedure or product.
3.2 Isolation and Manipulation of Fungal Pure Cultures,

Nutrient Media
Isolation, cultivation, and maintenance of ECM fungal cultures are crucial phases in
artificial mycorrhization of forest tree seedlings (Molina and Palmer 1982). Many,
but far from all, fungi can be cultivated in pure culture in the laboratory under
controlled conditions. The fungal tissues are most commonly isolated from young
and healthy fruit bodies as soon as possible after collection but can also be isolated
from surface sterilized ectomycorrhizae, sclerotia, and rhizomorphs (Molina and
Palmer 1982; Obase et al. 2009). The pieces of the tissues are usually transferred to
sterile glass tubes on agar nutrient media. Tissue transfers and fungal cultures are
incubated in the dark at 21–24 C for 2–6 weeks. Between the cultivation cycles the
stock cultures are stored approximately for 2 months at 2–4 C. Higher rates of agar
cultures are cultivated in Petri dishes and submerged cultures in Erlenmeyer flasks.
One of the most common nutrient media was the Modess modification of the
Hagem semisynthetic formula (Molina and Palmer 1982). Modess medium was
further modified (e.g., amended with thiamine by Timonen et al. 1997) and is still
used (e.g., Niemi et al. 2006). Modified Melin–Norkrans medium (MMN) (Marx
1969) is probably the most used worldwide for experimental procedures (e.g. Fortin
et al. 1980; Wong and Fortin 1989; Repáč 1996a; Gáper et al. 1999; Parladé et al.
1999). The original solution was a synthetic solution (no malt extract) with only
25 mg of thiamine and 2.5 g of glucose. MMN altered by Vrålstad et al. (2002)
contained only 1.0 g of glucose to reduce possible adverse effects on ECM
formation caused by high levels of exogenous carbon. MMN was also modified
by Niemi et al. (2002) and Langer et al. (2008). Pachlewski medium (Pachlewski
and Pachlewska 1974) was used, e.g., by Duponnois and Garbaye (1991) and
Parladé et al. (1999). Moore et al. (1989) cultivated cultures of fungus Cantharellus
cibarius on special species-specific C-medium. In the study of Guerin-Laguette
et al. (2003), stock cultures were maintained on agar medium proposed by Ohta
(1990), and mycelium was grown on Tween- or olive oil-supplemented A medium.
Fungal biomass increased up to 15-fold as a result of olive oil incorporation.
Basic components of natural media are leachates of various crops such as potato,
cabbage, wheat, and others. Although use of natural formulations is less frequent
than semi-synthetic or synthetic ones, potato dextrose agar (PDA) was used in the
past years, e.g., by Parladé et al. (1996a) and Giomaro et al. (2002). MMN (Marx
1969), BAF (Moser 1960), KHO (Šašek and Musı́lek 1967), and malt–peptone
(50 ml of brewery wort + 5.0 g of peptone to 1,000 ml of H2O) media are used for
manipulation of fungal cultures in our laboratory of Department of Silviculture,
3 Ectomycorrhizal Inoculum and Inoculation Techniques 45
Forestry Faculty, Technical University in Zvolen, Slovakia (project VEGA 1/0516/09).

The media are changed in recultivation cycles to support the vitality and resistance
of cultures.
3.3 Synthesis of Ectomycorrhizae in Controlled Conditions
Syntheses of ECM in controlled conditions are important techniques for verification

of symbiotic relationships (compatibility) between intended fungi and host trees,
structural, physiological and biochemical studies, and ultimately the genetic control
of ECM formation. Depending on the research goal and synthesizing apparatus
either sterile, semi-sterile, or non-sterile techniques can be used. The purpose of this
Section (and also Sect. 3.4) is not a comprehensive categorization, but just a review
of some of the most often reported or meaningful inoculum types, methods of
inoculum preparation, and inoculation procedures. For better understanding, see
illustrations of the apparatus of synthesizing techniques, e.g., in the works of
Peterson and Chakravarty (1991), Duponnois and Garbaye (1991), and other related
literature cited.
3.3.1 Fungal Cultures Preparation
Fungi are applied into synthesis system most often as agar culture transfers.
Usually, agar cultures are grown on nutrient agar in Petri plates for 3–4 weeks,
then transferred onto fresh solid medium, and incubated for a few days. Several
plugs (5–8 mm diameter) are then aseptically removed and placed around the roots
in the synthesis apparatus. Mycelial agar plugs served as inoculum in Petri dish
synthesis systems, e.g., in experiments of Wong and Fortin (1989), Brunner (1993),
Gáper et al. (1999), Bending et al. (2002), Kr€uger et al. (2004), and Sarjala and
Taulavuori (2004). Fortin et al. (1980) used plugs of mycelium in ECM synthesis in
growth pouches, H€ ogberg et al. (1999) and Giomaro et al. (2002) in vessels, and
Vrålstad et al. (2002) in conical flasks. In Petri dishes, Herrmann et al. (2004)
inoculated roots with a nylon sheet carrying 7–8 fungal plugs and Niemi et al.
(2006) using two filter paper strips covered by mycelium.
Apart from agar cultures, fungi are usually introduced into synthesis system
from liquid cultures (slurry of mycelium). Because the liquid cultures are shaken
just before inoculation, the numerous mycelial fragments will yield rapid and
uniform colonization of the synthesis substrate (Molina and Palmer 1982). Molina
(1979) cultivated mycelium in glass tubes filled with MMN nutrient solution and
small chips of broken glass; cultures were shaken periodically to fragment the
mycelium. In experiments of Duponnois and Garbaye (1991), Brunner (1993),
Parladé et al. (1996a), and Obase et al. (2009), fungal mycelium was grown for
3–4 weeks on a shaker in Erlenmeyer flasks containing liquid medium. The
46 I. Repáč
mycelium was then washed in tap water to remove residual nutrients, homogenized
in a blender for a few seconds, and resuspended in distilled water. Guerin-Laguette
et al. (2003) collected mycelium of Tricholoma matsutake grown in liquid medium
over nylon mesh filter, washed it thoroughly with and resuspended in 100 ml of
a modified nutrient medium (NM). Flores et al. (2008) reported cultivation of
Laccaria bicolor in flasks with semi-liquid MMN medium (1.5 g agar.l-1) in the
dark at 23 C. The mycelia obtained were filtered through a 65-mm net, washed
in sterile water to eliminate sugars and to reduce the growth of contaminants in
the seedling root systems, fragmented and homogenized by manual agitation,
and then resuspended in distilled water (70–80 g mycelium.l-1).
Vermiculite–peat (Marx and Bryan 1975) and alginate-bead (Le Tacon et al.
1983) inocula can be used in ECM synthesis at controlled conditions, even though
they are primarily used in the operational inoculations. Preparation of these inocu-
lum types is described in detail in Sect. 3.4.1.3. Mycelial plugs were laid on top of
vermiculite–peat mixture (4:5–1:5, v:v) moistened to field capacity with modified
liquid Pachlewski medium to prepare vermiculite–peat inoculum for synthesis in
sterile conditions (Duponnois and Garbaye 1991). Corrêa et al. (2006) and Martı́n-
Pinto et al. (2006) inoculated this substrate mixture with liquid fungal cultures.
Alginate-bead inoculum of Laccaria laccata was used to synthesize ectomycor-
rhizae in aseptic conditions of glass test tubes (Duponnois and Garbaye 1991) and
inoculum of Amanita rubescens and Hebeloma sinapizans in polyethylene pots in a
plant growth cabinet (Kozdrój et al. 2007).
3.3.2 Inoculation Techniques
3.3.2.1 Sterile Techniques
Petri Dishes
Petri plates are probably the most extensively used method of ectomycorrhiza
synthesis. Wong and Fortin (1989) described a Petri dish technique that avoids
limitations of previous ones. In Petri dish filled with sugar-free agar medium, two
sheets of nylon membrane sandwiched the root and were overlaid with a sheet of
filter paper to keep the exposed surface of the roots moist. Cotton rolls were placed
along the opposite edge of the Petri dish to absorb water which condensed during
incubation. The filter paper was removed and the fungal plugs were placed on the
membrane beside root laterals. Peterson and Chakravarty (1991) differentiated
between simple system, divided Petri plates, sandwich technique, and nylon mesh
method. Sandwich technique (mycelial plugs placed on cellophane sheets) was a
method of synthesis experiments conducted by Langer et al. (2008) on Populus
tremula plantlets. Mixture of vermiculite and peat seems to be a suitable substrate
for Petri dish synthesizing system (Duponnois and Garbaye 1991; Bending et al.
2002; Sarjala and Taulavuori 2004). Duponnois and Garbaye (1991) observed pure
culture synthesis of Pseudotsuga menziesii–Laccaria laccata in autoclaved soil or

silica sand. Gáper et al. (1999) studied synthesis of Picea abies–Inocybe lacera and
Suillus bovinus in a mixture of perlite, peat, florex, and keramzit (7:2:2:1). Of
course, substrate is always autoclaved or sterilized regardless of the purpose of
ECM synthesis; in most instances substrate is disinfected or fumigated in other
inoculation experiments, including operational conditions. To protect fungus and
root system from direct illumination, Niemi et al. (2006) placed a semi-circle brown
paper on the lower part of the lid of 14-cm Petri dish. To reduce the loss of plants
during acclimatization, Kr€ uger et al. (2004) placed a 90-mm Petri dish system in
a 140-mm Petri dish in which humidity was regulated with moistened paper.
Herrmann et al. (2004) transferred rooted micropropagated oak plantlets into
90-mm Petri dish rhizotrons. In this system, the roots grew two dimensionally
inside the dish, whereas the shoot developed outside. Between inoculation and
transfer to the growth chamber, the plants were kept in plastic bags moistened with
wet cotton sheets to reduce adaptation shocks for the shoots.
Flasks and Jars
Some of the early attempts to synthesize ectomycorrhizae under sterile conditions

were made by Melin using Erlenmeyer flasks containing sterile sand moistened
with a nutrient solution into which an aseptically germinated seedling and single
fungus culture were introduced (Peterson and Chakravarty 1991). Hacskaylo
improved the method by using vermiculite instead of sand (Molina and Palmer
1982). Peterson and Chakravarty (1991) described Mason jars and Leonard jars, in
which shoot of seedling remains in the environment and roots in aseptic conditions
inside the jars. Fungal inoculum can be added into flasks as plugs of actively
growing mycelium (Vrålstad et al. 2002) or as slurry of fungal hyphae (Brunner
et al. 1992) either before, at time, or after seedling introduction. Vermiculite, peat,
and particularly their mixture are mostly used as substrate since their advantages
had been detected and confirmed for ECM synthesis. Martı́n-Pinto et al. (2006)
transferred 7-day-old Pinus nigra seedlings to 45-ml sterile flasks containing 30 ml
of substrate (MMN, peat or vermiculite) colonized by the ECM fungi.
Test Tubes
Pachlewski and Pachlewska (1974) achieved good results to synthesize ectomycor-

rhizae between Pinus sylvestris and more ECM fungi in test tubes containing water
agar amended with thiamine. Molina (1979) used 300 38-mm glass test tubes
filled with 110 ml of vermiculite and 10 ml of peat moss moistened with 70 ml of
MMN nutrient solution to test a wide range of ECM fungi on Alnus species entirely
enclosed in tubes. Technique described by Molina (1979), modified with double the
amount of peat, was used by Parladé et al. (1996a). Within series of experiments
48 I. Repáč
studying the biology of Pseudotsuga menziesii–Laccaria laccata symbiosis, Dupon-

nois and Garbaye (1991) used glass test tubes (3 15 cm) filled with autoclaved
peat–vermiculite (1:1, v:v) moistened to field capacity with modified Shemakanova
mineral nutrient solution. Inoculation was carried out either with (1) peat–vermiculite
inoculum mixed with substrate (1:10, v:v) or laid on top of the tube (1–2 cm), (2)
alginate-bead inoculum (five beads laid on top of the tube), or (3) mycelial suspen-
sion (injected with a syringe or deposited with a pipette). The roots were maintained
in axenic conditions, while the top of the plant developed outside the tube.
Polycarbonate Boxes
Guerin-Laguette et al. (2003) introduced mycorrhizal synthesis between Tricho-

loma matsutake and Pinus densiflora in the following substrate and apparatus: dry
soil, vermiculite, and moist Pinus densiflora bark (pieces of about 1 cm2) were
mixed together (1:1:1/v:v:v) before addition of distilled water supplemented with
olive oil. Wet substrate was autoclaved twice before distribution into sterile 920-ml,
wide-mouth, polycarbonate boxes (9.5 9.5 11.5 cm) and inoculation with
mycelial slurry (1 volume of slurry into 5 volumes of substrates). The substrate
(approximately 300 ml per box) was applied as five layers (60 ml each), each layer
being inoculated with the slurry before application of the following layer, i.e., 10 ml
of slurry for the first two layers, 20 ml for the next two, 0 ml for the last one. Five
sterile 12-day-old pine seedlings were then aseptically introduced into each box.
The boxes were maintained 4 months at 23 C under a photosynthetic active
radiation with a 16-h photoperiod.
3.3.2.2 Semi- and Non-sterile Techniques
Growth Pouches
Growth pouches were first introduced in detail by Fortin et al. (1980). Flat,
transparent polyester pouches were used for synthesis of ectomycorrhizae between
Pinus strobus and several ECM fungi. Fifteen milliliters of MMN solution was
added, and a piece of glass tube was placed along the side of each pouch to add
nutrient solution and water. The root system was laid directly onto the paper pad. As
soon as formation of short roots initiated, plugs of actively growing mycelium were
placed on the paper pad 3–5 mm from a short root primordium. Brunner (1993) used
autoclaved polyethylene pouches 13 16 cm including a glass fiber paper with an
activated charcoal paper disk attached. Pouches were filled with 10 ml of MMN
liquid medium and one Picea abies seedling. After 2 months, mycelial disks were
placed in the vicinity of the short roots. Two strips of foam were inserted to provide
air space. Advantages of this technique are that numerous seedlings can be grown
in a very small space, root system can be viewed without disturbing the roots or
the fungus, and roots are clean since substrate is not involved (Peterson and
Chakravarty 1991).
Pots, Containers, Vessels
A brief review of several experiments can illustrate a variety of vessels, substrates,

inoculum types, and methods of their applications used in non-sterile ECM synthe-
sis techniques. Duponnois and Garbaye (1991) used transparent boxes, rootrainers,
Hiko and “M” containers filled with soil or vermiculite–peat mixture (1:1, v:v) for
growing Douglas fir seedlings in the glasshouse. Liquid, vermiculite–peat or algi-
nate-bead inoculum were applied either by mixing with the substrate before filling
the containers or by spreading the inoculum on the well-developed root system.
Brunner (1993) synthesized ectomycorrhizae between Picea abies and Hebeloma
crustuliniforme in autoclaved cuvettes of stainless steel (15 12 2 cm) filled
with a vermiculite–peat moss mixture. Homogenized mycelium grown in liquid
solution was introduced into cuvettes using an inverted pipette. H€ogberg et al.
(1999) planted Pinus sylvestris seedlings in 0.5-l plastic pots filled with sand
inoculated with ten mycelial agar plugs placed below the pine roots. Tissue blocks
of Tuber brumale mycelium served for inoculation of micropropagated plantlets of
Tilia americana and Quercus pubescens in the vessels (12 cm diameter 18 cm
height) filled with 800 ml of peat–vermiculite mixture (1:30, v:v) in experiment of
Giomaro et al. (2002). Timonen et al. (1997) planted sterile Pinus sylvestris
seedlings in plastic pots enclosed in sterile plastic bags to maintain moisture and
prevent aerial contamination. After 25 days, the roots of one seedling were inocu-
lated with three agar plugs (5 mm3) of mycelium. Bogeat-Triboulot et al. (2004)
and Flores et al. (2008) used mycelial suspension to inoculate Pinus pinaster with
Hebeloma cylindrosporum in 0.4-1 pots filled with soil and Abies guatemalensis
with Laccaria bicolor in 300-cm3 containers filled with peat moss/vermiculite (1:1,
v:v), respectively. Obase et al. (2009) soaked roots of Populus maximowiczii in
MMN liquid medium containing cultured ECM fungal mycelia. Each seedling was
planted in a plastic pot (60 60 100 mm) filled with heat-sterilized volcanic
debris.
Aspray et al. (2006) and Corrêa et al. (2006) inoculated seedlings of Pinus
sylvestris and P. pinaster with vermiculite–peat inoculum of Laccaria bicolor,
Lactarius rufus, and Pisolithus tinctorius in 80-ml and 350-ml pots, respectively.
Kozdrój et al. (2007) transferred Pinus sylvestris seedlings to polyethylene pots
containing 150 g of autoclaved soil thoroughly mixed with alginate-bead inocu-
lum of Amanita rubescens or Hebeloma sinapizans in ratio 15:1 (v:v). The
inoculated seedlings were grown after inoculation in either growth (climatic)
chamber (Brunner 1993; H€ ogberg et al. 1999; Corrêa et al. 2006; Flores et al.
2008; Obase et al. 2009), culture room (Giomaro et al. 2002), growth cabinet
(Bogeat-Triboulot et al. 2004; Kozdrój et al. 2007), or glasshouse (Timonen et al.
1997; Aspray et al. 2006).
50 I. Repáč
3.4 Greenhouse and Nursery Inoculation
3.4.1 Inoculum Types
3.4.1.1 Natural Inoculum (Soil, Humus, Ectomycorrhizae)
Various organic matters, such as soil, litter, variable forms of humus, rotten wood,
and ectomycorrhizae collected from forest plantations or mature stands were
exploited especially in the beginning of the effort to inoculate forest tree seedlings.
Collection of a large amount of viable inoculum of fungi adapted to the sites from
which they were taken is relatively reliable and easy. A major drawback of natural
inoculum is that the species of ECM fungi in the inoculum cannot be controlled.
This inoculum may also contain harmful microorganisms and weeds in addition to
the ECM fungi. Forest soil was used as a source of indigenous ECM fungi in
experiments of Borchers and Perry (1990), Querejeta et al. (1998), and Wallander
et al. (2005). Litter and diverse forms of humus from the forest floor including a
range of materials from recently fallen needles on the surface to well-decomposed
humus overlying the mineral soil were added to pot substrate (Parke et al. 1983;
Repáč 1996a). Aucina et al. (2007) used pine and oak litter to affect development of
bareroot seedlings. Rotten wood was collected from a Douglas-fir stand and
fragmented by hand into small pieces about 1 cm in diameter for mixing in a
growing medium (Kropp 1982). Natural inoculum is usually collected from stand or
under individual tree of species which is inoculated. Ectomycorrhizae excised from
root systems of trees were also used as inoculum on a limited basis in research trials
(Marx and Kenney 1982). A great deal of time and care is required to obtain a
sufficient quantity of viable ectomycorrhizae.
3.4.1.2 Basidiospores
Sporocarps and spores of various fungi were used as inoculum to form specific
ectomycorrhizae on various forest tree species. Sporocarps are essentially spore
inoculum, since their vegetative matrix is killed by dessication during drying or by
decomposition when added to soil (Marx and Kenney 1982). Gastromycete, such as
genera Rhizopogon, Scleroderma, and Pisolithus, produces numerous basidiospores
that are easier to collect in large quantities than those of mushroom-produced ECM
fungi. Advantages of using spores for inoculation are that spores require no
extended growth phase under aseptic conditions like vegetative inoculum, spore
inoculum is very light, and spores are able to survive storage from one season to the
next. Major disadvantages are the lack of standard laboratory tests to determine
spore viability, insufficiency of sporocarps of many fungi in any year, delay in
ectomycorrhiza formation as compared with vegetative inoculum, and lack of
genetic definition.
Basidiospores can be obtained and stored before application in principle either

in dry state or blended with water. Basidiospores were obtained by crushing and
sieving basidiocarps through a mesh screen (Rincón et al. 2001; Chen et al. 2006)
in a closed plastic bag (Marx and Bryan 1975). Using these techniques, well over
1 kg (fresh weight) of basidiospores of Pisolithus tinctorius was collected in less
than 3 h (Marx and Bryan 1975). The dry spores were stored in either small
plastic bags or amber glass bottles in darkness at 5 C for 10 days before use
(Marx and Bryan 1975), mixed with vermiculite for seedling inoculation (Rincón
et al. 2001) or stored at 4 C for 1–18 months before blended with distilled
deionized water (1:10, v:v) for 5 s on low speed (Chen et al. 2006). In experi-
ments of Parladé et al. (1996b, 1999), Rincón et al. (2001), Hortal et al. (2008),
and Becerra et al. (2009), spore suspensions were prepared by blending sporo-
carps with distilled or tap water using a blender at low or high speed until the
spores were released. After collection, Parladé et al. (1996b) and Chen et al.
(2006) dried sporocarps at 30–40 C for 48 h before blending and sieving,
respectively. Núñez et al. (2006) cleaned and sterilized sporocarps of Tuber
melanosporum by brief superficial flaming. To prepare the inoculum, Roldan
et al. (1996), Querejeta et al. (1998), and Núñez et al. (2006) suspended spores
in distilled water and stored the suspensions at 2–4 C until use. Required spore
concentrations are prepared by serial dilution of spore suspension with water.
Spore concentrations are counted using haemocytometer. The approximate
number of spores contained per gram of dried sporocarp tissue was 107–1010
(Parladé et al. 1996b; Rincón et al. 2001) or 1.1 million basidiospores per
milligram (Marx and Bryan 1975). Chen et al. (2006) obtained a concentration
of 105 spores.ml-1 and Rincón et al. (2001) 103–108 spores.10 ml-1.
3.4.1.3 Vegetative (Mycelial) Inoculum
Mycelial Suspension (Slurry)
This type of vegetative inoculum is more often used in a small-scale synthesis in

controlled conditions than in nursery experiments (see Sect. 3.3.1). Mycelial slurry
of Pisolithus tinctorius, Suillus granulates, and Rhizopogon luteolus was prepared
by blending mycelial mats from liquid cultures with distilled water at high speed for
less than 3 s (Cline and Reid 1982). One liter of sterile water contained 40 g of
(fresh weight) mycelium. Gagnon et al. (1991, 1995) produced mycelial suspension
of Laccaria bicolor in a fermentor. Starter content of living propagules was diluted
five times with water and mixed with the substrate in a cement mixer to get a final
content approximately 6.1 104 living propagules or 5.68 mg of dried mycelium
per seedling. Gange et al. (2005) prepared mycelial slurry of Laccaria laccata
maintained in sterile liquid culture on MMN medium by fragmenting the mycelium
in a blender for 30 s.
52 I. Repáč
Vermiculite–Peat (Substrate Carrier) Inoculum
Moser (1958) in Austria was one of the first to make a serious attempt to produce
vegetative inoculum of ECM fungi. For production of inoculum, mycelium of
Suillus plorans was first grown in liquid culture and then in sterile peat moss.
Moser and other workers tested various organic materials as the final inoculum
substrate, e.g., forest litter, sawdust, grain of cereals, corn, bark, and found that they
were not as effective as peat moss (Marx and Kenney 1982). Although mycelium
tends to grow around rather than into the particles of perlite substrate, Repáč
(1996b) and H€onig et al. (2000) reported that a perlite–peat mixture appears to be
a possible form of mycelial carrier. Vozzo and Hacskaylo (1971) grew mycelium of
several fungi in polypropylene cups containing a 2:1 ratio of sterile peat moss and
vermiculite moistened with nutrient solution. In the fundamental study of Marx and
Bryan (1975), the inoculum containers were 2-L jars containing the mixture of
1,400 ml of vermiculite, 50 ml of finely divided peat moss, and 750 ml of liquid
MMN medium with glucose. The containers were autoclaved for 30 min and each
was inoculated with eight mycelium-agar disks of Pisolithus tinctorius. After 15
weeks at room temperature, the vermiculite particles were permeated with myce-
lium. To prepare mass inoculum for infestation of soil, mycelium was removed
from the jars, passed through a 5-mm mesh screen, and held with two layers of
cheesecloth while being leached with cool running tap water to remove nonassimi-
lated nutrients.
Novel formulations of vermiculite–peat inoculum did not require leaching and
drying before use (Garbaye et al. 1988; Marx and Cordell 1989). An important
modification of original inoculum formulation was carried out on vermiculite:peat
ratio of inoculum. Marx et al. (1982) reported that vermiculite–peat inoculum
mixture produced by solid-substrate fermentation contained 5–10% peat moss by
volume. Other vermiculite:peat ratios used were, e.g., 4:5–1:5 (Duponnois and
Garbaye 1991), 9:1 (Garbaye et al. 1988; Baum et al. 2009), 10:1 (Machón et al.
2006), and 11:1 (Rincón et al. 2001). Blended mycelial starter cultures mixed with
the substrate will reduce the time of incubation by half (Marx and Kenney 1982;
Rincón et al. 2001). Depending on fungal properties, inoculum is incubated for 4–8
weeks at 24–25 C in the dark. The attempts to measure the quantity of mycelium in
the vermiculite–peat inoculum did not give reliable results because of the peat that
interferes with colorimetric measurements (Duponnois and Garbaye 1991).
Alginate-Bead Inoculum
Le Tacon et al. (1983) and Mauperin et al. (1987) have shown that mycelium grown
in a liquid medium and entrapped in calcium alginate gel is a very efficient
inoculum for ECM development and can be used as an alternative to the classical
vermiculite–peat mixture. Mycelium in alginate-bead inoculum is better protected,
survives longer, and has a longer lasting effect than when grown on a vermiculite–
peat mixture (Mortier et al. 1988). For production of alginate-bead inoculum,
fungul cultures are grown in liquid medium. The mycelial pellets are washed in tap
water, homogenized in a blender for 5–10 s, and resuspended in distilled water
containing 10 g.l 1 of sodium alginate and 30 g.l 1 of powdered sphagnum peat.
This suspension is pumped through a pipe with 5-mm holes above a 100 g.l 1 CaCl2
solution, each drop forming a bead of reticulated calcium alginate gel 3–4 mm in
diameter. The beads are cured in CaCl2 for 24 h at room temperature (for ensuring
complete reticulation of the gel), washed in tap water to remove NaCl, stored in air-
tight containers at room temperature to prevent drying, and used in the nursery
(Mauperin et al. 1987).
Procedure of the French authors was modified by Kropáček and Cudlı́n (1989).
In their work, sodium alginate and peat were substituted by agents Agricol and
perlite, respectively, to obtain alginate paste containing the mycelium. Beads were
cured in 5% calcium chloride for 30 min, rinsed with distilled water, dried to
surface dryness (about 30 min in air filtered box), and stored in air-tight containers
at 4 C until use. Alginate-bead inoculum was further used for inoculation of
seedlings, e.g., in experiments of Duponnois and Garbaye (1991), Gagnon et al.
(1991), Baum et al. (2000, 2002), and Repáč (2007). Parladé et al. (1999) prepared
alginate-bead inoculum containing spores of Rhizopogon and mycelium of Lac-
caria bicolor. Mixed inoculum was prepared by adding spores plus fragmented
mycelium (blended in autoclaved water) at different proportions to Pyrex flasks
containing 20 g.l 1 of an autoclaved water solution of sodium alginate. The content
of flask was gently mixed, dropped into a 0.3-M water solution of CaCl2 to
polymerize, then washed with sterile distilled water, and kept at 4 C in plastic
bags for 1 week.
3.4.2 Methods of Inoculum Application
Fumigation of nursery soil before inoculation improves ECM development because

it lowers populations of soil microorganisms that can colonize introduced inocula,
feeder root pathogens that damage roots and thus reduce ECM development, and/or
indigenous competing ECM fungi. The seedlings can be inoculated with ECM
inoculum during one of the following stages:
– Before seeds are sown or seedlings planted
– When seeds are sown or seedlings planted
– After seedlings emerge or seedlings planting
3.4.2.1 Basidiospores
Several inoculation techniques have been used:

– Mixing either crushed sporocarps or dry spores directly into soil or container
medium
54 I. Repáč
– Mixing spores with a moistened carrier, such as vermiculite, kaolin or sand,

broadcasting onto soil and then mixing into the nursery soil or the growing
medium of containers
– Dusting dry spores onto soil around young seedlings and leaching them into the
root zone
– Suspending in water and drenching, irrigating, or injecting into growth substrate
– Dusting or spraying on roots of nonmycorrhizal seedlings
– Mixing with the pelletizing matrix and encapsulating or coating seeds before
sowing
Marx et al. (1978) broadcast spores of Pisolithus tinctorius added to moist
vermiculite onto soil and mixed inoculum with the soil using handtools. The
spore quantities were 108, 324, or 648 mg of spores.m 2 of soil surface. Rincón
et al. (2001) mixed dry spores of P. tinctorius and Scleroderma verrucosum
included in vermiculite with potting substrate and filled containers with 103–108
spores per 175 ml of substrate.
Perhaps the most used method is application of spore water suspension to growth
substrate. Marx and Bryan (1975) poured basidiospores of P. tinctorius (10 g
suspended in 500 ml of distilled water with 1 drop of Tween 20) into microplots
which were heavily watered to wash the basidiospores into the soil. Theodorou
(1984) inoculated seeds of Pinus radiata with basidiospores (5.15 104 spores per
seed) of Rhizopogon luteolus 1–2 days before sowing. Suspension of spores of
R. luteolus (4.46 107 spores.ml 1) was sprayed onto soil, and the spores were
mixed with the soil by raking and rolling and by watering 1 day after sowing of
inoculated seeds (i.e., 2 days after soil inoculation).
In experiments of Roldan et al. (1996) and Querejeta et al. (1998), water
suspension of spores of Pisolithus arhizus was applied three times, 1 month apart,
12 weeks after sowing of Pinus halepensis to 300-ml plastic bags filled with 3:1
soil/peat mixture. Each plant received 5 105 spores per application. Molina et al.
(1997) applied spore slurries of six Rhizopogon spp. to container-grown Pseudot-
suga menziesii and Pinus ponderosa seedlings over two inoculations. For each
inoculation, 10 ml of diluted spore suspension was pipetted onto the peat–
vermiculite substrate. Chen et al. (2006) inoculated Eucalyptus urophylla seedlings
2 weeks after transplanting to plastic pots with 10 ml of spore suspension at a rate
106 spores per seedling. The spore slurry was added to a 2–3 cm deep hole near the
plant using a 5-ml pipette. Parladé et al. (1996b), Rincón et al. (2001), and Hortal
et al. (2008) inoculated 1-month-old container-grown seedlings with spore suspen-
sion of several ECM fungi at the rate of 102–108 spores per seedling. Núñez et al.
(2006) applied spore suspension of Tuber melanosporum by injecting manually into
each seedling’s pot substrate (400 ml of light and dark peat and vermiculite, 2:1:1)
approximately 7.5 105 spores at 3–8 cm depth. In the work of Becerra et al.
(2009), 1 ml of spore suspension of two Alpova species containing 106 spores was
inoculated at the base of Alnus acuminata seedlings.
3.4.2.2 Natural and Vegetative Inoculum
In inoculation programs, since hyphae cannot grow from the inoculum to roots,
inoculum must be placed in the rooting zone of seedlings where roots can grow into
the inoculum (Marx and Kenney 1982). In principle, natural and vegetative inocu-
lum can be applied by three methods:
– Mixed with the rooting medium
– Banded or layered below seeds or seedlings
– Suspended in water and poured onto seedlings or dipping seedlings into the
slurry before planting (except of beads)
Bareroot and container-grown seedlings and cuttings of numerous tree species
were inoculated with vegetative inocula of many ECM fungi in greenhouse and
nursery experiments. Pisolithus tinctorius (Marx and Bryan 1975; Marx et al. 1982;
Marx and Cordell 1989; Vijaya and Srivasuki 1999; Rincón et al. 2001) and genus
Laccaria (Mortier et al. 1988; Kropáček and Cudlı́n 1989; Gagnon et al. 1995;
Garbaye and Churin 1997; Parladé et al. 1999; Baum et al. 2002; Gange et al.
2005; Machón et al. 2006) have been the most often tested fungi in inoculation
experiments. ECM seedlings or cuttings are produced through inoculation of dif-
ferent growing substrates. Soil and peat mixed with vermiculite or other substrate
components – the standard substrates for production of planting stock of forest tree
species, are convenient and most frequently used for ECM inoculation.
Unequivocally, the most preferred technique of application of organic matter
and vegetative inoculum is mixing it with a growth substrate. Amount of inoculum
mixed with substrate in pot experiments is most commonly expressed as a ratio of
inoculum and substrate by volume. Organic matter (humus, litter, rotten wood) is
added to substrate in a larger ratio (1:4 to 1:1) (Kropp 1982; Parke et al. 1983;
Repáč 1996a) than vegetative inoculum. Vermiculite–peat inoculum was mixed
with potting substrate at the proportion 1:4 (Rincón et al. 2001), 1:10 (Hortal et al.
2009), as well as 1:64 (Rincón et al. 2001). Baum et al. (2009) expressed a ratio of
soil:vermiculite-peat inoculum mixture (10:1) by weight. Parladé et al. (1999) and
Baum et al. (2000, 2002) carried out inoculation by mixing alginate beads with
potting substrate in the proportion 1:20 (v:v). Before filling containers, Gagnon
et al. (1991) mixed alginate beads with the substrate so that each seedling received a
volume of 17 ml of beads. Gagnon et al. (1995) mixed liquid inoculum with
substrate in a cement mixer to get a final volume of 30 ml of mycelial slurry per
seedling.
In nursery bed experiments, vegetative inoculum was broadcast evenly over
nursery bed (0.5–2.8 l.m 2) and incorporated into the upper 10–12 cm of substrate
with handtools (Marx et al. 1978; Mortier et al. 1988; Duponnois and Garbaye
1991; Repáč 1996b; Garbaye and Churin 1997). Marx and Bryan (1975) reported
application of P. tinctorius inoculum to fumigated soil at a ratio 1:8 (v:v) in upper
10 cm layer of plot.
Cline and Reid (1982) injected 20 ml of mycelial slurry into a root zone of
container-grown seedlings at a depth of 6–8 cm using a glass syringe. Garbaye et al.
56 I. Repáč
(1988), Machón et al. (2006), and Gange et al. (2005) added 50 ml of vermiculite–
peat inoculum and 20 ml of mycelial slurry, respectively, into containers in contact
with the roots of transplanted seedlings. Aucina et al. (2007) placed a layer of pine
or oak litter on the surface of the nursery bed soil with seedlings to influence the
growth and ECM communities of seedlings. Kropáček and Cudlı́n (1989) and
Repáč (2007) evenly spread alginate-bead inoculum below surface of substrate
immediately before seed sowing.
Perhaps the most capable way of expression of amount of vegetative inoculum is
in a dry weight of mycelium, enabling the comparison of inoculum rate between
experiments of different patterns. Unfortunately, to ascertain quantity of mycelium
in vermiculite–peat carrier is almost impossible. Nevertheless, Mortier et al. (1988)
reported 1–2 g of mycelium (dry weight) per m2 in this type of inoculum. Mortier
et al. (1988), Duponnois and Garbaye (1991), Vijaya and Srivasuki (1999), and
Repáč (2007) referred 2 g, 2–10 g, 4 g, and 5 g of mycelium in dry weight per m2,
respectively, in alginate-bead inoculum. In the pot experiments, each seedling
received either 6.82 mg (Gagnon et al. 1991) or 12 mg (Gagnon et al. 1995) of
dried mycelium or 20–80 mg of mycelium in fresh weight (Parladé et al. 1999).
3.5 Field Inoculation
Inoculation of seedlings at the time of field planting is time consuming, requires

more inoculum, and the introduced fungus must be compatible with native micro-
organisms and climatic conditions of the planting site (Riffle and Maronek 1982).
Thus, nursery inoculated seedlings are more frequently outplanted (Roldan et al.
1996; Garbaye and Churin 1997; Núñez et al. 2006; Rincón et al. 2007) than
inoculated at the time or after field planting. Castellano (1996) reviewed most of
available literature (including unpublished data) on outplanting performance of
ECM-inoculated seedlings and provided insight to the fungus-inoculum type-
host-location combinations in field experiments. Inoculum types and application
methods in the field are very similar to nursery inoculation of container-grown
seedlings. Various natural organic materials and organic wastes are often used and
may be considered as a simple source of ECM fungi propagules. Hallsby (1995)
planted Norway spruce seedlings to mounds containing forest floor material from
the F- and H-layers. Querejeta et al. (1998) added 150 ml of pine forest soil (top
20 cm of mineral soil) to the planting holes of Pinus halepensis seedlings at the
time of planting. Organic materials, particularly decayed wood or mixtures contain-
ing decayed wood were equal or superior to mineral substrates for supporting
ECM activity on planted seedlings (Harvey et al. 1997). Larcheveque et al.
(2006) incorporated fresh co-composted sewage sludge and greenwastes (20 or
40 kg.m 2 of compost) into the soil at each stem of 1-year-old seedlings of Quercus
ilex, Pinus halepensis, and P. pinea.
In field experiments with laboratory produced inoculum, e.g., Baum et al. (2002)
applied 50 ml of alginate beads containing mycelium of Laccaria laccata around
each Populus trichocarpa cutting (1.5-m long annual shoots, diameter 13–17 mm)
in 50-mm soil depth. Duñabeitia et al. (2004) inoculated seedlings few days after
outplanting (and reinoculated 5 months later) by watering at 5 cm around the stem
with 200 ml of spore slurry of Scleroderma citrinum, Pisolithus arhizus, and
Leccinum scabrum. Menkis et al. (2007) wrapped root systems of Pinus sylvestris
and Picea abies seedlings in a filter paper containing mycelium of Cenococcum
geophilum, Piceirhiza bicolorata or Hebeloma crustuliniforme, overlaid with
damp peat–sand mixture, wrapped in a paper towel, and planted seedlings on
poor sandy soil.
3.6 Commercial Inoculum
It is relatively simple to produce sufficient volumes of inoculum for small research

studies, but it is extremely difficult to produce sufficient quantities of inoculum to
support commercial nursery inoculation. Since 1976, several formulations of vege-
tative and spore inocula of “super-strain” of Pisolithus tinctorius were produced
commercially (e.g., inoculum trademarked MycoRhiz®) and used in large scale for
inoculation of millions of bareroot and containerized pine and oak seedlings in USA
(Marx 1991). In France, a company called Somycel produced vegetative beads
inoculum, which substantially increased reforestation effort of coniferous trees
followed by nursery inoculation (Kropp and Langlois 1990).
Numerous entities of international, national, or local importance produce and
offer ECM fungal inocula for commercial purposes. For example, several European
producers of mycorrhizal fungi inocula constituted organization called “Federation
of European Mycorrhizal Fungi Producers” (FEMFiP) in 2003, aimed to achieve
and maintain high standards for these products in Europe (Federation of European
Mycorrhizal Fungi Producers 2010). The most products of FEMFiP contain arbus-
cular fungi and are intended to horticulture, agriculture, and landscape sectors. The
companies PlantWorks Ltd. (Great Britain, product TerraVital), Biorize (France,
product Ectorize), and Symbio-m (Czech Republic, product Ectovit) offer ECM
inocula for inoculation of forest tree species.
Spore and mycelial inocula are used in operational scale to match the ECM fungi
to the host and environment in which they are required to thrive. Natural biostimu-
lants and ingredients supporting the development of ECM symbiosis (natural
humates, sea-grass extracts, ground minerals) are usually added to commercial
inoculum. Inoculation techniques are in principle the same as described in
Sects. 3.4.2 and 3.5. Compared to research-scale inoculum formulations, gel for-
mulation (slurry), prepared by diluting ECM inoculum containing naturally degrad-
able granules of water-retaining gel in water, is more frequently used in a
commercial scale. The slurry can be sprayed onto the substrate before seed sowing,
into the rooting zone of transplanted seedlings, or root systems of seedlings can be
dipped into the slurry. The production and marketing of inocula on a commercial
scale increased recently and further potential appears to be quite large.
58 I. Repáč
3.7 Conclusion
There are a variety of ECM inoculum types, inoculum preparation methods, and
inoculation techniques to initiate the development of ectomycorrhizae on forest tree
seedlings. The selection of fungi, inoculum type, and inoculation method all
depends on the intended purpose of inoculation. Better understanding of the
structure and functioning of ECM symbiosis and better performance of inoculated
seedlings under nursery and field conditions are the ultimate goals of research on
artificial mycorrhization, and therefore further development of inoculation methods
in operational conditions is desirable. Although sterile conditions are a faraway
from natural ones, techniques of pure culture synthesis are necessary for compati-
bility, structural, physiological, molecular, and other studies.
Application of ECM inoculum to substrate does not guarantee that ectomycor-
rhizae will develop on a host plant. Success of inoculation depends on the type and
age of inoculum used, inoculum dose, timing of inoculation, inoculum placement in
the growing medium, etc. (Riffle and Maronek 1982; Mortier et al. 1988; Rodrigues
et al. 1999). Besides inoculum and inoculation pattern, interspecific and intraspe-
cific host–fungus variation, environmental conditions, seedling production prac-
tices, and other factors are responsible for seedling response to inoculation (Kropp
and Langlois 1990; Castellano 1996; Menkis et al. 2007; Duponnois et al. 2008).
Despite of (or just because of) inconsistency of effects of introduced fungi on
performance of inoculated seedlings, more research is needed on the screening of
potential host–fungus species and genotype combinations and the host–fungus–
environment interactions to optimize the effect of fungi on plants. Also, researchers
might be able to make much progress in simplifying the application of ECM fungi
in seedling inoculation. Because of natural complexity and diversity of ECM
relationships, we have to realize that their understanding will neither be completed
nor their use completely reliable. Nevertheless, when we consider extensive area of
treeless lands and adverse forest sites required artificial regeneration, the impor-
tance of artificial mycorrhization of seedlings as reforestation and afforestation
management tool is obvious.
References
Aspray TJ, Frey-Klett P, Jones JE, Whipps JM, Garbaye J, Bending GD (2006) Mycorrhization
helper bacteria: a case of specifity for altering ectomycorrhiza architecture but not ectomycor-
rhiza formation. Mycorrhiza 16:533–541
Aucina A, Rudawska M, Leski T, Skridaila A, Riepsas E, Iwanski M (2007) Growth and
mycorrhizal community structure of Pinus sylvestris seedlings following the addition of forest
litter. Appl Environ Microb 73:4867–4873
Baum C, Schmid K, Makeschin F (2000) Interactive effects of substrates and ectomycorrhizal
colonization on growth of poplar clone. J Plant Nutr Soil Sci 163:221–226
Baum C, Stetter U, Makeschin F (2002) Growth response of Populus trichocarpa to inoculation by
the ectomycorrrhizal fungus Laccaria laccata in a pot and a field experiment. For Ecol Manage
163:1–8
Baum C, Toljander YK, Eckhardt KU, Weih M (2009) The significance of host-fungus combina-
tions in ectomycorrhizal symbioses for the chemical quality of willow foliage. Plant Soil
323:213–224. doi:10.1007/s111040099928x
Becerra AG, Menoyo E, Lett I, Li CY (2009) Alnus accuminata in dual symbiosis with Frankia
and two different ectomycorrhizal fungi (Alpova austroalnicola and Alpova diplophloeus)
growing in soilless growth medium. Symbiosis 47:85–92
Bending GD, Poole EJ, Whipps JM, Read DJ (2002) Characterisation of bacteria from Pinus
sylvestris – Suillus luteus mycorrhizas and their effects on root-fungus interactions and plant
growth. FEMS Microbiol Ecol 39:219–227
Bogeat-Triboulot MB, Bartoli F, Garbaye J, Marmeisse R, Tagu D (2004) Fungal ectomycorrhizal
community and drought affect root hydraulic properties and soil adherence to roots of Pinus
pinaster seedlings. Plant Soil 267:213–223
Borchers SL, Perry DA (1990) Growth and ectomycorrhiza formation of Douglas-fir seedlings
grown in soils collected at different distances from pioneering hard-woods in southwest
Oregon clear-cuts. Can J For Res 20:712–721
Brunner I (1993) Production of ectomycorrhizal Picea abies – Hebeloma crustuliniforme seed-
lings for ecological studies: effects of synthesis techniques on the morphology of the symbio-
sis. Water Air Soil Poll 68:231–240
Brunner I, Amiet R, Zollinger M, Egli S (1992) Ectomycorrhizal syntheses with Picea abies and
three fungal species: a case study on the use of an in vitro technique to identity naturally
occurring ectomycorrhizae. Mycorrhiza 2:89–96
Castellano MA (1996) Outplanting performance of mycorrhizal inoculated seedlings. In: Mukerji
KG (ed) Concepts in mycorrhizal research. Kluwer Academic Publishers, Netherlands,
pp 223–301
Chen YL, Kang LH, Dell B (2006) Inoculation of Eucalyptus urophylla with spores of Sclero-
derma in a nursery in south China: comparison of field soil and potting mix. For Ecol Manage
222:439–449
Cline ML, Reid CPP (1982) Seed source and mycorrhizal fungus effects on growth of contain-
erized Pinus contorta and Pinus ponderosa seedlings. For Sci 28:237–250
Corrêa A, Strasser RJ, Martins-Loução MA (2006) Are mycorrhiza always beneficial? Plant Soil
279:65–73
Duñabeitia M, Rodrı́guez N, Salcedo I, Sarrionandia E (2004) Field mycorrhization and its
influence on the establishment and development of the seedlings in a broadleaf plantation in
the Basque country. For Ecol Manage 195:129–139
Duponnois R, Garbaye J (1991) Techniques for controlled synthesis of Douglas-fir – Laccaria
laccata ectomycorrhizal symbiosis. Ann Sci For 48:641–650
Duponnois R, Kisa M, Prin Y, Ducousso M, Plenchette Ch, Lepage M, Galiana A (2008) Soil
factors influencing the growth response of Acacia holosericea A. Cunn. ex G. Don to
ectomycorrhizal inoculation. New For 35:105–117
Federation of European Mycorrhizal Fungi Producers (2010) http://www.fenfip.com. Cited 15 Feb
2010
Flores R, Dı́az G, Honrubia M (2008) Mycorrhizal synthesis and basidioma primordium formation
between Abies guatemalensis and Laccaria bicolor. Nova Hedwigia 87:153–160
Fortin JA, Piché Y, Lalonde M (1980) Technique for the observation of early morphological
changes during ectomycorrhiza formation. Can J Bot 58:361–365
Gagnon J, Langlois CG, Garbaye J (1991) Growth and ectomycorrhiza formation of container-
grown red oak seedlings as a function of nitrogen and inoculum type of Laccaria bicolor. Can J
For Res 21:966–973
Gagnon J, Langlois CG, Bouchard D, Le Tacon F (1995) Growth and ectomycorrhizal formation
of container-grown Douglas-fir seedlings inoculated with Laccaria bicolor under four levels of
nitrogen fertilization. Can J For Res 25:1953–1961
Gange AC, Gane DRJ, Chen Y, Gong M (2005) Dual colonization of Eucalyptus urophylla S.T.
Blake by arbucular and ectomycorrhizal fungi affects levels of insect herbivore attack. Agric
For Entomol 7:253–263
60 I. Repáč
Gáper J, Repáč I, Cudlı́n P (1999) In vitro mycorrhizae syntheses of Inocybe lacera and Suillus
bovinus with Picea abies. In: Moreno G et al (eds) Book of abstracts of the XIII Congress of
European Mycologists, Alcalá de Henares (Madrid), p 41
Garbaye J, Churin JL (1997) Growth stimulation of young oak plantations inoculated with the
ectomycorrhizal fungus Paxillus involutus with special reference to summer drought. For Ecol
Manage 98:221–228
Garbaye J, Delwaulle JC, Diangana D (1988) Growth response of Eucalypts in the Congo to
ectomycorrhizal inoculation. For Ecol Manage 24:151–157
Giomaro G, Sisti D, Zambonelli A, Amicucci A, Cecchini M, Comandini O, Stocchi V (2002)
Comparative study and molecular characterization of ectomycorrhizas in Tilia americana and
Quercus pubescens with Tuber brumale. FEMS Microbiol Lett 216:9–14
Guerin-Laguette A, Vaario LM, Matsushita N, Shindo K, Suzuki K, Lapeyrie F (2003) Growth
stimulation of Shiro-like mycorrhiza forming mycelium of Tricholoma matsutake on solid
substrates by non-ionic surfactants or vegetable oil. Mycol Prog 2:37–44
Hallsby G (1995) Field performance of outplanted Norway spruce – effects of organic-matter
amendments and site preparation. Can J For Res 25:1356–1367
Harvey AE, Page-Dumroese DS, Jurgensen MF, Graham RT, Tonn JR (1997) Site preparation
alters soil distribution of roots and ectomycorrhizae on outplanted western white pine and
Douglas-fir. Plant Soil 188:107–117
Herrmann S, Oelm€uller R, Buscot F (2004) Manipulation of the onset of ectomycorrhiza formation
by indole-3-acetic acid, activated charcoal or relative humidity in the association between oak
microcuttings and Piloderma croceum: influence on plant development and photosynthesis.
J Plant Physiol 161:509–517
H€ogberg P, H€ogberg MN, Quist ME, Ekblad A, N€asholm T (1999) Nitrogen isotope fractionation
during nitrogen uptake by ectomycorrhizal and non-mycorrhizal Pinus sylvestris. New Phytol
142:569–576
onig K, Riefler M, Kottke I (2000) Survey of Paxillus involutus (Batsch) Fr. inoculum and
H€
fruitbodies in a nursery by IGS-RFLPs and IGS sequences. Mycorrhiza 9:315–322
Hortal S, Pera J, Parladé J (2008) Tracking mycorrhizas and extraradical mycelium of the edible
fungus Lactarius deliciosus under field competition with Rhizopogon spp. Mycorrhiza
18:69–77
Hortal S, Pera J, Parladé J (2009) Field persistence of the edible ectomycorrhizal fungus Lactarius
deliciosus: effects of inoculation strain, initial colonization level, and site characteristics.
Kozdrój J, Piotrowska-Seget Z, Krupa P (2007) Mycorrhizal fungi and ectomycorrhiza associated
bacteria isolated from an industrial desert soil protect pine seedlings against Cd(II) impact.
Ecotoxicology 16:449–456
Kropáček K, Cudlı́n P (1989) Preparation of granulated mycorrhizal inoculum and its use in forest
nurseries. In: Vančura V, Kunc F (eds) Interrelationships between microorganisms and plants
in soil. Academia, Praha, pp 177–182
Kropp BR (1982) Rotten wood as mycorrhizal inoculum for containerized western hemlock. Can J
For Res 12:428–431
Kropp BR, Langlois CG (1990) Ectomycorrhizae in reforestation. Can J For Res 20:438–451
Kr€uger A, Peškan-Bergh€ ofer T, Frettinger P, Herrmann S, Buscot F, Oelm€uller R (2004) Identifi-
cation of premycorrhiza-related plant genes in the association between Quercus robur and
Piloderma croceum. New Phytol 163:149–157
Langer I, Krpata D, Peintner U, Wenzel WW, Schweiger P (2008) Media formulation influences
in vitro ectomycorrhizal synthesis on the European aspen Populus tremula L. Mycorrhiza
18:297–307. doi:10.1007/s0057200801825
Larcheveque M, Ballini C, Korboulewsky N, Montes N (2006) The use of compost in afforestation
of Mediterranean areas: effects on soil properties and young tree seedlings. Sci Total Environ
369:220–230
Le Tacon F, Jung G, Michelot P, Mugnier M (1983) Efficacité en pépiničre forestiére d’un

inoculum de champignon ectomycorhizien produit en fermenteur et inclus dans une matrice
de polyméres. Ann Sci For 40:165–176
Machón P, Santamarı́a O, Pajares JA, Alves-Santos FM, Diez JJ (2006) Influence of the ectomy-
corrhizal fungus Laccaria laccata on pre-emergence, post-emergence and late damping-off by
Fusarium moniliforme and F. oxysporum on Scots pine seedlings. Symbiosis 42:153–160
Martı́n-Pinto P, Pajares J, Dı́ez J (2006) In vitro effects of four ectomycorrhizal fungi, Boletus
edulis, Rhizopogon roseolus, Laccaria laccata and Lactarius deliciosus on Fusarium damping
off in Pinus nigra seedlings. New For 32:323–334. doi:10.1007/s1105600690067
Marx DH (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to
pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil
bacteria. Phytopathology 59:153–163
Marx DH (1991) The practical significance of ectomycorrhizae in forest establishment. In:
H€agglund B (ed) Ecophysiology of ectomycorrhizae of forest trees. The Marcus Wallenberg
foundation symposia proceedings: 7, Stralins Tryckeri AB, Grycksbo, Sweden, pp 54–90
Marx DH, Bryan WC (1975) Growth and ectomycorrhizal development of Loblolly pine seedlings
in fumigated soil infested with the fungal symbiont Pisolithus tinctorius. For Sci 21:245–254
Marx DH, Cordell CE (1989) The use of specific ectomycorrhizas to improve artificial forestation
practices. In: Whipps JM, Lumsden RD (eds) Biotechnology of fungi for improving
plant growth. Press Syndicate of the University of Cambridge, British Mycological Society,
New York, pp 1–26
Marx DH, Kenney DS (1982) Production of ectomycorrhizal fungus inoculum. In: Schenck NC
(ed) Methods and principles of mycorrhizal research. American Phytopathology Society, St.
Paul, MN, pp 131–146
Marx DH, Morris WG, Mexal JG (1978) Growth and ectomycorrhizal development of Loblolly
pine seedlings in fumigated and nonfumigated nursery soil infested with different fungal
symbionts. For Sci 24:193–203
Marx DH, Ruehle JL, Kenney DS, Cordell CE, Riffle JW, Molina RJ, Pawuk WH, Navratil S,
Tinus RW, Goodwin OC (1982) Commercial vegetative inoculum of Pisolithus tinctorius and
inoculation techniques for development of ectomycorrhizae on container-grown tree seedlings.
For Sci 28:373–400
Mauperin C, Mortier F, Garbaye J, Le Tacon F, Carr G (1987) Viability of an ectomycorrhizal
inoculum produced in a liquid medium and entrapped in a calcium alginate gel. Can J Bot
65:2326–2329
Menkis A, Vasiliauskas R, Taylor AFS, Stenlid J, Finlay R (2007) Afforestation of abandoned
farmland with conifer seedlings inoculated with three ectomyorrhizal fungi – impact on plant
performance and ectomycorrhizal community. Mycorrhiza 17:337–348
Molina R (1979) Pure culture synthesis and host specificity of red alder mycorrhizae. Can J Bot
57:1223–1228
Molina R, Palmer JG (1982) Isolation, maintenance, and pure culture manipulation of ectomycor-
rhizal fungi. In: Schenck NC (ed) Methods and principles of mycorrhizal research. American
Phytopathology Society, St. Paul, MN, pp 115–129
Molina R, Smith JE, McKay D, Melville LH (1997) Biology of the ectomycorrizal genus,
Rhizopogon. New Phytol 137:519–528
Moore LM, Jansen AE, Van Griensen LJLD (1989) Pure culture synthesis of ectomycorrhizas with
Cantharellus cibarius. Acta Bot Neerl 38:273–278
Mortier F, Le Tacon F, Garbaye J (1988) Effects of inoculum type and inoculation dose on
ectomycorrhizal development, root necrosis and growth of Douglas fir seedlings inoculated
with Laccaria laccata in a nursery. Ann Sci For 45:301–310
Moser M (1958) Die k€ unstliche Mykkorhizaimpfung an Forstpflanzen. I. Erfahrungen bei der
Reinkulture von Mykorrhizapilzen. Forstwiss Centralbl 77:32–40
Moser M (1960) Die Gattung Phlegmacium (Schleimk€opfe). Verlag Julius Klinkhardt, Bad
Heilbrunn, Germany, 440 pp
62 I. Repáč
Niemi K, Vuorinen T, Ernstsen A, H€aggman H (2002) Ectomycorrhizal fungi and exogenous

auxins influence root and mycorrhiza formation of Scots pine hypocotyl cuttings in vitro. Tree
Physiol 22:1231–1239
Niemi K, Julkunen-Titto R, H€aggman H, Sarjala T (2006) Suillus variegatus causes significant
changes in the content of individual polyamines and flavonoids in Scots pine seedlings during
mycorrhiza formation in vitro. J Exp Bot 58:391–401. doi:10.1093/jxb/erl209
Núñez JAD, Serrano JS, Barreal JAR, González JAS (2006) The influence of mycorrhization with
Tuber melanosporum in the afforestation of a Mediterranean site with Quercus ilex and
Quercus faginea. For Ecol Manage 231:226–233
Obase K, Tamai Y, Yajima T, Miyamoto T (2009) Mycorrhizal synthesis of four ectomycorrhizal
fungi in potted Populus maximowiczii seedlings. Mycoscience 50:143–145
Ohta A (1990) A new medium for mycelial growth of mycorrhizal fungi. T Mycol Soc JPN
31:323–334
Pachlewski R, Pachlewska J (1974) Studies on symbiotic properties of mycorrhizal fungi of pine
(Pinus sylvestris L.) with the aid of the methods of mycorrhizal synthesis in pure cultures on
agar. For Res Inst Press, Warsaw, Poland, 228 pp
Parke JL, Linderman RG, Trappe JM (1983) Effects of forest litter on mycorrhiza development
and growth of Douglas-fir and western red cedar seedlings. Can J For Res 13:666–671
Parladé J, Álvarez IF, Pera J (1996a) Ability of native ectomycorrhizal fungi from northern Spain
to colonize Douglas-fir and other introduced conifers. Mycorrhiza 6:51–55
Parladé J, Pera J, Alvarez IF (1996b) Inoculation of containerized Pseudotsuga menziesii and
Pinus pinaster seedlings with spores of five species of ectomycorrhizal fungi. Mycorrhiza
6:237–245
Parladé J, Alvarez IF, Pera J (1999) Coinoculation of containerized Douglas-fir (Pseudotsuga
menziesii) and maritime pine (Pinus pinaster) seedlings with the ectomycorrhizal fungi
Laccaria bicolor and Rhizopogon spp. Mycorrhiza 8:189–195
Peterson RL, Chakravarty P (1991) Techniques in synthesizing ectomycorrhiza. In: Norris JR,
Read DJ, Varma AK (eds) Methods in microbiology, vol 23. Academic Press Limited, London,
pp 75–106
Querejeta JI, Roldán A, Albaladejo J, Castillo V (1998) The role of mycorrhizae, site preparation,
and organic amendment in the afforestation of a semi-arid mediterranean site with Pinus
halepensis. For Sci 44:203–211
Repáč I (1996a) Inoculation of Picea abies (L.) Karst. seedlings with vegetative inocula of
ectomycorrhizal fungi Suillus bovinus (L.:Fr.) O. Kuntze and Inocybe lacera (Fr.) Kumm.
New For 12:41–54
Repáč I (1996b) Effects of forest litter on mycorrhiza formation and growth of container-grown
Norway spruce (Picea abies [L.] Karst.) seedlings. Lesnictvı́ 42:317–324
Repáč I (2007) Ectomycorrhiza formation and growth of Picea abies seedlings inoculated with
alginate-bead fungal inoculum in peat and bark compost substrates. Forestry 80:517–530
Riffle JW, Maronek DM (1982) Ectomycorrhizal inoculation procedures for greenhouse and
nursery studies. In: Schenck NC (ed) Methods and principles of mycorrhizal research. Ameri-
can Phytopathology Society, St. Paul, MN, pp 147–155
Rincón A, Alvarez IF, Pera J (2001) Inoculation of containerized Pinus pinea L. seedlings with
seven ectomycorrhizal fungi. Mycorrhiza 11:265–271
Rincón A, de Felipe MR, Fernández-Pascual M (2007) Inoculation of Pinus halepensis Mill. with
selected ectomycorrhizal fungi improves seedling establishment 2 years after planting in a
degraded gypsum soil. Mycorrhiza 18:23–32
Rodrigues LS, Kasuya MCM, Borges AC (1999) Viability of ectomycorrhizal fungus mycelium
entrapped in calcium alginate gel. Mycorrhiza 8:263–266
Roldan A, Querejeta I, Albaladejo J, Castillo V (1996) Growth response of Pinus halepensis to
inoculation with Pisolithus arhizus in a terraced rangeland amended with urban refuse. Plant
Soil 179:35–43
Sarjala T, Taulavuori K (2004) Fluctuation in free and conjugated polyamines in Scots pine
seedlings after changes in temperature and daylength. Acta Physiol Plant 26:271–279
Šašek V, Musı́lek V (1967) Cultivation and antibiotic activity of mycorrhizal basidiomycetes.
Folia Microbiol 12:515–523
Theodorou C (1984) Mycorrhizal inoculation of pine nurseries by spraying basidiospores onto soil
prior to sowing. Aust For 47:76–78
Timonen S, Tammi H, Sen R (1997) Outcome of interactions between genets of two Suillus spp.
and different Pinus sylvestris genotype combinations: identity and distribution of ectomycor-
rhizas and effects on early seedlings growth in N-limited nursery soil. New Phytol
137:691–702
Vijaya T, Srivasuki KP (1999) Effect of inoculum type and inoculation dose on ectomycorrhizal
development and growth of Acacia nilotica seedlings inoculated with Pisolithus tinctorius in a
nursery. Biologia 54:439–442
Vozzo JA, Hacskaylo E (1971) Inoculation of Pinus caribaea with ectomycorrhizal fungi in Puerto
Rico. For Sci 17:239–245
Vrålstad T, Schumacher T, Taylor AFS (2002) Mycorrhizal synthesis between fungal strains of the
Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts. New Phytol
153:143–152
Wallander H, Fossum A, Rosengren U, Jones H (2005) Ectomycorrhizal fungal biomass in roots
and uptake of P from apatite by Pinus sylvestris seedlings growing in forest soil with and
without wood ash amendment. Mycorrhiza 15:143–148
Wong KKY, Fortin JA (1989) A Petri dish technique for the aseptic synthesis of ectomycorrhizae.
Can J Bot 67:1713–1716
.
Part II
Biotechnological Aspect of ECM
.
Chapter 4
Systematics and Ecology of Tropical
Ectomycorrhizal Fungi Using Molecular
Approaches
Rivière-Dobigny Taiana
4.1 Evaluation of Tropical ECM Species Composition
4.1.1 Introduction
Traditionally, surveys on ECM species communities rely on morphological

descriptions of sporocarps due to previous species collections. Such macroscopic
approaches are inaccurate since sporocarp identifications only provide an incom-
plete picture of ECM fungal diversity (Egger 1995; Gardes and Bruns 1996; Grogan
et al. 2000; Jonsson et al. 2000). Indeed, while sporocarps are necessarily associated
with ectomycorrhizas, a fungus forming ectomycorrhizas may not always form
sporocarps (Horton and Bruns 2001). Furthermore, investigations on ECM fungi
are delicate as morphological characters of root tips are usually not sufficient for
species recognition (Bruns et al. 1998). This has long represented a considerable
limitation to our understanding of ECM diversity, and thus an adequate understand-
ing of ECM ecology (Debaud et al. 1999; Kretzer et al. 2003). Fortunately,
molecular tools have proved to be of great help to solve taxonomic discrepancies
and have, therefore, considerably improved our knowledge on their ecology
(Horton and Bruns 2001). Indeed, molecular techniques enable identification of
ECM genera or species through analyses conducted on DNA extracted from
root tips. The latter approach is often achieved through comparisons with DNA
sequences database of ribosomal internal transcribed spacer (ITS) or mitochondrial
sequences (Gardes et al. 1991; Kendall 2007). Also, cloning and sequencing T-RFLP
(Terminal Restriction Fragment Polymorphism) and DGGE (Denaturing Gradient
Gel Electrophoresis) strategies have been employed to describe ECM communities in
soil (Landeweert et al. 2003; Koide et al. 2005).
R-D. Taiana
IRD, avenue de Maradi, BP 11416 Niamey, Niger
e-mail: taianariviere@yahoo.co.uk

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_4,
68 R.-D. Taiana
To our knowledge, the mycorrhizal status of numerous tropical indigenous tree

species remains undocumented even if large areas of tropical and subtropical
forests are dominated by ECM trees (Redhead 1980; Alexander and H€ogberg
1986), suggesting a key role of these symbiosis in the functioning of some tropical
forest ecosystems (Onguene and Kuyper 2001). For instance, the Dipterocarpaceae
family in South East Asia comprises 470 tree species, which largely dominate
tropical rainforests and represent a major source of commercial timber (Maury-
Lechon and Curtet 1998). These tree species have been shown to be associated with
various fungal genera such as Russula, Boletus, Cortinarius, Lactarius, Laccaria,
Pisolithus, Amanita, Scleroderma, Suillus, Strobilomyces, and Cantharellus (Smits
1992; Watling and Lee 1998; Natarajan et al. 2005; Rivière et al. 2007; Peay et al.
2010). In the same manner, surveys in African dry woodlands and tropical rain-
forests revealed that the dominant group of ECM trees includes caesalpinioid
legumes (12 genera in the Amherstieae and one genus, Afzelia, in the Detarieae)
and Phyllanthaceae (one genus, Uapaca) (Thoen and Bâ 1989; Newbery et al. 1997;
Sanon et al. 1997; Onguene and Kuyper 2001), and associated ECM fungi belong to
several genera, mainly Russula, Lactarius, Amanita, Boletus, and Cantharellus
(Buyck et al. 1996; Eberhardt and Verbeken 2004). ECM trees can form local
patches that contribute from 45 to 70% of the basal rainforest area, such as in the
Korup National Park in Cameroon, where three species Microberlinia bisulcata,
Tetraberlinia bifoliolata, and Tetraberlinia moreliana are dominant (Newbery et al.
1997).
South American tropical rainforests are reported to be mainly dominated by
vesicular-arbuscular mycorrhizal associations (Picone 2000). For example, in
Brazil, the mycorrhizal status of the Araucaria forest and the Atlantic rainforest
true 29 native species belonging to 19 families revealed no evidence of ectomycor-
rhizal colonization (Andrade et al. 2000). Some authors reported ECM occurrence
in native Brazilian trees such as Thomazini (1974) and Singer and Araujo (1979) on
Caesalpinioidae species. Surveys in the Pakaraima Mountains of western Guyana
revealed previously undocumented forests dominated by ECM leguminous trees,
with a rich assemblage of ECM mycobionts. These fungi belonged to the Boleta-
ceae, Amanitaceae, Russulaceae, Cortinariaceae, Cantharellaceae, Clavulinaceae,
and Entolomataceae Basidiomycete families (Henkel et al. 2002).
There is increasing evidence that some tropical ECM communities are more
diversified than previously supposed, and so, many questions are still unstudied.
Indeed, ECM diversity is still poorly documented in tropical rainforests and so, with
regards to forests destructions going on, sampling efforts are urgently needed. In
this study, we examined ECM Basidiomycete species’ taxonomic structure in
natural tropical rainforests located in West Africa and Western India. We used
both morphological and molecular methods to identify ECM fungal species occur-
ring on the root tip, and importantly, in order to link the above- and belowground
ECM diversity, sporocarp and ectomycorrhizas sequences were compared. In order
to propose species’ identification through molecular typing of sporocarps vs.
ectomycorrhiza, 40 reference sequences from available databases were included
in our phylogenetic analyses.
4 Systematics and Ecology of Tropical Ectomycorrhizal Fungi 69
4.1.2 ECM Systematics and Diversity Assessment
ECM species diversity is poorly documented in natural tropical rainforests. The

primary difficulty lies in the identification of the species. Indeed, many species’
descriptions are still going on mainly based on morphological descriptions of the
sporocarps, and so, tropical investigations, which forget root tip morphotypes, are
most of the time incomplete. In this context, molecular methods offer a great
opportunity to evaluate ECM taxonomic diversity including ECM root DNA
extracts. We present here a survey of ECM Basidiomycete communities conducted
in two tropical rainforests, one located in Africa (Western Upper Guinea) and the
other in Asia (Western Ghats, India). To solve taxonomic discrepancies, molecular
analysis involved sequencing a fragment of the mitochondrial large subunit rRNA
gene and comparative phylogenetic analysis using sequences from European speci-
mens as taxonomic benchmarks. Here, we focus on ML5/ML6 sequences for three
main reasons. We are aware that the ML5/ML6 region often provides identical
sequences for closely related species, thus limiting the interest of this conservative
DNA fragment at the species level. The ITS fragment, on the contrary, is more
accurate for species identification (Bruns et al. 1998). Here, we had to choose ML5/
ML6 fragment for three main reasons (1) with regards to the high morphological
diversity of sporocarps and numerous ectomycorrhizas found in the field, it first
appeared pivotal to obtain rapid but nonambiguous sequences allowing to identify
the samples at the genus level, and yet many sequences are available on Genbank;
(2) as recommended by Gardes and Bruns (1993), we previously sequenced the
ITS fragment of 37 Guinean Russula and 15 Indian Russula specimens, but
the sequences were too variable to be unambiguously aligned and none of the
sequences matched with known species; and (3) we consistently observed that ITS
primers often amplify plant DNA from ectomycorrhizas, whereas the ML5/ML6
primers never did.
Inventory of the plant species composition is a prerequisite to the study on ECM
fungal community; consequently, it is essential to describe flora diversity before
ECM fungal one. Study sites were located in Southern Guinea and in Western India.
Southern Guinea forests cover hills and mountains ranging in altitude from 500 m
in the Ziama forest (8 510 N, 9 310 W) to 1,752 m on the Mount Nimba forest
(7 600 N, 8 490 W). They are typical evergreen or semi-evergreen rainforests with
a mean annual rainfall of 2,500–3,000 mm and a dry season from January to March.
These Guinean forests shelter ECM trees belonging to the Caesalpiniaceae (Afzelia
bella, Paramacrolobium coeruleum, Anthonotha fragans, A. macrophylla, Crypto-
sepalum tetraphyllum, Pelligriniodendron diphyllum, Gilbertiodendron limba) and
the Phyllanthaceae (Uapaca heudelotii, U. esculenta, U. guineensis, and U. cheva-
lieri). Two additional species, Uapaca somon and Afzelia africana, were found in
the dryer and lower woodlands bordering the Mount Nimba rainforest.
The Kadamakal Reserve Forest is located in the Western Ghats, India, in the
district of Kodagu (Karnataka) near the village of Uppangala (12 300 N; 75 390 W).
Its altitude ranges from 400 to 600 m. Annual rainfall is about 5,200 mm with a
70 R.-D. Taiana
marked dry season from December to March. Vegetation is a dense moist evergreen
forest dominated by three species, Dipterocarpus indicus, Kingiodendron pinna-
tum, and Humboltia brunonis (Pascal 1984). Two ectomycorrhizal Dipterocarpa-
ceae species dominate the high canopy, Vateria indica and Dipterocarpus indicus,
which together represent 41.2% of the basal area (Pélissier et al. 1998). To optimize
the assessment of the floristic diversity, the Uppangala forest was sampled follow-
ing a previously designed transect (3 plots from 180 to 370 m long and 20 m wide;
see Pélissier et al. 1998).
Sporocarps belonging to Basidiomycete families that were typically ectomycor-
rhizal were collected each August during 4 successive years in Guinea. In India,
samples were collected during 2 successive years. In each spot, sporophores and
ectomycorrhizas were collected in order to subsequent DNA extraction, PCR, and
sequencing (see Rivière et al. 2007 for protocols). A total of 198 sequences were
edited and assembled. In addition, 40 reference fragments from known boreal and
temperate species were downloaded from NCBI databank. These data were used as
external taxonomic benchmarks as well as references to investigate any phylogeo-
graphic pattern. Most of these sequences were retrieved from the Bruns et al. (1998)
database (http://plantbio.berkeley.edu/~bruns/). Five sequences (Tulasnella irregu-
laris, Sebacina sp., and three Cantharellus spp.) were used as outgroups (Bruns
et al. 1998).
4.1.2.1 ECM Status of Tropical Trees
In Guinea, the presence of ectomycorrhizas was confirmed on six caesalpinioid

species, A. africana, A. bella, A. fragans, A. macrophylla, G. limba, and P.
coeruleum, and five Uapaca species (U. guineensis, U. esculenta, U. heudelottii,
U. somon, and U. chevalieri), as previously reported (Thoen and Bâ 1989). Fur-
thermore, we report for the first time the ECM status of two Caesalpinioideae
species of the Amherstieae tribe, i.e., Cryptosepalum tetraphyllum and Pelligrinio-
dendron diphyllum. In India, sporocarps were collected under the two species V.
indica and D. indicus, which are known as ECM tree species (Rivière 2004;
Natarajan et al. 2005; Rivière et al. 2007).
4.1.2.2 Ectomycorrhizas Diversity
Based on morphological identification, considering the diversity of both the

sites, sporocarps were assigned to five families (Amanitaceae, Russulaceae,
Sclerodermataceae, Tomentellaceae, and Tricholomataceae) and one superfami-
lial group (boletoids). Based on molecular typing, the most represented family
was the Russulaceae (75 samples of both sporocarps and ectomycorrhizas),
followed by the Amanitaceae (26), the boletoids and Chalciporus group (32),
including the genera Boletus, Xerocomus, Leccinum, Tubosaeta, Strobilomyces,
and Chalciporus, the Sclerodermataceae (32), the tricholomatoids (10), and the
telephoroids (21), of which only one sporocarp was identified as a Tomentella

species. Four subsequent phylogenies were reconstructed in order to independently
focus on the four previously identified clades in Fig. 4.1, i.e., (1) Russulaceae
Fig. 4.1 Maximum likelihood ML5-ML6 tree using a HKY85 þ g þ Inv model (a ¼ 0.5684,
proportion of invariable sites ¼ 0.3421, rate categories ¼ 4) for 160 sequences and 389 sites.
Bootstrap support values >50% are indicated at the relevant nodes. Main taxonomical groups
included in the phylogeny are indicated. Sebacina sp. and Tulanesnella irregularis were chosen as
outgroups according to Bruns et al. (1998)
72 R.-D. Taiana
Fig. 4.2 Russulaceae maximum likelihood ML5-ML6, using a HKY85 þ g þ Inv model
(a ¼ 0.5897, proportion of invariable sites ¼ 0.6539, rate categories ¼ 4) for 57 different
sequences and 391 sites. Bootstrap support values >50% are indicated at the relevant nodes.
Identical sequences are included in the same terminal node. The brackets to the right of the tree
indicate the clades including species of the same section and vertical lines indicate sections of the
same subgenera (in bold). Names and grouping follow Singer (1986), Romagnesi (1985), and
Miller and Buyck (2002) classifications. gn sample from Guinea, in Indian sample. Equality
between numbers means perfect homology between their sequences
(Fig. 4.2), (2) boletoids, (3) Scleroderma, and (4) Amanitaceae plus Tricholomata-
ceae groups (Rivière et al. 2007).
Among the 55 ectomycorrhizal sequences, a strict correspondence between
sporocarps and ectomycorrhizas was obtained for only 27 of them. This is most
probably due to the fact that many but not all ECM fungi produce fruitbodies, thus
being observable macroscopically. By way of illustration, remarkable percentage of
nonsporulating ECM species have been described by Jonsson et al. (1999a), where
up to 70% of the ECM fungal species were only sampled from root tips without
associated sporocarps in a Pinus sylvestris plantation. Moreover, sporocarp produc-
tion also varied over the season, thus escaping sampling (Erland et al. 1999; Kõljalg
et al. 2000). For instance, 20 ectomycorrhizas, but no sporocarp, were found to be
closely related to thelephoroid taxa. It is well-known that sporocarps are seldom
produced by Telephorales, but they commonly form mycorrhizas on roots of young
trees (Kõljalg et al. 2000). Our study has once again confirmed that belowground
fungal diversity is dissimilar from that of aboveground sporocarps (Gardes and
Bruns 1996; Dahlberg et al. 1997; Jonsson et al. 1999a, b), thus underlying the
importance of molecular analyses for the assessment of ECM fungal diversity.
The Russulaceae family, with 45 sequences, was the largest group sampled in
our study. Russulaceae includes two genera Lactarius and Russula. Lactarius
samples did not group together and appeared polyphyletic. At a lower taxonomical
level, the Eurussula subgenus appeared to be polyphyletic, too, whereas the subge-
nus Compactae and each section Heterophylla or Foetentinae were found mono-
phyletic. In the Scleroderma phylogeny, 32 sequences have been obtained
including 16 ectomycorrhizas, which represent the highest mycorrhizas vs. spor-
ocarps ratio in our sample. The boletoid group includes the genera Boletus, Bole-
tellus, Xerocomus, Leccinum, Tubosaeta, and Strobilomyces. All Leccinum
sequences as well as the two Strobilomyces sequences were grouped within a single
clade. All other sequences were intermingled in the phylogeny, including mainly
Boletus and Xerocomus species. The single Chalciporus sequence was located close
to Chalciporus piperatoides, and roots the rest of the boletoid group sensu stricto.
Finally, in the fourth analysis, all Amanitaceae cluster together in a highly sup-
ported monophyletic clade (Fig. 4.1), whereas Tricholomataceae (including Pluta-
ceae, Hydnum, and Tricholoma genera) were paraphyletic.
Some differences were noted in the fungal diversity between the two sites, as
well as between the tropical and more northern areas, as described in the literature.
Russulaceae genotypes sequenced from the Guinean site, which include vouchered
new species, are in accordance with the observations of Buyck (1994a, b, 1997),
suggesting a high species diversity of this family in Africa. However, no species
from India have been found to be common with those of Guinea. This lack of shared
species (or sequences) between Africa and India suggests that there has been no
recent gene flow between the two continents. Berbee and Taylor (1993, 2001)
suggested that the divergence within ECM fungi occurred around 180 Mya ago,
whereas separation of the Indian–Malagasy block from Africa is usually dated
around 120 Mya. Based on morphological identifications, Russulaceae described
in the Uppangala forest site may be related to known species from Europe
74 R.-D. Taiana
(Natarajan et al. 2005). However, none of the Russula or Lactarius species collected
in India share the same sequence with already known African or European taxon.
Enhanced molecular monitoring of tropical Russula diversity based on nuclear
sequences is urgently needed to resolve such an interesting ectomycorrhizal group.
A single Boletaceae sample was recovered from India (identified as Leccinum
sp.), whereas 17 genotypes were found in Guinea. Accordingly, Natarajan et al.
(2005) described only two Suillus and one Strobilomyces species in India. In
addition, very few Cortinariaceae specimens or species belonging to the Suilloid
group have been found so far in tropical forests (e.g., Cortinarius in Cameroon and
in India; Onguene and Kuyper 2001; Peintner et al. 2003; Natarajan et al. 2005).
Our study supports this trend, as none of the sequences fell within Cortinariaceae or
Suilloids available in Genbank database and used here as genetic benchmarks.
Nevertheless, tropical forests are still undersampled relative to northern boreal
forests, and further surveys are clearly needed to confirm this result.
In Guinea, a great diversity of sequences belonging to the Russulacae family
was obtained. Morphological taxonomies of this family (Romagnesi 1985; Singer
1986; Buyck 1994a, b, 1997) are rarely congruent, reflecting the ambiguities of
characters used in the classification of this group. In a similar way, some of the
sporocarps collected were morphologically identified as already described species,
but many others could not be linked to already known morphotypes. The latter
may represent putative new species and further studies are required [species
identification in progress, all specimens housed at the Natural History Museum,
Paris and CASB (Centre of Advanced Study in Botany, Madras)]. So far, three
specimens have been formally identified as new species, Russula sect. Archaeinae
sp. nov. (C53), Russula sp. nov. aff. sesenagula (C366), and Lactarius nov. (C13).
However, recognition of the remaining ones as new taxa would be premature at
this stage.
Here, Lactarius appears as a paraphyletic group, whereas Shimono et al. (2004)
support the monophyly of all Lactarius species based on LSU rDNA. Once again,
this discrepancy is probably due to the low variability of the ML5/ML6 fragment at
this level. Within the Russula genus, the subgenus Eurussula appears as a polyphy-
letic group, being split into four separated clades. This is not congruent with
phylogeny based on nuclear regions (Eberhardt 2002; Miller and Buyck 2002;
Eberhardt and Verbeken 2004). These differences can have several nonexclusive
origins (1) various resolution levels of the markers used among studies, due to
different rates and modes of molecular evolution, (2) complex relationships
between nuclear and mitochondrial genomes, or (3) the reduced level of resolution
at the species level of the locus used in our study (Doyle 1992; Bull et al. 1993;
Bruns and Szaro 1992). It is known that mitochondrial genomes evolved at least
partially independently from the nuclear genome, thus sometimes leading to incon-
gruent phylogenetic inferences (Moncalvo et al. 2000). Other potential sources of
incongruence between these genomes may be due to ancestral polymorphisms or
horizontal transfers (Wall 2003). Such phenomena are not rare in plants and may
obscure ECM Basidiomycete relationships as well. Unfortunately, too few molecu-
lar investigations have been performed so far to conclude (Hibbett et al. 2000;
Moncalvo et al. 2000; Binder and Hibbett 2002; Miller and Buyck 2002; den
Bakker et al. 2004).
Tropical ECM fungi diversity found is inseparable from tree species diversity
(Kabir et al. 2009), which is today endangered by strong human pressure that
threatens tropical rainforests. The composition of ECM associations and the
changes they undergo are still very poorly known in tropical regions and thus
suggesting that further investigations are required of both above- and belowground
species’ composition over extended periods of time.
4.2 Genet Distribution of Russula sp. foetentinae

in a Tropical Rainforest
4.2.1 Introduction
ECM fungi are symbiotic partners of most woody plants in forest ecosystems and
have a crucial role in terms of nutritional transfer between soil and host plant roots
(Smith and Read 1997). ECM symbiosis are characterized by bidirectional move-
ment of nutrients where carbon flows to the fungal partner while inorganic nutrients
move to the plant. The fungal vegetative network forms extensive mycelia that
radiate from ECM fungal root tips to explore the soil for resources and permit the
transfer of nutrients to associated plants (Finlay et al. 1998; Perez-Moreno and
Read 2000). Together with sexual sporocarps, ECM fungi use mycelial spread to
propagate and colonize new habitats. Therefore, the spatial distribution of individ-
ual mycelial systems is of considerable importance to understand ECM population
dynamics in forest ecosystems (Dahlberg and Stenlid 1990).
Molecular techniques, such as those described above, make it possible to
determine structure of fungal populations on a fine scale. Indeed, genotypes of
fungal “individuals” using DNA-fingerprinting techniques are commonly used in
population surveys (Burnett 2003). The concept of genet, a group of individuals of a
given genotype, is a useful tool to understand not only the spatial distribution of the
populations but also the dynamics of fungal succession of sequences (Smith et al.
1992). In general, genets can only be identified after isolating and further investi-
gating the properties of each species. Somatic incompatibility is the technique
traditionally used to resolve individual Basidiomycete genotypes (Dahlberg and
Stenlid 1994), but it is not feasible for all ECM species (e.g., Russulaceae) because
of its very limited growth in culture. Inter-simple sequence repeats (ISSRs) have
therefore been used to characterize the genetic variation within fungal populations
as it is a highly reproducible technique, which allows the detection of ECM fungal
genets from sporocarps (Hantula et al. 1996; Anderson et al. 1998; Sawyer et al.
1999; Zhou et al. 1999). ISSR polymorphism is a useful tool to distinguish the
otherwise morphologically indistinguishable individuals of fungi (Hantula et al.
76 R.-D. Taiana
1996). This technique was particularly appropriate in our study because the Russula
genus contains a large number of species notorious for exhibiting high phenotypic
plasticity (Miller and Buyck 2002). However, even when the individuals making up
a population can be defined phenotypically or genotypically, their breeding behav-
ior is rarely immediately obvious. In particular, the sampling of basidiocarps
aboveground may not be an adequate estimate of the size, frequency, or spatial
extension of genets belowground (Gardes and Bruns 1996; Jonsson et al. 1999a, b).
In investigations of community structure above- and belowground for ECM fungi,
the abundance of basidiocarps was not always indicative of the mycorrhizal mor-
photypes (Gardes and Bruns 1996; Dahlberg et al. 1997; Jonsson et al. 1999a).
Because root tip or soil DNA extract analyses were not feasible under our field
conditions, the information available on the distribution of basidiocarps was the
only possible indicator of the presence and activity of individual mycelia.
The genet size of ECM fungal populations is assumed to vary with species
identity, host forest age, and environmental conditions (Dahlberg and Stenlid
1995). For example, pioneer genera such as Hebeloma and Laccaria generally
show numerous small (<3.5 m2) and nonpersistent genets (Baar et al. 1994;
Gryta et al. 1997). In contrast, fungi appearing late in succession, such as Cortinar-
ius rotundisporus, spread primarily from hyphal networks, and their genets are
large (up to 30 m) and temporally persistent (Sawyer et al. 1999). However, the
overall picture is not that clear since certain genera such as Suillus may be found in
disturbed areas and also occur in mature forests, thus following a mixed strategy
(Dahlberg et al. 1997; Bonello et al. 1998). As a general principle, a high number of
genotypes would be expected as a result of reproduction primarily by spores,
whereas the formation of larger clones would be predicted if reproduction occurred
primarily by mycelial expansion (Dahlberg and Stenlid 1990).
The Russulaceae are of particular interest for examination of the genetic
structure and dynamics of late stage ECM populations. Despite the fact they are
diverse and abundant in many types of forest ecosystems (Mason et al. 1987), the
way they survive and spread in nature is still controversial. On the one hand, they
are believed to be typical protagonists of the late stage field succession (Deacon
and Fleming 1992; Keizer and Arnolds 1994) as they represent the majority of
basidiocarps found in mature stands of temperate forest. Although little is known
about the ability of Russulaceae to colonize tree seedlings in the field, laboratory
studies indicate difficulties in germination from spores (Redecker et al. 2001). On
the other hand, some studies have found small genets of Russula in late stage
forests, suggesting that the role of sporulation in the life history of the Russulaceae
growing on undisturbed forest may play a much more important role than previ-
ously recognized (Redecker et al. 2001; Bergemann and Miller 2002). Another
interesting aspect of Russulaceae is that they are often dominant in tropical rain-
forests of Africa, Asia, and Madagascar (Buyck et al. 1996; Lee et al. 1997;
Watling and Lee 1998). Once again, to our knowledge, genet distribution of
ECM comes essentially from temperate ecosystems and virtually nothing is
known for tropical rainforests.
4.2.2 Methods
In such a context, the aim of this study was to investigate the relative sizes of the
individual genotype of Russula sp. foetentinae in a primary rainforest dominated by
Dipterocarps species using ISSRs. To minimize the possibility of errors in assign-
ing genotypes to genets (see Redecker et al. 2001), we quantified the probability of
obtaining a given genotype by chance and calculated a Pairwise Jaccard’s similarity
among samples. The study site was located in a dense evergreen forest in the
Kadamakal Reserve (12 300 N; 75 390 E). The annual rainfall reaches 5,200 mm
with a marked dry season from December to March. The vegetation is dominated
by Vateria indica L. (Diperocarpaceae), Humboltia brunonis Wall. (Fabaceae),
Myristica dactyloı¨des Gaertn. (Myristicaceae), and Dipterocarpus indicus Bedd.
(Dipterocarpaceae). They represent more than 48% of the trees and 55% of the
basal area (Pascal and Pélissier 1996) with pioneer species accounting for only
1.1% of the trees. Dominant ECM tree species include V. indica and D. Indicus.
They represent 21% of the tree density (Pascal and Pélissier 1996). Basidiocarps of
Russula sp. foetentinae were collected and mapped to the nearest 0.1 m within a
study plot of 7,700 m2 (110 m 70 m), represented in Fig. 4.3.
DNA was extracted from each sample of basidiocarp. The molecular identifica-
tion of each Russula sp. foetentinae sporocrap was confirmed using both nuclear
and mitochondrial DNA fragments. The GenBank accession numbers for the
sequences described here are DQ093423 and DQ093424. Finally, ISSR-PCR reac-
tions were performed with three primers named ISSR1 (50 BDB (ACA)5), ISSR2
(50 DDB (CCA)5), and ISSR3 (50 DHB (CGA)5), where B is C, G, or T; D is A, G or
T; H for a A, C or T (Hantula et al. 1996; for protocols see details in Rivière et al.
2005).
4.2.2.1 Spatial Randomness
To determine whether the pattern of basidiocarp distribution was random, aggre-

gated, or regular, we used the first-order pair correlation function G(r). This
function gives the expected number of points at a distance r from an arbitrary
point, divided by the intensity l of the pattern (Diggle 1983; for details see Rivière
et al. 2005)
4.2.2.2 Genotype Identification
In order to test for nonidentity due to somatic, miss-scored gels or reproducibility

problems, we calculated Pairwise Jaccard’s similarity (Sj) between genotypes on
arcsin-transformed data using the following formula: Sj ¼ a/(aþbþc)<; Where a
is number of bands common to both genotypes and b and c the number of bands
present in only one of the two genotypes. All statistical analyses were performed
78 R.-D. Taiana
D. indicus
V. indica
Russula sp. 517a (cf.foentinae)
110
100
90
80
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70
Fig. 4.3 Spatial distribution of Russula sp. subfoetens sporocarps, Dipterocarpus indicus, and
Vateria indica within the study plot of 7,700 m2 (110 m 70 m). Tree diameters are indicated
>200 cm; 100–200 cm, 50–100 cm; <50 cm
using R 2.0.1 (free software available online: http://www.r-project.org – The R

Development Core Team). The histogram of the pairwise Sj was obtained using
Grapher 4.00 (Golden software, Colorado).
4.2.3 Discussion
Forty five basidiocarps of Russula sp. foetentinae were mapped. Spatial analyses of
the point pattern revealed a significant aggregation of the basidiocarps in a central
zone of the study area, representing less than 30% of the total surface prospected.
The first paired correlation function, g(r), showed that 60% of all basidiocarps were
located at a distance lower than 1 m from the nearest basidiocarp. To test the
possibility of reproducibility errors in inferring genets from genotypes, we plotted
all pairwise Jaccard similarity coefficients of genotypes for statistical outliers.
Our results showed a bell-shaped distribution, typical of randomly mating popula-

tion (detail see Rivière et al. 2005). Identical genotypes were clearly separated from
the right tail of the distribution. Cluster analyses on the 45 ISSR patterns revealed a
total of 29 different genotypes with 18 genotypes represented by only one basidio-
carp and 11 genotypes represented by two to three basidiocarps. Basidiocarps
belonging to the same genet were not necessarily collected on the same day. The
eleven detected genets could be separated into two size classes (Fig. 4.4). Six genets
were large with a diameter ranging from 31 m (genet J) to 70 m (genet D). The five
other genets (A, C, G, I, and K) were of smaller size with diameters ranging from
0.5 m (Genet I) to 5 m (Genet G). ISSRs revealed the presence of large Russula sp.
foetentinae mycelial individuals in the mature Dipterocarps stand we studied. The
largest individual comprises three sporocarps, two of which were situated 70 m
from each other. We are aware that caution is needed in the interpretation of this
80
G
70
E
C
60
J
50
D
Distance (m)
I
F
40
30
B
20
H
10
A
K
0
0 10 20 30 40 50 60 70
Distance (m)
Fig. 4.4 Schematic relative positions and arbitrary sizes of Russula subsect. foetentinae geno-
types plotted on a portion of the Uppangala study site (5,600 m2). Each individual basidiocarp is
identified with an icon (open, sporocarps belonging to a genet; full, individual basidiocarps).
Dotted lines represent arbitrary surfaces of the separate genets
80 R.-D. Taiana
result. First, the size estimates are based only on the presence of aboveground
fruiting bodies. Second, no connecting sporocarps were found over this distance,
leaving the possibility of an error during handling of the specimens. Third, the
action of fungivores or physical constraints of soil may have separated ECM
mycelia into several genetically identical genotypes or ramets (Dahlberg and
Stenlid 1995; Griffiths et al. 1996). It is however not possible from our data to
assess the extent to which fragmentation has occurred within the mycelial indivi-
duals at the site. Despite all these potential limitations, we found five other large
genets (>30 m) in the study site, which suggests the capacity of Russula sp.
foetentinae individuals to spread over a long distance.
Russulaceae species are usually believed to spread via vegetative reproduction
and to form relatively small genets; e.g., Russula cremolicolor (1.1 m2, Redecker
et al. 2001), Russula brevipes (3–18 m, Bergemann and Miller 2002), Russula
vinosa (<1 m, Liang et al. 2004). Our data indicate genets that are much larger than
previously described for other Russulaceae species, but the presence of large genets
is not uncommon among ECM fungal species. Large genets have been found for
Suillus bovinus and S. variegatus (40 m, Dahlberg and Stenlid 1995; Dahlberg
1997), S. pungens (40 m, Bonello et al. 1998), Pisolithus tinctorius (30 m, Anderson
et al. 1998), and Xerocomus chrysenteron (110 m, Fiore-Donno and Martin 2001).
Comparison with data from the literature is however limited as the size and extent
of the mycelial phase can differ between genera and species or between different
genets of the same species (Bonello et al. 1998; Redecker et al. 2001; Bergemann
and Miller 2002). The presence of large genets may suggest that Russula sp.
foetentinae can colonize by mycelial expansion and may be indicative of more
mature mycelial systems that have grown from point sources of individual mating
events over a long period (Dahlberg and Stenlid 1990). Because the spatial extent of
genets has been correlated with age of host stands (Dahlberg and Stenlid 1994), our
main hypothesis to explain the presence of large genets of Russula sp. foetentinae
would be the absence of disturbance over a long period of time in the studied
primary forest (>100 years, Loffeier 1989). However, the presence of smaller
genets for 18 basidiocarps suggests that Russula sp. foetentinae could also spread
via sexual reproduction of basidiocarps.
Field knowledge is lacking on the ecology of tropical ECM symbiosis, particu-
larly in primary rainforest ecosystems. To date, most studies on ECM fungi in such
ecosystems have consisted in species inventories (Buyck et al. 1996; Béreau et al.
1997; Lee et al. 1997). There is little information about life history strategies of
tropical ECM fungi and only recently, Onguene and Kuyper (2001) revealed in a
tropical forest of Cameroon that ECM fungal mycelium might form important
networks acting as “nursery zones” for young trees. Our results give preliminary
information on reproductive strategies of Russula sp. foetentinae, which may have
important implications in terms of conservation of Asiatic primary rainforests.
Dipterocarps are one of the most important timber species of tropical rain forest
in Southeast Asia and are mostly ECM. Furthermore, they are mainly associated
with Russulaceae and Amanitaceae species (Alexander and H€ogberg 1986; Watling
and Lee 1998). The failure of dipterocarp regeneration in logged forests has been
related to inadequate or inefficient ECM formations (Smits 1983). The latter have
further been proved to enhance dipterocarps seedling growth both in nursery and in
natural habitats (Lee and Alexander 1994; Lee et al. 1997).
This study represents a first step in our understanding of ECM fungal population
dynamics in tropical primary forest ecosystems but only a snap shot of the genet
distribution of Russula sp. foetentinae. Our results suggest that mature stands may
shelter well-spread underground mycelium and may be crucial for durable interac-
tion with the plant partner. Although the consistency of these results has to be
confirmed on additional sites and over several years, they could be of particular
importance in the light of current destruction of tropical forest or degradation into
secondary stands. Further studies on the temporal persistence of these large genets
and on the consequences of human-induced changes on the dynamics of fungal
populations are urgently needed.
4.3 Conclusion
Nowadays, few field studies looking at ECM species composition and communities
are conducted in tropical rainforests, mainly due to the sites’ inaccessibility. So far,
the only method to link an ECM root tip to a tree has been to physically trace it back
to the stem. Due to molecular methods, it is now feasible to extract fungal and plant
DNA from a single root tip, which allow scientists to unambiguously assign a fungal
species to its specific plant host. Furthermore, molecular fingerprinting methods are
routinely used to identify individual host tree from ECM root tips in addition to
identifying the ECM fungus – from a single DNA extraction. Such efficient methods
are of great help to investigate a new fine scale in the mycorrhizal ecology: it is now
possible to describe the ECM communities between individual trees.
The first study indicates that there is still an unknown diversity of ECM fungi in
tropical rainforests, including new species. It also points to the fact that further
investigations are urgently required of both above- and belowground diversity over
extended periods of time and over larger areas of forest biomes. The second
objective was to evaluate the spatial distribution of individual mycelial systems
in an undisturbed tropical rainforest. The genets identified reveal an underlying vast
mycelium network. Due to unsustainable logging of tropical rainforests, the influ-
ence of destruction of such ECM network, upon subsequent forest restoration, must
be seriously considered.
References
Alexander IJ, H€ogberg P (1986) Ectomycorrhizas of tropical angiospermous trees. New Phytol
102:541–549
Anderson IC, Chambers SM, Cairney JWG (1998) Use of molecular methods to estimate the size
and distribution of mycelial individuals of the ectomycorrhizal basidiomycete Pisolithus
tinctorius. Mycol Res 102:295–300
82 R.-D. Taiana
Andrade ACS, Queiroz MH, Hermes RAL, Oliveira VL (2000) Mycorrhizal status of some
plants of the Araucaria forest and the Atlantic rainforest in Santa Catarina, Brazil. Mycorrhiza
10(3):131–136
Baar J, Ozinga WA, Kuyper TW (1994) Spatial distribution of Laccaria bicolor genets reflected by
basidiocarps after removal of litter and humus layers in a Pinus sylvestris forest. Mycol Res
98:726–728
Berbee ML, Taylor JW (1993) Dating the evolutionary radiations of the true fungi. Can J Bot
71:1114–1127
Berbee ML, Taylor JW (2001) Fungal molecular evolution: gene trees and geologic time. In:
McLaughlin DJ, McLaughlin RG, Lemke PA (eds) The Mycota VII. Part B. Systematics and
evolution. Springer-Verlag, Berlin, pp 229–245
Béreau M, Gazel M, Garbaye J (1997) Mycorrhizal symbiosis in trees of the tropical rainforest of
French Guiana. Can J Bot 75:711–716
Bergemann SE, Miller SL (2002) Size, distribution and persistence of genets in local populations
of the late-stage ectomycorrhizal basidiomycete, Russula brevipes. New Phytol 156:313–320
Binder M, Hibbett DS (2002) Higher-level phylogenetic relationships of Homobasidiomycetes
(Mushroom-forming fungi) inferred from four rDNA regions. Mol Phyl Evol 22:76–90
Bonello P, Bruns TD, Gardes M (1998) Genetic structure of a natural population of the ectomy-
corrhizal fungus Suillus pungens. New Phytol 138:533–542
Bruns TD, Szaro TM (1992) Rate and mode differences between nuclear and mitochondrial small-
subunit rRNA genes in mushrooms. Mol Biol Evol 9:836–855
Bruns TD, Szaro TM, Gardes M, Cullings KW, Pan JJ, Taylor DL, Horton TR, Kretzer A,
Garbelotto M, Li Y (1998) A sequence database for the identification of ectomycorrhizal
Basidiomycetes by phylogenetic analysis. Mol Ecol 7(3):257–272
Bull JJ, Huelsenbeck PP, Cunningham CW, Swofford DL, Waddell PJ (1993) Partitioning and
combining data in phylogenetic analysis. Sys Biol 42:384–397
Burnett J (2003) Fungal populations and species. Oxford University Press, Oxford, pp 1–368
Buyck B (1994a) Russula I (Russulaceae). Flore illustrée des champignons d’Afrique centrale
15:335–408
Buyck B (1994b) Russula II (Russulaceae). Flore illustrée des champignons d’Afrique centrale
16:411–542
Buyck B (1997) Russula III (Russulaceae). Flore illustrée des champignons d’Afrique centrale
17:545–597
Buyck B, Thoen D, Walting R (1996) Ectomycorrhizal fungi of the Guinea-Congo Region. Proc R
Soc Edinb B 104:313–333
Dahlberg A (1997) Population ecology of Suillus variegatus in old Swedish Scots pine forests.
Mycol Res 107:47–54
Dahlberg A, Stenlid J (1990) Population structure and dynamics in Suillus bovinus as indicated by
spatial distribution of fungal clones. New Phytol 115:487–493
Dahlberg A, Stenlid J (1994) Size, distribution and biomass of genets in populations of Suillus
bovinus (L.: Fr.) Roussel revealed by somatic incompatibility. New Phytol 128:225–234
Dahlberg A, Stenlid J (1995) Spatiotemporal patterns in ectomycorrhizal populations. Can J Bot
73(Suppl 1):1222–1230
Dahlberg A, Jonsson L, Nylund JE (1997) Species diversity and distribution of biomas above and
below-ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in South
Sweden. Can J Bot 75:1323–1335
Deacon JW, Fleming LV (1992) Interactions of ectomycorrhizal fungi. In: Allen MF (ed)
Mycorrhizal functionning: an integrative plant-fungal process. Chapman and Hall, New York,
pp 249–300
Debaud JC, Marmeisse R, Gay G (1999) Intraspecific genetic variation and populations of
ecomycorrhizal fungi. In: Varma AK, Hock B (eds) Mycorrhiza: structure, molecular biology
and function. Springer-Verlag, Berlin, pp 75–110
Den Bakker HC, Zuccarello GC, Kuyper THW, Noordeloos ME (2004) Evolution and host
specificity in the ectomycorrhizal genus Leccinum. New Phytol 163:201–215
Diggle PJ (1983) Statistical analysis of spatial point patterns. Academic Press, London, pp 1–444
Doyle JJ (1992) Gene trees and species trees: molecular systematics as one-character taxonomy.
Sys Bot 17:144–163
Eberhardt U (2002) Molecular analyses of the agaricoid Russulaceae: correspondence with
mycorrhizal and sporocarp features in the genus Russula. Mycol Prog 1(2):201–224
Eberhardt U, Verbeken A (2004) Sequestrate Lactarius species from tropical Africa: L. angio-
carpus sp. nov. and L. dolichocaulis comb. nov. Mycol Res 108:1042–1052
Egger KN (1995) Molecular analysis of ectomycorrhizal fungal communities. Can J Bot
73:S1415–S1422
Erland S, Jonsson T, Mahmood S, Finlay RD (1999) Below-ground ectomycorrhizal community
structure in tow Picea abies forests in southern Sweden. Scand J For Res 14:209–217
Finlay RD, Ek H, Odham G, S€ oderstr€
om B (1998) Mycelial uptake, translocation and assimilation
of nitrogen from 15N-labelled ammonium by Pinus sylvestris plants infected with four different
ectomycorrhizal fungi. New Phytol 110:59–66
Fiore-Donno AM, Martin F (2001) Populations of ectomycorrhizal Laccaria amesthystina and
Xerocomus spp. show contrasting colonization patterns in a mixed forest. New Phytol
152:533–542
Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for Basidiomycetes –
application for the identification of mycorrhizas and rusts. Mol Ecol 2:113–118
Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata
forest: above and below-ground views. Can J Bot 74:1572–1583
Gardes M, White TJ, Fortin J, Bruns TD, Taylor JW (1991) Identification of indigenous and
introduced symbiotic fungi in ectomycorrhizas by amplification of nuclear and mitochondrial
ribosomal DNA. Can J Bot 69:180–190
Griffiths RP, Bradshaw GA, Marks B, Lienkaemper GW (1996) Spatial distribution of ectomycor-
rhizal mats in coniferous forests of the Pacific Northwest, USA. Plant Soil 180:147–158
Grogan P, Baar J, Bruns TD (2000) Below-ground ectomycorrhizal community structure in a
recently burned bishop pine forest. J Ecol 88:1051–1062
Gryta H, Bebaud JC, Effosse A, Gay G, Marmeisse R (1997) Fine scale structure of populations of
the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune ecosystems. Mol
Ecol 6:353–364
Hantula J, Dusabenyagasani M, Hamelin RC (1996) Random Amplified Microsatellites (RAMS),
a novel method for characterizing genetic variation within fungi. Eur J Path 26:159–166
Henkel TW, Terborgh J, Vilgalys RJ (2002) Ectomycorrhizal fungi and their leguminous hosts in
the Pakaraima Mountains of Guyana. Mycol Res 106:515–531
Hibbett DS, Gilbert LB, Donoghue MJ (2000) Evolutionary instability of ectomycorrhizal sym-
bioses in Basidiomycetes. Nature 407:506–508
Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into
the black-box. Mol Ecol 10:1855–1871
Jonsson L, Dahlberg A, Nilsson M, Zackrisson O, Karen O (1999a) Ectomycorrhizal fungal
communities in late-successional Swedish boreal forests, and their composition following
wildfire. Mol Ecol 8:205–215
Jonsson L, Kokalj S, Finlay R, Erland S (1999b) Ectomycorrhizal community structure in a limed
spruce forest. Mycol Res 103:501–508
Jonsson L, Dahlberg A, Brandrud TE (2000) Spatiotemporal distribution of an ectomycorrhizal
community in an oligotrophic Swedish Picea abies forest subjected to experimental nitrogen
addition: above and below-ground views. For Ecol Man 132:143–156
Keizer PJ, Arnolds E (1994) Succession of ectomycorrhizal fungi in roadside verges planted with
common oak (Quercus robur L.) in Drenthe, The Netherlands. Mycorrhiza 4:147–159
84 R.-D. Taiana
Kendall JM (2007) Introduction to molecular analysis of ectomycorrhizal communities. Invited

mini-review for the molecular based approaches to soil microbiology symposium, vol 71,
pp 601–610
Koide RT, Xu B, Sharda J (2005) Contrasting below-ground views of an ectomycorrhizal fungal
community. New Phytol 166:251–262
Kõljalg U, Dahlberg A, Taylor AFS, Larsson E, Hallenberg N, Stenlid J, Larsson KH, Fransson
PM, Kåren O, Jonsson L (2000) Diversity and abundance of resupinate thelephoroid fungi as
ectomycorrhizal symbionts in Swedish boreal forests. Mol Ecol 9:1985–1996
Kretzer AM, Dunham S, Molina R, Spatafora JW (2003) Microsatellite markers reveal the below
ground distribution of genets in two species of Rhizopogon forming tuberculate ectomycor-
rhizas on Douglas fir. New Phytol 161:313–320
Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003)
Molecular identification of ectomycorrhizal mycelium in soil horizons. Appl Environ Micro-
biol 69:327–333
Lee SS, Alexander IJ (1994) The response of seedlings of two dipterocarp species to nutrient
additions and ectomycorrhizal infection. Plant Soil 163:299–306
Lee SS, Alexander IJ, Watling R (1997) Ectomycorrhizas and putative ectomycorrhizal fungi of
Shorea leprosula Miq. (Dipterocarpaceae). Mycorrhiza 7:63–81
Liang Yu, Guo Liang-dong, Ma Ke-ping (2004) Genetic structure of a population of the ectomy-
corrhizal fungus Russula vinosa in subtropical woodlands in southwest China. Mycorrhiza
14:235–240
Loffeier ME (1989) Sylviculture et sylvigénèse en forêt sempervirente du Coorg (sud-ouest de
l’Inde). Travaux de la section Scientifique et Technique, Tome XXVI, Institut Français de
Pondichéry, Inde.1–21
Mason PA, Last FT, Wilson J, Deacon JW, Fleming LV, Fox FM (1987) Fruiting and succession of
ectomycorrhizal fungi. In: Pegg GF, Ayres PG (eds) Fungal infection of plant. Cambridge
University Press, Cambridge, pp 253–268
Maury-Lechon G, Curtet L (1998) Biogeography and evolutionary systematics of Dipterocarpa-
ceae. In: Appanah S, TurnbullU MJ (eds) A review of dipterocarps taxonomy, ecology and
silviculture. CIFOR, Jakarta, pp 5–44
Miller SL, Buyck B (2002) Molecular phylogeny of the genus Russula in Europe with a compari-
son of modern infrageneric classifications. Mycol Res 106:259–276
Moncalvo JM, Drehmel D, Vilgalys R (2000) Variation in modes and rates of evolution in nuclear
and mitochondrial ribosomal DNA in the mushroom genus Amanita (Agaricales, Basidiomy-
cota): phylogenetic implications. Mol Phyl Evol 16:48–63
Natarajan K, Senthilarasu G, Kumaresan V, Rivière T (2005) Diversity in ectomycorrhizal fungi
of a dipterocarp forest in Western Ghats. Curr Sci 88(12):1893–1895
Newbery DM, Alexander IJ, Rother LA (1997) Phosphorus dynamics in lowland African rain-
forest: the influence of ectomycorrhizal trees. Ecol Monogr 67:367–409
Onguene NA, Kuyper TW (2001) Mycorrhizal associations in the rainforest of South Cameroon.
For Ecol Man 140:277–287
Pascal JP (1984) Les forêts denses humides sempervirentes des Ghâts Occidentaux de l’Inde.
Tome XX, travaux de la section scientifique et technique, Institut Français de Pondichéry,
Pondicherry, pp 1–365
Pascal JP, Pélissier R (1996) Structure and floristic composition of a tropical evergreen forest in
south-west India. J Trop Ecol 12:191–211
Peay KG, Kennedy PG, Davies SJ, Tan S, Bruns TD (2010) Potential link between plant and
fungal distributions in a dipterocarp rainforest: community and phylogenetic structure of
tropical ectomycorrhizal fungi across a plant and soil écotone. New phytol 182(2):529–542
Peintner U, Moser MM, Thomas KA, Manimohan P (2003) First records of ectomycorrhizal
Cortinarius species (Agaricales, Basidiomycetes) from tropical India and their phylogenetic
position based on rDNA ITS sequences. Mycol Res 107(4):485–494
Pélissier R, Pascal JP, Houllier F, Laborde H (1998) Impact of selective logging on the dynamics
of a low elevation dense moist evergreen forest in the Western Ghats (South India). For Ecol
Man 105:107–119
Perez-Moreno J, Read DJ (2000) Mobilization and transfer of nutrients from litter to tree seedlings
via the vegetative mycelium of ectomycorrhizal plants. New Phytol 145:301–309
Picone C (2000) Diversity and abundance of arbuscular-mycorrhizal fungus spores in tropical
forest and pasture. Biotropica 32(4a):734–750
Redecker D, Szaro TM, Bowman RJ, Bruns TD (2001) Small genets of Lactarius xanthogalactus,
Russula cremoricolor and Amanita francheti in late-stage ectomycorrhizal successions.
Mol Ecol 10:1025–1034
Redhead JF (1980) Mycorrhiza in natural tropical forests. In: Mikola P (ed) Tropical mycorrhiza
research. Clarendon Press, Oxford, pp 127–142
Rivière T (2004) Biodiversity, molecular ecology and phylogeography of tropical ectomycorrhizal
symbiosis. PhD Université de Montpellier II, France, pp 1–256
Rivière T, Senthilarasug V, Kumaresan V, Natarajan K, Dreyfus B (2005) Spatial distribution of
ectomycorrhizal Basidiomycete Russula sp. foetentinae populations in a primary Dipterocarp
rainforest. Mycorrhiza 7:124–127
Rivière T, Senthilarasug V, Kumaresan V, Natarajan K, Dreyfus B (2007) Genetic diversity of
ectomycorrhizal Basidiomycetes from African and Indian tropical rainforests. Mycorrhiza
17:415–428
Romagnesi H (1985) Les Russules d’Europe et d’Afrique du Nord. Reprint with supplement.
J. Cramer, Lehre
Sanon KB, Bâ AM, Dexheimer J (1997) Mycorrhizal status of some fungi fruiting beneath
indigenous trees in Burkina Faso. For Ecol Man 98:61–69
Sawyer NA, Chambers SM, Cairney JWG (1999) Molecular investigation of genet distribution and
genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests. New
Phytol 142:561–568
Shimono Y, Kato M, Takamatsu S (2004) Molecular phylogeny of Russulaceae (Basidiomycetes;
Russulales) from the nucleotide sequences of nuclear large subunit rDNA. Mycoscience
45:303–316
Singer R (1986) The Agaricales in modern taxonomy, 4th edn. Koeltz Scientific Books, Koening-
stein, pp 1–981
Singer R, Araujo IJS (1979) Litter decomposition and ectomycorrhiza in Amazonian forests: 1. A
comparison of litter decomposition and ectomycorrhizal Basidiomycetes in latosol-terra firme
rainforest and white podzol Campinarana. Acta Amaz 9:25–41
Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic Press, San Diego, CA
Smith ML, Bruhn JN, Anderson JB (1992) The fungus Armillaria bulbosa is among the largest and
oldest living organisms. Nature 356:428–431
Smits WTM (1983) Dipterocarps and mycorrhiza, an ecological adapation and a factor in forest
regeneration. Flora Mal Bul 36:3926–3937
Smits WTM (1992) Mycorrhizal studies in dipterocarp forests in Indonesia. In: Read DJ, Lewis
DH, Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. Cambridge University Press,
Cambridge, pp 283–292
Thoen D, Bâ AM (1989) Ectomycorrhizas and putative ectomycorrhizal fungi of Afzelia africana
Sm. and Uapaca guineensis M€ ull. Arg. in southern Senegal. New Phytol 113:549–559
Thomazini LI (1974) Mycorrhiza in plants of the “Cerrado”. Plant Soil 41(3):707–711
Wall JD (2003) Estimating ancestral population sizes and divergence times. Genetics
163:395–404
ceae in Peninsular Malaysia. J Trop For Sci 10:421–430
Zhou Z, Miwa M, Hogetsu T (1999) Analysis of genetic structure of a Suillus grevillei population
in a Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR). New
Phytol 144:55–63
.
Chapter 5
The Molecular Ectomycorrhizal Fungus Essence
in Association: A Review of Differentially
Expressed Fungal Genes During Symbiosis
Formation
Bartolomeu Acioli-Santos, Helder Elı́sio E. Vieira, Cláudia E.P. Lima,

and Leonor C. Maia
5.1 Introduction
The formation of ectomycorrhiza is a complex process governed by biochemical

and molecular interactions that start before the physical contact between potential
partners. This involves intense gene expression control in both partners that leads to
drastic morphological and physiological changes that are crucial to the develop-
ment of mutualism and symbiotic harmony.
The purpose of this chapter was to review the main genes (Expressed Sequence
Tags-ESTs, mRNAs or proteins) from ectomycorrhizal fungi (EMF) expressed in
association with roots or under mycorrhizal stimulus. It was possible to group
a large number of these genes/proteins into different physiological categories
according to probable functions within symbiotic associations. For data collection,
researchers looked at different EMF and host interactions, screened different
experimental conditions and techniques (microarray, DDRT-PCR, subtractive
suppression hybridization techniques, and other approaches). The data were analyzed
and discussed under current microbiological views.
In total 389 ESTs/proteins were listed from different fungal genus and species,
specifically in Pisolithus, Paxillus, Tuber, Suillus and Laccaria. Ectomycorrhizal
genes were grouped in eight physiological categories as follows: cell growth and
B. Acioli-Santos (*)
Departamento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n,
Cidade Universitária, Recife, PE 50670-420, Brasil
and
Departamento de Virologia e Terapia Experimental-LaViTE, Centro de Pesquisas Aggeu
Magalhães, Fundação Instituto Oswaldo Cruz, Recife, PE 50670-420, Brasil
e-mail: bartacioli@cpqam.fiocruz.br; bartacioli@yahoo.com
H.E.E. Vieira, C.E.P. Lima, and L.C. Maia
Departamento de Micologia, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n,
Cidade Universitária, Recife, PE 50670-420, Brasil

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_5,
88 B. Acioli-Santos et al.
organization, morphogenesis, energy metabolism, protein synthesis and protein inter-

action, transcriptional and translation regulation, ion transport, amino acid and
peptide transport, cell signaling and structural membrane proteins, RNA/DNA pro-
cessing and cell defense and apoptosis.
5.2 Ectomycorrhizal Genes Differentially Expressed During

Symbiosis Formation
5.2.1 Growth and Cell Organization Genes
Ectomycorrhiza formation requires intense cell activity that occurs both before and
during physical contact between potential partners. The class of growth and cell
organization genes encompassed 51 ESTs/proteins (Table 5.1).
The a-tubulin and actin proteins/ESTs were identified in Suillus variegatus,
Paxillus involutus, Tuber borchii and Pisolithus microcarpus mycelia under asso-
ciation. Studies revealed the a-tubulin, actin and associated proteins (actin-binding
proteins) were active in symbiotic tissues (Timonen and Peterson 2002). Also the
centractin-like protein, which functions similar to the tubulin (Menotta et al. 2004),
was isolated from T. borchii–Tilia americana mycelium at 30 days of development.
Expression of centractin is similar to the increased expression of E-MAP 115
(microtubule-associated protein) in Laccaria bicolor–Pinus resinosa mycelium
between 6 and 72 h of contact (Podila et al. 2002). This protein is necessary to
the microtubule complex, cytoskeleton formation, and cell polarity maintenance in
epithelial cells (Masson and Kreis 1993). Likewise, the GAS 2-homologue
(observed in 30-day-old associated T. borchii mycelium) has direct participation
in the reorganization of cytoskeleton and mammal cell exponential growth
(Manzow et al. 1996). The enzyme apparatus that supports these cytoskeleton
proteins is exemplified by the expression of Rho family mRNAs observed in
P. involutus–Betula pendula (repressed at 25 days), T. borchii–Tilia americana
(stimulated at 30 days) and P. microcarpus–Eucalyptus globulus (up-regulated at
12 days) mycelium. Rho family members are involved in molecular signaling
processes prior to cell fission in yeast (Hirata et al. 1998; Imai et al. 2002).
The transcription of elongation factor 1a (translation elongation factor 1a)
promotes ribosome/tRNA binding and regulates growing peptide fidelity. This
molecule was observed in quick-growing cell cultures and is directly related to
cell growth and proliferation in diverse eukaryotes (Condeelis 1995). Increased
transcription of elongation factor 1a was observed in 4-day-old Pisolithus
tinctorius–Eucalyptus globulus mycelium (Voiblet et al. 2001).
Positive expression of Sur4, P300/CBP and septin transcripts at 6–72 h in
L. bicolor–Pinus resinosa mycelium produces proteins important for bipolar
growth in yeast (Zahner et al. 1996), proliferation/cell differentiation (Chan and
La Thangue 2001), and cytokinesis (Tasto et al. 2003). The 26S proteasome
5 The Molecular Ectomycorrhizal Fungus Essence in Association 89
Table 5.1 Some growth and cell organization genes differentially expressed in ectomycorrizal
fungus
Gene Ectomycorrhiza Time/gene Reference
expression tendency
26 s Proteasome Tuber borchii–Tilia 30d" Menotta et al.
regulatory subunit americana (2004)
mts4
Actin Suillus variegatus–Pinus 1–60d" Timonen et al.
contorta (1996)
Actin Paxillus involutus–Pinus 1–60d" Timonen et al.
contorta (1996)
Actin Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Actin Pisolithus 4–7# 12" 21d# Duplessis et al.
microcarpus–Eucalyptus (2005)a
globulus
Actin binding protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Actin cytoskeleton- Tuber borchii–Tilia 30d" Menotta et al.
assiociated americana (2004)
Actin-binding protein Pisolithus 4d" Voiblet et al.
Sop2/arp2/Arp3 tinctorius–Eucalypthus (2001)
globulus
Actin-like protein 3 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Actin-organizing Paxillus involutus–Betula 2#14d# Le Quéré et al.
complex pendula (2005)
Alpha tubulin Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Alpha tubulin Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Alpha tubulin Suillus variegatus–Pinus 1–60d" Timonen et al.
contorta (1996)
Alpha tubulin Paxillus involutus–Pinus 1–60d" Timonen et al.
contorta (1996)
Alpha tubulin A Paxillus involutus–Betula 2#14d# Le Quéré et al.
pendula (2005)
Centractin-like protein Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Chitin synthase I Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Cofilin Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Cornichon homolog Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Cyclophilin Paxillus involutus–Betula 2–8#14d" Le Quéré et al.
pendula (2005)
E-MAP 115 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
(continued)

expression tendency
Endopolygalacturonase Terfezia boudieri–Cistus 4w" Zaretsky et al.
1 (pectinase) incanus (2006)
Eukariotic initiation Pisolithus 4–7# 12" 21d# Duplessis et al.
factor eif-3p66 microcarpus–Eucalyptus (2005)a
globulus
GAS-2 homologue Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Histidine-rich protein Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Lbras Laccaria bicolor–Pinus 48h" Sundaram et al.
resinosa (2001)
Lis1 homolog Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Matin-type switching Paxillus involutus–Betula 2–8"14d# Le Quéré et al.
protein pendula (2005)
Methionine aminopept. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Mitotic checkpoint Pisolithus 4" 7–21d# Duplessis et al.
protein microcarpus–Eucalyptus (2005)a
globulus
Myosin-IA Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
P300/CBP Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Rah1/Rad51 Pisolithus 4d# Voiblet et al.
tinctorius–Eucalypthus (2001)
globulus
Ras Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Ras related protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
RAS related protein Pisolithus 4# 7" 12–21d# Duplessis et al.
microcarpus–Eucalyptus (2005) a
globulus
Ras1p Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ras-related protein Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Rho 2 protein Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Rho gdp dissociation Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Rho gdp dissociation Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
(continued)

expression tendency
Rho gdp dissociation Tuber borchii–Tilia 30d" Menotta et al.
inhibitor. americana (2004)
Ribonucleotide Paxillus involutus–Betula 2–14d" Le Quéré et al.
reductase pendula (2005)
Ribonucleotide Tuber borchii–Tilia 30d" Menotta et al.
reductase americana (2004)
Septin Spn3 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Sur4 Laccaria bicolor–Pinus 6–24h" Kim et al.
resinosa (1999b)
Sur4 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Temperature sensitive Paxillus involutus–Betula 25d# Johansson et al.
suppressor Bem1/ pendula (2004)
bud5
Translation-elongation Pisolithus 4d" Voiblet et al.
fator 1 alpha tinctorius–Eucalypthus (2001)
globulus
Vacuolar H-ATPase Paxillus involutus–Betula 25d# Johansson et al.
assembly pendula (2004)
YlmF Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
a
Presented in additional material. In the time and expression tendency column, hyphen between
numbers indicates interval time, (h) hours, (d) days, and (w) weeks. " Indicates over expression
while # indicates down expression
regulatory subunit, mts4, is part of a proteolytic degradation complex and is related

to cytoplasmic and nuclear protein degradation, and was found in abundance in the
nuclear periphery during the yeast fission process (Wilkinson et al. 1998) and in
T. borchii–T. americana fungal cells at 30 days of development. The presence of
chitin synthase I transcripts in L. bicolor–P. resinosa may be related with chitin
reposition during cell replication (Santos and Snyder 2005). Similarly, the pectinase
activity observed in Terfezia boudieri mycelium (4 weeks after ectomycorrhizal
stimulation with Cistus incanus (Zaretsky et al. 2006) could be related to roots
tissue invasion during the early symbiosis process (Poinssot et al. 2003).
The observation of Ras family mRNAs in ectomycorrhizal mycelium is difficult
to interpret, due to the diverse functions of this protein family in cells (Lammers
2004). The varied functions of these proteins may explain the lack of a specific
pattern of regulation in different associations (Table 5.1).
Down-regulation of cell resistance and stress response elements, such as the
temperature sensitive suppressor Bem1/Bud5 (Bender and Pringle 1991) and RAH1
(Chowdhury et al. 1992) were identified in some symbiotic mycelia (Table 5.1).
5.2.2 Morphogenesis
Only four ESTs have been reported in this category and all of them have increased
expression in L. bicolor–P. resinosa at 6–72 h of development (Podila et al. 2002).
As observed in Table 5.2, all citations are related to homeotic genes.
5.2.3 Energy and Metabolism
In this category, 96 ESTs were recognized that are related to metabolic processes in
different symbiotic combinations (Table 5.3).
5.2.3.1 Carbohydrate Metabolism
In eukaryote cells, carbohydrate metabolism is one of the major bioenergetic path-

ways utilizing simple sugars through glycolysis, the tricarboxylic acid cycle (TCA)
and oxidative phosphorylation. The hexose transporters carry different hexoses
(including glucose) across cell membranes. ESTs of this transporter have been
identified in P. involutus–B. pendula association (2–14 days) potentially indicating
increased metabolic activity during the early physical contact stage (Le Quéré et al.
2005). Sorbitol dehydrogenase expression was repressed in P. involutus from the
second to the eighth day of interaction. Together with the aldose reductase, this
enzyme converts sorbitol (sugar alcohol) into fructose, allowing for further glucose
production without ATP consumption. Sorbitol dehydrogenase down-regulation
may indicate the fungus’s preference for the glucose pathway.
Table 5.2 Some morphogenesis genes differentially expressed in ectomycorrizal fungus

expression tendency
Homeobox b4 gene Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Homeobox genes Hox 2.6 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Teashirt patterning gene Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
You-too homeotic gene Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
In the time and expression tendency column, hyphen between numbers indicates interval time, (h)
hours. " Indicates over expression while # indicates down expression
Table 5.3 Some energy and metabolism genes differentially expressed in ectomycorrizal fungus
expression tendency
a-ketoglutarate Tuber borchii–Tilia 5m" Polidori et al.
sulfonate platyphyllos (2002)
dioxygenase
b-keto thiolase 1 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
3-GPD-glyceraldehyde Laccaria bicolor–Pinus 6–72h" Podila et al.
3 phosphate resinosa (2002)
dehydrogenase
Acetyl CoA acetyl Laccaria bicolor–Pinus 6–72h" Podila et al.
transferase resinosa (2002)
Acid phosphatase Pisolithus 4d" Lei et al. (1990)
tinctorius–Eucalyptus
urophylla
Acyl CoA oxidase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Adenylylsulfate kinase Paxillus involutus–Betula 2# 14d# Le Quéré et al.
pendula (2005)
Adrenodoxin Laccaria bicolor–Pinus 6–24h" Kim et al.
resinosa (1999b)
Aldehyde Pisolithus 4–7# 12" 21d# Duplessis et al.
dehydrogenase microcarpus–Eucalyptus (2005)a
globulus
Alpha mannosidase Paxillus involutus–Betula 12w" Morel et al.
pendula (2005)
Alpha/beta-gliadin Tuber borchii–Tilia 30d" Menotta et al.
precursor americana (2004)
Alpha-L-rhamnosidase Tuber borchii–Tilia 30d" Menotta et al.
A precursor americana (2004)
Argininosuccinate Paxillus involutus–Betula 2#14d# Le Quéré et al.
lyase pendula (2005)
Aryl-alcohol Laccaria bicolor–Pinus 6–72h" Podila et al.
dehydrogenase resinosa (2002)
Asparagine synthase Tuber borchii–Tilia 30d" Menotta et al.
Asn2p americana (2004)
Aspartate Pisolithus 4# 7" 12–21d# Duplessis et al.
aminotransferase microcarpus–Eucalyptus (2005)a
globulus
Aspartic protease Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
ATP synthase subunit Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
ATP synthase subunit f Paxillus involutus–Betula 2# 4–8" 14d# Le Quéré et al.
pendula (2005)
ATPase F0 subunit 9 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
B2-aldehyde-forming Paxillus involutus–Betula 2–8#14d" Le Quéré et al.
enz. pendula (2005)
(continued)

expression tendency
CG15406-PA Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Choline-P-cytidylylt. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Chorismate synthase Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Citrate synthase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
COX1-i1 protein Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Cyanate lyase Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Cystahione beta Laccaria bicolor–Pinus 6–72h" Podila et al.
synthase resinosa (2002)
Cysteine proteinase Pisolithus 4–12# 21d" Duplessis et al.
globulus
Cyt ochrome c Paxillus involutus–Betula 2–8" 14d# Le Quéré et al.
reductase pendula (2005)
Cytochrome c oxidase Paxillus involutus–Betula 2–14d" Le Quéré et al.
pendula (2005)
Cytochrome c oxidase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Delta-1-pyrroline-5- Pisolithus 4–7# 12" 21d# Duplessis et al.
carboxilate microcarpus–Eucalyptus (2005)a
synthetase globulus
Dihydroorotase Paxillus involutus–Betula 12w" Morel et al.
pendula (2005)
Elastinolytic Pisolithus 4d" Voiblet et al.
metalloprotease tinctorius–Eucalyptus (2001)
globulus
Enolase Tuber borchii–Tilia 5m" Polidori et al.
platyphyllos (2002)
F1-ATP synthase delta Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ferrodoxin Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Glucoamylase-related Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Glycogen branching Terfezia boudieri–Cistus 4w" Zaretsky et al.
enzyme incanus (2006)
Glycosyl hydrolase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
(continued)

expression tendency
Hemolysin Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Hexokinase Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Hexokinase Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Hexose transporter Paxillus involutus–Betula 2–14d" Le Quéré et al.
pendula (2005)
Histidine kinase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Homoserine kinase Th. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Inorganic Tuber borchii–Tilia 30d" Menotta et al.
pyrophosphatase americana (2004)
Isoamyl alcohol Paxillus involutus–Betula 4–14d" Le Quéré et al.
oxidase pendula (2005)
Isocitrate Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Kynurenine 3- Pisolithus 4–7# 12" 21d# Duplessis et al.
monoxygenase microcarpus–Eucalyptus (2005)a
globulus
Lactonohydrolase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Malate dehydrogenase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Malate dehydrogenase Pisolithus 4d" Voiblet et al.
Mitochondrial tinctorius–Eucalyptus (2001)
globulus
Malate synthase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Mandelate racemase/ Pisolithus 12h# Acioli-Santos
muconate tinctorius–Castanea et al. (2008)
lactonizing sativa
Methylamine Pisolithus 4–12# 21d" Duplessis et al.
globulus
Mg2+ chelatase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Mitochondrial rib. Paxillus involutus–Betula 25d# Johansson et al.
protein 15.5 kDa pendula (2004)
YmL31
N. enase (ubiquinone) Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
(continued)

expression tendency
NAD-GDH Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
NADH dehydrogenase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
NAD-malate Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
NADP GDH Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
NADP-dep. Alc. Paxillus involutus–Betula 2–8d# Le Quéré et al.
Dehydrogenase pendula (2005)
Nar1p Tuber borchii–Tilia 30d" Menotta et al.
americana (2004)
Nitrilase Nit1 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Nucleoside Tuber borchii–Tilia 5m" Polidori et al.
diphosphate kinase platyphyllos (2002)
O-acetil-L-homoserine Paxillus involutus–Betula 25d" Johansson et al.
sulfhydrilase pendula (2004)
P-1P-4-tetraphosphat Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
P-2-dehydro-3- Paxillus involutus–Betula 4–14d" Le Quéré et al.
deoxyheptonate pendula (2005)
aldolase
PEP carboxykinase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Peroxisomal glycolat. Pisolithus 4–12# 21d" Duplessis et al.
globulus
Pho88p Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Phosphorylcholine Paxillus involutus–Betula 25d# Johansson et al.
transferase Pct1p pendula (2004)
P-hydroxybenzoate po. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Proline iminopeptida. Pisolithus 4–7# 12" 21d# Duplessis et al.
met. microcarpus–Eucalyptus (2005)a
globulus
Purine nucleotide-bi. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Putative diphthine Paxillus involutus–Betula 25d# Johansson et al.
synthase pendula (2004)
(continued)

expression tendency
Putative GDP- Tuber borchii–Tilia 30d" Menotta et al.
mannose americana (2004)
pyrophosphorylase
Pyruvate Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Pyruvate kinase Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Riboflavin synthase Paxillus involutus–Betula 2# 14d# Le Quéré et al.
pendula (2005)
Ring-box protein 1 Pisolithus 4d# Voiblet et al.
tinctorius–Eucalyptus (2001)
globulus
rRNA mitocondrial, Pisolithus 12h# Acioli-Santos
Large sub tinctorius–Castanea et al. (2008)
sativa
S-adenosyl-L- Pisolithus 12h" Acioli-Santos
homocysteine tinctorius–Castanea et al. (2008)
hydrolase sativa
S-adenosylmethionine Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Shp1 protein Pisolithus 4d" Voiblet et al.
phosphatase tinctorius–Eucalyptus (2001)
globulus
Sorbitol Paxillus involutus–Betula 2–8d# Le Quéré et al.
dehydrogenase pendula (2005)
Squalene Laccaria bicolor–Pinus 6–72h" Podila et al.
monooxygenase resinosa (2002)
Stearoyl-Coa Paxillus involutus–Betula 25d# Johansson et al.
desaturase pendula (2004)
Sterol 14 alpha Pisolithus 4d" Voiblet et al.
demethylase tinctorius–Eucalyptus (2001)
globulus
Tyrosinase precursor Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Ubiquinol-cytochrome Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
UMP-CMP kinase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Vacuolar ATP Pisolithus 4–7# 12" 21d# Duplessis et al.
synthase microcarpus–Eucalyptus (2005)a
globulus
a
numbers indicates interval time, (h) hours, (d) days, (w) weeks, and (m) month. " indicates over
expression while # indicates down expression
Hexokinase (the first enzyme of the glycolysis pathway) was up-regulated in

P. involutus–B. pendula (25 days) and in P. microcarpus–E. globulus mycelium
(only on the seventh day).
Positive enolase expression was found in 5-month-old T. borchii–Tilia platy-
phyllos mycelium (Polidori et al. 2002). This enzyme promotes the reversible
conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) (Babbitt et al.
1996). Pyruvate kinase, which is up-regulated in 7-day-old P. microcarpus–E.
globulus fungal cells, transforms phosphoenolpyruvate into pyruvate. Between 6
and 72 h of L. bicolor–P. resinosa interaction (Podila et al. 2002) differentially
expressed PEP carboxykinase ESTs were identified. PEP carboxykinase catalyzes
the decarboxylation of GTP (guanidine triphosphate) and oxaloacetate into
phosphoenolpyruvate, GDP (guanidine diphosphate) and CO2.
The conversion of isocitrate into a-ketoglutarate is performed by isocitrate
dehydrogenase within the TCA cycle. This enzyme was identified as up-regulated
in 7-day-old P. microcarpus mycelium. Malate dehydrogenase promotes the
malate–oxaloacetate conversion; the ESTs for this enzyme were determined to be
up-regulated at fourth day of P. tinctorius–E. globulus association. Moreover, in
P. microcarpus, the malate dehydrogenase expression was increased between 7 and
12 days of association with the same host. The malate synthase is indirectly linked
to the Krebs cycle by the glyoxylate pathway (Smith et al. 2003). This enzyme was
up-regulated in L. bicolor–P. resinosa mycelium (6–72 h).
In the respiratory chain, several enzymes were found differentially expressed in
ectomycorrhizal association. The ubiquinol (Q oxidoreductase complex) repre-
sents the second complex of the respiratory chain and is the entry point of
electrons from FADH2 molecules. Transcripts of this ubiquinol were stimulated
in P. microcarpus mycelium at 12 days of ectomycorrhizal development with
E. globulus. Cytochrome c reductase is part of complex III. This enzyme promotes
the electron transfer from coenzyme Q to a cytochrome c molecule (reduction
reaction) and pumps protons out of the mitochondrial matrix (Berg et al. 2004).
The cytochrome c oxidase is the last enzyme of the electron transport chain
(complex IV). The cytochrome c oxidase receives four electrons from cytochrome
c and transfers them to an oxygen molecule. The addition of four H+ protons
converts O2 into two H2O molecules (Berg et al. 2004). Expression of both
enzymes, cytochrome c reductase and cytochrome c oxidase, was increased in
P. involutus–B. pendula (2–14 days) and E. globulus–P. microcarpus associations
(12 days, only cytochrome c oxidase). This expression is consistent with the ATP
synthase subunit activation in both P. involutus and P. microcarpus mycelium.
The ATP synthase complex performs the final ATP formation in the respiratory
chain.
Interestingly in two different situations, mRNAs encoding structural elements of
mitochondria were identified as “repressed” in symbiotic or free-living fungus.
ESTs that encode a 15.5-kDa mitochondrial ribosomal protein YmL31 was isolated
from 25-day-old P. involutus–B. pendula mycelium, while mRNAs encoding
the large mitochondrial subunit, rRNA were observed in P. tinctorius–C. sativa
preinfection mycelium (12 h) (Acioli-Santos et al. 2008).
5.2.3.2 Lipid Metabolism
In this category, we can exemplify the negative expressions of enzymes that act in
fatty acids, sterols, and hormone synthesis in ectomycorrhizal fungus. Both the
phosphorylcholine transferase, related to the glycerophospholipide and aminopho-
sphate pathways, and stearoyl-CoA desaturase transcript, an enzyme involved in
fatty acids synthesis (Ntambi et al. 1988), was down-regulated in P. involutus
associated with B. pendula (25 days).
The ESTs encoding the sterol-14-alpha-demethylase, involved in sterol synthesis
(Bellamine et al. 1999), was reported as up-regulated in 4-day-old P. tinctorius–
E. globulus mycelium. Others lipid related enzyme transcripts were identified in
L. bicolor associated with P. resinosa. Among these, were mRNA of adrenodoxin,
a gene that produces a protein that acts as an electron carrier in the transformation
of cholesterol to steroid hormones (Lambeth et al. 1979), the acetyl CoA acetyl
transferase, involved in glycerophospholipide metabolism, squalene monooxy-
genase, important in sterol and terpene biosynthesis, and acetyl CoA oxidase,
an enzyme involved in fatty acid oxidation (Emanuelsson et al. 2003). We found
either of the transcripts encoding b-ketothiolase, a participant in fatty acid, pyruvate
and ketone body metabolic pathways (Slater et al. 1998). The b-ketothiolase
expression may indicate the fungal physiology preference towards the fatty acid
catabolism to minimize energy waste. This enzyme was identified in L. bicolor
mycelium with P. resinosa interaction between 6 and 72 h.
5.2.3.3 Amino Acid and Protein Metabolism
In this category, we found ESTs of O-acetyl-L-homoserine sulfhydrylase and

S-adenosyl-L-homocysteine hydrolase, important enzymes in the methionine path-
way (Yamagata 1989), and chorismate synthase, functionally related to production
of aromatic amino acid compounds (phenylalanine, tyrosine and tryptophan) in
bacteria, fungi and plants (Schmid and Amrhein 1995; Macheroux et al. 1999).
Only chorismate synthase was repressed. S-adenosyl-L-homocysteine hydrolase
was identified as over expressed in P. tinctorius–C. sativa preinfection mycelium
(12 h) (Acioli-Santos et al. 2008). In the methionine pathway, the S-adenosyl-
L-homocysteine hydrolase promotes S-adenosyl-L-homocysteine hydrolysis into
adenosine and homocysteine. To the homocysteine, a methyl group is added to
promote methionine formation (Kawalleck et al. 1992). In addition, the ESTs for
cystathione b-synthase were found in L. bicolor–P. resinosa fungal cells (6–72 h),
potentially indicating the involvement of methionine and cysteine metabolism in
ectomycorrhizal associations (Bydlowiski et al. 1998). ESTs for a-ketoglutarate
sulfonate dioxygenase were identified in mycelium of T. borchii–T. platyphyllos
after 5 months of contact. In Saccharomyces cerevisiae, this enzyme can act in the
alternative sulfonated compound pathway (e.g., taurine and isotianato as a sulfur
source) when cells are grown in the absence of sulfate (Hogan et al. 1999).
The a-mannosidase is involved in glycoprotein synthesis and has been observed

in Aspergillus nidulans (Eades et al. 1998) and Amanita muscaria (Kong 1995).
Transcripts of a-mannosidase were stimulated in P. involutus associated with
B. pendula during 12 weeks of development.
5.2.3.4 Nucleic Acids Metabolism
The dihydroorotase mRNA, involved in the pentose and pyrimidine pathways

(Porter et al. 2004), was identified in 12-week-old P. involutus–B. pendula myce-
lium. The nucleoside diphosphate kinase catalyzes, in turn, the nucleoside diphos-
phate phosphorylation to nucleoside triphosphate; this is a reversible reaction
regulating the nucleotides supplied to the cell (Hasunuma et al. 2003). The nucleo-
side diphosphate kinase was reported in T. borchii–T. platyphyllos association at
5 months of development.
5.2.4 Protein Synthesis and Interaction, Transcriptional

and Translational Regulation
A total of 104 ESTs were grouped in this category, 57 representing genes associated
with the ribosomal machinery, i.e., they encode for ribosomal subunit proteins
(Table 5.4). We observed that the 19 ribosomal genes found in the P. involutus–B.
pendula fungal cells were repressed in 2 to 8 day old symbiosis (Le Quéré et al.
2005), while eight of them were stimulated later (Johansson et al. 2004; Morel et al.
2005). In the P. microcarpus–E. globulus ectomycorrhiza 15 ribosomal genes were
stimulated at 12 days of development. Interestingly, ribosomal P. tinctorius ESTs
were also repressed at the first 12 h of interaction (preinfection) with C. sativa
(Acioli-Santos et al. 2008).
5.2.5 Ions, Amino Acids and Peptides Transports
36 ESTs/genes were categorized into four main groups in this category delineated
as vesicular transport, traffic mitochondrial molecules, ion channels and transpor-
ters and protein transport (Table 5.5). The vesicular transport genes are involved in
aspects of cellular vesicle formation for transport and secretion. As for example,
coatomer zeta subunit, COPII and NIPSNAPI, are genes that produce proteins
directly related to the vesicle formation and solutes secretory transport in the
endoplasmic reticulum and Golgi complex (Kuge et al. 1993; Campbell and
Scheckman 1997; Seroussi et al. 1998), respectively; although the function of
NIPSNAPI is still under speculation. The ESTs for COPII and NIPSNAPI were
Table 5.4 Some protein synthesis and interaction, transcriptional and translational regulation
genes differentially expressed in ectomycorrizal fungus
expression tendency
Bystin Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Calmodulin A Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Clathrin adapter protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
COP9 subunit 7ª Pisolithus 4" 7–21d# Duplessis et al.
globulus
Deoxyhypusine synthase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Dipeptidyl-peptidase IV Terfezia boudieri–Cistus 4w" Zaretsky et al.
incanus (2006)
Disulfide isomerase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Elongation factor EF Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Elongation factor EF-1 Paxillus involutus–Betula 2–8#14d" Le Quéré et al.
gamma pendula (2005)
Elongation factor EF1a Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Eukariotic initiation Pisolithus 4d" Voiblet et al.
factor eIF4 A tinctorius–Eucalyptus (2001)
globulus
Eukariotic initiation Pisolithus 4–7# 12" 21d# Duplessis et al.
factor eIF-5 microcarpus–Eucalyptus (2005)a
globulus
Eukariotic initiation Paxillus involutus–Betula 2–8d# Le Quéré et al.
factor eIF-6 pendula (2005)
GPI anchor transamidase Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Isopeptidase t Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Kexin protein Pisolithus 4d" Voiblet et al.
globulus
Peptidyl-prolyl cis/ Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Problabe translation Paxillus involutus–Betula 12S" Morel et al.
release fator Erf3 pendula (2005)
Proteasome component Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Proteasome p44.5 26S Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
(continued)

expression tendency
Proteasome subunit a Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
PWP2 GTP protein Pisolithus 4d" Voiblet et al.
globulus
PWP2 periodic trypto.. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
RE63412p Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Rex p27 protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribophorin II Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Ribosomal protein S2 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Ribosomal Acid protein Paxillus involutus–Betula 25d" Johansson et al.
P2 pendula (2004)
Ribosomal protein Pisolithus 4" 7–21d# Duplessis et al.
globulus
Ribosomal protein 136 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Ribosomal protein 22 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Ribosomal protein 4 S1 Pisolithus 4" 7–21d# Duplessis et al.
globulus
Ribosomal protein 4.S1 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
globulus
globulus
globulus
Ribosomal protein 40s Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Ribosomal protein 40s Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Ribosomal protein 40S Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
(continued)

expression tendency
Ribosomal protein 40S Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
Ribosomal protein 40s Paxillus involutus–Betula 12w" Morel et al.
S10 pendula (2005)
Ribosomal protein 40s Paxillus involutus–Betula 2–8d# Le Quéré et al.
S16 pendula (2005)
Ribosomal protein 50S Pisolithus 4–12# 21d" Duplessis et al.
globulus
Ribosomal protein 6. l Pisolithus 4–7# 12d" 21d# Duplessis et al.
globulus
Ribosomal protein 6. L15 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Ribosomal protein 6..L1 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein 6.L2 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein 60S Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Ribosomal protein 60S Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein E L3 Pisolithus 4" 7–21d# Duplessis et al.
globulus
Ribosomal protein E. L2 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Ribosomal protein E. L4 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Ribosomal protein E.S15 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein L10 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Ribosomal protein L10 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein L11 Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
pendula (2005)
(continued)

expression tendency
Ribosomal protein L13A Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
pendula (2005)
pendula (2005)
Ribosomal protein L23a Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
pendula (2005)
pendula (2005)
Ribosomal protein L32 Pisolithus 4–7# 12d" 21d# Duplessis et al.
globulus
pendula (2005)
pendula (2005)
pendula (2005)
Ribosomal protein L41 Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Ribosomal protein L41 Paxillus involutus–Betula 2–8# 14d" Le Quéré et al.
pendula (2005)
Ribosomal protein r.L32 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein r.S25 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ribosomal protein rs6/l7a Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Ribosomal protein S11 Paxillus involutus–Betula 25d" Johansson et al.
homologue pendula (2004)
pendula (2005)
Ribosomal protein S14 Paxillus involutus–Betula 2–8# 14d" Le Quéré et al.
pendula (2005)
pendula (2005)
Ribosomal protein S2 Pisolithus 4" 7–21d# Duplessis et al.
globulus
Ribosomal protein S28 Paxillus involutus–Betula 12w" Morel et al.
pendula (2005)
Ribosomal protein S29A Paxillus involutus–Betula 2–14d" Le Quéré et al.
pendula (2005)
(continued)

expression tendency
pendula (2005)
Ribosomal RNA 18S Pisolithus 12h# Acioli-Santos
tinctorius–Castanea et al. (2008)
sativa
sativa
sativa
Serine/threonine protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Shp1 protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Subtilisin–like serine Paxillus involutus–Betula 12w" Morel et al.
protease pendula (2005)
SUG1 26S protein reg sub Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Threonyl-tRNA synthe. Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Transcription elongation Pisolithus 4" 7–21d# Duplessis et al.
factor tef1-beta microcarpus–Eucalyptus (2005)a
globulus
Transcription elongation Pisolithus 4–7# 12" 21d# Duplessis et al.
factor tef1-g microcarpus–Eucalyptus (2005)a
globulus
Transcription elongation Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
Transcription elongation Pisolithus 4–12# 21d" Duplessis et al.
globulus
Transcription factor Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Transcription initiation Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Translation initiation fator Paxillus involutus–Betula 25d# Johansson et al.
3 Sui1p pendula (2004)
Translation initiation Paxillus involutus–Betula 2–8# 14d" Le Quéré et al.
SUI1 pendula (2005)
Ubiquitin Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Ubiquitin / ribosomal Paxillus involutus–Betula 2–8d# Le Quéré et al.
S27a pendula (2005)
(continued)

expression tendency
Ubiquitin C-terminal Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ubiquitin like protein Paxillus involutus–Betula 12S" Morel et al.
pendula (2005)
Ubiquitin-conjugatin Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Ubiquitin-conjugating Paxillus involutus–Betula 2–8d# Le Quéré et al.
enz. pendula (2005)
WD40 domain Terfezia boudieri–Cistus 4S" Zaretsky et al.
incanus (2006)
WD-repeat protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
a
numbers indicates interval time, (h) hours, (d) days, (w) weeks, and (m) month. " Indicates over
down-regulated in P. involutus–B. pendula (25 days) and P. tinctorius–Eucalyptus

globulus (4 days) mycelium, respectively. ESTs encoding for the endosomal Vps
protein complex subunit were isolated from Terfezia boudieri–Cistus incanus
fungal cells. Vps proteins act in particle transport from the Golgi complex to the
cytoplasm vacuole and have been identified in S. cerevisiae cells (Tatsumi et al.
2007). In addition, the Lb-AUT-7 gene is related to the cell mechanisms of phago-
cytosis (Podila et al. 2002).
Mitochondrial trafficking genes identified encode for proteins found in the
mitochondrial inner membrane, which promote nuclear protein transport to mito-
chondria (Rehling et al. 2003; Palmieri 1994), and the mitochondrial carrier
protein, which participates in phosphate anion transport (Palmieri 1994). The
ADP/ATP carrier protein, also a mitochondrial carrier protein, performs transloca-
tion of ADP/ATP to the mitochondrial membranes, and here the protein catalyzes
the reduction reaction of internal ADP to ATP by translocation of a radical
phosphate. Interestingly, the ADP/ATP carried gene was repressed in 25-day-old
P. involutus and B. pendula mycelium. The ADP/ATP carrier protein was also
observed in P. tinctorius associated with E. globulus, both as repressed in the early
and in the late development stages (i.e., at 4 and 21 days of ectomycorrhizal
development) (Table 5.5).
Ion channel proteins were represented by calcium channels (specifically, Al
voltage dependent calcium channels) and acid-sensitive K+ channels. The detection
of a putative Ca2+ transporting ATPase molecule as a calcium carrier was
also reported. Genes for all of these were up-regulated in L. bicolor mycelium
associated with P. resinosa (Kim et al. 1999b; Podila et al. 2002). The plasma
Table 5.5 Some ion, amino acids and peptide transport genes differentially expressed in ecto-
mycorrizal fungus
expression tendency
b-importin Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
A1 voltage dependente calcium
Channel Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Acid sensitive K+ channel Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Adenine nucleotide Paxillus involutus–Betula 25d# Johansson
translocation pendula et al.
(2004)
ADP/ATP carrier protein Paxillus involutus–Betula 25d# Johansson
pendula et al.
(2004)
ADP-ATP carrier protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
BIP protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Brefeldin A resistant. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Calcium channel Laccaria bicolor–Pinus 6–24h" Kim et al.
resinosa (1999b)
Endosomal Vps protein Terfezia boudieri–Cistus 4w" Zaretsky et al.
complex subunit incanus (2006)
Glutamate transporter Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Golgi memb. sorting protein Paxillus involutus–Betula 12w" Morel et al.
pendula (2005)
Huntingtin interacti. Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Import protein Tim9p Paxillus involutus–Betula 2–14d" Le Quéré et al.
pendula (2005)
Lb-AUT7 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Lb-AUT7-autophagocitose Laccaria bicolor–Pinus 6–24h" Kim et al.
resinosa (1999a)
Mir1p Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Mitochondrial carrier Paxillus involutus–Betula 25d# Johansson
protein pendula et al.
(2004)
Mitochondrial Carrier Paxillus involutus–Betula 2–8# 14d" Le Quéré et al.
YHM1 pendula (2005)
Mitochondrial inner memb. Paxillus involutus–Betula 25d# Johansson
Translocase subunit pendula et al.
Tim17 homologue (2004)
(continued)

expression tendency
MSF Transporter Paxillus involutus–Betula 25d# Johansson
pendula et al.
(2004)
NIPSNAPI Pisolithus 4d# Voiblet et al.
globulus
Nuclear Transport factor 2 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Outer membrane protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Oxaloacetate transport Paxillus involutus–Betula 2–8" 14d# Le Quéré et al.
Peptide transport protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Plasma membrane H(+)- Paxillus involutus–Betula 2d# Le Quéré et al.
ATPase pendula (2005)
Probable mitochondrial Paxillus involutus–Betula 25d# Johansson
carrier protein pendula et al.
(2004)
Putaive Coatomer zeta Paxillus involutus–Betula 25d" Johansson
subunit pendula et al.
(2004)
Putaive syntaxin Paxillus involutus–Betula 25d" Johansson
pendula et al.
(2004)
Putative Ca 2+ transporting Laccaria bicolor–Pinus 6–72h" Podila et al.
ATPase resinosa (2002)
Putative component of Paxillus involutus–Betula 25d# Johansson
COPII-coated vesicle pendula et al.
(2004)
Ran interactin giant Laccaria bicolor–Pinus 6–72h" Podila et al.
nucleopore protein resinosa (2002)
Ryanodine receptor Laccaria bicolor–Pinus 6–24h" Kim et al.
resinosa (1999b)
Urea active transport Paxillus involutus–Betula 12w" Morel et al.
Zn transporter Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
a
membrane H(+)-ATPase is involved in inorganic phosphorus transport especially in

low availability conditions (Shen et al. 2006). ESTs for these genes were repressed
in a 2-day-old P. involutus–B. pendula mycelium.
In the protein transport class of altered genes, the peptide transporter (stimulated
between 6 and 72 h of L. bicolor and P. involutus mycelium), the nuclear transport
factor 2 (repressed between 2 and 8 days of Paxillus involutus–Betula pendula
association) and ß-importin gene were found. The ß-importin gene encodes for a
protein that translocates histones from the cytoplasm to the nucleus through the
nuclear channel (Jakel et al. 1999); and here it was observed as up-regulated in L.
bicolor–P. resinosa mycelium. The Bip protein, which is related to increased
cellular protein excretion via cellular vesicle, was up-regulated in L. bicolor–
P. resinosa mycelium (Punt et al. 1998).
5.2.6 Signaling and Structural Membrane Proteins
Two groups of molecules are in evidence in this category: the SRAPs (symbiosis-
regulated acidic polypeptides) and hydrophobins (Table 5.6). SRAPs are commonly
observed to be stimulated in P. tinctorius–E. globulus ectomycorrhiza formation
and considered to be a strong association marker for this interaction (Voiblet et al.
2001). However, P. microcarpus–E. globulus mycelium demonstrated several
ESTs that encode for SRAPs that were suppressed in early ectomycorrhizal stages
(Duplessis et al. 2005).
Hydrophobins are small molecular weight peptides that are moderately hydro-
phobic proteins containing eight conserved cysteine residues. They are found in the
hypha cell wall or are secreted by hyphae (Kershaw and Talbot 1998; W€osten
and Vocht 2000; Tagu et al. 2001). Several hydrophobins have been reported in
ectomycorrhiza and many of them are stimulated in fully developed or advanced
stages of symbiosis (Table 5.6). However, recently, a few different hydrophobin
transcripts (hydrophobin 2 and 3) were determined to be repressed during
P. tinctorius – C. sativa preinfections mycelium indicating a difference in hydro-
phobin gene regulation during the early interaction stages (Acioli-Santos et al.
2008).
5.2.7 DNA/RNA Processing
Thirteen ESTs were related to DNA replication and nucleic acid maintenance/
processing. Interestingly, most of these genes were repressed in early ectomy-
corrhizal association (Table 5.7). Maintenance and processing of DNA genes
mostly showed increased expression in the intermediate ectomycorrhiza formation
stage. Helicases, important enzymes during DNA replication, were up-regulated in
P. microcarpus associated with E. globulus at 12 days of association. The coiled
coil domain is related to DNA a-helix folding, and works as a gene expression
controller element (Yu 2002); this was observed in P. involutus associated with B.
pendula (up-regulated between 4 and 8 days).
Table 5.6 Some signaling and structural membrane protein genes differentially expressed in
ectomycorrizal fungus
expression tendency
b-transducin Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
CFTR Laccaria bicolor–Pinus 6–24 h" Kim et al.
resinosa (1999b)
Cornichon homolog Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Extensin like plant Amanita muscaria–Picea 2m" Nehls et al.
protein abies (1999)
FUN34/ATO protein Pisolithus 4–12# 21d" Duplessis et al.
globulus
GNS1/SUR4 family Paxillus involutus–Betula 2–14d" Le Quéré et al.
GPR/FUN34 family Paxillus involutus–Betula 2–8d# Le Quéré et al.
GTP bindin protein Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Het-c Paxillus involutus–Betula 2#14d# Le Quéré et al.
pendula (2005)
Hydorphobin hydPt-8 Pisolithus 4d" Voiblet et al.
globulus
HydPt-1 prom Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Hydrofobin hydPt-1 Pisolithus 0.5–7d" Tagu et al.
globulus
Hydrofobin hydPt-2 Pisolithus 0.5–7d" Tagu et al.
globulus
Hydrofobin hydPt-5 Pisolithus 4d" Voiblet et al.
globulus
Hydrofobin hydPt-6 Pisolithus 4d" Voiblet et al.
globulus
Hydrophobin-3 precursor Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Hydrophobin 1 Paxillus involutus–Betula 4–14d" Le Quéré et al.
pendula (2005)
Hydrophobin hydPt-4 Pisolithus 4d" Voiblet et al.
globulus
Hydrophobin-2 Pisolithus 12h# Acioli-Santos
sativa
(continued)

expression tendency
Hydrophobin-2 Pisolithus 4" 7–21d# Duplessis et al.
globulus
Hydrophobin-2 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Hydrophobin-3 Pisolithus 12h# Acioli-Santos
sativa
Hydrophobin-3 Pisolithus 4" 7–21d# Duplessis et al.
globulus
Hydrophobin-3 Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
Hydrophobin-3 Pisolithus 4–12# 21d" Duplessis et al.
globulus
IRS1 like protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
LZK protein kinase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Malignant T cell Paxillus involutus–Betula 2#14d# Le Quéré et al.
amplified protein pendula (2005)
Probable membrane Laccaria bicolor–Pinus 6–72h" Podila et al.
protein YOL 063e resinosa (2002)
Profilaggrin Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Receptor kinase like Laccaria bicolor–Pinus 6–72h" Podila et al.
protein resinosa (2002)
S receptor kinase Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
S.ar ribonucleoprotein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Small ribonucleoprotein Paxillus involutus–Betula 2–14d" Le Quéré et al.
pendula (2005)
Snod Prot1 Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
Spil-GTP-biding protein Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
SRAP17 Pisolithus 4d" Voiblet et al.
globulus
SRAP17 Pisolithus 4" 7–21d# Duplessis et al.
globulus
(continued)

expression tendency
SRAP17 Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
SRAP17 Pisolithus 4–12# 21d" Duplessis et al.
globulus
SRAP17 Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
SRAP32 Type I Pisolithus 4" 7–21d# Duplessis et al.
globulus
SRAP32 Type II Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
SRAP32-1 Pisolithus 4d" Voiblet et al.
globulus
SRAP32-3 Pisolithus 4d" Voiblet et al.
globulus
Steroid binding protein Paxillus involutus–Betula 2#14d# Le Quéré et al.
pendula (2005)
Transmembrane FUN 34 Pisolithus 4d" Voiblet et al.
protein tinctorius–Eucalyptus (2001)
globulus
a
In RNA processing, five ESTs showed repressed expression during short inter-
action periods. These ESTs include transcripts that encode for RNA binding motif
protein, PolyA-binding protein, pre-rRNA processing, mRNA maturase bl2 and U6
snRNA-associated Sm-like protein Lsm8. The mRNA maturase bl2 participates in
mRNA splicing (Szczepaneck and Lazowska 1996) and U6 snRNA-associated/
Sm-like protein Lsm8 is a protein within the mRNA decay enzyme complex
(He and Parker 2000).
5.2.8 Cell Defense and Apoptosis
Table 5.8 shows some oxidative defense genes observed in EMF. Brain selenopro-
tein, glutathione peroxidase and thiol-specific antioxidant are enzymes that act to
protect organisms against reactive oxygen species (Moon et al. 1994, Koga et al.
1998). These genes were observed in L. bicolor and P. involutus mycelium.
Table 5.7 Some DNA/RNA processing genes differentially expressed in ectomycorrizal fungus
expression tendency
Coiled-coil protein Paxillus involutus–Betula 2# 4–8" 14d# Le Quéré et al.
pendula (2005)
GATA zinc finger protein Pisolithus 4# 7" 12–21d# Duplessis
microcarpus–Eucalyptus et al.
globulus (2005)a
Helicase homolog Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
Histone Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
Histone gene complex Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
mRNA maturase bl2 Pisolithus 4d# Voiblet et al.
globulus
PolyA-binding protein Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
Pre-rRNA processing Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
Rad51 Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
RNA binding motif protein Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
Sin3 associated poly. Pisolithus 4–7# 12" 21d# Duplessis
globulus (2005)a
ssDNA biding protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
U6 snRNA-associated Pisolithus 4d# Voiblet et al.
Sm-like protein Lsm8 tinctorius–Eucalyptus (2001)
globulus
a
The metallothionein mRNA identified in P. involutus and P. microcarpus

mycelium and the arsenite ATPase mRNA both have increased expression in
advanced stages of development in their respective ectomycorrhizal systems.
These genes encode for enzymes important for toxic metal tolerance (Haerslev
et al. 1995) and resistance to arsenic (Silver et al. 1989), respectively. Aldose
reductase acts on the metabolism of carbohydrates (such as fructose, mannose,
Table 5.8 Some cell defense and appopitosis genes differentially expressed in ectomycorrizal
fungus
expression tendency
ABC transporter Pisolithus 4" 7–21d# Duplessis et al.
MSBA microcarpus–Eucalyptus (2005)a
globulus
Aldose reductase Paxillus involutus–Betula 12w" Morel et al.
pendula (2005)
Arsenite- Paxillus involutus–Betula 12w" Morel et al.
translocating pendula (2005)
ATPase
Bap31 protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Brain seleno protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Calcineurin B Pisolithus 4–7# 12" 21d# Duplessis et al.
subunit microcarpus–Eucalyptus (2005)a
globulus
Cycloheximide Pisolithus 4–7# 12–21d" Duplessis et al.
resist. microcarpus–Eucalyptus (2005)a
globulus
Cytochrome P450 Paxillus involutus–Betula 25d# Johansson et al.
pendula (2004)
Cytochrome P450 Paxillus involutus–Betula 2–8" 14d# Le Quéré et al.
pendula (2005)
Cytochrome P450 Tuber borchii–Tilia americana 30d" Menotta et al.
(2004)
Dna J protein Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
Dna J Protein Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Glutathione Laccaria bicolor–Pinus 6–72h" Podila et al.
peroxidase resinosa (2002)
Glutathione Laccaria bicolor–Pinus 6–72h" Podila et al.
S-transferase III resinosa (2002)
Glutathione- Paxillus involutus–Betula 25d# Johansson et al.
S-transferase pendula (2004)
Glutathione- Paxillus involutus–Betula 2–8d# Le Quéré et al.
Glutathione- Paxillus involutus–Betula 2–14d" Le Quéré et al.
HSP 104 Pisolithus tinctorius–Castanea 12h# Acioli-Santos
sativa et al. (2008)
HSP 70 Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
HSP 70-related Pisolithus tinctorius–Castanea 12h# Acioli-Santos
protein HSS1 sativa et al. (2008)
HSP 90 BB, 1 beta Pisolithus tinctorius–Castanea 12h# Acioli-Santos
isoform sativa et al. (2008)
(continued)

expression tendency
HSP 10 Pisolithus 4–7# 12" 21d# Duplessis et al.
mitochondrial microcarpus–Eucalyptus (2005)a
globulus
LEA protein Pisolithus 4–7# 12" 21d# Duplessis et al.
homolog microcarpus–Eucalyptus (2005)a
globulus
Macrolide-binding Pisolithus 4–7# 12–21d" Duplessis et al.
globulus
Metallothionein Paxillus involutus–Betula 25d" Johansson et al.
pendula (2004)
Metallothionein Pisolithus 4–12# 21d" Duplessis et al.
globulus
PAPS reductase Paxillus involutus–Betula 2# 14d# Le Quéré et al.
pendula (2005)
PCAR apoptotic Laccaria bicolor–Pinus 6–72h" Podila et al.
related resinosa (2002)
Rehydrin-like Paxillus involutus–Betula 25d" Johansson et al.
Related to Tuber borchii–Tilia 30d" Menotta et al.
trichodiene americana (2004)
oxygenase
Cytochrome
P450
TBP like Cyp450 Laccaria bicolor–Pinus 6–72h" Podila et al.
resinosa (2002)
Thiol-spec. Paxillus involutus–Betula 2–8# 14d" Le Quéré et al.
antioxidant pendula (2005)
protein
Thioredoxin Paxillus involutus–Betula 2–8d# Le Quéré et al.
pendula (2005)
Thioredoxin Pisolithus 4d" Voiblet et al.
reductase tinctorius–Eucalyptus (2001)
globulus
Thioredoxin Pisolithus 4–7# 12" 21d# Duplessis et al.
reductase microcarpus–Eucalyptus (2005)a
globulus
Tpo3 MFS multidrug Paxillus involutus–Betula 12w" Morel et al.
efflux transporter pendula (2005)
Txl protein Pisolithus 4# 7" 12–21d# Duplessis et al.
globulus
WSC4 homolog Pisolithus 4–7# 12" 21d# Duplessis et al.
globulus
a
and galactose) and, in yeast, is related to several stress response mechanisms

such as NaCl, H2O2 and carbon unavailability (Aguilera and Prieto 2001). Aldose
reductase ESTs were identified as up-regulated in 12-week-old P. involutus–
B. pendula mycelium (Morel et al. 2005).
Glutathione S transferase acts to detoxify the cell (Hayes and Pulford 1995) and
was identified in ectomycorrhiza mycelium. Podila et al. (2002) observed up
regulation of Glutathione S transferase III in L. bicolor associated with P. resinosa
between 6 and 72 h of contact. However, Johansson et al. (2004) identified the same
gene repressed in mycelium from P. involutus–B. pendula symbiosis at 25 days of
association.
The cytochrome P450 family acts broadly in many oxidative functions including
defense mechanisms and stress resistance (Chapple 1998). Cytochrome P450 ESTs
were identified in different ectomycorrhizal combinations (Table 5.8). Rehydrin
ESTs were observed overexpressed after 25 days in P. involutus–B. pendula
mycelium potentially relating to dehydration resistance (Velten and Oliver 2001).
The HSP (heat shock proteins) are stress cellular response proteins with the major
cellular HSPs including HSP60, HSP70 and HSP90 (molecular weights of 60 kD,
70 kD and 90 kD, respectively). These proteins play an essential chaperone role
facilitating intracellular transport and peptide interactions (Gupta 1995). Five HSP
ESTs were observed as differentially expressed in mycelium associated (or in the
process of association) with different host plants. In P. tinctorius–C. sativa system
(Acioli-Santos et al. 2008); the mycelium showed three HSP transcripts down
regulated after 12 h (HSP90BB 1 beta isoform, HSP70 HSS1-related protein, and
HSP104) in response to mycorrhizal stimulus. The HSP70 EST was also observed
repressed in P. involutus–B. pendula fungal cells (2–8 days). Finally, the HSP10
was repressed in P. microcarpus–E. globulus during early and then late symbiosis
development. These data suggest a HSP repression trend in short interaction/
formation intervals.
The PCAR apoptotic gene was observed in L. bicolor–P. resinosa fungal cells
(Podila et al. 2002) and may be related to the hyphal apoptosis processes because
apoptotic genes have been identified in yeast (Madeo et al. 2002).
5.3 Conclusions
Symbiotic ectomycorrhizal final tissue results from roots and fungal cells interaction
under temporal/spatial regulation by both partner genetic programs. This process
involves environmental sensing and cell–cell communication that culminates in the
construction of new symbiotic structures. The reviewed data indicates the use of
new genetic programs during different ectomycorrhizal phases. Thus, related genes
change expression levels during formation and maintenance of symbiotic relation-
ships; however, all of them are basally expressed in most symbiotic conditions.
There seems to be a tendency for gene expression suppression (down-regulation)
during short association periods or even the preinfection interaction. This tendency
can be observed in protein synthesis genes, carbon metabolism genes and mem-
brane proteins like SRAPs and hydrophobins, which are proteins believed to be
important agents for ectomycorrhiza formation. If these findings are confirmed, the
research focus for association genetic markers should be directed toward studying
shorter interaction intervals to identify potentials key elements of ectomycorrhizal
development.
However, in several experiments not discussed in this chapter, genes of unknown
function that could be potential participants in ectomycorrhizal associations (Podila
et al. 2002; Menotta et al. 2004; Johansson et al. 2004; Acioli-Santos et al. 2008;
Martin et al. 2008) were observed. This fact highlights a knowledge gap concerning
fungal origin sequences in symbiosis and the urgent emerging need to encourage
future studies in molecular ectomycorrhizal associations.
References
Acioli-Santos B, Sebastiana M, Pessoa F, Sousa L, Figueiredo A, Fortes AM, Balde A, Maia LC,
Pais MS (2008) Fungal transcript pattern during the preinfection stage (12 h) of ectomycorrhiza
formed between Pisolithus tinctorius and Castanea sativa roots, identified using cDNA
microarrays. Curr Microbiol 57:620–625
Aguilera J, Prieto JA (2001) The Saccharomyces cerevisiae aldose reductase is implied in the
metabolism of methylglyoxal in response to stress conditions. Curr Genet 39:273–283
Babbitt PC, Hasson MS, Wedekind JE, Palmer DJ, Barrett WC, Reed GH, Rayment I, Ringe D,
Kenyon GL, Gerlt JA (1996) The enolase superfamily: a general strategy for enzyme-catalyzed
abstraction of the r-protons of carboxylic acids. Biochemistry 35:16489–16501
Bellamine A, Mangla AT, Nes WD, Waterman MR (1999) Characterization and catalytic
properties of the sterol 14 a-demethylase from Mycobacterium tuberculosis. Biochemistry
96:8937–8942
Bender A, Pringle JR (1991) Use of a screen for synthetic lethal and multicopy suppressed mutants
to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell
Biol 11:1295–1305
Berg JM, Tymoczko JL, Stryer L (2004) Bioquı́mica 5ª Ed. Guanabara Koogan S.A., Rio de
Janeiro, p p1059
Bydlowiski P, Magnannelli AC, Chamone DAF (1998) Hyper-homocistenemia e doenças vaso-
oclusivas. Arquivos Brasileiros de Cardiologia 71:69–76
Campbell JL, Scheckman R (1997) Selective packaging of cargo molecules into endoplasmic
reticulum-derived COPII vesicles. Proc Natl Acad Sci 94:837–842
Chan HM, La Thangue NB (2001) P300/CBP proteins: HATs for transcriptional bridges and
scaffolds. J Cell Sci 114:2363–2373
Chapple C (1998) Molecular-genetics analysis of plant cytochrome P450-dependent monoxi-
genases. Annu Rev Plant Physiol Plant Mol Biol 49:311–343
Chowdhury S, Smith KW, Gustin MC (1992) Osmotic stress and the yeast cytoskeleton: pheno-
type-specific suppression of an actin mutation. J Cell Biol 118:561–571
Condeelis J (1995) Elongation factor 1a, translation and the cytoskeleton. Trends Biochem Sci
20:169–170
Duplessis S, Courty P, Tagu D, Martim F (2005) Transcript patterns associated with ectomy-
corrhiza development in Eucalyptus globulus and Pisolithus microcarpus. New Phytol
165:599–611
Eades CJ, Gilbert A, Goodman CD, Hintz WE (1998) Identification and analysis of a class 2 alpha-
mannosidase from Aspergillus nidulans. Glycobiology 8:17–33
Emanuelsson O, Elofsson A, Von Heijne G, Cristobal S (2003) In silico prediction of the
peroxisomal proteome in fungi, plants and animals. J Mol Biol 330:443–456
Gupta RS (1995) Phylogenetic analysis of 90kD heat shock family of protein sequences and an
examination of the relationship among animals, plants and fungi species. Mol Biol Evol
12:1063–1073
Haerslev T, Jacobson GK, Zedeler K (1995) The prognostic significance of immunohisto-
chemically detectable metallothionein in primary breast carcinomas. Acta Pathol Microbiol
Immunol Scand 103:279–285
Hasunuma K, Yabe N, Yoshida Y, Ogura Y, Hamada T (2003) Putative functions of nucleoside
diphosphate kinase in plants and fungi. J Bioenerg Biomembr 35:57–65
Hayes JD, Pulford DJ (1995) The glutathione s-transferase supergene family: regulation of GST
and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev
Biochem Mol Biol 30:445–600
He W, Parker R (2000) Functions of Lsm proteins in mRNA degradation and splicing. Curr Opin
Cell Biol 12:346–350
Hirata D, Nakano K, Fukui M, Takenaka H, Miyakawa T, Mabuchi I (1998) Genes that cause
aberrant cell morphology by overexpression in fission yeast: a role of a small GTP-binding
protein Rho2 in cell morphogenesis. J Cell Sci 111:149–159
Hogan DA, Auchtung TA, Hausinger RP (1999) Cloning and characterization of a sulfonate/
a-ketoglutarate dioxygenase from Saccharomyces cerevisiae. J Bacteriol 181:5876–5879
Imai K, Kijima T, Noda Y, Sutoh K, Yoda K, Adachi H (2002) A Rho GDP-dissociation
inhibitor is involved in cytokinesis of Dictyostelium. Biochem Biophys Res Commun
296:305–312
Jakel S, Albig W, Kutay U, Bischoff FR, Schwamborn K, Doenecke D, Gorlich D (1999) The
importin ß/importin 7 heterodimer is a functional nuclear import receptor for histone H1. Eur
Mol Biol Organ J 18:2411–2423
Johansson T, Le Quéré A, Ahren D, S€oderstr€
om B, Erlandsson R, Lundeberg J, Uhlén M, Tunlid A
(2004) Transcriptional responses of Paxillus involutus and Betula pendula during formation of
ectomycorrhizal root tissue. Mol Plant Microbe Interact 17:202–205
Kawalleck P, Plesh G, Hahlbrock K, Somssich IE (1992) Induction by fungal elicitor of
S-adenosyl-L-methionine synthetase and S-adenosyl-L-homocysteine hydrolase mRNAs in
cultured cell and leaves of Petroselinum crispum. Proc Natl Acad Sci 89:4713–4717
Kershaw MJ, Talbot NJ (1998) Hydrophobins and repellents: proteins with fundamental roles in
fungal morphogenesis. Fungal Genet Biol 23:18–33
Kim S, Bernreuther D, Thumm M, Podila G (1999a) LB.AUT7, a novel symbisis-regulated gene
from an ectomycorrhizal fungus, Laccaria bicolor, is functionally related to vesicular transport
and autophagocytosis. J Bacteriol 181:1963–1967
Kim S, Shivanand T, Podila G (1999b) Cloning and identification of symbiosis-regulated genes
from the ectomycorrhizal Laccaria bicolor. Mycol Res 103:168–172
Koga M, Tanaka H, Yomogida K, Tsuchida J, Uchida K, Kitamura M, Sakoda S, Matsumiya K,
Okuyama A, Nishimune Y (1998) Expression of selenoprotein-p messenger ribonucleic acid in
the rat testis. Biol Reprod 58:261–265
Kong FK (1995) Influence of copper, manganese and pH on the growth and several enzyme
activities in mycorrhizal fungus Amanita muscaria. Hemosphere 30:199–207
Kuge O, Hara-Kuge S, Orci L, Ravazzola M, Amherdt M, Tanigawa G, Wieland FT, Rothman JE
(1993) z-COP, a subunit of coatomer, is required for COP-coated vesicle assembly. J Cell Biol
123:1727–1734
Lambeth JD, Seybert DW, Kamin H (1979) Ionic effects on adrenal steroidogenic electron
transport. The role of adrenodoxin as an electron shuttle. J Biol Chem 254:7255–7264
Lammers PJ (2004) Symbiotic signalling: new functions for familiar proteins. New Phytol
161:324–326
Le Quéré A, Wright DP, S€ oderstr€om B, Tunlid A, Johansson T (2005) Global patterns of gene
regulation associated with the development of ectomycorrhiza between Birch (Betula pendula
Roth.) and Paxillus involutus. Mol Plant Microbe Interact 18:659–673
Lei J, Lapayerie F, Malajczuk N, Dexheimer J (1990) Infectivity of pine and eucalypt isolates of
Pisolithus tinctorius (Pers.) Coker & Couch on toots of Eucalyptus urophylla S. T. Blake
in vitro. II. Ultrastructural and biochemical changes at the early stage of mycorrhizal formation.
Macheroux P, Schmid J, Amrhein N, Schaller A (1999) A unique reaction in a common pathway:
mechanism and function of chorismate synthase in the shikimate pathway. Planta 207:325–334
Madeo F, Herker E, Maldener C, Wissing S, L€aChelt S, Herlan M, Fehr M, Lauber K, Sigrist SJ,
Wesselborg S, Fr€ohlich KU (2002) A caspase-related protease regulates apoptosis in yeast.
Mol Cell 9:911–917
Manzow S, Brancolini C, Marks F, Richter KH (1996) Expression of growth arrest-specific (Gas)
genes in murine keratinocytes: Gas2 is specifically regulated. Exp Cell Res 224:200–203
Martin F, Aerts A, Ahrén D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E,
Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canb€ack B,
Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, Difazio S, Duplessis S,
Tachet LF, Lucic E, Klett PF, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P,
Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D, Montanini B,
Muratet M, Nehls U, Hirzel HN, Le Secq MPO, Peter M, Quesneville H, Rajashekar B,
Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, K€ues U, Lucas S, Peer YV,
Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR, Stajich JE, Tunlid A,
Tuskan G, Grigoriev IV (2008) The genome of Laccaria bicolor provides insights into
mycorrhizal symbiosis. Nature 452:88–92
Masson D, Kreis TE (1993) Identification and molecular characterization of E-MAP -115, a novel
microtubule-associated protein predominantly expressed in epithelial cells. J Cell Biol 123:
357–371
Menotta M, Amicucci A, Sisti D, Gioacchini AM, Stocchi V (2004) Differential gene expression
during pre-symbiotic interaction between Tuber borchii Vittad. and Tilia Americana L. Curr
Genet 46:158–165
Moon BY, Ho ZC, Sue GR, Chock PB, Earl RS (1994) On the protective mechanism of the thiol-
specific antioxidant enzyme against the oxidative damage of biomacromolecules. J Biol Chem
269:1621–1626
Morel M, Jacob C, Kohler A, Johansson T, Martin F, Charlot M, Brun A (2005) Indentification of
genes differentially expressed in extraradical mycelium and ectomycorrizal roots during
Paxillus involutus–Petula pendula ectomycorrhizal symbiosis. Appl Environ Microbiol 71:
382–391
Nehls U, Mikolajewski S, Ecke M, Hampp R (1999) Identification and expression analysis of
two fungal cDNA regulated by ectomycorrhiza and fruit body formation. New Phytol 144:
195–202
Ntambi JM, Buhrow SA, Kaestner KH, Christy RJ, Sibley E, Kelly TJ Jr, Lane MD (1988)
Differentiation-induced gene expression in 3T3-Ll preadipocytes. Characterization of a differ-
entially expressed gene encoding steaoryl-coa desaturase. J Biol Chem 263:17291–17300
Palmieri F (1994) Mitochondrial carrier proteins. FEBS Lett 346:48–54
Podila GK, Zheng J, Balasubramanian S, Sundaram S, Hiremath S, Brand JH, Hymes MJ (2002)
Fungal gene expression in early symbiotic interactions between Laccaria bicolor and red pine.
Plant Soil 244:117–128
Poinssot B, Vandelle E, Bentéjac M, Adrian M, Levis C, Brygoo Y, Garin J, Sicilia F, Thévenot
PC, Pugin A (2003) The Endopolygalacturonase 1 from Botrytis cinerea activates grapevine
defense reactions unrelated to its enzymatic activity. Mol Plant Microbe Interact 16:553–564
Polidori E, Agostini D, Zeppa S, Potenza L, Palma F, Sisti D, Stocchi V (2002) Identification of
differentially expressed cDNA clones in Tilia platiphyllos-Tuber borchii ectomycorrhizae
using a differential screening approach. Mol Genet Genomics 266:858–864
Porter TN, Li Y, Raushel FM (2004) Mechanism of the dihydroorotase reaction. Biochemistry

43:16285–16292
Punt PJ, Van Gemeren IA, Drint-Kuijvenhoven J, Hessing JGM, Van Muijlwijk-Harteveld GM,
Beijersbergen A, Verrips CT, Van Den Hondel CAMJJ (1998) Analysis of the role of the gene
bipA, encoding the major endoplasmic reticulum chaperone protein the secretion of homolo-
gous and heterologous proteins in black Aspergilli. Appl Microbiol Biotechnol 50:447–454
Rehling P, Model K, Brandner K, Kovermann P, Sickmann A, Meyer HE, K€uhlbrandt W, Wagner
R, Truscott KN, Pfanner N (2003) Protein insertion into the mitochondrial inner membrane by
a twin-pore translocase. Science 299:1747–1751
Santos B, Snyder M (2005) Targeting of chitin synthase 3 to polarized growth sites in yeast
requires Chs5p and Myo2p. J Cell Biol 136:95–110
Schmid J, Amrhein N (1995) Molecular organization of the shikimate pathway in higher plants.
Phytochemistry 39:737–749
Seroussi F, Pan HQ, Kedra D, Roe BA, Dumanski JP (1998) Caracterization of human NIPSNAP1
gene from 22q12: a member of novel gene family. Gene 212:13–20
Shen H, Chen J, Wang Z, Yang C, Sasaki T, Yamamoto Y, Matsumoto H, Yan X (2006) Root
plasma membrane H1-ATPase is involved in the adaptation of soybean to phosphorus starva-
tion. J Exp Bot 57:1353–1362
Silver S, Misra TK, Laddaga RA (1989) DNA sequence analysis of bacterial toxic heavy metal
resistances. Biol Trace Elem Res 21:145–163
Slater S, Houmiel KL, Tran M, Mitski TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple
b-ketothiolases mediate poly(b-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha.
J Bacteriol 180:1979–1987
Smith CV, Huang C, Miczak A, Russell DG, Sacchettini JC, Bentrup KH (2003) Biochemical and
structural studies of malate synthase from Mycobacterium tuberculosis. J Biol Chem 278:
1735–1743
Sundaram S, Kim SJ, Suzuki H, Mcquattie CJ, Hiremath ST, Podila G (2001) Isolation and
characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria
bicolor. Mol Plant Microbe Interact 14:618–628
Szczepaneck T, Lazowska J (1996) Replacement of two non-adjacent amino acid in the S.
serevisiae bi2 intron-incoded RNA maturase is sufficient to gain a homing- endonuclease
activity. Eur Mol Biol Organ J 15:3758–3767
Tagu D, Nasse B, Martin F (1996) Cloning and caracterization of hydrophobins-enconding cDNAs
from the ectomycorrhizal Basidiomycete Pisolithus tinctorius. Gene 168:93–97
Tagu D, De Bellis R, Balestrini R, De Vries OMH, Piccolli G, Stocchi V, Bonfante P, Martin F
(2001) Immuno localization of hydrophobin HYDPt-1 from de ectomycorrhizal basidiomycete
Pisolithus tinctorius during colonization of Eucalyptus globulus roots. New Phytol 149:
127–135
Tasto JJ, Morrell JL, Gould KL (2003) An anillin homologue, Mid2p, acts during fission yeast
cytokinesis to organize the septin ring and promote cell separation. J Cell Biol 160:1093–1103
Tatsumi A, Shoji J, Kikuma T, Arioka M, Kitamoto K (2007) Aggregation of endosomal-vacuolar
compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae.
Biochem Biophys Res Commun 362:474–479
Timonen S, Peterson L (2002) Cytoskeletons in mycorrhizal symbioss. Plant Soil 244:199–210
Timonen S, S€oderstr€ om B, Raudaskoski M (1996) Dynamics of cytosckeletal proteins in
developing pine ectomycorriza. Mycorrhiza 6:423–429
Velten J, Oliver MJ (2001) Tr288, a rehydrin with a dehydrin twist. Plant Mol Biol 45:713–722
Voiblet C, Duplessis S, Encelot L, Martin F (2001) Identifications of symbiosis-reguleted genes in
Eucalyptus globulus-Pisolitus tinctorius ectomycorrhiza by differential hybridization of
arrayed cDNAs. Plant J 25:181–191
Wilkinson CRM, Wallace M, Morphew M, Perry P, Allshire R, Javerzat JP, Mcintosh JR, Gordon
C (1998) Localization of the 26S proteasome during mitosis and meiosis in fission yeast.
EMBO J 17:6465–6476
W€osten HAB, Vocht ML (2000) Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta
1469:79–86
Yamagata S (1989) Roles of O-acetyl-L-homoserine sulfhydrylases in micro-organisms. Biochimie
71:1125–1143
Yu YB (2002) Coiled-coils: stability, specificity, and drug delivery potential. Adv Drug Deliv Rev
54:1113–1129
Zahner HA, Harkins JE, Pringle JR (1996) Genetic analysis of the bipolar pattern of bud site
selection in the yeast Saccharomyces cerevisiae. Mol Cell Biol 16:1857–1870
Zaretsky M, Sitrit Y, Mills D, Roth-Bejerano N, Kagan-Zur V (2006) Differential expression of
fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates
and Cistus incanus hairy root clones. New Phytol 171:837–846
.
Chapter 6
Agrobacterium tumefaciens-Mediated
Transformation of Ectomycorrhizal Fungi
Minna J. Kemppainen, Maria C. Alvarez Crespo, and Alejandro G. Pardo
6.1 Introduction
Introduction of homologous or heterologous genes into filamentous fungi requires

DNA-mediated transformation systems. The breakthrough of efficient fungal trans-
formation took place with the yeast Saccharomyces cerevisiae (Hinnen et al. 1978).
This advance rapidly led to the development of DNA-transformation techniques for
ascomycete filamentous model species such as Neurospora crassa and Aspergillus
nidulans (Case et al. 1979; Ballance et al 1983; Tilburn et al. 1983; Yelton et al.
1984) which later on were used for genetic transformation of basidiomycete
dimorphic pathogenic species such as Ustilago maydis and Cryptococcus neofor-
mans (Wang et al. 1988; Edman and Kwon-Chung 1990) and filamentous sapro-
trophic basidiomycetes such as Schizophyllum commune, Phanerochaete
crysosporum, Coprinus spp. and Agaricus bisporus (Binninger et al. 1987; Specht
et al. 1988; Alic et al. 1989; van de Rhee et al. 1996). Most of these methodologies
are based on permeabilization of cell membranes with polyethylene glycol (PEG)
or electroporation, or a combination of both (Punt et al. 1987; Richey et al. 1989;
Ward et al. 1989; Chakraborty and Kapoor 1990; Kuwano et al. 2008) and generally
require preparation of fungal protoplasts. Introduction of DNA can be accompanied
with restriction enzyme-mediated integration (REMI) for increasing transformation
frequency (Kahmann and Basse 1999). Also direct introduction of DNA into intact
cells by particle bombardment has been used for transforming filamentous fungi
(Lorito et al. 1993; Christiansen et al. 1995). Transformation of filamentous fungi is
traditionally carried out with linear or circular DNA that integrates into the fungal
genome by nonhomologous or homologous integration mechanisms. The frequency
M.J. Kemppainen, M.C. Alvarez Crespo, and A.G. Pardo (*)

Laboratorio de Micologı́a Molecular, Departamento de Ciencia y Tecnologı́a, Universidad Nacio-
nal de Quilmes, y Consejo Nacional de Investigaciones Cientı́ficas y Tecnicas (CONICET), Roque
Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina
e-mail: apardo@unq.edu.ar

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_6,
124 M.J. Kemppainen et al.
of homologous vs. nonhomologous integration varies and it is highly dependent on

the fungal species. The massive genetic research over the last 30 years on model
filamentous ascomycetes has led to the availability of several integrative transfor-
mation vectors and genetic tools for this group of fungi. Also auto-replicative DNA
elements are available for transformation of some fungal species such as
A. nidulans and U. maydis (Tsukuda et al. 1988; Aleksenko 1994). Transformants
selection of filamentous fungi is performed either with nutritional markers, requir-
ing auxotrophic strains, or with dominant antibiotic markers. The most common
antibiotic markers used for fungal transformation are hygromycin B, phelomycin,
bialophos, sulfonylurea, nourseothricin, carboxin, blasticidin S and benomyl
(Weld et al. 2006).
The transformation efficiency of filamentous fungi by traditional transformation
techniques is often limited by the researcher’s capacity to produce the necessary
quantity of viable protoplasts. This is rather challenging and it is carried out by
enzymatic degradation of the fungal cell wall. Optimizing the cell wall degradation
protocol can be complicated by differences in the physiological state of the starting
fungal material and batch variations in commercially available enzyme prepara-
tions. Successful protoplast preparation also generally requires young homogenous
hyphal material. This is preferably obtained by freshly germinating sexual or
asexual spores, oidia, or the yeast-phase of dimorphic filamentous fungi. If these
are not produced by the fungus under study, vegetative mycelium can also be used
for protoplast production. This is however not an optimal material due to wide
physiological variation between different parts of the mycelium. Also PEG is
known to cause fusion of protoplasts in filamentous fungi and fungal cells used to
prepare protoplasts of A. nidulans usually contain multiple nuclei (Fiddy and Trinci
1976; Kuwano et al. 2008).
6.2 Genetic Modification of ECM Fungi with Traditional

Transformation Methods
Basidiomycete ECM fungi have turned out to be especially difficult to modify

via traditional genetic transformation methods. This is partly due to the fact that
spore production cannot be obtained under laboratory conditions and production of
viable protoplast needed for many transformation techniques has been difficult and
requires working with rather slowly growing vegetative mycelia. On the other hand,
regeneration of protoplast is extremely difficult for many ECM fungi, presenting
another obstacle for genetic transformation. Furthermore, many antibiotic markers
used for ascomycetes cannot be used in basidiomycetes due to natural resistance.
Despite these difficulties some ECM fungi such as Hebeloma cylindrosporum
and Laccaria laccata have been transformed via protoplast (Barret et al. 1990;
Marmeisse et al. 1992). Particle bombardment mediated transformation, avoiding
tedious protoplast production, has been reported for Paxillus involutus, Pisolithus
6 Agrobacterium tumefaciens-Mediated Transformation of Ectomycorrhizal Fungi 125
tinctorius and Suillus spp. (Bills et al. 1995; Rodrı́guez-Tovar et al. 2005;
Sunagawa et al. 2007) and also for Laccaria bicolor (Bills et al. 1999). However,
the transformation efficiencies of ECM fungi have been rather low and obtaining
transformants has been irregular and time consuming. Therefore, these original
reports have not led to the efficient and widespread use of genetic fungal transfor-
mation in mycorrhizal research. On the other hand, the lack of functional genetic
tools adapted to filamentous basidiomycetes (transformation plasmids adapted to
ECM fungi, availability of selection markers, etc.) partly due to the low number
of researchers dedicated to the subject has slowed down the advances in genetic
transformation of ECM fungi.
6.3 Agrobacterium-Mediated Transformation of Fungi
Agrobacterium tumefaciens and A. rhizogenes are Gram-negative bacteria which

cause crown gall tumors and hairy root disease in their natural hosts, dicotyledon-
ous plants. These diseases are caused by the transfer of plant hormone biosynthesis
genes and genes responsible for production of opine from bacteria to the plant.
These genes are located in Ti-(tumor inducing) and Ri (root inducing)-plasmids
and transferred as single-stranded T-DNA (ssT-DNA or T-strand), via a type IV
secretion system to the host cell where then double-stranded T-DNA (dsT-DNA or
T-DNA) incorporates in the host genome (Ream 2009). Opines produced by the
plant in tumor tissue are used by the bacteria as C and N sources. For a long time
Agrobacterium was thought to be the only example of horizontal interkingdom
gene transfer in nature but also some other bacteria such as Rhizobium sp. NGR234,
Sinorhizobium meliloti and Mesorhizobium loti were demonstrated to be capable
of genetically transforming different plant tissues and plant species (Broothaerts
et al. 2005).
The natural capacity of Agrobacterium to transfer genes to intact plant cells has
been exploited by plant researchers to genetically modify both dicotyledonous and
monocotyledonous plants for decades. Agrobacterium-mediated transformation
(AMT) had already become a routine tool in plant sciences in the 1980s. The
finding that the natural oncogenes in the T-DNA could be replaced by sequences
of interest, and that these were equally transferred to the host plant for trans-genesis
revolutionized plant research. Development of binary vector systems, where the
T-DNA and virulence genes in a large size Ti-plasmid (~200 kb) were separated
into two different replicons, an easily handled T-DNA containing binary vector and
a virulence Ti-helper plasmid, also greatly increased the use of Agrobacterium in
plant transformation (de Framond et al. 1983; Hoekema et al. 1983).
In 1995 Bundock et al. published the first report on AMT of Saccharomyces
cerevisiae demonstrating that A. tumefacies is capable of transferring T-DNA not
only to its natural hosts, plants, but also to fungi. This finding initiated a true
revolution in fungal research. Demonstration of AMT of filamentous ascomycetes
(de Groot et al. 1998) has led to the widespread use of Agrobacterium as a tool
for fungal transformation. Several ascomycetes, basidiomycetes and also zygomy-

cetes have been shown to be susceptible to this transformation methodology
(Michielse et al. 2005b) and new fungal species are joining this group with
increasing speed. The great benefit of AMT of fungi is that there is no need for
complicated protoplast preparation. Intact fungal cells can be transformed via
Agrobacterium and these cells can originate from different sources available:
spores, germlings, vegetative mycelium or even fruiting body tissue. Many fungal
species, before recalcitrant to or presenting very low transformation efficiencies
with traditional gene transformation methods, have been efficiently modified via
AMT. These also include several filamentous basidiomycetes. Despite the wide
success of AMT on fungi not all species are transformed with this technique.
Furthermore, the fungal susceptibility to AMT has been show to vary between
different isolates of the same species (Covert et al. 2001; Sullivan et al. 2002;
Fitzgerald et al. 2003). The precise reasons for this variation are not known but
strain-specific variations on cell wall structures could be involved. Also several
studies have shown that Agrobacterium strain/binary vector combination used is a
fundamental factor for fungal transformation efficiency (Vijn and Govers 2003;
Park and Kim 2004).
6.4 AMT of ECM Fungi
Successful Agrobacterium transformation of the homobasidiomycete Agaricus

bisporus with hygromycin B resistance (de Groot et al. 1998; Chen et al. 2000)
gave the first insight that AMT could also be used for genetic transformation of
basidiomycete ECM fungi. This transformation method was soon proved functional
by transforming vegetative mycelia of the ECM fungi Suillus bovinus, Paxillus
involutus and Hebeloma cylindrosporum with the Streptoalloteicus hindustanus
phleomycin resistance gene (Sh BLE) and the Escherichia coli hygromycin resis-
tance gene (hph) as selection markers (Pardo et al. 2002; Hanif et al. 2002; Combier
et al. 2003). Other AMT-modified ECM fungi are Pisolithus tinctorius (Pardo et al.
2005; Rodrı́guez-Tovar et al. 2005) and Laccaria bicolor (Kemppainen et al. 2005).
Tuber borchii, the model ascomycete ECM fungus, has also been transformed with
Agrobacterium. This transformation, however, did not lead to stable genomic
integrations of the transgenes (Grimaldi et al. 2005). A similar situation has been
shown in ECM basidiomycetes with phleomycin resistance as selection marker
(Pardo et al. 2002, 2005). Also the ECM fungus Amanita muscaria has not been
successfully transformed with Agrobacterium (Nehls, personal communication).
It is clear that susceptibility of EMC fungi to AMT shows species variation
and setting-up an AMT-based transformation system for a new species requires
optimization.
Figure 6.1 shows a comparison between different transformation methods of
fungi.
cost/difficulty: low/high low/high high/high low/low
PEG/CaCl2-mediated transformation
AMT
electroporation particle bombardment

intact cells
protoplasts
fungal starting material:

spores vegetative mycelia
fruiting body
protoplast preparation challenging, tissue
mycelia regeneration often difficult yeast cells
germlings
typically multiple predominantly single
transgene copy number:
transgene integrity after

variable highly conserved
genomic integration
Fig. 6.1 A comparison of traditional genetic transformation methods and AMT of fungi. Most of
the traditional methods require tedious preparation of protoplast, which is not the case for AMT.
Also the number of AMT integrated transgenes is generally low and their integrity highly
conserved making analysis of transformed strains simple. The cost/difficulty comparison includes
both the price of reagents and special equipments needed, and the overall efficiency of each fungal
transformation method
6.5 AMT-Mechanisms: T-DNA Transfer to the Host Cell
Agrobacterium natural hosts are plants but it can also transfer the T-DNA element
to other eukaryotic cells such as fungi and also, under laboratory conditions to,
mammalian cells (Kunik et al. 2001). The T-DNA transfer has been exhaustively
studied in plant cells (McCullen and Binns 2006; Citovsky et al. 2007; Dafny-Yelin
et al. 2008; Gelvin 2009; Ream 2009). How horizontal gene transfer between
Agrobacterium and other nonplant hosts is carried out and which are the funda-
mental processes shared between all the hosts is not yet known (Lacroix et al.
2006). AMT is already a well established and a routine transformation method for
many fungal species. Despite its popular use the knowledge on AMT of fungi at the
molecular level is minimal and should receive more research interests in future.
The T-DNA transfer between bacteria and the plant host is a multistep process
and it can be divided into three fundamental phases: (1) pretransfer bacterial
processes, (2) T-DNA transfer and (3) host-cell events leading to T-DNA integra-
tion in the host genome. Steps 1 and 2 are mostly controlled by the bacterial protein-
machinery but step 3 depends both on imported bacterial proteins and host cellular
mechanisms. Agrobacterium-infection also activates several plant-host defense
mechanisms such as production of reactive oxygen species, systemic acquired
resistance processes and RNA silencing during early stages of the interaction but
these are attenuated afterwards probably by direct repression of defense genes by

the bacterium. Even though several factors participating in T-DNA mobilization
and integration are known, the full understanding of AMT on plant cells is still
incomplete and under active research.
6.5.1 Pretransfer Bacterial Processes Leading to T-DNA

Mobilization
These events depend on the activity of bacterial chromosomal virulence genes (chv)
and tumor-inducing Ti plasmid virulence genes (vir) which control host recogni-
tion, attachment and T-DNA production.
Host cell recognition starts by sensing the vicinity of a wounded plant. Wounded
plant cells release phenolic compounds that are detected by the Agrobacterium two-
component regulatory system of VirA and VirG and this induction leads to activa-
tion of other vir genes responsible for T-DNA mobilization. Acetosyringone
(3,5-Dimethoxyacetophenone) a commercially available phenolic molecule, is a
potent inducer of Agrobacterium and is used for inducing bacteria for transforming
nonplant hosts or increasing plant transformation efficiency. The inner-membrane
protein VirA, a kinase present as a dimer, detects the phenolic compound and this
leads to its autophosphorylation. This phosphorylation is further transferred to
VirG, a transcriptional activator protein with DNA-binding capacity. The activated
VirG causes transcriptional activation of itself and other vir-genes in the Ti plasmid
[such as virB operon genes (1–11), virD1, virD2, virD4, virE1, virE2, virE3]. The
chromosomally encoded ChvE protein, present in the periplasmic space further
increases the vir-gene induction level by interacting with VirA in the presence of
aldose monosaccharides, low pH and low phosphorus conditions. Activity of virD1
and virD2 proteins leads to excision and liberation of a single-stranded DNA
molecule, T-strand, from the T-region of the Ti plasmid. This region is surrounded
by 24-bp border repeats called left- and right border (LB and RB respectively)
forming the T-DNA. The VirD1-VirD2 heterodimer nicks the T-DNA at the border
repeats and VirD2 covalently binds to the 50 end of it. The VirD2/T-strand is further
mobilized towards the bacterial membrane for horizontal transfer.
6.5.2 T-Strand Transfer
VirB proteins (1–11) and virD4 are structural compounds of a specialized trans-
membrane type IV secretion system (T4SS) apparatus with a T-pilus. These
generally localize to cell poles of Agrobacterium. The T4SS is used for transferring
VirD2/T-strand to the host cell. Also several virulence proteins (VirE2, VirE3, VirF
and VirD5) are transported via T4SS to the host. VirD4 of the transporter has ATP
binding domains and it has been shown to interact with transported virulence
proteins and T-strand indicating that VirD4 physically mediates and, most likely
also provides energy for their transport. The export of VirE2 needs a chaperon
protein VirE1 that avoids VirE2 to form filamentous aggregates with itself.
Attachment of Agrobacterium cells to the host cell is fundamental for T-strand
transfer. The nature of how bacteria interact with plant host cell wall is not clear.
Bacterial chromosomal genes seem to be needed for this step. Several putative
bacterial receptors and host proteins are postulated to be involved, such as victro-
nectin-like protein, rhicadhesin protein, a cellulose synthatase-like protein, and
several VirB2-interacting proteins. The attachment of Agrobacterium to the host
cell is not Ti-plasmid dependent, ruling out attachment via T-pilus. On the other
hand, chromosomal genes chvA, chvB and pscA(exoC), involved in synthesis and/or
localization of b 1-2-glucan are required even though the role of this compound in
attachment is not clear.
Exactly how the VirD2/T-strand conjugate and Vir-proteins are transferred to
the host cell cytoplasm is not yet understood. In T4SS-directed plasmid conjuga-
tions between bacteria the role of the pilus seems to be pulling the cells close
together forming the so called mating junction. This is characterized in Gram-
negative bacteria by fusion of outer membranes and there is no evidence of pilus in
the sites of genetic material transfer. Whether similar membrane unions are taking
place between Agrobacterium and the recipient eukaryotic cell is not known.
6.5.3 Host Cell Events Leading to T-DNA Integration in the Host

Genome
Once within the host cell VirD2/T-strand conjugate is believed to be covered with
VirE2 to form the T-complex. VirE2 is believed to protect the T-strand from
nuclease attacks because in the absence of this protein T-DNA integrations are
often truncated at their left ends. Recent data is, however, indicating that the
covering of T-strand by VirE2 would be taking place in the nucleus and not in
the cytoplasm (Ream 2009). VirD2 has a nuclear localization signal and T-strand
is moved, probably by an active dynein motor mechanism towards host cell
nucleus. Host proteins would be involved in this intracellular transport. Also
VirE2 promotes localization of T-strand to the nucleus. VirD5 might function in
plants to aid nuclear localization of VirE2.
Plant proteins VIP1 and a-importin proteins are involved in nuclear import of
VirE2. VirE3 can mimic the function of VIP1 and probably participate in the
nuclear import of VirE2. VirD2-T-DNA conjugate is most probably transported
across the nuclear membrane by an a-importin 1 protein. Nuclear localization
signal of VirD2 is fundamental for T-strand entrance to the nucleus.
Once inside the nucleus the T-complex may be directed to chromatin with the
assistance of plant VIP2 protein for integration. Also VIP1 has been proposed to
play role in T-DNA integration by interacting with histone H2A. VIP2 seems to be
required for stable, but not transient transformation of plants. A targeted proteolysis
of VirE2 takes place with the assistance of nuclear localizing VirF protein and prior
to integration in host genome the ssT-DNA (T-strand) is completed to the ds-form
(T-DNA) by host DNA repair mechanisms. Later on DNA breaks are produced in
the host genome and the dsT-DNA is ligated to these breaks. Complete understand-
ing of these integration events is still missing. Most likely double-stranded breaks
are involved.
Integration events are conducted by host proteins and the T-DNA can be
integrated via nonhomologous end joining (NHEJ) or homologous recombination
(HR) mechanisms depending on the predominant host mechanism of DNA integra-
tion. HR can take place if T-DNA shares homology with the recipient genome. In
plants T-DNA integration occurs predominantly via NHEJ while in other nonplant
hosts also HR-integration can take place.
6.6 Molecular Mechanism of AMT in Fungi
AMT of fungi depends on acetosyringone induction of Agrobacterium indicating

participation of vir-genes and T4SS in T-strand transfer to a nonplant host (de Groot
et al. 1998). The bacterial pretransfer steps are therefore most probably identical
during transformation of plant and nonplant hosts. However, the attachment,
T-strand transfer and host-cell directed events of T-DNA integration show some
differences between different eukaryotic hosts. Most information for these differ-
ences comes from AMT of S. cerevisieae and Aspergillus awamori with Agrobac-
terium strains mutated in different virulence genes (Bundock et al. 1995; Piers et al.
1996; Michielse et al. 2004a, b). A different requirement for VirE protein has been
reported. While lack of this protein almost inhibits transformation of plants, fungal
transformation efficiency is only reduced. Also transformation of yeast with Agro-
bacterium mutated in chvA, chvB and exoC is as efficient as with a wild type strain.
Activity of these chromosomally encoded proteins is shown to be fundamental for
attachment and transformation of plant cells but does not seem to play an important
role in AMT of fungi. Interestingly, ChvA and ChvB are needed for attachment
to human cells (Kunik et al. 2001) demonstrating that variation exist in require-
ments of AMT on different nonplant hosts. Yeast and human cells also lack plant
type VIP1 protein which is involved in nuclear import of VirE2. However,
bacterial origin VirE3 can mimic the VIP1 function and could partly explain the
functionality of AMT in these nonplant hosts.
The T-DNA integration step is conducted mainly by the host-cell protein
machinery. Therefore, it shows significant difference between plant and nonplant
hosts and is also dependent on the nucleotide sequence of the T-DNA. While in
plants T-DNA, even in the presence of long flanks homologous to plant genome,
integrates via a nonhomologous end-joining mechanism, in nonplant species, such
as fungi, both homologous (HR) and nonhomologous recombination (NHR)
mechanisms can participate in T-DNA integration. Which cellular mechanism is

dominant depends on the organism, length of homologous sequence in the T-DNA
and the degree of homology. The main molecular knowledge of T-DNA integration
comes from S. cerevisiae where both NHEJ and HR take place. Several DNA-repair
genes have been demonstrated to be essential or participate in HR and NHR of
T-DNA (van Attikum et al. 2001; van Attikum and Hooykaas 2003). In yeast the
Ku70 encoding gene is determinant for NHR and Rad52 for HR, and double
mutants of these genes are completely blocked in T-DNA integration (van Attikum
and Hooykaas 2003). The information on HR in yeast has been exploited by
plant researchers to improve low HR rates presented in these organisms. The
heterologous expression of another yeast DNA-repair protein, Rad54 involved in
HR, in plant cells has resulted in increased homologous T-DNA integration in this
system (Shaked et al. 2005).
6.7 Use of AMT in Fungal Genetics
The capacity of Agrobacterium to transfer genetic material to fungi has been

increasingly used for genetic modification of this group of eukaryotes (Michielse
et al. 2005b; Weld et al. 2006). It has proven to be a powerful tool for filamentous
fungi overcoming problems rising from traditional DNA based transformation
techniques such as the need for protoplast preparation. AMT has also resulted in
higher transformation efficiencies in many fungal species for which other transfor-
mation methods already existed resulting in less complex transgene integration
patterns. Traditional transformation methods, such as PEG mediated transforma-
tion, electroporation and particle bombardment, generally produce a high percent-
age of multiple and often tandem transgene integrations. This has complicated the
interpretation of produced fungal phenotypes. AMT on the other hand results in
predominantly single integration of the T-DNA in the fungal genome and this
integration, in absence of homologous sequences, occurs randomly enough for
making random gene tagging possible. Sequencing of fungal genomes has initiated
a true interest for functional genomics research in filamentous fungi. This requires
the optimization of high-throughput transformation and gene disruption tools such
as AMT.
6.8 AMT-Mediated Random Gene Tagging
Traditional fungal transformation methods generally produce multiple transgene

integration events. The transgene copy number can be reduced by incorporating a
REMI step but the use of restriction enzymes can lead to genomic reorganizations
and phenotypes not linked to the integration site of the transgene. The number of
T-DNAs integrated in the fungal genome via AMT can also vary. The number of
integrations can be influenced by factors such as cocultivation time, concentration

of acetosyringone or bacterial/fungal cell ratio during transformation. Despite the
species and transformation protocol a high percentage (50–80%) of Agrobacterium
transformed fungal strains are reported to carry a single T-DNA integration
(Michielse et al. 2005b). This is a much higher percentage than with AMT of plants
which generally produces multilocus and/or multicopy integrations. Even though
small deletions in the border sequences of the integrated T-DNAs are common
in fungi, as in transformed plants, the T-DNAs of known sequence are usually
quite complete making integration site analysis by PCR-based methods such as
TAIL-PCR or inverted PCR possible. Due to this predominant single transgene
copy integration nature, AMT has been the preferred methodology for producing
random insertional fungal libraries of Magnaporthe grisea, Cryptococcus neofor-
mans, Leptosphaeria maculans and Colletotrichum higginsianum (Tsuji et al. 2003;
Idnurm et al. 2004; Walton et al. 2005; Betts et al. 2007; Blaise et al. 2007; Jeon
et al. 2007; Li et al. 2007; Talhinhas et al. 2008; Huser et al. 2009). These random
gene disruption collections have been used for successful identification of novel
gene functions involved in fungal pathogenesis.
The AMT random tagging methodology can also be used for detecting promo-
ters or increase transcription of contiguous genes. These can be done by enhancer
trap constructions where a minimal promoter is linked to a reporter gene, with a
promoterless reporter gene construct or placing a strong promoter close to the
border of the T-DNA. These approaches have been used in fungi with traditional
transformation methods but not yet with AMT. There is no reason to believe
that these genetic modification techniques are not compatible with AMT. Also
incorporation of autoreplicative elements in transferred T-DNAs has been shown to
enhance transformation efficiency in S. cerevisiae. In this case the T-DNA does not
need to integrate in the recipient genome (Bundock et al. 1995). Even though
autoreplicative sequences are available for some filamentous fungi, these have
not been used in combination with AMT.
6.9 Is Random T-DNA Tagging of Fungal Genome

Really at Random?
The randomness of T-DNA integration in the recipient genome has been a matter
of ongoing scientific debate which has direct implications on the use of AMT
in saturated mutagenesis. Several plant studies have indicated a bias towards
transcriptionally active sites, especially in regulatory regions (Koncz et al. 1989;
Brunaud et al. 2002; Alonso et al. 2003; Sallaud et al. 2004). The transcriptional
status and the open chromatin structure have been proposed to determine this
T-DNA attraction to gene-rich zones. However, recent studies on plants are indi-
cating that this observed bias has been an artifact of T-DNA libraries generated
under selection pressure. Analysis of plant T-DNA target sites under selection-free
conditions indicates that the integration profile of T-DNAs in the plant genome is
more random than previously believed (Kim et al. 2007). Recovery of transgenic
lines under selection pressure requires a strong expression of the selection marker
carried by the T-DNA. This is more probable in transcriptionally gene-rich zones
than in heterochromatin integrations where transcription of the selection marker
can be hindered by the epigenetic status of the integration site (Francis and
Spiker 2005).
Similar biased T-DNA integration has been reported for filamentous fungi. A
preference of T-DNA to integrate in fungal promoters and gene rich regions has
been reported for M. grisea and C. neoformans (Walton et al. 2005; Choi et al.
2007; Li et al. 2007; Meng et al. 2007). The T-DNA tagged analyzed fungal strains
in these studies were all originated from selective conditions. Therefore, it is highly
probable that T-DNA integration is also more random in fungi as now postulated for
plants. From the point of view of use of AMT for random mutagenesis and
transgene expression this bias towards gene rich zone is however more a benefit
than a problem. Higher percentage of tagging of active genes can be expected with
this genetic transformation method. Studies carried out in our group on gene
transfer from Agrobacterium and recovery of the transgene by plasmid rescue
show that T-DNA integration in the genome of the ectomycorrhizal model fungus
L. bicolor occurs mainly in genes and as single copy. These indicate that T-DNA
insertion by AMT would be very useful for high-throughput tagged mutagenesis in
ectomycorrhizal fungi (Kemppainen et al. 2008).
6.10 Targeted Gene Disruption by AMT
DNA integration into the genome via HR is highly rare in plants but more common
in fungi. Therefore, the integration of T-DNA harboring homology with the fungal
genome can also occur via HR. This has made possible the use of AMT not only for
random gene tagging but for targeted gene disruption. In gene targeting, a selection
marker cassette in the T-DNA is surrounded by DNA flanks homologous to the
target gene (or a zone around it). The successful integration of T-DNA via HR
results in interruption of the target gene by the selection cassette. This approach has
been used for gene inactivation already in several ascomycete species such as
S. cerevisiae, Monilinia fructicola, Verticillium dahliae, Aspergillus fumigatus,
A. awamori, Metarhizium anisopliae and Mycosphaerella graminicola (Zwiers and
de Waard 2001; van Attikum and Hooykaas 2003; Michielse et al. 2005a; Rauyaree
et al. 2005; Sugui et al. 2005; Lee and Bostock 2006; Staats et al. 2007). However,
the frequency of HR events is a fungal species specific character and it is also highly
dependent on the length of the homologous flanks used. While HR is the dominant
pathway in S. cerevisiae and just 100 bp homology flanks can be used for targeting
genes in this yeast, in filamentous fungi generally at least 1 kb flanks or even longer
homologous sequences are needed for gene disruption (Wilson et al. 2002;
Michielse et al. 2005b). Even in the presence of long homologous sequences HR
rates in filamentous fungi are variable and generally low. For example in wild-type
strains of Acremonium chrysogenum, Aspergillus nidulans, N. crassa, Sordaria

macrospora and M. grisea HR rates are as low as 0.1–7% (Chaveroche et al.
2000; Liu et al. 2001; Talbot and Foster 2001; Colot et al. 2006; P€oggeler and
K€ uck 2006). AMT when compared to other traditional genetic transformation
methods has been shown to increase this generally low HR rate both in yeast and
filamentous fungi (Bundock et al. 1999; Michielse et al. 2005a, b). Also the degree
of dependency on the length of homologous sequence flanks is lower with AMT
(Michielse et al. 2005a, b). This increased HR rate is proposed to depend on the ssT-
DNA nature or the active host protein assisted localization into the nucleus. Even
though AMT has been shown to increase HR, the difficulties in gene inactivation of
filamentous fungi and in basidiomycete fungi in particular have led to the genera-
tion of mutant strains affected in their NHEJ-pathway. NHEJ is shown to depend on
the activity of Ku70-Ku80 heterodimer that binds to broken DNA ends. These Ku70
and Ku80 proteins were also identified in fungi (Hefferin and Tomkinson 2005).
Mutations in these genes result in inactivation of NHEJ and leaves HR as the
dominant pathway for DNA integration. HR efficiency of 70–100% has been
reported for ku70 or ku80 mutants of Aspergillus oryzae, A. fumigatus, A. nidulans,
N. crassa, S. macrospora, C. neoformans and M. grisea (Ninomiya et al. 2004; da
Silva Ferreira et al. 2006; Goins et al. 2006; Nayak et al. 2006; P€oggeler and K€uck
2006; Takahashi et al. 2006; Villalba et al. 2008).
6.11 Laccaria bicolor Represents a Special Challenge

for AMT as a Reverse Genetic Tool
The lack of an efficient transformation method for the genome-sequenced ECM

fungus L. bicolor (Martin et al. 2008) was a major obstacle in ECM research. Even
though this species was reported to be transformed via particle bombardment (Bills
et al. 1999) and its closely related species L. laccata via PEG mediated protoplast
transformation (Barret et al. 1990) both of these traditional genetic transformation
techniques are time consuming and complicated, show low transformation effi-
ciency and reproducibility, are of high costs and generally result in multiple
transgene copies in the recipient genome. AMT on the other hand overcomes
these limitations but not all fungal species are as susceptible to this method or do
not produce stable integrations for still unknown reasons. We developed an efficient
AMT system for L. bicolor (Kemppainen et al. 2005). However, Laccaria shows
some special features that may or will complicate the use of AMT or any other
genetic transformation method for reverse functional genetic studies in ECM.
Unlike filamentous ascomycetes the symbiotic phase of the fungus is the dikaryon
where two compatible nuclei coexist in each hyphal compartment. The life-cycle
and basidiospore production cannot be completed without ECM formation with the
plant host. Furthermore, fruiting body production is unpredictable and requires a
long time and therefore cannot be achieved under axenic conditions. The dikaryotic
nature of Laccaria generates a technical obstacle for the use of AMT as a random
gene tagging tool for searching gene functions relevant in ECM. An interruption of
a gene copy in one of the nuclei may not be enough for altering gene expression and
thus produce functional phenotypes. Also a gene interruption may lead to increased
transcription from the intact allele in the untransformed nucleus. This compensation
may result in unaltered gene expression level in tagged dikaryons. Tagging of
Laccaria monokaryons could however be used for studying general fungal gene
function and especially for identifying genes involved in control of dikaryotization.
The same dilemma of the dikaryon also affects the use of AMT for targeted
inactivation of ECM-regulated genes. Targeted disruption of these genes should be
carried out independently in two compatible monokaryons which could form
functional double knock-out dikaryotic strains by further crossing. Homobasidio-
mycetes usually show extremely low HR rates. Working with two Laccaria mono-
karyotic strains for producing knock-out dikaryons can be predicted to be extremely
A. tumefaciens L. bicolor
growing in liquid (monokaryotic or
induction medium (with dikaryotic strain)
AS) at 28 °C growing on solid medium at 24 °C
Co-cultivation
Fungal mycelia (on cellophane
membranes) plus bacteria, on
solid medium supplemented with AS.
Incubated 3-5 days at 22 °C
LB RB Nucleus
Binary T-DNA
VirD1/D2
vector Transfer of fungal colonies to
5´ selection medium
VirD2 VirE2 (with hygromicin and cefotaxime).
Vir Ti-helper Predominantly Incubated 7-10 days
plasmid 3´ ? single integration
region
3´ Host
VirB1-11/ Selection of fungal growing points
VirE2 proteins?
VirG VirD4
= T4SS and transfer to 2nd round of selection
VirA
A. tumefaciens cell L. bicolor cell 3nd round of selection
AS
Laccaria trasnformants
LB 35S-3’ hph Pgpd Gene construct RB
Molecular analysis
T-DNA
Fig. 6.2 Schematic representation of Laccaria bicolor transformation mediated by Agrobacter-

ium tumefaciens. 35S-30 : cauliflower mosaic virus 35S terminator. AS: acetosyringone. Cefotax-
ime: cephalosporin antibiotic which inhibits the final transpeptidation step of peptidoglycan
synthesis in bacterial cell walls. Used for killing Agrobacterium cells. Gene construct: different
gen constructs can be transferred via Agrobacterium and integrated in Laccaria genome (i.e.,
plasmid rescue T-DNAs, RNAi-triggers, heterologous genes of interest, reporters, etc.). hph: hph
gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to hygro-
micin B (fungal selection marker) and structurally related antibiotics. LB T-DNA left border of
pCAMBIA1300. Pgpd glyceraldehide-3-phosphate dehydrogenase promoter of Agaricus bis-
porus. RB T-DNA right border of pCAMBIA1300. T4SS type IV secretion system. For details
on Agrobacterium-mediated transformation of Laccaria see Kemppainen et al. (2005, 2008)
challenging and time consuming if not impossible. The use of the RNA-silencing
technology could however represent an alternative and more straightforward
approach for studying ECM-regulated Laccaria genes (Kemppainen et al. 2009;
Kemppainen and Pardo 2010; this book, Chap. 9). A diagram showing the use of
Agrobacterium for genetic transformation of Laccaria is presented in Fig. 6.2.
6.12 Conclusion
Despite the great ecological and economic importance of ECM the current compre-
hension of host-fungus recognition, establishment and functions of ECM is still
rather limited. During the last ten years the use of novel molecular methods such as
EST-libraries and cDNA micro– and macro arrays has dramatically increased the
knowledge on genetic regulation underlying the ECM interaction. More impor-
tantly, the decision of the Department of Energy Joint Genome Institute (JGI) to
sequence the genomes of micobionts of the first genome-sequenced tree, poplar,
took the mycorrhizal research towards the genomic era. As a result the full genomic
sequence of the basidiomycete ECM fungus Laccaria bicolor strain S238N-H82
was resolved in collaboration between JGI and the Laccaria Genome Consortium
which joined the efforts of several ECM research laboratories around the world.
The Laccaria genome sequence is currently been used for genome-wide expression
profiles at different stages of ECM development. This approach is generating an
immense amount of valuable data. However, the current ECM research faces a
serious technical obstacle. Further studies for resolving the biological relevance of
the identified symbiosis-regulated genes depend on the availability of reverse
genetic tools, among them an efficient genetic transformation technique. We
established a high throughput AMT based on hygromycin B resistance for Laccaria
dikaryotic and monokaryotic strains. This gene transfer from Agrobacterium and
recovery of the transgene by plasmid rescue showed that T-DNA integration in the
genome of L. bicolor occurs mainly in genes and as single copy. These indicate that
T-DNA insertion by AMT would be very useful for high throughput tagged
mutagenesis in ectomycorrhizal fungi. AMT has opened a new era for genetic
studies in mycorrhizal research and its use in combination with the RNA silencing
technology will certainly help in elucidating the function of symbiosis-regulated
genes in ECM development.
References
Aleksenko AY (1994) Cointegration of transforming DNAs in Aspergillus nidulans: a model using

autonomously-replicating plasmids. Curr Genet 26:352–358
Alic M, Kornegay JR, Pribnow D, Gold MH (1989) Transformation by complementation of an
adenine auxotroph of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl
Environ Micobiol 55:406–411
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J,
Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H,
Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C,
Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE,
Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003)
Genome wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657
Ballance DJ, Buxton FP, Turner G (1983) Transformation of Aspergillus nidulans by the
orotidine-5-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Com-
mun 112:284–289
Barret V, Dixon RK, Lemke PA (1990) Genetic transformation of a mycorrhizal fungus. Appl
Microbiol Biotechnol 33:313–316
Betts MF, Tucker SL, Galadima N, Meng Y, Patel G, Li L, Donofrio N, Floyd A, Nolin S, Brown
D, Mandel MA, Mitchell TK, Xu JR, Dean RA, Farman ML, Orbach MJ (2007) Development
of a high throughput transformation system for insertional mutagenesis in Magnaporthe
oryzae. Fungal Genet Biol 44:1035–1049
Bills SN, Richter DL, Podila GK (1995) Genetic transformation of the ectomycorrhizal fungus
Paxillus involutus by particle bombardment. Mycol Res 99:557–561
Bills S, Podila G, Hiremath S (1999) Genetic engineering of a fungus Laccaria bicolor for use as a
biological control agent. Mycologia 91:237–242
Binninger DM, Skrzynia C, Pukkila PJ, Casselton LA (1987) DNA-mediated transformation of the
basidiomycte Coprinus cinereus. EMBO J 6:835–840
Blaise F, Remy E, Meyer M, Zhou L, Narcy JP, Roux J, Balesdent MH, Rouxel T (2007) A critical
assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity
gene discovery in the phytopathogenic fungus Leptosphaeria maculans. Fungal Genet Biol
44:123–138
Broothaerts W, Mitchell HJ, Weir B, Kaines S, Smith LM, Yang W, Mayer JE, Roa-Rodrı́guez C,
Jefferson RA (2005) Gene transfer to plants by diverse species of bacteria. Nature
433:583–584
Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C,
DeRose R, Pelletier G, Lepiniec L, Caboche M, Lecharny A (2002) T-DNA integration into the
Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep 12:1152–1157
Bundock P, Dulk-Ras A, Beijersbergen A, Hooykaas PJ (1995) Trans-kingdom T-DNA transfer
from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214
Bundock P, Mroczek K, Winkler AA, Steensma HY, Hooykaas PJ (1999) T-DNA from
Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis.
Mol Gen Genet 261:115–121
Case ME, Schweizer M, Kushner SR, Giles NH (1979) Efficient transformation of Neurospora
crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263
Chakraborty BN, Kapoor M (1990) Transformation of filamentous fungi by electroporation.
Nucleic Acids Res 18:6737
Chaveroche MK, Ghigo JM, d’Enfert C (2000) A rapid method for efficient gene replacement in
the filamentous fungus Aspergillus nidulans. Nucleic Acid Res 28:E97
Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method
for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ
Microbiol 66:4510–4513
Choi J, Park J, Jeon J, Chi MH, Goh J, Yoo SY, Park J, Jung K, Kim H, Park SY, Rho HS, Kim S,
Kim BR, Han SS, Kang S, Lee YH (2007) Genome-wide analysis of T-DNA integration into
the chromosomes of Magnaporthe oryzae. Mol Microbiol 66:371–382
Christiansen SK, Knudsen S, Giese H (1995) Biolistic transformation of the obligate plant
pathogenic fungus, Erysiphe graminis f.sp. hordei. Curr Genet 29:100–102
Citovsky V, Kozlovsky SV, Lacroix B, Zaltsman A, Dafny-Yelin M, Vyas S, Tovkach A, Tzfira T
(2007) Biological systems of the host cell involved in Agrobacterium infection. Cell Microbiol
9:9–20
Colot HV, Parks G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich K,
Dunlap JC (2006) A high-throughput gene knockout procedure for Neurospora crassa reveals
functions for multiple transcription factors. Proc Natl Acad Sci USA 103:10353–10357
Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R (2003) Agrobacterium tumefaciens-
mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal
fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 220:141–148
Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium-mediated transformation
of Fusarium circinatum. Mycol Res 105:259–264
da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T,
Brakhage AA, Goldman GH (2006) The akuB (KU80) mutant deficient for nonhomologous
end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot
Cell 5:207–211
Dafny-Yelin M, Levy A, Tzfira T (2008) The ongoing saga of Agrobacterium–host interactions.
Trends Plant Sci 13:102–105
de Framond AJ, Barton KA, Chilton MD (1983) Mini-Ti: a new vector strategy for plant genetic
engineering. Biotechnol 5:262–269
de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-
mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842
Edman JC, Kwon-Chung KJ (1990) Isolation of the URA5 gene from Cryptococcus
neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell
10:4538–4544
Fiddy C, Trinci AP (1976) Mitosis, septation, branching and the duplication cycle in Aspergillus
nidulans. J Gen Microbiol 97:169–184
Fitzgerald AM, Mudge AM, Gleave AP, Plummer KM (2003) Agrobacterium and PEG-mediated
transformation of the phytopathogen Venturia inaequalis. Mycol Res 107:810–813
Francis KE, Spiker S (2005) Identification of Arabidopsis thaliana transformants without selection
reveals a high occurrence of silenced T-DNA integrations. Plant J 41:464–477
Gelvin SB (2009) Agrobacterium in the genomics age. Plant Physiol 150:1665–1676
Goins CL, Gerik KJ, Lodge JK (2006) Improvements to gene deletion in the fungal pathogen
Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and
co-transformation of independent DNA molecules allows rapid complementation of deletion
phenotypes. Fungal Genet Biol 43:531–544
Grimaldi B, de Raaf MA, Filetici P, Ottonello S, Ballario P (2005) Agrobacterium-mediated gene
transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete
Tuber borchii: a first step towards truffle genetics. Curr Genet 48:69–74
Hanif M, Pardo AG, Gorfer M, Raudaskoski M (2002) T-DNA transfer and integration in the
ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker. Curr Genet
41:183–188
Hefferin ML, Tomkinson AE (2005) Mechanism of DNA doublestrand break repair by
non-homologous end joining. DNA Repair 4:639–648
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA
75:1929–1933
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy
based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature
303:179–180
Huser A, Takahara H, Schmalenbach W, O’Connell R (2009) Discovery of pathogenicity genes in
the crucifer anthracnose fungus Colletotrichum higginsianum, using random insertional muta-
genesis. Mol Plant Microb Interact 22:143–156
Idnurm A, Reedy JL, Nussbaum JC, Heitman J (2004) Cryptococcus neoformans virulence gene
discovery through insertional mutagenesis. Eukaryot Cell 3:420–429
Jeon J, Park S-Y, Chi M-H, Choi J, Park J, Rho H-S, Kim S, Goh J, Yoo S, Choi J, Park J-Y, Yi M,
Yang S, Kwon M-J, Han S-S, Kim BR, Khang CH, Park B, Lim S-E, Jung K, King S,
Karunakaran M, Oh H-S, Kim H, Kim S, Park J, Kang S, Choi W-B, Kang S, Lee Y-H
(2007) Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat
Genet 39:561–565
Kahmann R, Basse C (1999) REMI (Restriction enzyme mediated integration) and its impact on
the isolation of pathogenicity genes in fungi attacking plants. Eur J Plant Pathol 105:221–229
Kemppainen M, Pardo AG (2010) pHg/pSILBAg vector system for efficient gene silencing in
homobasidiomycetes: optimization of ihpRNA-triggering in the mycorrhizal fungus Laccaria
bicolor. Microb Biotechnol 3:178–200
Kemppainen M, Circosta A, Tagu D, Martin F, Pardo AG (2005) Agrobacterium-mediated
transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N. Mycorrhiza
16:19–22
Kemppainen M, Duplessin S, Martin F, Pardo AG (2008) T-DNA insertion, plasmid rescue and
integration analysis in the model mycorrhizal fungus Laccaria bicolor. Microb Biotechnol
1:259–269
Kemppainen M, Duplessin S, Martin F, Pardo AG (2009) RNA silencing in the model mycorrhizal
fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbi-
osis with Populus. Environ Microbiol 11:1878–1896
Kim SI, Veena HJ, Gelcin SB (2007) Genome-wide analysis of Agrobacterium T-DNA integra-
tion sites in the Arabidopsis genome generated under non-selective conditions. Plant J
51:779–791
Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Korber H, Redei GP, Schell J (1989) High-
frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86:8467–8471
Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V (2001) Genetic transformation of
HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 98:1871–1876
Kuwano T, Shirataki C, Itoh Y (2008) Comparison between polyethyleneglycol- and polyethyle-
neimine-mediated transformation of Aspergillus nidulans. Curr Genet 54:95–103
Lacroix B, Tzfira T, Vainstein A, Citovsky V (2006) A case of promiscuity: Agrobacterium’s
endless hunt for new partners. Trends Genet 22:29–37
Lee MH, Bostock RM (2006) Agrobacterium T-DNA-mediated integration and gene replacement
in the brown rot pathogen Monilinia fructicola. Curr Genet 49:309–322
Li G, Zhou W, Liu G, Zheng F (2007) Characterization of T-DNA insertion patterns in the genome
of the rice blast fungus Magnaporthe oryzae. Curr Genet 51:233–243
Liu G, Casqueiro J, Bañuelos O, Cardoza RE, Gutiérrez S, Martı́n JF (2001) Targeted inactivation
of the mecB gene, encoding cystathione-gamma-lyase, shows that the reverse transsulfuration
pathway is required for high-level cephalosporin biosı́ntesis in Acremonium chysogenum C10
but not for methionine induction of the cephalosporin genes. J Bacteriol 183:1765–1772
Lorito M, Hayes CK, Di Pietro A, Harman GE (1993) Biolistic transformation of Trichoderma
harzianum and Gliocladium virens using plasmid and genomic DNA. Curr Genet 24:349–356
Marmeisse R, Gay G, Debaud JC, Casselton LA (1992) Genetic transformation of the symbiotic
fungus Hebeloma cylindrosporum. Curr Genet 22:41–45
Martin F, Aerts A, Ahren D, Brun A, Danchin EGJ, Duchaussoy F, et al (2008) The genome of
Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452:88–92
McCullen CA, Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and
activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol
22:101–127
Meng Y, Patel G, Heist M, Betts MF, Tucker SL, Galadima N, Donofrio NM, Brown D, Mitchell
TK, Li L, Xu JR, Orbach M, Thon M, Dean RA, Farman ML (2007) A systematic analysis of
T-DNA insertion events in Magnaporthe oryzae. Fungal Genet Biol 44:1050–1064
Michielse CB, Ram AFJ, Hooykaas PJJ, van den Hondel CAMJJ (2004a) Agrobacterium-
mediated transformation of Aspergillus awamori in the absence of full length VirD2, VirC2
or VirE2 leads to insertion of aberrant T-DNA structures. J Bacteriol 186:2038–2045
Michielse CB, Ram AFJ, Hooykaas PJJ, van den Hondel CAMJJ (2004b) Role of bacterial
virulence proteins in Agrobacterium mediated transformation of Aspergillus awamori. Fungal
Genet Biol 45:571–578
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2005b) Agrobacterium-
mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1–17
Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA,
Oakley BR (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans.
Genetics 172:1557–1566
Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora
strains deficient for non-homologous end joining. Proc Natl Acad Sci USA 101:12248–12253
Pardo AG, Hanif M, Raudaskoski M, Gorfer M (2002) Genetic transformation of ectomycorrhizal
fungi mediated by Agrobacterium tumefaciens. Mycol Res 106:132–137
Pardo AG, Kemppainen M, Valdemoros D, Duplessis S, Martin F, Tagu D (2005) T-DNA transfer
from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus. Rev
Argent Microbiol 37:69–72
Park SM, Kim DK (2004) Transformation of a filamentous fungus Cryphonectria parasitica using
Agrobacterium tumefaciens. Biotechnol Bioprocess Eng 9:217–222
Piers KL, Heath JD, Liang X, Stephens KM, Nester EW (1996) Agrobacterium tumefaciens-
mediated transformation of yeast. Proc Natl Acad Sci USA 93:1613–1618
P€
oggeler S, K€uck U (2006) Highly efficient generation of signal transduction knockout mutants
using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of
Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene
56:117–124
Rauyaree P, Ospina-Giraldo MD, Kang S, Bhat RG, Subbarao KV, Grant SJ, Dobinson KF (2005)
Mutations in VMK1, a mitogen activated protein kinase gene, affect microsclerotia formation
and pathogenicity in Vericillium dahliae. Curr Genet 48:109–116
Ream W (2009) Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport
bacterial DNA into the plant cell nucleus. Microb Biotechnol 2:416–427
Richey MG, Marek ET, Schardl CL, Smith DA (1989) Transformation of filamentous fungi with
plasmid DNA by electroporation. Phytopathology 79:844–847
Rodrı́guez-Tovar AV, Ruiz-Medrano R, Herrera-Martı́nez A, Barrera-Figueroa BE, Hidalgo-Lara
ME, Reyes-Márquez BE, Cabrera-Ponce JL, Valdés M, Xoconostle-Cázares B (2005) Stable
genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius. J Microbiol
Methods 63:45–54
Sallaud C, Gay C, Larmande P, Bès M, Piffanelli P, Piégu B, Droc G, Regad F, Bourgeois E,
Meynard D, Périn C, Sabau X, Ghesquière A, Glaszmann JC, Delseny M, Guiderdoni E (2004)
High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse
genetics. Plant J 39:450–464
Shaked H, Melamed-Bessudo C, Levy AA (2005) High-frequency gene targeting in Arabidopsis
plants expressing the yeast RAD54 gene. Proc Natl Acad Sci USA 102:12265–12269
Specht CA, Muňoz-Rivas A, Novotny CP, Ullrich RC (1988) Transformation of Schizophyllum
commune; and análisis of parameters for improving transformation frequencies. Exp Mycol
12:357–366
Staats CC, Junges A, Fitarellim M, Fulaneto MC, Henning Vainstein M, Schrank A (2007) Gene
inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium
anisopliae. Appl Microbiol Biotechnol 76:945–950
Sugui JA, Chan YC, Kwon-Chung KJ (2005) Agrobacterium tumefaciens-mediated transforma-
tion of Aspergillus fumigatus: and efficient tool for insertional mutagenesis and targeted gene
disruption. Appl Environ Microbiol 71:1798–1802
Sullivan TD, Rooney PJ, Klein BS (2002) Agrobacterium tumefaciens integrates transfer DNA
into single chromosomal sites of dimorphic fungi and yields homokaryotic progeny from
multinucleate yeast. Eukaryot Cell 1:895–905
Sunagawa M, Murata H, Miyazaki Y, Nakamura M (2007) Transformation of the mycorrhizal
basidiomycetes. Suillus grevillei and S. bovinus, by particle bombardment. Biosci Biotechnol
Biochem 71:47–50
Takahashi T, Masuda T, Koyama Y (2006) Enhanced gene targeting frequency in ku70 and ku80
disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Genet Genomics
275:460–470
Talbot NJ, Foster AJ (2001) Genetics and genomics of the rice blast fungus Magnaporthe grisea:
developing an experimental model for understanding fungal diseases of cereals. Adv Bot Res
34:263–287
Talhinhas P, Muthumeenakshi S, Neves-Martins J, Oliveira H, Sreenivasaprasad S (2008) Agro-
bacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum
for investigating varied pathogenicity lifestyles. Mol Biotechnol 39:57–67
Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983)
Transformation by integration in Aspergillus nidulans. Gene 26:205–221
Tsuji G, Fujii S, Fujihara N, Hirose C, Tsuge S, Shiraishi T, Kubo Y (2003) Agrobacterium
tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum
lagenarium. J Gen Plant Pathol 69:230–239
Tsukuda T, Carleton S, Fotheringham S, Holloman WK (1988) Isolation and characterization of an
autonomously replicating sequence from Ustilago maydis. Mol Cell Biol 8:3703–3709
van Attikum H, Hooykaas PJ (2003) Genetic requirements for the targeted integration of Agro-
bacterium T-DNA in Saccharomyces cerevisiae. Nucleic Acid Res 31:826–832
van Attikum H, Bundock P, Hooykaas PJ (2001) Non-homologous end-joining proteins are
required for Agrobacterium T-DNA integration. EMBO J 20:6550–6558
van de Rhee MD, Graça PM, Huizing HJ, Mooibroek H (1996) Transformation of the
cultivated mushroom, Agaricus bisporus, to hygromycin B resistance. Mol Gen Genet 250:
252–258
Vijn I, Govers F (2003) Agrobacterium tumefaciens mediated transformation of the oomycete
plant pathogen Phytophthora infestans. Mol Plant Pathol 4:459–467
Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, Morin D, Bruel C, Beffa R,
Lebrun MH (2008) Improved gene targeting in Magnaporthe grisea by inactivation of
MgKU80 required for non-homologous end joining. Fungal Genet Biol 45:68–75
Walton FJ, Idnurm A, Heitman J (2005) Novel functions required for melanization of the human
pathogen Cryptococcus neoformans. Mol Microbiol 57:1381–1386
Wang J, Holden DW, Leong SA (1988) Gene transfer system for the phytopathogenic fungus
Ustilago maydis. Proc Natl Acad Sci USA 85:865–869
Ward M, Kodama KH, Wilson LJ (1989) Transformation of Aspergillus awamori and A. niger by
electroporation. Exp Mycol 13:289–293
Weld RJ, Lady CC, Ridgway HJ (2006) Agrobacterium-mediated transformation of Sclerotinia
sclerotiorum. J Microbiol Methods 65:202–207
Wilson LM, Idnurm A, Howlett BJ (2002) Characterization of a gene (sp1) encoding a secreted
protein from Leptosphaeria maculans, the blackleg pathogen of Brassica napus. Mol Plant
Pathol 3:487–493
Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a
trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474
Zwiers LH, De Waard MA (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption
in the phytopathogen Mycosphaerella graminicola. Curr Genet 39:388–393
.
Chapter 7
Biotechnological Processes Used in Controlled
Ectomycorrhizal Practices
Par Duponnois Robin, Bâ Amadou, Mousain Daniel, Galiana Antoine,

Baudoin Ezékiel, Dreyfus Bernard, and Prin Yves
7.1 Introduction
Excessive industrial exploitation, clearing for industrial purposes and collection of

firewood, has led to a dramatic deforestation during recent decades in Mediterra-
nean and tropical areas (Piéri 1991). One of the critical issues of deforestation is the
acceleration of soil degradation and desertification processes that involved a loss or
reduction of major physico-chemical and biological soil properties (Requena et al.
2001). This lack or scarcity of plant cover largely contributed to soil erosion
increase and consequently, to the decrease of soil fertility and soil microbial
activities (Garcia et al. 1997). Numerous studies have reported that in such condi-
tions, indigenous inoculum levels of mycorrhizal fungi were significantly reduced
(Duponnois et al. 2001; Azcon-Aguilar et al. 2003). The mycorrhizal symbiotic
P.D. Robin (*)

IRD, UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire des Symbioses Tropicales
et Méditerranéennes (LSTM), TA A-82/J, Campus International de Baillarguet, Montpellier,
Cedex 5, France
IRD, Laboratoire Commun de Microbiologie IRD/ISRA/UCAD, Centre de Recherche de Bel Air,
BP 1386 Dakar, Sénégal
e-mail: Robin.Duponnois@ird.fr
B. Amadou, B. Ezékiel and D. Bernard
Cedex 5, France
M. Daniel
INRA, UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire des Symbioses Tropicales
Cedex 5, France
G. Antoine and P. Yves
Cedex 5, France

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_7,
144 P. Duponnois Robin et al.
process mobilizes and transports nutrients to roots, improves soil aggregation in

eroded soils, and reduces water stress (Smith and Read 1997).
Mycorrhizal fungi are ubiquitous components of most ecosystems throughout
the world and are considered key ecological factors in governing the cycles of
major plant nutrients and in sustaining the vegetation cover (van der Hejden et al.
1998; Requena et al. 2001; Schreiner et al. 2003). Two major forms of mycorrhizas
are usually recognized: the arbuscular mycorrhizas (AM) and the ectomycorrhizas
(ECM). AM symbiosis is the most widespread mycorrhizal association type with
plants that have true roots, i.e., pteridophytes, gymnosperms, and angiosperms
(Read et al. 2000). These fungal symbionts affect about 80–90% land plants in
natural, agricultural, and forest ecosystems (Brundrett 2002). ECMs affect trees and
shrubs, gymnosperms (Pinaceae), and angiosperms and are usually the result of the
association of Homobasidiomycetes with about 20 families of mainly woody plants
(Smith and Read 1997). These woody species are associated with a larger
(compared to the AM symbiosis) diversity of fungi, comprising 4,000 to 6,000
species, mainly Basidiomycetes and Ascomycetes (Allen et al. 1995; Valentine
et al. 2004). The distribution of ectomycorrhizal fungi is not uniform in terms of
their presence, abundance, or community composition. This has important implica-
tion for reforestation in areas with no native ectomycorrhizal vegetation (Marx
1991) or where ectomycorrhizal fungi are absent resulting from natural (Terwilliger
and Pastor 1999) or anthropogenic disturbance (Jones et al. 2003).
The lack of mycorrhizal fungi on root systems is a leading cause of poor plant
establishment and growth in a variety of forest landscapes. Numerous studies have
shown that specific ectomycorrhizal fungi are able to improve the survival and early
growth of various tree species in the field (Castellano and Molina 1989; Kropp and
Langlois 1990; Marx et al. 1991; Castellano 1996; Roldan et al. 1996; Garbaye and
Churin 1997; Duponnois et al. 2005, 2007). Since mycorrhizal association is
estimated to occur in 95% of native undisturbed vegetation, whereas it occurs in
less than 1% of vegetation from disturbed sites, mycorrhizal symbiosis has to be
reestablished in order to benefit from the mycorrhizal effect on plant growth.
Hence, this topic can be achieved by inoculating seedlings before they are trans-
planted to disturbed sites. However, different fungal associations do not lead to the
same benefit to the host plant. A significant variability in response linked with the
nature of the fungal–plant association has been reported (Guehl et al. 1990; Bâ et al.
2002; Duponnois and Plenchette 2003). These variations generally result from
different factors such as the degree of host–fungus compatibility, mycorrhizal
dependency of the host, fungal effectiveness in relation to biotic and abiotic site
conditions, and the abundance and effectiveness of indigenous fungi (Garbaye
1988). In order to ensure the performance of afforestation, it is necessary that
nurseries produce tree seedlings associated with mycorrhizal fungi that are ecolo-
gically compatible with the tree species and the planting sites. According to these
conditions that have to be taken in account, different methods of controlled inocu-
lation have been identified to optimize the effect on plant growth.
The main objective of this chapter was to describe some methods required to
produce large quantities of efficient fungal inoculums and to present some tree
7 Biotechnological Processes Used in Controlled Ectomycorrhizal Practices 145
growth data resulting from the use of controlled ectomycorrhization in nursery and
field conditions in tropical and Mediterranean areas.
7.2 Criteria to Adopt an Inoculum Formulation
The main criteria that must be taken into account can be summarized as follows:
– The positive impact of the selected ectomycorrhizal fungi on the growth and the
survival of tree species targeted for plantation.
– The viability of the fungal propagules which has to be maintained in storage to
ensure the fungal efficiency on plant growth after inoculation.
– Ectomycorrhizal fungal inoculums have to be cost-effective to produce.
7.3 Spore-Based Fungal Inoculum
Spores collected from fruiting bodies can serve as a natural source of inoculum.
This inoculation practice has been widely used in forest nurseries (Castellano
1994). However, this inoculation technique is more effective with fungal species
that produce abundant sporocarps with large spore numbers (i.e., Pisolithus and
Scleroderma).
7.3.1 Formulation of Spore Inoculums and Effect

on Plant Growth
There are numerous formulations of spore inoculums. In a first step, sporocarps are
collected, carefully identified, and kept in paper bags. Then, they are brushed free of
adhering soil. These kinds of inoculum are frequently based on dry spores obtained
by air-drying fruit bodies at temperatures below 35 C. Then dried sporocarps are
crushed in plastic bags and sieved through 200–500 mm sieve. Then dried spores
can be used in different formulations described as follows:
– Spore powder can be mixed with sterilized fine sand (1:100, w/w). Then
containers are inoculated by mixing the soil substrate with this fungal mixture
or by adding a small quantity of the fungal inoculum at the base of seedlings.
– Spore powder can be included into an inert carrier (i.e., clay) to form pellets
which are applied at the base of seedlings (de la Cruz et al. 1990). According to
Turjaman et al. (2005), in an experiment conducted to determine the effect of
ectomycorrhizal fungi on the growth of dipterocarp species in peat soils, the
sporocarps were crushed manually in plastic bags to ensure minimal loss of
spores and cross-contamination between fungi (de la Cruz et al. 1990). Crushed
sporocarps and clay were mixed and formed into pellets with a ratio of 1:100 (w/
w). Inoculation was performed out 10 days after seed germination. One hole was
made in each pot, and a tablet (0.4 g) of fungal inoculum was applied to a potted
seedling 1 cm below the soil surface, at the proximity of the root.
– Another process of ectomycorrhizal spore inoculation is realized by coating seeds
with a mixture of spores and a binding agent such as clay (Marx et al. 1984).
The beneficial effects of ectomycorrhizal spore inoculation on the plant growth
in nursery conditions have been frequently reported (Table 7.1). Several disadvan-
tages have also been recorded with this type of fungal inoculum:
– Difficulties to collect large quantities of fruit bodies with some ectomycorrhizal
fungal species.
– Low efficiency of the fungal inoculum due to slow germination or low spore
viability ; this inoculation process is easy to apply and is an efficient method for
storage and transport of spores for some mycorrhizal fungi such as Pisolithus or
Scleroderma.
7.4 Mycelium-Based Fungal Inoculums
These inoculum forms require the isolation of fungal symbionts in axenic condi-
tions in order to obtain a purified living material of a single fungal strain. These
pure cultures are often obtained from fruit bodies of ectomycorrhizal fungi col-
lected in the field but they can also be performed by using mycorrhizal roots,
sclerotia, rhizomorphs, and spores (Molina and Palmer 1982). In this chapter, the
fungal isolation from fruiting bodies will be described.
7.4.1 Fungal Isolation from Fruit Bodies
This procedure is usually considered as the most successful method to obtain pure
fungal culture. Sporocarps are brushed free of adhering soil and fractured carefully
in a laminar flow hood. A small amount of tissue is then removed with a fine forceps
and placed on a nutrient medium agar. As growth requirements vary between
ectomycorrhizal fungi, different nutrient media can be used (Table 7.2). The fungal
cultures are incubated at 25 C in the dark and subcultured until all the contaminat-
ing microorganisms are eliminated. Fungal cultures are usually maintained in Petri
dishes over a required nutrient agar medium and at 20–25 C. Fresh subcultures are
made every 6–12 weeks depending on the fungal growth rates.
Table 7.1 Effect of ectomycorrhizal spore inoculation on plant growth in nursery conditions
References Fungal strain Plant species Inoculum formulation Effects on plant growth parameters (%) and
ectomycorrhizal colonization
Height Shoot Root Ecto. colonization
biomass biomass (%)
Turjaman et al. (2005) Pisolithus arhizus Shorea pinanga Ectomycorrhizal spore +46.1a +66.7 ndb 87
pellets
Turjaman et al. (2005) Scleroderma sp. S. pinanga Ectomycorrhizal spore +41.3 +60.5 nd 86
pellets
Aggangan et al. (2010) P. tinctorius Acacia mangium Ectomycorrhizal spore nd +29.1 +40.7 52
tablets
Rincon et al. (2007) Rhizopogon Pinus halepensis Spore suspension 30.1 nd nd 48
roseolus
Rincon et al. (2007) Suillus collinitus Pinus halepensis Spore suspension +27.8 nd nd 75
Chen et al. (2006) Scleroderma Eucalyptus Spore suspension +32.1 +19.7 +42.5 nd
albidum globulus
Chen et al. (2006) S. areolatum E. globulus Spore suspension +17.5 +9.1 +33.5 nd
Chen et al. (2006) S. cepa E. globulus Spore suspension +30.8 +8.8 +2.0 nd
Chen et al. (2006) S. albidum E. urophylla Spore suspension 3.6 +3.1 0.7 nd
Chen et al. (2006) S. areolatum E. urophylla Spore suspension +7.2 +4.9 +4.0 nd
Chen et al. (2006) S. cepa E. urophylla Spore suspension +6.3 +13.0 +1.4 nd
Torres and Honrubia (1994) P. tinctorius P. halepensis Spore suspension +37.1 +44.9 +41.7 55.4
Torres and Honrubia (1994) R. roseolus P. halepensis Spore suspension +38.3 +44.6 +28.6 39.5
7 Biotechnological Processes Used in Controlled Ectomycorrhizal Practices
Torres and Honrubia (1994) Suillus collinitus P. halepensis Spore suspension +35.1 +28.2 +30.6 28.9
a
(mean value of ectomycorrhizal plants – mean value of the nonectomycorrhizal plants) 100)/(mean value of the ectomycorrhizal plants)
b
nd: not determined
147
Table 7.2 Composition of some nutrient media commonly used for the isolation and culture of
ectomycorrhizal fungi (from Brundrett et al. 1996)
Components Nutrient media
MMNa Pachlewskib FDAc
1
Mineral nutrients (mg l )
(NH4)2HPO4 250
NH4Cl 500
C4H12N2O6d 500
KH2PO4 500 1000 500
MgSO4 7H2O 150 500 500
CaCl2 2H2O 50 50
NaCl 25
Fe EDTA 20 20
H3BO3 2.8
MnCl2 2H2O 3.0
ZnSO4 7H2O 2.3
CuCl2 2H2O 0.63
Na2Mo4 2H2O 0.27
Carbohydrate source (g l 1)
Maltose 5
Glucose 10 20 20
Malt extract 3
Vitamins (mg l 1)
Thiamine HCl 0.1 0.1
Agar (g l 1) 20 20 20
pH
Adjusted pH to 5.8 5.4 5.0
a
MMN: Modified Menin Norkrans medium (Marx 1969)
b
Pachlewski medium (Pachlewski and Pachlewski 1974)
c
Ferry and Das (1968)
d
Ammonium tartrate
7.4.2 Formulation of Mycelium-Based Inoculums and Effect

on Plant Growth
These procedures are only suitable with some fungal species that are able to grow in
culture for the economical production of fungal inoculums. Two main supports are
usually used to produce fungal inoculums: the multiplication of the fungal strain on
a peat/vermiculite substrate and the production of an ectomycorrhizal inoculum
entrapped in a hydrogel (i.e., calcium alginate gel).
7.4.2.1 Vermiculite–Peat Inoculums (Marx and Bryan 1975)
Glass jars (1.6 l) containing 1.3 l expanded vermiculite–sphagnum peat mixture

(4:5–1:5, v:v, pH 5.5) are autoclaved (120 C, 20 min). Another ratio of vermiculite–
peat can be used (2:3–1:3) but the peat can release some substances which could be
toxic for the fungal development. For this reason, the first ratio mixture is usually
used. Then the mixture is moistened to field capacity with 600 ml modified as a liquid
nutrient medium (see Table 7.2). The jars are blocked with lids with a 1-cm diameter
hole. This hole is fitted with a 4-cm long tube filled with cotton wool. The jars are then
autoclaved a second time (120 C for 20 min). After cooling, about eight mycelial
plugs are laid on top of the substrate. Mycelium grows down into the substrate, which
is completely colonized after 6–10 weeks at 25 C depending on the fungal growth
rate. For faster growth, jars can be filled with a smaller quantity of substrate and
shaken after mycelia have colonized a few centimeters: in this manner, mycelium is
evenly distributed throughout the substrate and incubation time is shortened. This
inoculum can be stored at 4 C for up to 6 months.
7.4.2.2 Encapsulation of Hyphal Fragments Within Beads of Alginate Gel

(Mauperin et al. 1987)
The process of including fungal mycelium in polymeric gels (especially calcium

alginate) has been previously described by Dommergues et al. (1979) and Le Tacon
et al. (1983, 1985). The inoculum prepared in this manner is more efficient than
the vermiculite–peat formulation (Mortier et al. 1988) because the mycelium is
protected in the gel from physical stresses (e.g., water stress) and from competitor
microorganisms. With this process, it is also possible to accurately determine the
weight of mycelium or the number of living propagules contained in the inoculum.
Different methods have been tested to measure the quantity of mycelium in the
vermiculite–peat inoculum:ergosterol assay (Martin et al. 1990); chitin assay
(Vignon et al. 1986), but none of them gave reliable results (Mortier et al. 1988)
because of the peat which interferes with colorimetric measurements. Using fungal
strains that are able to grow from fragmented hyphae, a mycelial suspension is
produced in Erlenmeyer flasks or in fermentor filled with the required liquid
nutrient medium. The mycelium is then washed in tap water in order to remove
residual nutrients, homogenized in a Waring blender for about 10 s, and resus-
pended in distilled water. This kind of fungal inoculum is quantified by measuring
the fungal dry weight per milliliter or by counting living propagules (determination
of colony-forming units by spreading 1 ml of suspension on a 6-cm Petri dish with
nutrient agar). Then the hyphal fragment suspension is mixed (1:1, v:v) with
distilled water containing 20 g l 1 sodium alginate and 50 g l 1 autoclaved dry
powdered sphagnum peat. The final solution is pumped throughout a pipe with
2-mm holes. The drops fall into a 100 g l 1 CaCl2 solution and form beads of
reticulated calcium alginate gel (Mauperin et al. 1987). The beads are kept in CaCl2
for 24 h at room temperature in order to ensure complete reticulation. Then they
are washed with tap water to eliminate NaCl and CaCl2 and stored in air-tight
containers at 4 C in order to prevent drying. This type of inoculum can be kept up
to 9 months in these conditions. The beads are prepared with 1–2 g mycelium
(dry weight) per liter of final solution (Mortier et al. 1988).
7.4.2.3 Controlled Ectomycorrhization in Glasshouse, Nursery,

and Field Conditions
The aims of these procedures are to screen fungal isolates for their compatibility
with host plants and to determine plant growth promotion in order to select the best
fungus to be inoculated in nurseries. This topic will be mainly illustrated by some
data resulting from controlled ectomycorrhization trials with Australian Acacia
species and tropical fungal strains.
Screening for Efficient Fungal Isolates in Controlled Conditions
Seedlings can be planted in soil (disinfected or not after autoclaving) or nondisin-

fected vermiculite–peat mixture (1:1, v:v). Several kinds of containers can be used
for growing seedlings in the glasshouse depending on the aim of the experiment.
Then the ectomycorrhizal inoculation was performed by mixing the fungal inocu-
lum (10/1; v/v) with the soil. The treatment without fungus (control) received an
autoclaved mixture of vermiculite and peat moss moistened in nutrient medium at
the same rate. Then the pots were arranged in a randomized complete block design.
Numerous studies have reported the beneficial effects on the growth and nutrient
status of tree seedlings in controlled conditions with the ultimate goal of selecting
the most promising and potential mycorrhizal inoculants for large-scale inoculation
programs in degraded lands in the tropics and Mediterranean areas (Table 7.3).
Although several methods of inoculation can be used in practice, only some species
of the genera Laccaria, Hebeloma, Paxillus, Pisolithus, Scleroderma, Suillus, or
Rhizopogon have been routinely used (Castellano 1996).
Controlled Inoculation in Field Conditions
Numerous studies have shown that controlled mycorrhizal symbiosis can signifi-
cantly improve the growth of tree species in degraded soils. However, most of these
experiments have been performed in controlled glasshouse conditions, hence data
on the sustainability of the positive effect of mycorrhiza on plant growth in field
conditions are lacking, especially in Sahelian areas. Controlled ectomycorrhization
experiments have been recently set up in Senegal using an Australian Acacia
species, A. holosericea, and an ectomycorrhizal fungus, Pisolithus albus strain
IR100 (Duponnois et al. 2007). A positive effect of mycorrhizal inoculation on A.
holosericea development has been recorded in all of the experiments (Fig. 7.1).
These results suggest that without suitable symbionts, this species has poor ability
to scavenge for P under P-limiting conditions (Dommergues et al. 1999) and clearly
shows that the controlled mycorrhization of A. holosericea could be a beneficial
tool in improving the survival and productivity of Acacia species and consequently
for the reafforestaion of Sahelian regions. However, in order to evaluate the
possible economic advantage of ectomycorrhizal inoculation, it is essential
to carry out field experiments in a range of situations representative of major
plantations.
Table 7.3 Growth of some Australian Acacia species inoculated with different ectomycorrhizal
fungal strains (peat–vermiculite formulation) after 4 months culturing in glasshouse conditions
Fungal species Acacia species Effects on plant growth parameters (%) References
and ectomycorrhizal colonization
Shoot Root Ectomycorrhizal
biomass biomass colonization (%)
Pisolithus albus A. auriculiformis +42.1a +38.6 45.2 Duponnois and
IR100 Plenchette
(2003)
P. albus IR100 A. mangium +35.9 +44.1 20.1 Duponnois and
Plenchette
(2003)
P. albus IR100 A. platycarpa +43.1 +9.8 31.6 Duponnois and
Plenchette
(2003)
Pisolithus sp. A. holosericea +56.7 +10.6 48.3 Duponnois and
SL2 Plenchette
(2003)
P. albus COI007 A. holosericea +55.6 +25.3 43.8 Duponnois and
Plenchette
(2003)
P. albus COI024 A. holosericea +50.4 +17.1 10.8 Duponnois and
Plenchette
(2003)
Pisolithus sp. A. holosericea +54.9 +12.6 15.0 Duponnois and
COI032 Plenchette
(2003)
P. albus IR100 A. holosericea +57.1 +48.9 25.2 Duponnois and
Plenchette
(2003)
P. tinctorius A. holosericea +57.8 +14.1 43.1 Duponnois and
GEMAS Plenchette
(2003)
Scleroderma A. holosericea +52.9 +21.0 53.4 Duponnois and
dictyosporum Plenchette
IR109 (2003)
S. verrucosum A. holosericea +64.4 +14.1 13.8 Duponnois and
IR500 Plenchette
(2003)
P. tinctorius A. crassicarpa +77.1 +52.3 49.4 Lesueur and
GEMAS Duponnois
(2005)
P. albus COI024 A. mangium +54.5 +52.1 41.7 Duponnois et al.
(2002)
Scleroderma sp. A. holosericea +82.1 +89.6 13.8 Duponnois et al.
IR408 (2006)
S. dictyosporum A. holosericea +72.9 +82.6 12.5 Duponnois et al.
IR412 (2006)
a
(mean value of ectomycorrhizal plants – mean value of the nonectomycorrhizal plants) 100)/
(mean value of the ectomycorrhizal plants)
Experiment EC8 Experiment EC9
2 2
1.5 1.5
1 1
0.5 0.5
0 0
Control IR 100 Control IR 100
Experiment EC10 Experiment EC13
2 8
7
1.5 6
5
1 4
3
0.5 2
1
0 0
Control IR 100 Control IR 100
Fig. 7.1 Field performance of A. holosericea trees (expressed in tons of wood biomass per ha with
1,200 planted trees per ha) inoculated or not with Pisolithus albus strain IR100 in field trials
conducted in Senegal after 18 month plantation (from Duponnois et al. 2007)
7.5 Conclusion
Numerous studies have reported the benefits that result from the use of fungal
inoculants, which can improve the development of tree seedlings in glasshouse and
nursery conditions. In the same way, a lot of technical procedures have been
elaborated to produce cost-effective mycorrhizal inoculum. Unfortunately, mycor-
rhizal inoculation remains underexploited in nursery cultural practices although the
management of tree mycorrhizal status could have very important implications for
tropical reforestation programs in the developing countries. The results presented in
this chapter suggest that inoculation with ectomycorrhizal fungi can improve the
early growth of the main tree species in tropical and Mediterranean forests and that
this technique will accelerate the rehabilitation of degraded forests.
References
Aggangan NS, Moon HK, Han SH (2010) Growth response of Acacia mangium Willd. seedlings to
arbuscular mycorrhizal fungi and four isolates of the ectomycorrhizal fungal Pisolithus
tinctorius (Pers.) Coker and Couch. New For 39:215–230
Allen EB, Allen MF, Helm DJ, Trappe JM, Molina R, Rincon E (1995) Patterns and regulation of
mycorrhizal plant and fungal diversity. Plant Soil 170:47–62
Azcon-Aguilar C, Palenzuela J, Roldan A, Bautista S, Vallejo R, Barea JM (2003) Analysis of the

mycorrhizal potential in the rhizosphere of representative plant species from desertification-
threatened Mediterranean shrublands. Appl Soil Ecol 14:165–175
Bâ AM, Sanon KB, Duponnois R (2002) Influence of ectomycorrhizal inoculation on Afzelia
quanzensis Welw. Seedlings in a nutrient-deficient soil. For Ecol Manage 161:215–219
Brundrett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytol
154:275–304
Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in
Forestry and Agriculture. ACIAR Monograph, 373p
Castellano MA (1994) Current status of outplanting studies using ectomycorrhiza-inoculated
forest trees. In: Pfleger F, Linderman B (eds) A reappraisal of mycorrhizae in plant health.
The American Phytopathological Society, St Paul, pp 261–281
Castellano MA (1996) Outplanting performance of mycorrhizal inoculated seedlings. In: Mukerji
KG (ed) Concepts in mycorrhizal research. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp 223–301
Castellano MA, Molina R (1989) Mycorrhizae. In: Landis TD (ed) The biological component:
nursery pest and mycorrhizae manual. Agric. Handbook 674, vol 5. USDA Forest Service,
Washington, DC, pp 101–167
Chen YL, Dell B, Malajczuk N (2006) Effect of Scleroderma spore density and age on mycorrhiza
formation and growth of containerized Eucalyptus globulus and E. urophylla seedlings. New
For 31:453–467
de la Cruz RE, Lorilla EB, Aggrangan NS (1990) Ectomycorrhizal tablets for Eucalyptus species.
In: Werner D, Muller P (eds) Fast growing trees and nitrogen fixing trees. Gustav Fisher
Verlag, Stuttgard, p 371
Dommergues R, Diem HG, Divies C (1979) Microbiological process for controlling the produc-
tivity of cultivated plants. US Patent No 4.155.737, 22 May 1979
Dommergues YR, Duhoux E, Diem HG (1999) Les arbres fixateurs d’azote, Editions Espaces 34.
ORSTOM, FAO, Montpellier, 501pp
Duponnois R, Plenchette C (2003) A mycorrhiza helper bacterium (MHB) enhances ectomycor-
rhizal and endomycorrhizal symbiosis of Australian Acacia species. Mycorrhiza 13:85–91
Duponnois R, Plenchette C, Thioulouse J, Cadet P (2001) The mycorrhizal soil infectivity and
arbuscular mycorrhizal fungal spore communities in soils of different aged fallows in Senegal.
Appl Soil Ecol 17:239–251
Duponnois R, Founoune H, Lesueur D (2002) Influence of the controlled dual ectomycorrhizal and
rhizobial symbiosis on the growth of Acacia mangium provenances, the indigenous symbiotic
microflora and the structure of plant parasitic nematode communities. Geoderma 109:85–102
Duponnois R, Founoune H, Masse D, Pontanier R (2005) Inoculation of Acacia holosericea with
ectomycorrhizal fungi in a semi-arid site in Senegal: growth response and influences on the
mycorrhizal soil infectivity after 2 years plantation. For Ecol Manag 207:351–362
Duponnois R, Assigbetse K, Ramanankierana H, Kisa M, Thioulouse J, Lepage M (2006) Litter-
forager termite mounds enhance the ectomycorrhizal symbiosis between Acacia holosericea
A. Cunn. Ex G. Don and Scleroderma dictyosporum isolates. FEMS Microbiol Ecol
56:292–303
Duponnois R, Plenchette C, Prin Y, Ducousso M, Kisa M, Bâ AM, Galiana A (2007) Use of
mycorrhizal inoculation to improve reafforestation process with Australian Acacia in Sahelian
ecozones. Ecol Eng 29:105–112
Ferry BW, Das M (1968) Carbon nutrition of some mycorrhizal Boletus species. Trans Br Mycol
Soc 51:795–798
Garbaye J (1988) Les plantations forestières tropicales: un champ d’application privilégié pour la
mycorhization contrôlée. Revue Bois et Forêts des Tropiques 216:23–34
Garbaye J, Churin JL (1997) Growth stimulation of young oak plantations inoculated with the
ectomycorrhizal fungus Paxillus involutus with special reference to summer drought. For Ecol
Manag 98:221–228
Garcia C, Roldan A, Hernandez T (1997) Changes in microbial activity after abandonment of

cultivation in a semi-arid Mediterranean environment. J Environ Qual 26:285–291
Guehl JM, Mousain D, Falconnet G, Gruez J (1990) Growth, carbon dioxide assimilation capacity
and water-use efficiency of Pinus pinea L. seedlings inoculated with different ectomycorrhizal
fungi. Ann Sci For 47:91–100
Jones MD, Durall DM, Cairney JWG (2003) Ectomycorrhizal fungal communities in young forest
stands regenerating after clearcut logging. New Phytol 157:399–422
Kropp BR, Langlois CG (1990) Ectomycorrhizae in reforestation. Can J For Res 20:438–451
Le Tacon F, Jung G, Michelot P, Mugnier J (1983) Efficacité en pépinière forestière d’un inoculum
de champignon ectomycorhizien produit en fermenteur et inclus dans une matrice de poly-
mères. Ann Sci For 40:165–176
Le Tacon F, Jung G, Mugnier J, Michelot P, Mauperin C (1985) Efficiency in a forest nursery of an
ectomycorhizal fungus inoculums produced in a fermentor and entrapped in polymeric gels.
Can J Bot 63:1664–1668
Lesueur D, Duponnois R (2005) Relations between rhizobial nodulation and root colonization of
Acacia crassicarpa provenances by an arbuscular mycorrhizal fungus, Glomus intraradices
Schenk and Smith or an ectomycorrhizal fungus, Pisolithus tinctorius Coker & Couch. Ann For
Sci 62:467–474
Martin F, Delaruelle C, Hilbert JL (1990) An improved ergosterol assay to estimate the fungal
biomass in ectomycorrhizas. Mycol Res 94:1069–1074
Marx DH (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to
pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil
bacteria. Phytopathology 59:153–163
Marx DH (1991) The practical significance of ectomycorrhizae in forest establishment. In:
Ecophysiology of ectomycorrhizae of forest trees, Marcus Wallenberg Foundation symposia
proceedings, vol 7, pp 54–90
Marx DH, Bryan WC (1975) Growth and ectomycorrhizal development of Loblolly pine seedlings
Marx DH, Jarl K, Ruehle JL, Bell W (1984) Development of Pisolithus tinctorius ectomycorrhizae
on pine seedlings using basidio-encapsulated seed. For Sci 30:897–907
Marx DH, Ruehle JL, Cordell CE (1991) Methods for studying nursery and field response of trees
to specific ectomycorrhiza. In: Norris JR, Read DJ, Varma AK (eds) Methods in microbiology,
vol 23, Techniques for the study of mycorrhiza. Academic Press, London, pp 383–411
Mauperin C, Mortier F, Garbaye J, Le Tacon F, Carr G (1987) Viability of an ectomycorrhizal
inoculum produced in a liquid medium and entrapped in a calcium alginate gel. Can J Bot
65:2326–2329
Molina R, Palmer JG (1982) Isolation, maintenance and pure culture manipulation of ectomycor-
rhizal fungi. In: Schenck NC (ed) Methods and principles of mycorrhizal research. The
American Phytopathological Society, St Paul, pp 115–129
Mortier F, Le Tacon F, Garbaye J (1988) Effect of inoculum type and inoculation dose on
ectomycorrhizal development, root necrosis and growth of Douglas fir seedlings inoculated
with Laccaria laccata in a nursery. Ann Sci For 45:301–310
Pachlewski R, Pachlewski J (1974) Studies on symbiotic properties of mycorrhizal fungi of pine
(Pinus silvestris L.) with the aid of the method of mycorrhizal synthesis in pure culture on agar.
Forest Research Institute, Warsaw, p 228
Piéri C (1991) Les bases agronomiques de l’amélioration et du maintien de la fertilité des terres de
savanes au sud Sahara. In: Savanes d’Afrique, terre fertile? Actes des Rencontres Internatio-
nales, 10–14 Dec 1990, Montpellier, France, pp 43–74
Read DJ, Duckett JG, Francis R, Ligrone R, Russell A (2000) Symbiotic fungal associations in
“lower” land plants. Philos Trans R Soc Lond B Biol Sci 355:815–830
Requena N, Perez-Solis E, Azcon-Aguilar C, Jeffries P, Barea JM (2001) Management of
indigenous plant–microbe symbioses aids restoration of desertified ecosystems. Appl Environ
Rincon A, de Felipe MR, Fernandez-Pascual M (2007) Inoculation of Pinus halepensis Mill. with
selected ectomycorrhizal fungi improves seedling establishment 2 years after planting in a
degraded gypsum soil. Mycorrhiza 18:23–32
Roldan A, Querejeta I, Albadalejo J, Castillo V (1996) Growth response of Pinus halepensis to
inoculation with Pisolithus arhizus in a terraced rangeland with urban refuse. Plant Soil
179:35–43
Schreiner RP, Mihara KL, McDaniel KL, Bethlenfalvay GJ (2003) Mycorrhizal fungi influence
plant and soil functions and interactions. Plant Soil 188:199–209
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn. Academic Press, UK, p 605
Terwilliger J, Pastor J (1999) Small mammals, ectomycorrhizae, and conifer succession in beaver
meadows. Oikos 85:83–94
Torres P, Honrubia M (1994) Inoculation of containerized Pinus halepensis (Miller) seedlings
with basidiospores of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) and
Suillus collinitus (Fr) O Kuntze. Ann Sci For 51:521–528
Turjaman M, Tamai Y, Segah H, Limin SH, Cha JY, Osaki M, Tawaraya K (2005) Inoculation
with the ectomycorrhizal fungi Pisolithus arhizus and Scleroderma sp. improves early growth
of Shorea pinanga nursery seedlings. New For 30:67–73
Valentine LL, Fieldler TL, Hart AA, Petersen CA, Berninghausen HK, Southworth D (2004)
Diversity of ectomycorrhizas associated with Quercus garryana in southern Oregon. Can J Bot
82:123–135
van der Hejden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T,
Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity
ecosystem variability and productivity. Nature 396:69–72
Vignon C, Plassard C, Moussain D, Salsac L (1986) Assay of fungal chitin and estimation of
mycorrhizal infection. Physiol Vég 24:201–207
.
Chapter 8
Signaling in Ectomycorrhizal Symbiosis
Establishment
Paula Baptista, Rui Manuel Tavares, and Teresa Lino-Neto
8.1 Introduction
A multiplicity of strategies has been developed by plants for their survival in a wide
range of biotic and abiotic stimuli. One successful approach is the development of
mutualistic associations, such as ectomycorrhizas (ECM) formed with soil-borne
fungi from Basidiomycota, Ascomycota, and Zygomycota. In forest ecosystems,
the ECM association is indeed the predominant form of mycorrhiza. The plant
species involved are diverse and belong mainly to Fagaceae, Betulaceae, Pinaceae,
and Dipterocarpaceae families (Smith and Read 2008). This symbiosis is charac-
terized by the presence of a fungal sheath around the fine root tips (the mantle) and
by the hyphae colonization of the intercellular space between root cells, the so-
called Hartig-net. From the mantle, hyphae spread over into the surrounding
substrate, increasing the root surface area. This represents an effective way to
enhance nutrient, carbon, and water exchanges between the fungus and the plant
roots. While the plant takes advantage from the improved nutrient uptake, the
fungus receives photosynthetic carbohydrates from plant (Smith and Read 2008).
Other advantages for the host plant include improved growth, enhanced water
acquisition and use, as well as enhanced soil stability (Smith and Read 2008;
Futai et al. 2008; Finlay 2008). In addition, plants displaying ECMs also exhibit
increased tolerance to pathogens (Marx 1972), heavy metals (Brunner and Frey
2000), or drought (Osonubi et al. 1991; Davies et al. 1996; Alvarez et al. 2009).
The ECM symbiosis results from the interaction between roots and fungi in
a time-regulated sequence of highly coordinated events. In this process, the
P. Baptista (*)
CIMO/School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia,
Apartado 1172, 5301-855 Bragança, Portugal
e-mail: pbaptista@ipb.pt
R.M. Tavares and T. Lino-Neto
Centre for Biodiversity Functional and Integrative Genomics (BioFIG), Plant Functional Biology
Centre, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
e-mail: tavares@bio.uminho.pt; tlneto@bio.uminho.pt

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_8,
158 P. Baptista et al.
mycobiont has the ability to recognize and escape the host defense surveillance,
being able to become associated with host roots and establish bidirectional nutrient
transfer. Exchange of signals between partners should occur not only to determine
their compatibility, but also to prepare them for further physiological and morpho-
logical changes for the formation of the symbiotic organ (Martin et al. 2001;
Voiblet et al. 2001; Podila 2002). Although representing an active research area,
little is known about the nature of the signaling molecules, and only few genes and
proteins associated to ectomycorrhizal symbiosis have been identified. The mecha-
nistic details of root–fungi perception and recognition are also basically unknown
or scarcely defined. Here, we review the recent results shedding light on the nature
of the molecular signals that determine ECM development.
8.2 Ectomycorrhiza Formation
The ECM development usually includes four stages: preinfection, initiation, differ-
entiation, and functioning (Table 8.1) (Martin and Tagu 1995; Le Quéré et al.
2005). In the preinfection stage, hyphae perceive the presence of host roots and the
potential partner recognition occurs. In the initiation stage, hyphae start to grow and
reach the root surface establishing physical contact. After attachment onto epider-
mal cells, hyphae proliferate in a series of layers that surround the roots and will
differentiate into the mantle. Their penetration between epidermal (angiosperms)
and also cortical (gymnosperms) root cells allows the differentiation of the Hartig-net.
Table 8.1 Time course developmental stages of ectomycorrhizal formation between Pinus
pinaster and Pisolithus tinctorius promoted under in vitro conditions
Preinfection Initiation Differentiation Functioning
0–12 h 12–24 h 2–5 days 5–15 days
a b c d e
20x 20x 20x 20x 100x
Recognition between Hyphae contact Hyphae growth around the Well-developed mantle
symbionts with the root roots (mantle is tightly adherent to
Fungal attachment formation) epidermal cells
to the root Hyphae penetration and Bidirectional
epidermis growth between translocation of
epidermal and cortical nutrients between
root cells (Hartig-net partners
formation)
The time required for attaining each stage and the principal events that occur are represented.
Figures a–d display the morphological aspects of root–fungus interaction and figure e depicts
a cross-section from a 15-day-old mycorrhiza. The arrow indicates an emerging lateral root from
P. pinaster
8 Signaling in Ectomycorrhizal Symbiosis Establishment 159
Finally, in the functioning stage, the bidirectional transfer of nutrients occurs

between both symbionts.
The time required for each stage of ECM development is variable and dependent
on the genetic compatibility between plant and fungi, as well on abiotic factors
(Smith and Read 2008). In addition, the method used for ECM establishment
(axenic or nonaxenic culture) could influence this process. The development of a
functional ECM association under axenic conditions occurs within 2–3 weeks. For
example, the development of Paxillus involutus–Betula pendula (Le Quéré et al.
2005) and Pisolithus microcarpus–Eucalyptus globulus (Duplessis et al. 2005)
ECM associations occurs within 21 days. When ectomycorrhizal development
was established between Pisolithus tinctorius and Pinus pinaster under in vitro
conditions, a mature mycorrhiza was observed 15 days postcontact (Table 8.1).
In this case, the preinfection and hyphae adherence onto root surface occurred
within the first 24 h. After 2 days of contact, the mantle started to be formed at the
apical region of the main root axis. After 5–15 days of contact, newly emerging
lateral roots were produced and became completely enveloped by the fungal
mantle. At the same time the Hartig-net became evident.
In diverse ECM associations, differential gene expression studies revealed
changes in the transcript levels of genes from both the fungus and host plant during
the interaction, resulting in the identification of several symbiosis-related (SR)
genes (Voiblet et al. 2001; Johansson et al. 2004; Duplessis et al. 2005; Le Quéré
et al. 2005; Acioli-Santos et al. 2008; Sebastiana et al. 2009). The identified genes
are involved in many cellular functions like fungal cell division and proliferation,
differentiation and signaling, synthesis of plant cell wall and extracellular matrices,
plant defense or stress responses, and primary metabolism (e.g., glycolysis, amino
acid biosynthesis, and transporter activity) (Martin et al. 2007). These results
suggest that ECM formation is a highly dynamic process, in which plant and
fungi are always receiving and sending signals and both are exposed to high stress
conditions (Martin et al. 2001).
8.3 Rhizospheric Signals Emitted During the Preinfection
The exchange of signals between the symbiotic partners starts long before the
occurrence of any physical contact, suggesting the involvement of diffusible elici-
tors in the early stages of ECM establishment (Fig. 8.1). Accordingly, previous to
any physical contact with the symbiotic host Tilia Americana, 58 genes of the
ectomycorrhizal fungus Tuber borchii were found to be differentially expressed
(Menotta et al. 2004). It was also observed that fungal genes encoding putative
signaling pathway components, such as ras (Martin et al. 2001; Sundaram et al.
2001) or PF6.2 (Kim et al. 1999), were induced prior to fungal contact with host
roots.
Pre-infection Initiation
Auxins Branching
Hypaphorine Adhesins
Lectins
Hydrophobins
Spore germination
Fungal growth
Elicitors
Binding
Rot cap
Abietic acid Zeatin
Rutin Rhizogenesis Induction of plant Acropetal mycorrhizal
Root exudates
… defence response extension
IAA plant regulation
Induction of lateral root formation Reduction of meristem activity

Chitinases Supression of plant defence response
Auxins
Inactivation of elicitors
(e.g. chitin) Radially elongated
epidermal cells Mantle Hartig net
Adhesins
SRAPs
Differentiation and functioning Hydrophobins
Fig. 8.1 Signals required for the ectomycorrhizal development. Root and fungal exudates are the
source of specific signals during the preinfection stage that lead to the perception and recognition
of compatible partners. The morphological changes that occur for forming the symbiotic structures
(in gray boxes) and the main signals that induce them are outlined. Signal molecules produced by
the plant are represented in dark gray bold and the developmental processes in which they are
involved are indicated by dashed gray arrows. Fungal signals are represented in light gray bold
and the processes controlled by them pointed out by dotted arrows
In the preinfection stage, hyphae sense the presence of a host root. Colonizer
hyphae may result from a germinated spore or from a mycelium already involved in
the development of other mycorrhizas (Tagu et al. 2002). When originating from
spore germination, the hyphae seem to sense the root system by the nutritional
composition changes that occur in the rhizosphere. The root nitrogen uptake and
secretion of sugars from the host plant turn the surroundings of the roots depleted of
nitrogen and enriched in carbon. These alterations could represent trophic signals
for fungus perception of the roots presence. However, as these trophic signals are
not specific, they could also be used by saprophytic or pathogenic fungi to recog-
nize the presence of roots (Tagu et al. 2002).
Root and fungal exudates seem to be the source of more specific signals for the
perception and recognition of a compatible association. The plant secretion of certain
signal molecules into the rhizosphere seems to promote spore germination and
hyphal growth (Barker et al. 1998; Barker and Tagu 2000). Root exudates and
extracts from Scotch pine (Pinus sylvestris) enhance spore germination of several
ectomycorrhiza-forming Suillus species (S. granulatus, S. grevillei, S. luteus and
S. variegatus) (Fries et al. 1987). The germination-induction compound was identi-
fied as abietic acid, which was described to confer selective advantage to Suillus spp.
against other fungal species present in the same rhizosphere. In root exudates of
Eucalyptus globulus ssp. bicostata the presence of flavonols, namely the rutin, was
also reported to promote the growth of several strains of Pisolithus tinctorius
(Lagrange et al. 2001). In addition, during the initial stages of Suillus tridentinus
colonization of in vitro Larix decidua roots, an increase in phenylpropanoids (mainly
flavonoids), benzoic, and cinnamic acids suggests their participation as signals in
ECM symbiosis (Weiss et al. 1997). However, even after the detection of their
effects, the chemical identification of inducing fungal growth molecules was not
always achieved. Although the diffusible molecules released by eucalyptus roots had
been described as chemoattractants towards Pisolithus tinctorius and Paxillus invo-
lutus mycelia, their chemical identification is still lacking (Horan and Chilvers 1990).
The ability of root extracts to regulate ectomycorrhizal fungal growth was also
confirmed during the early stages of Pisolithus tinctorius–Castanea sativa associa-
tion (Baptista et al. 2007). In this study, P. tinctorius growth was evaluated after
8 and 17 days of culture in the presence of crude extracts from P. tinctorius–elicited
C. sativa roots, prepared during the early stages of fungal contact (0–48 h)
(Fig. 8.2). In the first 8 days of culture, root-extracts inhibited P. tinctorius growth,
mainly after 3–9 h and 15 h of interaction (Fig. 8.2a). The enhanced fungal growth
in control (in the absence of root extracts) suggests that compounds from the
elicited plants could reduce the fungal growth. After 17 days of culture, the
inhibitory effect was not observed and an inducing effect became evident
(Fig. 8.2b). These results suggest that elicited plant could transiently restrain the
mycelium growth but the fungus is able to overcome the inhibitory effect. After
degradation/inactivation of the inhibitory compound(s), root extracts promote fun-
gal growth as previously described. Nevertheless, growth-stimulating activity
of root extracts was still reduced after 3, 9, and 15 h of root interaction with
P. tinctorius. As the H2O2 production was coincident with the inhibitory profile,
both phenomena may be related (Baptista et al. 2007).
The ectomycorrhizal fungi also produce signaling molecules that play a key role
in the early stages of ECM development (Barker and Tagu 2000; Martin et al.
2001). Several authors have attributed to the fungal auxin an important role as a
signaling molecule operating in the initial stages of the mycorrhiza formation (Gay
et al. 1994; Nehls et al. 1998, Martin et al. 1999; Rincón et al. 2001). Pine roots
inoculated with mutant strains of Hebeloma cylindrosporum overproducing indole-
3-acetic acid (IAA) develop more ectomycorrhizal roots than when inoculated with
wild-type mycelium (Gay et al. 1994). The stimulation of lateral roots by IAA was
suggested to create new targets for further fungal colonization, supporting the idea
that fungal IAA controls major anatomical features of pine ECM. The effect of
auxins on rhizogenesis can then be interpreted as a preparation of the root system
for a more efficient colonization by the mycelium (Barker et al. 1998). Accordingly,
the addition of an inhibitor of auxin transport (2,3,5-triiodobenzoic acid, TIBA)
blocked the colonization of the Norway spruce root by Laccaria bicolor, restricting
the hyphal growth between cortical cells and limiting the Hartig-net formation
(Karabaghli-Degron et al. 1998). The levels of IAA produced by the fungus also
seem to control the production of elicitors/signal molecules by the host plant
(Mensen et al. 1998; Rincón et al. 2001).
The fungal production of hypaphorine, another indolic compound, also appears
to influence the ECM establishment (Béguiristain et al. 1995; Béguiristain and
a
a a
Radial growth (mm day–1)

1.2 a
a control
aa
ab a
1.1
ab
abab ab
1.0
b
0.9
0.8
0 5 10 15 20 25 30 35 40 45 50
Time after plant elicitation (hours)
b
1.4
Radial growth (mm day–1)
a a
aa a
1.3 ab
ab
bc
1.2 c
c c c
c
control
1.1
1.0
0 5 10 15 20 25 30 35 40 45 50
Time after plant elicitation (hours)
Fig. 8.2 Radial growth of Pisolithus tinctorius after 8 days (a) and 17 days (b) of culture on agar
MMN media, in the presence of crude extracts obtained from elicited Castanea sativa roots with
P. tinctorius (from 0 to 48 h). Protein extraction buffer was used as control. Results are presented
as means SE (n ¼ 5). The letters above the columns indicate significant difference at p < 0.001
Lapeyrie 1997; Ditengou et al. 2000). This fungal alkaloid, present in P. tinctorius
mycelium axenically grown (Béguiristain et al. 1995), is highly produced during
ECM formation (Béguiristain and Lapeyrie 1997). In Eucalyptus globulus roots
inoculated with P. tinctorius, the fungal hypaphorine levels increased three- to five-
fold when compared to the pure mycelium culture (Béguiristain and Lapeyrie
1997). The host plant appears to stimulate the production of hypaphorine in the
fungus, which in turn seems to regulate the activity and levels of plant auxins (Nehls
et al. 1998; Jambois et al. 2005). The regulation of auxin levels is further supported
by the identification of several ECM-regulated genes in host tissues involved in
auxin metabolism (Voiblet et al. 2001; Johansson et al. 2004; Duplessis et al. 2005;
Le Quéré et al. 2005). According to Ditengou and Lapeyrie (2000), the hypapho-
rine could participate in plant IAA regulation by (1) regulating IAA transport;
(2) inducing a cellular IAA-specific detoxification mechanism; (3) competing with

IAA for some receptor; or (4) interfering with IAA signal transduction. Fungal
hypaphorine seems also to display a morphogenetic effect on Eucalyptus globulus
roots by reducing root hair elongation (Béguiristain and Lapeyrie 1997; Ditengou
et al. 2000; Ditengou and Lapeyrie 2000; Ditengou et al. 2003). The reduction in the
root hair growth is indeed a marker feature of ectomycorrhizal roots.
8.4 Proteins Involved in Fungal Attachment to the Root Host

Surface and Formation of Symbiotic Interface
The ectomycorrhizas are structurally distinguished by the presence of the mantle

and the Hartig-net, both involving the occurrence of physical contact between the
fungus and root surface (cell-to-cell contact). Soon after the initial contact, fungal
cells swell and adhere firmly to the root surface, which in turn induces rapid
modifications on fungal cell morphology. The fungal cells become enlarged and
intensively branched. These alterations are accompanied by the increase in nuclear
division (Martin and Tagu 1995). The signal or mechanism responsible for trigger-
ing such alterations is still not full elucidated. Nutrient limitation in the vicinity of
root system, root exudates, and the topographic sensing of root surface by fungus
could contribute to these morphologic alterations (Barker et al. 1998; Tagu et al.
2002). The fungal growth pattern as well as the hyphal branching seems also to be
controlled by the host roots through the production of cytokinins, namely zeatin
(Martin et al. 2001; Tagu et al. 2002). These early morphologic modifications seem
to constitute a reliable indicator of a compatible interaction between symbionts
(Peterson and Bonfante 1994).
During the process of ectomycorrhizal fungi adhesion to host roots, distinct
protein classes displaying cell adhesion properties are secreted by the fungi
(Fig. 8.1). Ultrastructural observations of fungus–root interface in the early stages
of colonization revealed the presence of fibrillar material rich in glycoproteins (Lei
et al. 1990a, b; Martin et al. 1999; Rincón et al. 2001). These proteins (adhesins) are
described as protein complexes able to recognize and bind to receptors located in
plant root cell surface (Martin et al. 1999; Gross et al. 2004). Adhesins are derived
from fungi cell wall and expand towards the root surface. Besides playing a role in
the adhesion of hyphae to root surface, these proteins are also important in myce-
lium aggregation during the mantle formation at the final stages of mycorrhization
(Martin and Tagu 1995).
The lectins seem also to be important in the fungi–root adhesion, not only in the
early stages by playing a similar role to adhesins, but also at the final stages of ECM
establishment. Receptor sites for the lectin produced by the specific symbiont of
spruce, Lactarius deterrimus, were found in the roots of Picea abies (Giollant et al.
1993). In this study, the lectin was mainly bounded to young tissues, such as root
hairs and tips of lateral roots. By contrast, no binding to the spruce root was
observed for the lectin isolated from Lactarius deliciosus, a symbiont of the pine.
This result suggests a role of the fungal lectin in the recognition and partner
specificity during the early stages of ECM development (Giollant et al. 1993).
However, the differential gene expression analysis performed during Betula pendula
and Paxillus involutus ECM development revealed a repression of fungal lectin-
encoding genes during the early stages of interaction and their induction 14 days
after inoculation (Le Quéré et al. 2005).
Search for differentially expressed genes during ECM interactions has contrib-
uted for the identification of additional gene products that could play a role in
fungi–host root adhesion. In Pisolithus–Eucalyptus ectomycorrhizal symbiosis,
genes encoding hydrophobins are up-regulated, suggesting the implication of the
gene products in fungi–cell adhesion (Tagu et al. 1996; Duplessis et al. 2001;
Voiblet et al. 2001; Peter et al. 2003; Duplessis et al. 2005). Hydrophobins exhibit
a conserved spacing of eight cystein residues which can form disulfide bonds
between themselves (Linder et al. 2005). These hydrophobic proteins are secreted
by the fungus into the medium but can also be found at the fungi cell wall. In this
case, they exhibit a hydrophilic domain through which they are linked to the
hydrophilic cell wall and a hydrophobic domain that could be exposed to a
hydrophobic external medium. Thus, these proteins have the ability to self-assemble
into an amphipathic membrane at a hydrophilic/hydrophobic interface. The hydro-
philic side of the amphipathic membrane orients and attaches to the fungal cell wall,
while the hydrophobic side becomes exposed to the hydrophobic environment, such
as the air or the hydrophobic surface of a host. In this way, the aerial hyphae and
spores become hydrophobic, whereas hyphae grown over a hydrophobic substrate
become attached to themselves (W€ osten 2001). As a result, hydrophobins seem to
play a dual role in ECM, not only in promoting the hyphae adhesion to the host
surface but also contributing to hyphae aggregation (Kershaw and Talbot 1998;
W€ osten 2001).
Hydrophobins are encoded by multigene families (Linder et al. 2005). For
example, in Pisolithus tinctorius seven different genes have already been identified
(hydPt-1 to hydPt-6 and hydPt-8), although the corresponding gene products
display minor structural differences (Tagu et al. 1996; Duplessis et al. 2001,
2005; Voiblet et al. 2001). As suggested by Linder et al. (2005), the presence of
multiple hydrophobin genes in an organism may be significant in two different
ways. They could complement each other by having differential expression in
distinct developmental stages or as a response to environmental conditions. Alter-
natively, they could fulfill different functional roles as a result of the minor
structural differences they exhibit.
Hydrophobin-like proteins have already been reported at least in 20 fungal
species belonging to ascomycetes, basidiomycetes, and zygomycetes (Wessels
1996). According to their structure, these proteins are described as being involved
in several fungal developmental processes, such as emergence of aerial hyphae,
hyphae aggregation during fruiting bodies (carpophores) development, sporulation
and spore dissemination, as well as during plant or insect infections caused by
pathogenic fungi (Wessels 1996; Kershaw and Talbot 1998; W€osten 2001; Linder
et al. 2005). In this case, hydrophobins role seems to be essential since the pathogen
must attach to the hydrophobic surface of the host before penetration and infection
can occur (Wessels 1996; W€ osten 2001). Besides their role in the adhesion of fungi
to the host root during ectomycorrhizal formation, hydrophobins have also been
described as being involved in the fungal recognition of the host plant, as well as in
the fungal specificity displayed by the host plant (Mankel et al. 2002).
During Pisolithus tinctorius ECM development, the expression analysis of
hydrophobin genes suggests the involvement of these proteins in the morphogenetic
events related to the fungus attachment to root surfaces, but not in the late stages of
fungal mantle or Hartig-net formation (Martin et al. 1999). In Pisolithus–Eucalyptus
association, hydrophobin gene expression was induced during the early steps of
root colonization, being repressed after 4 days of contact (Tagu et al. 1996; Martin
et al. 1999). During the first 12 h of Castanea sativa root–P. tinctorius contact, a
strong downregulation of two hydrophobin genes (HydPt-2 and HydPt-3) was
detected (Acioli-Santos et al. 2008). Other differential gene expression studies
performed in distinct ECM interactions describe different results concerning the
expression of hydrophobin genes (Mankel et al. 2002; Johansson et al. 2004; Le
Quéré et al. 2005). However, even when the hydrophobin gene expression is
transient, the gene product could be detected in late developmental stages. For
example, in the previously referred Pisolithus–Eucalyptus association, the hydro-
phobins were immunologically detected in hyphae forming the mantle and Hartig-
net (Tagu et al. 2001). The expression levels of hydrophobin genes could differ
depending on fungal species or host plants, or even be influenced by the presence of
certain nutrients in the substrate, suggesting a complex regulation of hydrophobin
gene expression during ECM development (Martin et al. 1999).
Another family of cell adhesion proteins was identified in P. tinctorius after
detection of gene overexpression in the early stages of the Eucalyptus globulus–
P. tinctorius interaction (Laurent et al. 1999). The identified protein family –
symbiosis-regulated acidic polypeptides (SRAP) – comprises at least six different
isoforms of 31–32 kDa and is characterized by the presence of Arg-Gly-Asp
tripeptide (RGD) motif (Martin et al. 1999). This motif is commonly associated to
cell adhesion in animals (Critchley et al. 1999) and plants (Mellersh and Heath 2001;
Meinhardt et al. 2002). Cellular localization of a 32-kDa SRAP (SRAP32) during
ECM development showed a preferential accumulation of this protein in fungal cell
walls, fungus–root interfaces, and in penetrating hyphae forming the Hartig-net
(Laurent et al. 1999). The suggestion that SRAP family could be involved in the
hyphae aggregation for the fungal mantle formation and for the Hartig-net develop-
ment is corroborated by differential expression studies during ECM development.
After 4 days of E. globulus–P. tinctorius interaction, an increase of SRAP32 tran-
script levels (4.1-fold) was detected, as well as the expression levels (4.7-fold) of
a new family member of low molecular weight (SRAP17) (Voiblet et al. 2001).
The participation of SRAP family members in the ectomycorrhizal development
seems to decrease during the final stages of the interaction. Genes encoding SRAP32
and SRAP17 are repressed after 7–12 days of E. globulus–Pisolithus microcarpus
interaction (Duplessis et al. 2005).
8.5 Ectomycorrhizal Morphogenesis
The development of a functional ECM involves large morphological alterations in

both partners. The major morphogenetic adaptations that occur in the symbiotic
fungus includes: the hyphae aggregation for the formation of the mantle envelop-
ing the root surface, and the hyphae penetration and growth between epidermal and
cortical root cells forming the Hartig-net (Smith and Read 2008). Development of
a mature mantle proceeds through a programmed and highly coordinated series of
events. After attachment onto root epidermal cells, hyphae multiply to form a
series of layers that will differentiate into the mature mantle (Martin et al. 2001).
The mantle morphology is dependent on both the plant and fungus genomes.
Indeed, the identification of mycobiont species of field-collected ectomycorrhizas
is often performed by observation of mantle morphology (Peterson and Bonfante
1994). The host plant seems to control the mantle formation by stimulating hyphal
growth and exerting a morphogenetic effect leading to compact hyphal mantle
development (Martin et al. 2001). In addition, several abiotic factors, such as
nutrients and oxygen availability, as well as the presence of a physical support,
are described to play a role in mantle formation (Peterson and Bonfante 1994;
Martin et al. 1999).
The hyphae that enter the intercellular spaces of the root will develop the Hartig-net.
The fungus penetration and growth within the apoplastic space is mainly accom-
plished by a mechanic process, but the production of lytic enzymes could also be
involved in the digestion of host cell walls (Cairney and Burke 1994). Once inside
the root, hyphae could only grow between epidermal cells (in the majority of
angiosperms) or could also penetrate between cortical cells up to the endodermis
(typical in gymnosperms) (Smith and Read 2008). The restriction of fungal ingress
into the cortical tissues seems to be due to the fungus inability to degrade the
suberin and lignin that are present in the endodermal cell walls (Barker et al. 1998).
As the same fungal species could only colonize the epidermal layer or also the
cortical layer, depending on the host plant, the hyphal growth was suggested to be
under the host plant control (Barker et al. 1998).
The progression of ectomycorrhizal fungus within intercellular spaces of root
epidermal cells promotes morphologic alterations in the majority of angiosperm
roots (Fig. 8.1). The physical contact with the fungus promotes a radial elongation
of the cell roots, thus changing their orientation of growth and shape (Peterson and
Bonfante 1994). Several studies have showed that these modifications are related to
cytoskeleton rearrangements (Timonen et al. 1993). Changes in the amount of actin
and tubulin proteins in root and/or fungal cells were observed during ECM forma-
tion, suggesting its involvement in the ECM morphogenesis (Timonen et al. 1993;
Carnero Diaz et al. 1996; Timonen et al. 1996; Johansson et al. 2004). In agreement,
fungal genes encoding proteins associated with actin and microtubules were found
to be differentially regulated in distinct ECM associations (Voiblet et al. 2001;
Timonen and Peterson 2002; Menotta et al. 2004; Le Quéré et al. 2005). However, it
still remains to be clarified if changes in cytoskeletal gene expression are the cause
or the consequence of mycorrhizal root morphogenesis (Barker et al. 1998).
In contrast to what is observed in the initial mycorrhization stages, after fungal
root colonization there is a reduction of plant meristem activity, as well as early
formation of lateral roots. In the case of Pinaceae ectomycorrhiza, dichotomous
branching of short roots occurs, resulting occasionally in the formation of coralloid
structures (Peterson and Bonfante 1994). These morphological modifications were
suggested to be caused in part by fungal auxins, which have been also related to the
Hartig-net formation. Several studies have demonstrated that fungal auxins are
morphogen factors in ECM development, playing a key role in inducing the fungal
proliferation alterations (Gay et al. 1994; Martin and Tagu 1995; Karabaghli-
Degron et al. 1998; Mensen et al. 1998; Rincón et al. 2001). The fungal production
of auxins in roots completely surrounded by ectomycorrhizal hyphae could alter the
internal plant auxin balance, and consequently promote typical ectomycorrhizal
root morphogenesis (Barker and Tagu 2000). Inhibition of auxin transporters has
shown that these morphogenetic effects, like the lateral root dichotomy, are depen-
dent on auxin concentration and distribution in the root meristem (Karabaghli-
Degron et al. 1998; Kaska et al. 1999). However, to our knowledge, the clear effect
of ectomycorrhizal fungi on concentration and/or distribution of auxin in the roots
still remains to be elucidated.
Differential gene expression studies have shown that the morphological and
physiological changes observed throughout ECM development are accompanied by
changes in gene expression in both partners (Heller et al. 2008). The number of
genes and the amplitude of their expression vary in time but seem to be more
significant at the early stages of development, soon after plant–fungus contact.
Accordingly, gene expression studies revealed that the gene overexpression levels
were more evident during the first 8 days of interaction being repressed in advanced
stages of mycorrhization (Duplessis et al. 2005; Le Quéré et al. 2005). In addition, it
was shown that the number of fungal differentially expressed genes is much higher
than the number of plant host genes.
In all the ECM associations studied up to now, ECM-specific genes were not yet
identified. Therefore, the ontogenic and metabolic programs that lead to the devel-
opment of symbiosis seem to be driven by the differential expression of preexisting
transcription factors and/or transduction pathways, rather than by the expression of
symbiosis-specific gene arrays (Martin et al. 2007). However, a more detailed study
encompassing all differentially expressed genes is needed in order to clarify this
question. Many up- or downregulated genes during ECM development have not
shown similarity to known sequences (Voiblet et al. 2001; Podila et al. 2002; Kr€uger
et al. 2004). Some of these genes may represent unidentified fungal mycorrhiza-
specific genes (Heller et al. 2008), or be unique to a particular ectomycorrhizal
fungus, or even represent very rare transcripts that have not been previously
identified and/or characterized (Podila et al. 2002).
8.6 Induction of Plant Defense Responses by Ectomycorrhizal

Fungi
When infected by pathogens, plants can activate a multitude of defense mechan-

isms (Dixon and Lamb 1990; Baker and Orlandi 1995; Doke et al. 1996; Mehdy
et al. 1996; Lamb and Dixon 1997). The recognition of pathogen molecules
(elicitors) by specific plant cell receptors could lead to the activation of kinases
and phosphorylation cascades, involving phosphatases, G proteins, and ionic fluxes.
These events induce a typical pattern of plant defense responses that includes (1)
the production of reactive oxygen species (ROS) in the so-called oxidative burst;
(2) the hypersensitive response; (3) the induction of defense response genes or
pathogen-related genes (PR genes); (4) the reinforcement of cell wall, including the
deposition of callose, lignin, or hydroxiproline-rich glycoproteins; (5) synthesis of
jasmonate, ethylene, and/or salicylic acid; (6) production of antimicrobial second-
ary metabolites, such as phytoalexins; and (7) production of hydrolytic enzymes,
such as chitinases and glucanases. Plants could also induce systemic-acquired
resistance and produce signals to other plants.
As observed for plant–pathogen interactions, the plant inoculation with ectomy-
corrhizal fungi comprises the induction/suppression of mechanisms associated to
plant defense responses (Fig. 8.1). These mechanisms seem to play a key role in
determining the symbiotic compatibility, as well as in ectomycorrhizal develop-
ment and functioning (Martin et al. 2001). Suspension-cultured cells of Picea abies
subjected to cell wall elicitors from the ectomycorrhizal fungi Amanita muscaria
(Schwacke and Hager 1992) and Hebeloma crustuliniforme (Schwacke and Hager
1992; Salzer et al. 1996) exhibit biochemical resistance responses similar to those
described for incompatible pathogen attack. Soon after elicitation, an efflux of Cl
and Kþ takes place, followed by an influx of Ca2þ into plant cells (Schwacke and
Hager 1992; Salzer et al. 1996). Four minutes after elicitation, protein phosphory-
lation and dephosphorylation (Salzer et al. 1996), alkalinization of the medium, and
transient accumulation of ROS, such as hydrogen peroxide (H2O2) (Schwacke and
Hager 1992; Salzer et al. 1996), can be detected. The activity of antioxidative
enzymes, such as superoxide dismutase (SOD) and catalase (CAT), seems to play a
role in regulating ROS levels in ECM interactions (Schwacke and Hager 1992).
During the first 48 h of contact between ectomycorrhizal fungus Pisolithus tinctorius
and Castanea sativa roots, three peaks of H2O2 production were observed, the first
two synchronized with O2 bursts (Baptista et al. 2007). The accumulation of
H2O2 was presumed to be the result of a regulated time-dependent stimulation of
ROS-generating systems and decrease of ROS-scavenging enzyme activities.
Accordingly, the first H2O2 production peak coincided with an increase in SOD
activity, while CAT activity seemed to be implicated in subsequent H2O2 scaveng-
ing (Baptista et al. 2007).
The similarity of signal transduction pathways in incompatible plant–pathogen
interactions and ECM symbiosis suggests that the fungus produces elicitors, at the
early stages of the symbiotic association that will induce typical plant defense
responses (Hebe et al. 1999). Ultrastructural observations revealed that, when
Pinus nigra was inoculated with Suillus collinitus, a defense reaction was activated
in the host plant with the formation of wall thickenings containing b-1,3-glucans, a
callose constituent (Bonfante et al. 1998). In addition, Norway spruce seedlings
inoculated with the ectomycorrhizal fungus Pisolithus tinctorius displayed
increased guaiacol peroxidase activity in the roots, which is involved in cell-wall
strengthening (Matevž and Regvar 2008). The accumulation of phenolics in plant
cell wall observed in distinct ECM associations was also suggested to restrain
fungal progression during the formation of ECM structures, thus limiting the
Hartig-net formation (Weiss et al. 1997, 1999; Feugey et al. 1999). During the
initial fungal progression into apoplastic root regions to form the Hartig-net, a
transient increase in the activity of the phenylpropanoid pathway enzyme phenyl-
alanine ammonia lyase was observed, together with the increase in transcripts of
PR-encoding genes (Feugey et al. 1999). Differential gene expression studies
during ectomycorrhiza formation have also confirmed the transient increase in
plant defense/stress responses which has been interpreted as an initial reaction of
plant to restrict fungal growth (Voiblet et al. 2001; Johansson et al. 2004; Morel
et al. 2005; Le Quéré et al. 2005; Duplessis et al. 2005, Sebastiana et al. 2009).
In contrast to what has been reported for incompatible plant–pathogen interac-
tions, in ectomycorrhizal symbiosis a rapid decline in plant defense responses
seems to occur (Salzer et al. 1996). In P. abies this response suppression seems to
be related to the production of chitinases by the plant that inactivates elicitors, like
chitin fragments produced by the ectomycorrhizal fungus (Salzer et al. 1997a, b).
These results suggest that elicitor inactivation could be a prerequisite for a compat-
ible interaction between plant and ectomycorrhizal fungus. Furthermore, the stim-
ulation of chitinase production in host plant could contribute to hyphae growth
within root intercellular spaces, promoting the formation of the ECM structures
(Sauter and Hager 1989). This hypothesis is supported by the increase in ECM root
number in the presence of high chitinase amounts (Albrecht et al. 1994). The auxins
produced by the ectomycorrhizal fungus during the colonization process also seem
to play a role in declining the defense capacity of plant cells (Mensen et al. 1998).
The induction of plant defense responses during the early stages of mycorrhiza-
tion is not always followed by its suppression at later stages. In the co-cultures of
P. abies callus and Lactarius deterrimus or Suillus variegatus the development of
typical hypersensitive response reactions was observed. Initially the spruce cells
become brownish and finally the whole callus became necrotic (Sirrenberg et al.
1995). Therefore, plant defense responses induced by ectomycorrhizal fungi seem
to be diverse and often contradictory in what concerns their attenuation during the
later stages of the symbiosis. According to Martin et al. (1999), depending on the
host and ectomycorrhizal fungus, different biochemical and molecular mechanisms
could exist, thus explaining the differences in the reported results. The environ-
mental factors occurring during plant–fungus interaction could also be decisive for
the defense response displayed by host plant.
8.7 Conclusions
The establishment of an ectomycorrhizal symbiosis is a complex process triggered

by signals produced by both partners (Fig. 8.1). The ECM development involves
five main events: hyphae survival in the rhizosphere; hyphae attachment to the host
roots; invasion of root tissues by fungi; plant–fungus coordinated formation of
symbiotic structures; and bilateral transfer of assimilates. During the preinfection,
diffusible signal molecules are exchanged between the host plant and the fungus.
The recognition of these signals by both partners determines the compatibility of
symbiosis. Although the nature of these signaling molecules is scarcely known,
diffusible molecules (such as abietic acid or rutin) are released by host plant and
would chemoattract, promote spore germination, or regulate ectomycorrhizal fun-
gal growth. The fungus also takes part in this signaling process through the
secretion of auxins and hypaphorin. Besides their signaling role, these compounds
have also a direct effect on rhizogenesis and ECM structure formation.
Once mutually recognized, root and fungus establish physical contact. During
the initiation, fungal proteins (such as adhesins, lectins, or hydrophobins) promote
fungus adhesion to host roots. The initial contact induces a defense response in host
plant that seems to restrain the fungal progression into the plant tissues. After the
attenuation/suppression of host surveillance mechanisms, drastic morphological
and physiological modifications occur in both fungal and root cells for forming
the symbiotic structures. Fungal tissues differentiate into the mantle and the Hartig-
net, whereas root cells change their shape and orientation, and root tips proliferate
and develop lateral roots. These auxin-regulated plant developmental processes are
induced by the mycobiont through the action of secreted auxins.
The ontogenesis of a functional ECM symbiosis requires a finely regulated
crosstalk between plant and fungi. Despite the contribution given by gene expres-
sion studies, the molecular information on ECM establishment is still scarce. A few
genes and proteins associated to ECM symbiosis have been recognized and a small
number of signaling molecules chemically identified. Further studies are needed to
understand the formation and functioning of ECM symbiosis.
References
Acioli-Santos B, Sebastiana M, Pessoa F, Sousa L, Figueiredo A, Fortes AM, Baldé A, Maia LC,
Pais MS (2008) Fungal transcript pattern during the preinfection stage (12 h) of ectomycorrhiza
formed between Pisolithus tinctorius and Castanea sativa roots, identified using cDNA
microarrays. Curr Microbiol 57:620–625
Albrecht C, Asselin A, Piché Y, Lapeyrie F (1994) Chitinase activities are induced in Eucalyptus
globulus roots by ectomycorrhizal or pathogenic fungi, during early colonization. Physiol Plant
91:104–110
Alvarez M, Huygens D, Fernandez C, Gacitúa Y, Olivares E, Saavedra I, Alberdi M, Valenzuela E
(2009) Effect of ectomycorrhizal colonization and drought on reactive oxygen species metab-
olism of Nothofagus dombeyi roots. Tree Physiol 29:1047–1057
Baker CJ, Orlandi EW (1995) Active oxygen in plant pathogenesis. Annu Rev Phytopathol 33:
299–321
Baptista P, Martins A, Pais MS, Tavares RM, Lino-Neto T (2007) Involvement of reactive oxygen
species during early stages of ectomycorrhiza establishment between Castanea sativa and
Pisolithus tinctorius. Mycorrhiza 17:185–193
Barker SJ, Tagu D (2000) The roles of auxins and cytokinins in mycorrhizal symbioses. J Plant
Growth Regul 19:144–154
Barker SJ, Tagu D, Delp G (1998) Regulation of root and fungal morphogenesis in mycorrhizal
symbioses. Plant Physiol 116:1201–1207
Béguiristain T, Lapeyrie F (1997) Host plant stimulates hypaphorine accumulation in Pisolithus
tinctorius hyphae during ectomycorrhizal infection while excreted fungal hypaphorine controls
root hair development. New Phytol 136:525–532
Béguiristain T, Côte R, Rubini P, Jay-Allemand C, Lapeyrie F (1995) Hypaphorine accumulation
in the hyphae of the ectomycorrhizal fungus Pisolithus tinctorius. Phytochemistry 40:
1089–1091
Bonfante P, Balestrini R, Martino E, Perotto S, Plassard C, Mousain D (1998) Morphological
analysis of early contacts between pine roots and two ectomycorrhizal Suillus strains. Mycor-
rhiza 8:1–10
Brunner I, Frey B (2000) Detection and localization of aluminum and heavy metals in ectomycor-
rhizal Norway spruce seedlings. Environ Pollut 108:121–128
Cairney J, Burke R (1994) Fungal enzymes degrading plant cell walls: their possible significance
in the ectomycorrhizal symbiosis. Mycol Res 98:1345–1356
Carnero Diaz E, Martin F, Tagu D (1996) Eucalypt alpha-tubulin: cDNA cloning and increased
level of transcripts in ectomycorrhizal root system. Plant Mol Biol 31:905–910
Critchley DR, Holt MR, Barry ST, Priddle H, Hemmings L, Norman J (1999) Integrin-mediated
cell adhesion: the cytoskeletal connection. Biochem Soc Symp 65:6579–6599
Ditengou F, Lapeyrie F (2000) Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius
counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt
seedlings. MPMI 13:151–158
Ditengou F, Béguiristain T, Lapeyrie F (2000) Root hair elongation is inhibited by hypaphorine,
the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by
indole-3-acetic acid. Planta 211:722–728
Ditengou F, Raudaskoski M, Lapeyrie F (2003) Hypaphorine, an indole-3-acetic acid antagonist
delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin
and the microtubule cytoskeleton in Eucalyptus globulus ssp. bicostata root hairs. Planta
218:217–225
Dixon RA, Lamb CJ (1990) Molecular communication in interactions between plants and micro-
bial pathogens. Annu Rev Plant Physiol Plant Mol Biol 41:339–367
Doke N, Miura Y, Sanchez LM, Park H-J, Noritake T, Yoshioka H, Kawakita K (1996) The
oxidative burst protects plants against pathogen attack: mechanism and role as an emergency
signal for plant bio-defence – a review. Gene 179:45–51
Duplessis S, Sorin C, Voiblet C, Palin B, Martin F, Tagu D (2001) Cloning and expression analysis
of a new hydrophobin cDNA from the ectomycorrhizal basidiomycete Pisolithus. Curr Genet
39:335–339
Duplessis S, Courty P-E, Tagu D, Martin F (2005) Transcript patterns associated with ectomycor-
rhiza development in Eucalyptus globulus and Pisolithus microcarpus. New Phytol 165:
599–611
Feugey L, Strullu D-G, Poupard P, Simoneau P (1999) Induced defence responses limit Hartig-net
formation in ectomycorrhizal birch roots. New Phytol 144:541–547
Finlay RD (2008) Ecological aspects of mycorrhizal symbiosis: with special emphasis on the
functional diversity of interactions involving the extraradical mycelium. J Exp Bot 59:1115–1126
Fries N, Serck-Hanssen K, Hall-Dimberg L, Theander O (1987) Abietic acid, an activator of
basidiospore germination in ectomycorrhizal species of the genus Suillus (Boletaceae). Exp
Mycol 11:360–363
Davies FT Jr, Svenson SE, Cole JC, Phavaphutanon L, Duray SA, Olalde-Portugal V, Meier CE,
Bo SH (1996) Non-nutritional stress acclimation of mycorrhizal woody plants exposed to
drought. Tree Physiol 16:985–993
Futai K, Taniguchi T, Kataoka R (2008) Ectomycorrhizae and their importance in forest ecosys-
tems. In: Mycorrhizae: sustainable agriculture and forestry. Springer, Netherlands, pp 241–285
Gay G, Normand L, Marmeisse R, Sotta B, Debaud JC (1994) Auxin overproducer mutants of
Hebeloma cylindrosporum Romagnesi have increased mycorrhizal activity. New Phytol
128:645–657
Giollant M, Guillot J, Damez M, Dusser M, Didier P, Didier E (1993) Characterization of a lectin
from Lactarius deterrimus. Research on the possible involvement of the fungal lectin in
recognition between mushroom and spruce during the early stages of mycorrhizae formation.
Plant Physiol 101:513–522
Gross E, Casagrande LIT, Caetano FH (2004) Ultrastructural study of ectomycorrhizas on
Pinus caribaea Morelet. var. hondurensis Barr. & Golf. seedlings. Acta Botanica Brasilica
18:1–7
Hebe G, Hager A, Salzer P (1999) Initial signalling processes induced by elicitors of ectomycorrhiza-
forming fungi in spruce cells can also be triggered by G-protein-activating mastoparan and
protein phosphatase-inhibiting cantharidin. Planta 207:418–425
Heller G, Adomas A, Li G, Osborne J, van Zyl L, Sederoff R, Finlay R, Stenlid J, Asiegbu F (2008)
Transcriptional analysis of Pinus sylvestris roots challenged with the ectomycorrhizal fungus
Laccaria bicolour. BMC Plant Biol 8:19
Horan DP, Chilvers GA (1990) Chemotropism: the key to ectomycorrhizal formation? New Phytol
116:297–301
Jambois A, Dauphin A, Kawano T, Ditengou FA, Bouteau F, Legue V, Lapeyrie F (2005)
Competitive antagonism between IAA and indole alkaloid hypaphorine must contribute to
regulate ontogenesis. Physiol Plant 123:120–129
ectomycorrhizal root tissue. MPMI 17:202–215
Karabaghli-Degron C, Sotta B, Bonnet M, Gay G, Le Tacon F (1998) The auxin transport inhibitor
2,3,5-triidobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and
the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the
ectomycorrhizal fungus Laccaria bicolor. New Phytol 140:723–733
Kaska DD, Myllyl€a R, J.B C (1999) Auxin transport inhibitors act through ethylene to regulate
dichotomous branching of lateral root meristems in pine. New Phytol 142:49–58
Kershaw MJ, Talbot NJ (1998) Hydrophobins and repellents: proteins with fundamental roles in
fungal morphogenesis. Fungal Genet Biol 23:18–33
Kim S-J, Hiremath ST, Podila GK (1999) Cloning and identification of symbiosis-regulated genes
from the ectomycorrhizal Laccaria bicolor. Mycol Res 103:168–172
Kr€uger A, Peskan-Bergh€ ofer T, Frettinger P, Herrmann S, Buscot F, Oelm€uller R (2004) Identifi-
cation of premycorrhiza-related plant genes in the association between Quercus robur and
Piloderma croceum. New Phytol 163:149–157
Lagrange H, Jay-Allemand C, Lapeyrie F (2001) Rutin, the phenolglycoside from Eucalyptus root
exudates, stimulates Pisolithus hyphal growth at picomolar concentrations. New Phytol
149:349–355
Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol
Plant Mol Biol 48:251–275
Laurent P, Voiblet C, Tagu D, Carvalho D, Nehls U, De Bellis R, Balestrini R, Bauw G, Bonfante P,
Martin F (1999) A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus
tinctorius. MPMI 12:862–871
Le Quéré A, Wright DP, S€ oderstr€
om B, Tunlid A, Johansson T (2005) Global patterns of gene
regulation associated with the development of ectomycorrhiza between birch (Betula pendula
Roth.) and Paxillus involutus (Batsch) Fr. MPMI 18:659–673
Lei J, Ding H, Lapeyrie F, Piché Y, Malajczuk N, Dexheimer J (1990a) Ectomycorrhizal formation

on the roots of Eucalyptus globulus and Pinus caribaea with two isolates of Pisolithus
tinctorius: structural and cytochemical observations. In: Nardon P, Gianinazzi-Pearson V,
Greiner AM, Margulis L, Smith DC (eds) Endocytobiology, vol IV. INRA, Paris, France,
pp 123–126
Lei J, Lapeyrie F, Malajczuk N, Dexheimer J (1990b) Infectivity of pine and eucalypt isolates of
Pisolithus tinctorius (Pers.) Coker & Couch on roots of Eucalyptus urophylla S.T. Blake
in vitro. II. Ultrastructural and biochemical changes at the early stage of mycorrhiza formation.
Linder MB, Szilvay GR, Nakari-Set€al€a TN, Penttil€a ME (2005) Hydrophobins: the protein-
amphiphiles of filamentous fungi. FEMS Microbiol Rev 29:877–896
Mankel A, Krause K, Kothe E (2002) Identification of a hydrophobin gene that is developmentally
regulated in the ectomycorrhizal fungus Tricholoma terreum. Appl Environ Microbiol
68:1408–1413
Martin F, Tagu D (1995) Ectomycorrhiza development: a molecular perspective. In: Varma A,
Hock B (eds) Mycorrhiza: structure, function, molecular biology and biotechnology. Springer,
Berlin, pp 30–58
Martin F, Laurent P, de Carvalho D, Voiblet C, Balestrini R, Bonfante P, Tagu D (1999) Cell wall
proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function,
and expression in symbiosis. Fungal Genet Biol 27:161–174
Martin F, Duplessis S, Ditengou F, Lagrange H, Voiblet C, Lapeyrie F (2001) Developmental
cross talking in the ectomycorrhizal symbiosis: signals and communication genes. New Phytol
151:145–154
Martin F, Kohler A, Duplessis S (2007) Living in harmony in the wood underground: ectomycor-
rhizal genomics. Curr Opin Plant Biol 10:204–210
Marx DH (1972) Ectomycorrhizae as biological deterrents to pathogenic root infections. Annu
Rev Phytopathol 10:429–454
Matevž L, Regvar M (2008) Early defence reactions in Norway spruce seedlings inoculated with
the mycorrhizal fungus Pisolithus tinctorius (Persoon) Coker & Couch and the pathogen
Heterobasidion annosum. Trees Struct Funct 22:861–868
Mehdy M, Sharma YK, Sathasivan K, Bays NW (1996) The role of activated oxygen species in
plant disease resistance. Physiol Plant 98:365–374
Meinhardt SW, Cheng W, Kwon SY, Donohue CM, Rasmussen JB (2002) Role of the Arginyl-
Glycyl-Aspartic motif in the action of Ptr ToxA produced by Pyrenophora tritici-repentis.
Mellersh DG, Heath MC (2001) Plasma membrane–cell wall adhesion is required for expression of
plant defence responses during fungal penetration. Plant Cell 13:413–424
Menotta M, Amicucci A, Sisti D, Gioacchini AM, Stocchi V (2004) Differential gene expression
during pre-symbiotic interaction between Tuber borchii Vittad. and Tilia americana L. Curr
Genet 46:158–165
Mensen R, Hager A, Salzer P (1998) Elicitor-induced changes of wall-bound and secreted
peroxidase activities in suspension-cultured spruce (Picea abies) cells are attenuated by auxins.
Physiol Plant 102:539–546
Morel M, Jacob C, Kohler A, Johansson T, Martin F, Chalot M, Brun A (2005) Identification of
genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during
Paxillus involutus-Betula pendula ectomycorrhizal symbiosis. Appl Environ Microbiol
71:382–391
Nehls U, Béguiristain T, Ditengou F, Lapeyrie F, Martin F (1998) The expression of a symbiosis-
regulated gene in eucalypt roots is regulated by auxins and hypaphorine, the tryptophan betaine
of the ectomycorrhizal basidiomycete Pisolithus tinctorius. Planta 207:296–302
Osonubi O, Mulongoy K, Awotoye OO, Atayese MO, Okali DUU (1991) Effects of ectomycor-
rhizal and vesicular-arbuscular mycorrhizal fungi on drought tolerance of four leguminous
woody seedlings. Plant Soil 136:131–143
Peter M, Courty P-E, Kohler A, Delaruelle C, Martin D, Tagu D, Frey-Klett P, Duplessis S, Chalot M,
Podila G, Martin F (2003) Analysis of expressed sequence tags from the ectomycorrhizal
basidiomycetes Laccaria bicolor and Pisolithus microcarpus. New Phytol 159:117–129
Peterson RL, Bonfante P (1994) Comparative structure of vesicular-arbuscular mycorrhizas and
ectomycorrhizas. Plant Soil 159:79–88
Podila GK (2002) Signalling in mycorrhizal symbioses – elegant mutants lead the way. New
Phytol 154:541–545
Podila GK, Zheng J, Balasubramanian S, Sundaram S, Hiremath S, Brand JH, Hymes MJ (2002)
Fungal gene expression in early symbiotic interactions between Laccaria bicolor and red pine.
Rincón A, Gérard J, Dexheimer J, Le Tacon F (2001) Effect of an auxin transport inhibitor on
aggregation and attachment process during ectomycorrhiza formation between Laccaria
bicolor S238N and Picea abies. Can J Bot 79:1152–1160
Salzer P, Hebe G, Reith A, Zitterell-Haid B, Stransky H, Gaschler K, Hager A (1996) Rapid
reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma
crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes. Planta
198:118–126
Salzer P, Hebe G, Hager A (1997a) Cleavage of chitinous elicitors from the ectomycorrhizal
fungus Hebeloma crustuliniforme by host chitinases prevents induction of Kþ and Cl release,
extracellular alkalinization and H2O2 synthesis of Picea abies cells. Planta 203:470–479
Salzer P, H€ubner B, Sirrenberg A, Hager A (1997b) Differential effect of purified spruce chitinases
and beta-1,3-glucanases on the activity of elicitors from ectomycorrhizal fungi. Plant Physiol
114:957–968
Sauter M, Hager A (1989) The mycorrhizal fungus Amanita muscaria induces chitinase activity in
roots and in suspension-cultured cells of its host Picea abies. Planta 179:61–66
Schwacke R, Hager A (1992) Fungal elicitors induce a transient release of active oxygen species
from cultured spruce cells that is dependent on Ca2þ and protein-kinase activity. Planta
187:136–141
Sebastiana M, Figueiredo A, Acioli B, Sousa L, Pessoa F, Baldé A, Pais MS (2009) Identification
of plant genes involved on the initial contact between ectomycorrhizal symbionts (Castanea
sativa – European chestnut and Pisolithus tinctorius). Eur J Soil Biol 45:275–282
Sirrenberg A, Salzer P, Hager A (1995) Induction of mycorrhiza-like structures and defence
reactions in dual cultures of spruce callus and ectomycorrhizal fungi. New Phytol 130:149–156
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic Press, London
Sundaram S, Kim SJ, Suzuki H, Mcquattie CJ, Hiremath ST, Podila GK (2001) Isolation and
characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria
bicolor. MPMI 14:618–628
Tagu D, Nasse B, Martin F (1996) Cloning and characterization of hydrophobins-enconding
cDNAs from the ectomycorrhizal basidiomycete Pisolithus tinctorius. Gene 168:93–97
Tagu D, De Bellis R, Balestrini R, De Vries OMH, Piccoli G, Stocchi V, Bonfante P, Martin F
(2001) Immunolocalization of hydrophobin HydPt-1 from the ectomycorrhizal basidiomycete
Pisolithus tinctorius during colonization of Eucalyptus globulus roots. New Phytol 149:
127–135
Tagu D, Lapeyrie F, Martin F (2002) The ectomycorrhizal symbiosis: genetics and development.
Timonen S, Peterson RL (2002) Cytoskeleton in mycorrhizal symbiosis. Plant Soil 244:199–210
Timonen S, Finlay RD, S€ oderstr€
om B, Raudaskoski M (1993) Identification of cytoskeletal
components in pine ectomycorrhizas. New Phytol 124:83–92
Timonen S, S€oderstr€om B, Raudaskoski M (1996) Dynamics of cytoskeletal proteins in developing
pine ectomycorrhiza. Mycorrhiza 5:423–429
Voiblet C, Duplessis S, Encelot N, Martin F (2001) Identification of symbiosis-regulated genes in
Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of
arrayed cDNAs. Plant J 25:181–191
Weiss M, Mikolajewski S, Peipp H, Schmitt U, Schmidt J, Wray V, Strack D (1997) Tissue-

specific and development-dependent accumulation of phenylpropanoids in Larch Mycorrhizas.
Weiss M, Schmidt J, Neumann D, Wray V, Christ R, Strack D (1999) Phenylpropanoids in
mycorrhizas of the Pinaceae. Planta 208:491–502
Wessels JGH (1996) Fungal hydrophobins: proteins that function at an interface. Trends Plant Sci
1:9–15
W€
osten HAB (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646
.
Chapter 9
RNA Silencing in Ectomycorrhizal Fungi
Minna J. Kemppainen and Alejandro G. Pardo
9.1 Introduction
RNA was for a long time believed to be a rather passive molecule. The only
function was to transfer coding information from genomic DNA to the protein
translation machinery. However, during the last 10 years this role of RNA has gone
through a radical transformation which has in turn revolutionized the way the
eukaryotic cells are understood to function today. Several types of small nonprotein
encoding RNAs are shown to be highly important as controllers of gene expression
and genome stability in plants, animals and fungi. These RNA molecules originate
predominantly from double-stranded RNA precursors (dsRNA) and they can affect
the gene transcript level both transcriptionally and post-transcriptionally. They also
modify and control the epigenetic state of nuclear DNA by participating in the
formation and maintenance of heterochromatin. These RNA-dependent eukaryotic
regulatory networks are now referred to as RNA interference (RNAi). RNAi is
believed to have evolved as a protection mechanism against invading nucleic acids
such as viruses that would have later become involved in general genetic control of
cells. The cellular mechanisms in which RNAi is currently known to participate are
multiple and the small-RNA type is organism specific. However, they all seem to
share a highly controlled processing of larger RNAs into small effector RNAs
which via sequence specific binding target homologous RNAs (or probably also
DNA). The outcome of RNAi pathways is usually a reduction of gene expression
and this phenomenon is therefore also called RNA silencing.
The finding that an artificial introduction of genetic components (DNA or RNA)
can trigger cell’s RNAi and lead to targeted modification of gene activity has made
the RNA-research one of the most blooming fields of investigation in biosciences.
Besides its use as a basic tool for studying gene function, RNAi has numerous
M.J. Kemppainen and A.G. Pardo (*)

Laboratorio de Micologı́a Molecular, Departamento de Ciencia y Tecnologı́a, Universidad
Nacional de Quilmes, y Consejo Nacional de Investigaciones Cientı́ficas y Tecnicas (CONICET),
Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina
e-mail: apardo@unq.edu.ar

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_9,
178 M.J. Kemppainen and A.G. Pardo
biotechnological and therapeutic applications. The discovery of the RNAi mecha-

nism in 1998 (Fire et al. 1998) was awarded with the Nobel Prize for Medicine or
Physiology in 2006. The cellular mechanisms connected to and controlled by RNAi
are under massive scientific research. Even though many aspects of RNAi are still
poorly understood the basic steps of various pathways have already been estab-
lished. With increasing information it is however becoming evident that previous
classifications of RNAi dependent mechanisms have been oversimplifications.
Different small RNA dependent pathways are now known to work in a simulta-
neous and interconnected manner and new RNAi regulatory networks are also
identified with increasing speed.
9.2 RNAi Mechanisms
Several gene inactivation phenomena described separately such as transgene-

induced cosuppression of endogenes (Jorgensen 1990) and virus-induced gene
silencing (VIGS) (Ruiz et al. 1998) in plants as well as quelling in Neurospora
crassa (equivalent to cosuppression in plants) (Romano and Macino 1992), are now
known to be mediated by small 20–30 nt cellular RNAs originating from dsRNA.
The RNAi mechanism was first described in the model nematode Caenorhabditidis
elegans (Fire et al. 1998). It is now known to be a conserved eukaryotic pathway
which affects genes and genomes by controlling chromatin structure, chromosome
segregation, protection against invading viruses, transposon inactivation, gene
transcription, RNA processing, RNA stability and translation in plants, fungi and
animals (Carthew and Sontheimer 2009; Moazed 2009).
RNAi pathways are shown to act in cells both in cytosolic and nuclear environ-
ments and even though highly variable in their actions and outcomes they all rely on
the sequence complementary of the small regulatory RNAs to their target tran-
scripts. Three main classes of cellular small regulatory RNAs have been described
this far: small interfering RNAs (siRNAs), micro-RNAs (miRNAs) and PIWI-
interacting RNAs (piRNAs).
The siRNAs act at several regulatory levels and via variable RNAi target
mechanisms in plants, animals and fungi while the miRNA-dependent translational
silencing pathway is detected both in animals and plants but it seems to be missing
in the fungal kingdom. The biogenesis of siRNAs and miRNAs requires dsRNA
precursors and strictly depends on activities of two RNAi proteins: ribonuclease
(RNase) Dicer (Bernstein et al. 2001; Carmell and Hannon 2004) and Argonaute
(Hammond et al. 2001; Hutvagner and Simard 2008). The piRNAs are the newest
and less understood class of regulatory small RNAs. They silence transposons in
animal germline cells and their biosynthesis is not dsRNA dependent and neither
does it need Dicer activity. They however interact with PIWI-clade Argonaute
proteins (Girard et al. 2006). This classification of small regulatory RNA is based
on the biogenesis and mechanisms of action and is to some extent artificial though
some biogenesis classes can perform different organism-specific target activities.
9 RNA Silencing in Ectomycorrhizal Fungi 179
Many of the proteins involved in RNAi are present as multigene families in

eukaryotes. The homologous proteins are demonstrated to have different RNAi
pathway specificities and/or several pathways can be also interconnected by their
protein or RNA components. The facts that many of the already known protein
components are still uncharacterized and many of the detected RNA-dependent
cellular mechanisms are not fully resolved, indicate that there are certainly other
important regulatory roles for RNAs to be identified in future.
Also while the basic RNAi mechanisms are shown to be conserved, each
eukaryotic group has had its own evolutionary diversification resulting in distinc-
tive characteristics of RNA silencing pathways. Because siRNAs are the only class
of small regulatory RNAs described in fungi till today their biogenesis and target
actions are discussed in more detail. The use of RNAi as a genetic tool for fungi also
depends on the artificial introduction of this type of small regulatory RNAs. The
biogenesis and action of another important group, miRNAs, which are responsible
for gene regulation of at least 30% of human genes (Lewis et al. 2005) are also
presented as their target functions may overlap with siRNAs.
9.3 siRNA-Dependent RNA Silencing
9.3.1 siRNAs
The majority of information on basic RNAi mechanism originates from studies on

siRNAs (Carthew and Sontheimer 2009). siRNAs are double stranded small regu-
latory RNAs of 21–25 nt which are processed from dsRNA precursors in plants,
animals, fungi and ciliates cells. Several siRNA-mediated RNA silencing pathways
have been detected and they act both in cytosol and nucleus. In cytosol siRNAs
mediate post-transcriptional gene silencing (PTGS) predominantly via target mRNA
degradation but they can also cause translational arrest. In the nucleus siRNAs have
been connected to chromatin-dependent gene silencing (CDGS) both by transcrip-
tional gene silencing (TGS) and cotranscriptional gene silencing (CTGS) mechan-
isms (Moazed 2009).
SiRNAs were first detected in plants where they were believed to be of exoge-
nous origin and degradation products of invading viral, transgene or transposon
RNA (Mello and Conte 2004). However, several classes of siRNAs homologous to
centromeric and other natural genome repetitive sequences were detected indicat-
ing that siRNAs were processed in cells also from endogenous RNA sources in
plants (Lippman and Martienssen 2004). This led to the classification of exo-
siRNAs and endo-siRNAs. Endo-siRNA production has an obligate nuclear step
while exo-siRNA production can take place directly in cytosol. Besides plants
various classes of endo-siRNAs have also been detected in animals and fungi
proving that nuclear DNA is a natural source of siRNAs in different eukaryote
kingdoms (Golden et al. 2008; Moazed 2009).
9.3.2 Sources of dsRNA in Cells
Long dsRNA precursors of siRNAs can be a result of direct invasion of dsRNAs

into cells such as viral RNA. Also exogenously introduced single-stranded RNAs
(ssRNA) can be turned into dsRNA in cytosol by RNA dependent RNA poly-
merases (RdRP), enzymes present in plants and fungi but absent in insects and
vertebrates. Also some noncoding or aberrant ssRNAs (such as mRNAs lacking
proper processing signals) originating from nuclear transcription can be detected as
a template for RdRPs directly in the nucleus or in cytosol. dsRNA molecules can
also be a direct result of nuclear transcription without RdRP activity. Antisense
transcription can result in dsRNAs by binding with sense transcripts. Also tran-
scripts of repetitive sequences, inverted repeats, and transposons can produce
dsRNAs (Moazed 2009).
Once dsRNA is present in the cell, either in the nucleus or in cytoplasm, it is
detected by the RNAi machinery. The cytosolic production of siRNAs and PTGS
cascade is best characterized in Drosophila, plant and human cells, while nuclear
siRNA activities involved in CDGS and heterochromatin control is profoundly
studied in the model fission yeast Shizosaccharomyces pombe. Today the artificial
triggering of cytosolic siRNA-dependent PTGS pathway is by far the most common
approach in altering gene expression by the RNA silencing technology in filamen-
tous fungi.
9.3.3 Cytosolic siRNA-Mediated PTGS
When dsRNA is detected in cytosol it initiates a multistep RNAi cascade that is

characterized by (1) digestion into double-stranded 21–25 nt primary-siRNAs by
the RNase III domain of the Dicer protein, (2) loading of one of the siRNA strands
(guide strand) into a multiprotein siRNA-induced silencing complex (siRISC)
including Argonaute protein, (3) sequence specific targeting of siRISC to a homol-
ogous ssRNA by guide-siRNA base paring and destruction of the target by the
slicing activity of RNase H-like fold of Argonaute, (4) dissociation of the siRISC
from the cut target RNA and targeting a new mRNA.
Dicer activity on dsRNAs is characterized by 2 nt 30 overhangs leaving a 50
monophosphate in siRNA-ends. Binding of Dicer to dsRNA is also assisted by
proteins with dsRNA-binding domains (Tomari and Zamora 2005). If an organism
has several Dicer-like proteins these are generally specialized to process a certain
type of dsRNA templates (siRNA precursors or miRNA precursors respectively).
The role of Dicer as a siRNA-cutter can also be extended to RISC assembly as
especially Dicer-associated dsRBPs are shown to be important in the early steps of
RISC-formation (so called pre-RISC).
Argonaute (AGO) is a core silencing protein and many organism have several
AGO proteins specialized for acting in different RNAi pathways. While S. pombe
has only one Argonaute, five, eight and twenty-seven paralogs exist in Drosophila,
humans and C. elegans (Carthew and Sontheimer 2009). All Argonautes do not
have endonucleolytic capacity and these might be important in RNAi pathways not
leading to direct target RNA destruction. The election of the guide-siRNA strand in
siRISC was for a long time a mystery but now it is known that the less thermody-
namically stable 50 -terminus is chosen as a guide. siRNAs from different sources
such as viral siRNAs and transgene siRNAs in plants and endo- and exo-siRNAs in
Drosophila have different requirements for RISC-assembly depending on different
dsRBD (dsRNA binding domain) proteins (Golden et al. 2008). The precise
mechanisms leading to such a precise discrimination of different origin siRNAs is
not yet known. The slicing activity of Argonaute requires a perfect match in base
paring between the siRNA-guide strand and the target RNA. The AGO slicing
generates 50 -monophosphate and 30 -hydroxyl termini in the RNA fragments pro-
duced. These are attacked by cellular exonucleases and degraded further (Orban
and Izaurralde 2005). RISC is demonstrated to localize in cytoplasm in special
structures, the processing bodies (P bodies), which are implicated in the regulation
of mRNA translation, storage, and degradation (Jagannath and Wood 2009).
Eukaryote genomes generally encode for several Dicer and Argonaute proteins.
Some isoforms have been linked to separate RNAi pathways such as virus defense,
chromatin modifications, miRNA processing or siRNA synthesis while some have
overlapping functions and are functional in several pathways. How different iso-
forms discriminate their dsRNA-targets and how they are engaged in specific RNAi
pathways is still poorly understood and under vigorous scientific investigation.
9.4 miRNA-Mediated Translational Arrest
If the base pairing with the target RNA is not perfect enough or the RISC contains
an Argonaute protein without slicing activity the direct degradation cannot take
place. However, these siRISCs can theoretically still act on target RNAs via a
second PTGS mechanism characteristic of another class of small regulatory RNAs,
miRNAs.
miRNAs were originally detected in viruses as sequences that affected expres-
sion of specific host mRNAs (Pfeffer et al. 2004). Later on endogenous miRNAs of
21–25 nt were described in plants and animals and they are now known to play a
fundamental role in gene expression regulation in these organisms. The majority of
miRNAs originate from nuclear transcriptional units of nonprotein encoding RNAs
and they are processed from 65–70 nt sequence that forms a ~33 bp mismatch stem-
loop precursor (pri-miRNAs) in nucleus by Dicer (plants) or the specific Dicer-like
protein Drosha (animals) to pre-miRNAs (Kim 2005). The pre-miRNAs with a
stem-loop structure are further processed in nucleus (plants) or in cytosol (animals)
to a mature ~22 bp miRNAs by Dicer and the mature guide-strand of miRNA
is incorporated into miRISC and targeted to a homologous RNA in cytosol. The

animal miRNAs generally target gene transcripts at their 30 UTR (untranslated
region) and this base paring is imperfect. Plant miRNAs however show nearly
perfect paring and they target coding sequences. In the case that the Argonaute
slicing activity is not compromised and the target binding is perfect or nearly
perfect, miRISC act in a similar manner as siRISC and degrades the target. This
often happens in plants where miRNAs can act in a siRNA manner. However, an
imperfect match, typical of animal miRNAs, inhibits slicing and leads to prolonged
binding of miRISC to the target and translational repression of the mRNA. The
mechanisms behind the translational repression are not yet clear but it might
involve various pathways that affect translation and direct mRNA to degradation
by deadenylation (Petersen et al. 2006; Wu et al. 2006).
9.5 RdRP Amplification of the Silencing Trigger
Organisms which have RdRP activity can amplify the primary siRNA trigger and
generate the so-called secondary siRNA cascade. This amplification of the original
effector molecule in plants, animals and fungi is a key for understanding how a
small amount of dsRNA can result in a strong and persistent RNA silencing effect
in cells. The amplification of the silencing signal is also behind the systemic
spreading of locally initiated RNAi in multicellular organisms. Furthermore, it
explains how the silencing signal can spread its effect and result in Argonaute-
mediated slicing of the whole target mRNA, and not only the RNA site sharing
sequence homology, with the primary siRNAs. The latter is known as transitive
silencing and it can have important implications for the use of RNA silencing as a
reverse genetic tool in organisms with RdRP activity.
The production of secondary siRNAs depends on RdRPs and this process is
shown to differ between plants and C. elegans (Petersen and Albrechtsen 2005;
Axtell et al. 2006; Ruby et al. 2006; Sijen et al. 2007; Pak and Fire 2007). In
C. elegans transitive silencing spreads toward the 50 but in plants to both 50 and 30 of
target mRNAs. In plants secondary siRNAs are also efficiently produced when the
target mRNA is already cut by Argonaute and these cleaved molecules are then
recognized by RdRP that generates more dsRNA molecules. These new dsRNAs
are further digested by Dicer into secondary siRNAs which can target homologous
RNAs. In C. elegans the primary siRNAs can directly lead to RdRP activity on
target mRNA and produce 22–23 nt dsRNA which are later cut by a still unchar-
acterized endonuclease activity into secondary siRNAs. The secondary siRNA
pools in plants and C. elegans differ also in their 50 -end due their different origins.
The plant secondary siRNA carry 50 -monophosphates while C. elegans siRNAs
have 50 -di or triphosphates. 50 -monophosphate cleavage is a typical Dicer cut
siRNA character. The secondary siRNAs in C. elegans differ also in a detected
strand-bias. They are predominantly antisense to the target RNA. They are also in
phase respect to each other. The first secondary siRNA carries a sequence starting
close to the primary siRNA and the following one cover the next 22 bases and so on.
This dsRNA synthesis in the nematode seems not to be primed by the primary
siRNAs and how these exact 22 nt-long secondary siRNA synthesis are controlled
and how they are cleaved is not yet understood. The characteristics and extent of
secondary siRNA cascade in different fungal taxa has been rather poorly studied.
However, this cascade must be widespread as many fungi posses RdRPs.
The RdRP-dependent amplification of the original silencing signal is behind the
systemic silencing phenomenon observed in multicellular organisms. Introduction
of a silencing trigger in one part of an organism with RdRP activity can lead to
spreading of silencing to the whole plant, nematode or fungal colony. The molecu-
lar identity of the cell-to-cell mobile signal in RNAi is however not yet clear but it
is believed to be siRNA (Kalantidis et al. 2008). As there is no secondary siRNA
amplification in mammals or in Drosophila like in plants, fungi and nematodes
(Cogoni and Macino 1999; Dalmay et al. 2000; Sijen et al. 2001) the direct
introduction of synthetic siRNAs to these organisms can cause only transient
silencing effect.
Besides the systemic silencing, another important aspect of transitive silencing is
that in these organisms the RNA silencing effect may not be strictly primary trigger
specific. Secondary cascade siRNAs may act on other mRNAs that share homolo-
gous sequences with the original target outside the primary silencing trigger
binding site. These are called off-target effects and cellular silencing pathways
responsible for them can vary according to the grade of homology between the
secondary siRNAs and the RNA targets. Perfect matches can produce Argonaute-
mediated slicing and the imperfect base pairing can lead to miRNA-type transla-
tional arrest and RNA decay. The majority of transitive silencing linked off-target
effects are believed to rise via the latter mechanism due to the predominantly
imperfect base paring nature of the secondary siRNAs. Interestingly, no plant or
animal type miRNAs have been detected in fungi and whether the imperfectly
paired secondary siRNAs in these eukaryotes can act via translational arrest and
mRNA decay is not clear.
However, these silencing off-target effects can also be a benefit. Transitive
silencing makes possible more efficient simultaneous silencing of homologous
members in multigene families. This can be highly useful in polyploid plants.
However, species specific differences for transitive silencing and efficiency
depending whether the target gene is an endo- or a transgene have been reported
(Miki et al. 2005; Bleys et al. 2006a, b). The molecular mechanisms leading to such
sequence discrimination are not yet understood but as plants and fungi generally
have a numerous set of RdRPs this suggest a specialized activity of different
isoforms on specific RNA substrates. Studies on some of the Arabidopsis six
RdRPs have in fact revealed the specificity of independent enzymes, some partici-
pating in sense transgene-triggered RNA silencing but not in RNA silencing
activated by inverted repeated transgenes or RNA viruses while others are respon-
sible for viral defense (Yu et al. 2003).
9.6 siRISC Can Operate on Target mRNAs in Nuclear

Environment Also
siRNA-dependent slicing of target mRNAs was believed to occur only in cytosol.

However, recent data strongly indicates that Argonaute-mediated slicing takes
place also in the nuclear environment. Nuclear RISC (nRISC) has been detected
in human cells and it is loaded with siRNAs in cytosol. This nRISC is later imported
to the nucleus where it targets homologous mRNAs either by slicing or directing
them to other decay pathways (Ohrt et al. 2008; Weinmann et al. 2009). The nRISC
appears to be a conserved part of the eukaryotic silencing machinery. A nuclear
RNAi–specific Argonaute protein has also been described in C. elegans. Its locali-
zation into the nucleus is siRNA-binding dependent and this binding takes place in
cytosol (Guang et al. 2008).
The existence of cytosol-loaded nRISC is now explaining some long known
silencing phenomena such as transcriptional inactivation of host genes via VIGS.
This nuclear effect was detected in plants when infected with cytosolic dsRNA
viruses (Wassenegger et al. 1994). Equal to siRNAs, other types of small regulatory
RNAs are also now proposed to function both in cytosolic and nuclear environ-
ments. Besides the direct post-transcriptional silencing effect on target mRNAs in
cytosol and also in nucleus these small RNAs can also be involved in epigenetic
modification of homologous DNA sequences and control gene expression at the
transcriptional level.
9.7 Epigenetic Effects Associated with RNAi
RNAi is connected to epigenetic modifications of the host genome. These include

direct DNA methylation and/or histone modifications depending on the organism. It
is increasingly being recognized that all types of small regulatory RNAs have some
epigenetic nuclear effects and that the basic mechanisms responsible for them seem
to be conserved within the eukaryotic domain (Djupedal and Ekwall 2009; Verdel
et al. 2009). These chromatin modifications are complex, long lasting, even herita-
ble, and they can affect gene expression as well as be implicated in different
diseases. Epigenetics along with RNAi are the current hot spots of biological
research.
Majority of RNAi-mediated DNA modifications can be seen as auto-defense
responses which secure genome integrity by inactivating transposable elements and
imbedding repetitive sequences into heterochromatin. Also some genes can be
under RNAi-dependent epigenetic control. siRNA-mediated heterochromatin con-
trol and the histone methylation of the pericentromeric repeats has been exhaus-
tively studied in the fission yeast S. pombe. Heterochromatin, a traditionally
transcriptionally inactive part of the genome is now, paradoxically, shown to
depend on active transcription for its formation and maintenance (B€uhler 2009).
A nascent-transcript model has been proposed where siRNAs, originating from

transcription of genome repetitive elements and produced via RdRP and Dicer
activities in nucleus, are incorporated into RITS (RNA-induced transcriptional
silencing complex) which binds to nascent homologous RNA transcripts at their
site of synthesis. This RITS-binding serves as a mark for histone modifying proteins
(S. pombe does not have direct DNA methylation response) which initiate hetero-
chromatin formation and maintain it in repetitive genomic loci (Moazed 2009).
Equivalent mechanisms based on active transcription and nuclear siRNA–RNA
binding are proposed to control epigenetic modifications present in natural or
introduced repetitive sequences in plants, fungi and animals. These effects are
called CTGS.
Heterochromatin formation can reduce transcription of genes embedded in the
region. Such a heterochromatization leading to TGS can be a result of RNAi
mechanisms and to be mediated via siRNAs. If dsRNA precursor in the cell carries
a sequence homologous to a gene promoter, siRNAs processed from such a
precursor can be engaged in a RNAi pathway targeting homologous DNA in
nucleus. This results in the inhibition of the target gene transcription and this
reduced transcription correlates with an altered epigenetic status (increased DNA
and histone methylation) of the targeted promoter sequences. TGS has been
demonstrated in plants, fungi, insects and also in mammalian cells (Weinberg
2006; Verdel et al. 2009). The function of siRNAs in TGS is not yet totally resolved
but they seem to target homologous promoter sequences by RNA–RNA, and not via
direct RNA–DNA binding, probably according to the nascent transcript model. The
production of primary siRNAs involved in TGS can, however, also be cytosolic and
not only nuclear as postulated for S. pombe heterochromatin formation. A cytosolic
nRISC formation and nuclear import may therefore take place before homologous
DNA sequence targeting in nucleus. Besides siRNAs, both plant and animal
miRNAs are also now known to have the capacity to target, not only mRNAs in
cytosol, but to act on homologous DNA sequences in nucleus (Gonzalez et al. 2008;
Kim et al. 2008). This targeting, probably via RNA–RNA interactions, causes
epigenetic modification and can result in TGS. Epigenetic effects linked to RNAi
are highly complex and a field of massive scientific research.
A scheme showing different RNA silencing pathways demonstrated to be active
in eukaryotic cells is presented in Fig. 9.1.
9.8 Use of RNAi as a Genetic Tool
RNAi technology has offered an exceptional tool for functional genomic studies.
RNAi can be launched in different organisms by artificial introduction of RNAi
inducing RNA (or DNA) triggers. Because RNAi results in reduced mRNA levels
but does not completely abolish gene function it has also made possible to study
genes whose knock-outs would be lethal. Due to the cytosolic nature of PTGS it
offers a direct and fast approach to alter gene expression in dikaryotic, diploid and
Fig. 9.1 Schematic representation of different conserved siRNA- and miRNA-dependent cytosolic and nuclear RNA silencing pathways demonstrated to be
active in eukaryotic cells. The figure shows the key processing steps leading to RNAi-dependent post-transcriptional (PTGS), transcriptional (TGS) gene
silencing and siRNA-dependent detection and epigenetic modification of repetitive genomic sequences. Not all the protein components demonstrated to
participate in each step of the different pathways have been illustrated. The figure presents the cytosolic PTGS pathway. However similar mechanisms are now
known to act also in nuclear environment. I-V: siRNA- and miRNA-mediated cytosolic PTGS. I Different dsRNAs of endo- or exogenous origin or aberrant
RNAs turned into dsRNAs via RdRP-activity (in plants, fungi and C. elegans) can be recognized by ribonuclease protein Dicer. Dicer’s RNase activity leads to
cutting of dsRNA into 21–24 bp dsRNAs, the so called primary (1 ) cascade siRNAs. II One strand of the 1 cascade siRNAs is loaded into a multiprotein
complex (siRISC) which contains a core RNAi protein, Argonaute (AGO). SiRISC can bind to homologous mRNA sequences by siRNA base paring. III The
degree of sequence homology of siRISC binding to mRNA can result in two silencing outcomes: IIIa A perfect mach permits slicing activity of Argonaute and
the mRNA is cut. The siRISC dissociates from the cut mRNA and returns to act on new homologous mRNAs. This siRISC-mediated slicing leads to reduced
cytosolic concentration of target mRNA and gene silencing. Also plant miRNAs produced by nuclear transcription and Dicer activity (IVa) share this siRISC-
pathway as they typically act via perfect match of miRISC producing target mRNA slicing. IIIb In the case of imperfect match with the target (or when
associated with AGO without slicing activity) Argonaute slicing of mRNA cannot occur. The PTGS however takes place as this type of siRISC binding results
in translational arrest and exonucleolytic degradation of the target mRNA. Animal miRNAs which are produced via nuclear transcription and further
processed by the nuclear Dicer-like protein Drosha and cytosolic Dicer (IVb) regulate their target mRNAs in cytosol via the imperfect match strategy. The
9 RNA Silencing in Ectomycorrhizal Fungi
majority of off-target effects caused by artificially initiated silencing in different eukaryotes are proposed to be a result of this RNAi pathway as siRNA
binding to homologous non-target mRNAs can be expected to be predominantly imperfect. V In organisms with RdRP activity (plants, fungi, C. elegans) the
initial silencing trigger can be further amplified resulting in the so-called transitive silencing. This is characterized by the production of a secondary (2 )
siRNA cascade, not homologous to the initial Dicer-cut dsRNA-silencing trigger. The siRISC-cut mRNAs (or intact mRNAs) can act as templates for RdRP-
catalyzed dsRNA synthesis which are processed into siRNAs and further incorporated into RISC. The synthesis of these 2 siRNAs is demonstrated to show
some fundamental differences between plants and C. elegans. While in plants siRNAs are a product of Dicer cut of dsRNAs in C. elegans they are produced by
RdRP using the mRNA as template toward the 50 -end but no Dicer-slicing takes place. Transitive silencing potentially increases the off-target effects as the
silencing is not strictly primary dsRNA trigger-dependent. However, this RdRP-activity and silencing amplification also explains the long lasting and systemic
silencing signal in the case of local or transitive dsRNA triggering in these organism. The degree of transitive silencing is organism-dependent but it also
shows target gene specific behavior. In plants endogenes seem to act as weaker templates for transitive silencing than heterologous transgenes. VI: The recent
demonstrations of cytosolic-loading of nuclear targeting siRISC (nRISC) confirms that cytosol-produced or introduced siRNAs can also potentially act in
nuclear environment either directly on homologous target mRNAs by Argonaute-mediated slicing and transcript decay or by mediating epigenetic effects on
DNA sequences. NRISC is a link with potential to connect the cytosolic and nuclear RNAi pathways. Both primary and secondary siRNAs might participate in
nRISC. The presence of nRISC in fungi has not been demonstrated yet. 1 and 2: siRNA-mediated nuclear RNAi can result in TGS and heterochromatization of
repetitive genomic sequences by DNA methylation and/or histone modifications. 1. siRNAs with homologous sequences to gene promoters can initiate nuclear
Dicer/AGO-dependent RNAi pathway leading to direct DNA methylation and/or histone modifications in these genomic sequences. As a result the
transcriptional activity from the targeted promoters is reduced causing transcriptional gene silencing (TGS). The siRNAs linked to TGS can be a result of
nuclear transcription, artificially-introduced to nucleus, or of cytosolic origin and entering to nucleus via mechanisms such as nRISC. How these siRNAs can
187
polyploid organisms. RNAi has been successfully used for studying specific gene
functions in a wide range of eukaryotes (Travella and Keller 2009). Today RNAi
research is mainly dedicated to the use of artificial miRNAs instead of siRNAs due
to their higher target specificity (Ossowski et al. 2008; Carthew and Sontheimer
2009). However, miRNA applications are functional in organisms which posses this
specific silencing pathway, these not including fungi.
The number of full genomic sequences is growing almost on a daily basis and
RNAi technology has made possible, with a relative low investment, to look for the
function of practically any gene of interest. Studies at full genome scale have
already been conducted in C. elegans. Of nematode’s approximately 19,000
genes 86% have been screened for their function with the RNAi feeding library
technology (Kamath et al. 2003). Of the genes analyzed 1,722 produced identified
phenotypes proving RNAi a highly efficient tool for reverse genetics in this
organism. RNAi has also been used for several genome wide studies in Drosophila
(Boutros et al. 2004; Chen et al. 2008; Cronin et al. 2009; Mummery-Widmer et al.
2009). Similar kinds of studies with the RNAi technology are now initiated in plants
(Ossowski et al. 2008). Moreover, RNAi is massively used for screening gene
functions is mammalian cells. These studies are headed for resolving the genetic
background of different diseases and developing RNA therapy drugs (Gobeil et al.
2008; Zhou et al. 2008; Krishnan et al. 2008; Castanotto and Rossi 2009).
Fig. 9.1 (continued) initiate de novo methylation in homologous genomic DNA sites is not yet
well understood but it seems to depend on nascent RNA-siRNA binding similarly to RNAi-
dependent maintenance of heterochromatin in pericentromeric repetitive sequences of S. pombe
(see below) 2. Nuclear siRNAs are also shown to participate in heterochromatization in different
eukaryotes. siRNAs can target both natural and transgenic genomic repetitions resulting in target
DNA methylation and/or histone modifications and this process involves Dicer, AGO and RdRP
proteins. The RNAi-dependent maintenance of cellular heterochromatin has been most thoroughly
studied in S. pombe where a nascent transcript model has been proposed: RNA transcripts
constantly produced from these repetitive genomic loci are responsible for the physical co-
localization of siRNAs-protein complexes (RITS) to these sites and also for the further production
of the homologous siRNAs. The RNA-RNA binding of RITS with the nascent transcripts serves as
a flag for nuclear histone modifying enzymes to maintain the heterochromatic state of these sites.
Also these same nascent transcripts are detected by RdRP to produce dsRNAs which are processed
by Dicer into siRNAs and loaded into RITS. Even though characterized in S. pombe the involve-
ment of RNAi and nascent RNA transcripts in heterochromatization of genomic sequence repeti-
tions seem to be a conserved cellular mechanism among different eukaryotes. To which extent
these nuclear siRNAs can initiate de novo chromatin modification however is not yet clear. Also
the participation of cytosol processed siRNAs in this nuclear RNAi–pathway and in initiation of
epigenetic modifications cannot be excluded. hpRNA hairpin RNA; dsRNA double-stranded RNA;
siRNA small interfering RNA; miRNA micro-RNA; RdRP RNA dependent RNA polymerase; AGO
Argonaute protein; siRISC siRNA-induced silencing complex; miRISC miRNA-induced silencing
complex; nRISC nuclear siRISC; RITS RNA-induced transcriptional silencing complex; DMT
DNA methylation; HMT histone methylation; RNAPol RNA polymerase; Prom gene promoter
region
9.9 RNA Silencing in Fungi
In fungi transgene induced endogene cosuppression was first reported in the

filamentous ascomycete N. crassa where the phenomenon was called quelling
(Romano and Macino 1992). Now it is known that quelling is a RNAi gene
inactivation process dependent on multiple transgene arrays. Further studies on
quelling have resulted in the discovery of many of the underlying mechanisms of
RNAi in eukaryotes and turned this fungus as one of the model organisms in RNAi
research (Fulci and Macino 2007). Also in the fission yeast S. pombe studies about
RNAi-mediated heterochromatin formation have resolved how nonprotein coding
natural small RNAs control genome integrity in eukaryotes (Moazed 2009; B€uhler
2009). Later on, the RNA silencing pathway has been shown to exist in many other
yeast and filamentous fungal species. Small nonprotein coding RNAs have been
shown to target homologous mRNAs by Dicer- and Argonaute- dependent slicing
in fungi, similar to other eukaryotes. RNAi has also been linked to cell defense
against viruses, stabilization of transposons, control of heterochromatin formation
and some programmed DNA elimination processes. Natural nonprotein coding
small RNAs originating from transposons and repetitive genome sequences have
recently been isolated in M. oryzae, N. crassa, and A. fumigatus demonstrating that
RNAi pathways are involved in the control of genome integrity in filamentous fungi
as well as in S. pombe (Murata et al. 2007; J€ ochl et al. 2008; Cecere and Cogoni
2009). However, no regulatory miRNAs have been reported yet and the absence of
this RNAi pathway could be characteristic of the fungal kingdom (Nakayashiki and
Nguyen 2008).
9.10 RNA Silencing Protein Machinery in Fungi
With growing genomic sequence information from diverse fungi, comparative

studies on evolution and diversification of RNA silencing proteins in this group
of eukaryotes have become possible. A phylogenetic analysis of fungal species
covering, ascomycetes, basidiomycetes and zygomycetes was carried out by
Nakayashiki and Kadotani (2006). These authors used sequences coding for
Dicer, RdRP and Argonaute proteins in order to establish the evolutionary history
of diversification of RNA silencing pathways between and within different fungal
taxa and also between fungal, plant (Arabidopsis) and animal (Drosophila) king-
doms. The existence of RNAi protein machinery has been demonstrated in asco-
mycetes, basidiomycetes and zygomycetes. However, different fungal taxa show an
unexpected wide diversification in their RNAi protein repertoire. Some parts of the
fungal RNAi machinery have gone through phases of expansion while a complete
or partial loss of the pathway has happened independently in different nonphylo-
genetically related species. It seems that the whole Saccharomyces complex,
including S. cerevisiae and Candida spp. has lost all Dicer and RdRP proteins
and the same has happened in the basidiomycete Ustilago maydis. However, in the
closely related species U. hordei the dsRNA-triggered RNAi pathway is functional
(Laurie et al. 2008). Similarly different orthologous proteins such as RdRP-1, 2 and
3, present in different fungal phyla, cluster together indicating that these duplication
events are ancestral and have occurred before fungal diversification. Schizosac-
charomyces pombe, which has just one of each RNAi proteins, would thus have lost
paralogous proteins during evolution.
A special RNAi gene expansion is detected among basidiomycetes where at least
three distinct classes of Dicer and RdRP proteins and two of Argonaute-like
proteins have been detected. This gene expansion has especially affected Argonaute
and RdRP proteins in homobasidiomycetes such as Phanerochaete chrysosporium
and Coprinus cinereus. The former has seven AGOs and nine RdRPs and the latter
eight AGOs and seven RdRPs. Moreover, both of these basidiomycetes have three
Dicer genes while filamentous ascomycetes usually have two. Cryptococcus neo-
formans, a basidiomycete yeast, seem to have evolved distinctly and its RNA
silencing proteins show characteristics not observed in other fungal proteins. For
example the Cryptococcus Dicer proteins lack the typical DEAD/DEAH box heli-
case, a feature that has also been reported in functional Tetrahymena Dicer-like
protein (Dcl1) involved in RNA silencing related pathway of internal elimination
sequences (IES) (Mochizuki and Gorovsky 2005). Also a comparison of RNAi
protein machinery of seven Aspergillus species has revealed that significant varia-
tion exists between them indicating both gene duplication and truncation events
(Hammond et al. 2008b).
These phylogenetic studies highlight some fundamental aspects of fungal RNAi.
Firstly, different fungal taxa have highly different RNAi protein repertoire and
secondly, some fungal species have lost all or are impaired in some RNAi path-
ways. Also closely related species can significantly differ in their RNAi capacity.
The evolutionary forces driving eukaryotic RNA silencing gene change are unclear
and in fungi in general, RNA silencing gene evolution appears to be more complex
than in any other type of eukaryotes. Due to this high variation, RNA silencing and
its functionality must be demonstrated in each fungal species of interest.
9.11 Viral Origin of RNAi in Fungi
RNAi mechanisms are activated in eukaryotes during viral infections. The evolu-
tionary origin of RNAi is believed to be linked to the protection of cells against
invading nucleic acids. Also both plant DNA and RNA viruses encode for suppres-
sor proteins which inactivate plant RNAi mechanisms supporting this hypothesis
further (Moissiard and Voinnet 2006; Dı́az-Pendón and Ding 2008). Similar
mechanisms are active also during viral infection in animal cells (Gitlin and Andino
2003; Li et al. 2004; Berry et al. 2009).
The first indications of the virus defense origin of RNAi pathways in fungi come
from a recent work on the chestnut blight fungus Cryphonectria parasitica. Viru-
lence attenuating hypoviruses of the species, Cryphonectria hypovirus 1 (CHV1),
encode a papain-like protease, p29, that shares similarities with the potyvirus-
encoded suppressor of RNA silencing HC-Pro. Expression of this protein in the
fungus was shown to suppress hpRNA (hairpin RNA) induced RNA silencing of
the reporter GFP gene (Segers et al. 2006). The direct link between RNA silencing
and virus defense was further demonstrated by disruption of two Dicer-like genes
of Cryphonectria, dcl1 and dcl2. While dcl1 disruption did not cause an obvious
phenotype, dcl2 or dcl1/dcl2 double mutants were highly susceptible to hypo-
virus CHV1-EP13 infection (Segers et al. 2007) The virus-derived small RNAs
(vsRNAs) accumulation, their dependency on DCL-2 activity and virus induced up-
regulation of Dicer expression was also recently reported in Cryphonectria (Zhang
et al. 2008). The activation of fungal RNAi during viral infection or when triggered
with long dsRNAs has been demonstrated in N. crassa and A. nidulans as well
(Choudhary et al. 2007; Hammond et al. 2008a).
9.12 RNA Silencing in Different Fungi
Description of the RNAi pathways and their efficient artificial triggering in the
filamentous ascomycetes N. crassa, M. oryzae (syn. M. grisea) and the fission yeast
S. pombe (Cogoni et al. 1996; Raponi and Arndt 2003; Kadotani et al. 2003) has led
to the increasing use of RNAi as a reverse genetic tool in fungi. Especially in
filamentous fungi, where gene knock-out experiments are often very challenging,
RNAi has offered a novel and fast way for modifying gene expression. In last years
siRNA-dependent silencing has been demonstrated and used for genetic studies in
several ascomycete species such as M. oryzae, Colletotrichum lagenarium, Ven-
turia inaequalis, Cladosporum fulvum, S. pombe, Aspergillus fumigatus, A. nidu-
lans, A. flavus, A parasiticus, Fusarium graminearum, Histoplasma capsulatum,
Sclerotinia sclerotiorum, Ophiostoma floccosum, O. piceae, Coniothyrium mini-
tans, Penicillium expansum, Acremonium chrysogenum (syn. Cephalosporium
acremonium), Trichoderma harzianum and in N. crassa (Hamada and Spanu
1998; Kadotani et al. 2003; Raponi and Arndt 2003; Schramke and Allshire
2003; Fitzgerald et al. 2004; Goldoni et al. 2004; Mouyna et al. 2004; Sigova
et al. 2004; McDonald et al. 2005; Nakayashiki et al. 2005; Rappleye et al. 2004;
Cardoza et al. 2006; Tanguay et al. 2006; Erental et al. 2007; Gong et al. 2007;
Janus et al. 2007; Sch€umann and Hertweck 2007). RNA silencing has been also
reported in the zygomycete Mucor circinelloides (Nicolás et al. 2003) and the
basidiomycetes Schizophyllum commune, Cryptococcus neoformans, Coprinus
cinereus, P. chrysosporium and Moniliophthora perniciosa (Schuurs et al. 1997;
Gorlach et al. 2002; Liu et al. 2002; Namekawa et al. 2005; de Jong et al. 2006;
W€alti et al. 2006; Matityahu et al. 2008; Caribé Dos Santos et al. 2009).
9.13 How RNAi Is Experimentally Initiated in Fungi
In fungi RNAi is generally launched by similar techniques used by plant research-

ers. These are characterized by a stable cellular production of RNAi trigger which
results in a stable and not transient RNAi phenotype. This is achieved by transform-
ing the fungus with a DNA construct which via host cell’s transcriptional machin-
ery produces dsRNAs and later siRNAs homologous to the target mRNA
predominantly via PTGS. All fungal studies this far have used gene coding
sequences for targeting homologous or heterologous mRNAs. Whether the trig-
gered reduction of the target mRNA has been purely due to PTGS or TGS
mechanisms have also contributed to the silencing phenotypes, has however not
been verified in the majority of these studies.
The RNAi triggering genetic DNA elements introduced in fungi can be integrative
or auto-replicative and the dsRNA in cells can be produced from different RNA
precursors. These can directly have a dsRNA structure or form dsRNA by binding
to cellular sense-transcripts. Fungal as well as plant cells tolerate long dsRNA
molecules unlike vertebrate and mammalian cells where longer than 30 nt dsRNA
launches strong interferon response (Stark et al. 1998). Therefore, long dsRNA hair-
pins (hpRNAs), antisense RNAs and expression of sense and antisense RNAs from
convergent promoters can be used for triggering RNA silencing in fungi. However, the
most potent and widely used RNAi triggers in fungi are promoter expressed hpRNAs
with homology arms of around 200–500 bp.
An efficient RNA silencing/transformation vector system adapted to fungi
however did not exist until Nakayashiki et al. (2005) published pSilent-1. This
cloning vector was developed for an easy PCR based cloning of intronic spacer
hairpin RNAs (ihpRNAs) to be expressed in ascomycetes. pSilent-1 was also
designed to function for direct plasmid transformation and carries a hygromycin
resistance cassette for antibiotic selection of the transformants. This cloning/trans-
formation vector has made RNA silencing studies in ascomycetes easy to perform
and has been successfully used, with or without modifications, in M. oryzae,
Colletotrichum lagenarium, Bipolaris oryzae and Acremonium chrysogenum
(Nakayashiki et al. 2005; Janus et al. 2007; Moriwaki et al. 2007). Now the second
generation of silencing vectors for direct hpRNA or convergent promoter RNA
expression is being designed. Further modifications of the pSilent-1 vector, pSilent-
Dual1 (Nguyen et al. 2008), pTroya (Shafran et al. 2008), and pFANTAi4
(Krajaejun et al. 2007) are adapted for high-throughput functional genomic studies
in ascomycetes, the latter two also incorporating the GATEWAY technology
(Invitrogen) in fungal silencing vectors. Also other hpRNA cloning vectors such
as pFIRD1, designed for A. niger and utilizing recombination in vitro (Oliveira
et al. 2008), and an inducible promoter system for RNAi in A. nidulans (Barton and
Prade 2008), have been released.
RNAi is already established as quite a standard genetic tool for working with
ascomycetes (Nakayashiki and Nguyen 2008). While the number RNA silencing
publications in ascomycetes is rapidly increasing, studies conducted in
basidiomycetes are still few. This low number of RNA silencing publications partly
reflects the complications with DNA-transformation techniques but most of all the
lack of molecular genetic RNAi tools adapted for this group of fungi.
In C. elegans an exogenous exposure of the organism to small dsRNA molecules
launches RNAi. A similar approach of a direct RNA triggers has not traditionally
been used for fungal RNAi. Nevertheless, in a recent work on A. nidulans the
cocultivation of the fungus with siRNAs targeted to the fungal key polyamine
biosynthesis gene ornithine decarboxylase (ODC) was shown to launch RNAi. As
a result target mRNA and cellular polyamine concentrations levels in the fungus
were reduced (Khatri and Rajam 2007). This has been the first demonstration of
uptake of siRNA by germlines from the culture medium and RNAi triggering in
fungi. Also direct incubation of A. niger protoplast with siRNAs has been shown to
launch transient RNAi (Barnes et al. 2008). These discoveries open the possibility
of using siRNAs for new applications such as antifungal drug development. Also a
direct electroporation of long dsRNAs have been recently shown to trigger long
lasting RNAi response in the basidiomycete Moniliophthora perniciosa (Caribé
Dos Santos et al. 2009). These reports strongly propose that, equal to animal
research, dsRNAs as directly introduced silencing triggers could be used in fungal
research as well.
Different DNA and RNA triggers used for initiating PTGS in fungi and the
proposed cellular RNAi pathways involved are summarized in Fig. 9.2. Also, the
possible epigenetic effects linked to silencing triggering are illustrated.
9.14 Simultaneous Silencing of Genes with a Single Trigger

in Fungi
A singular silencing trigger can lead to simultaneous silencing of several targets.

Genes belonging to multigene families sharing homologous sequences can theoreti-
cally be targeted with a single trigger. Also chimeric constructs carrying target
endogene and reporter gene sequences can lead to simultaneous silencing of both.
Such approach can be used for facilitating the screening of strongly silenced strains
as silencing of the marker and the target endogene generally coincides. An efficient
simultaneous silencing of two genes has been reported by a chimeric hairpin con-
struct in Venturia inaequalis. Transformation with a hairpin carrying both reporter
egfp and endogenous gene sequences resulted in reduced expression of both genes
(Fitzgerald et al. 2004). A similar approach has been used for cosilencing a dsRED-
maker gene in Acremonium chrysogenum (Janus et al. 2007), ade2 in Cryptococcus
neoformans (Liu et al. 2002) and egfp in M. oryzae (Nguyen et al. 2008).
RNA silencing of two fungal endogenes by a single hairpin has been demon-
strated in Coprinus cinereus. Simultaneous silencing of genes encoding isogalec-
tins (cgl1 and cgl2) sharing only 87% identity was produced by dsRNA carrying a
cgl2 sequence (W€alti et al. 2006). These results indicate that RNAi serves also for
Direct dsRNA introduction demonstrated Different dsRNA-trigger expression strategies used for fungi
194
to trigger RNAi in fungi (in integrative or autoreplicative DNA-elements)

5´ 3´ long hpRNA expression Pro
long dsRNAs
(electroporation) 3´ 5´
short hpRNA expression Pro
5´ 3´
short dsRNAs convergent promoter system for Pro
3´ 5´ Pro
(direct feeding) expression of long dsRNAs
anti-sense RNA expression Pro
5´ 3´ 5´ 3´
3´ 5´ 3´ 5´
5´ 3´
5´ 3´ AAAAAA
3´ 5´
3´ 5´ 5´
3´
dsRBP
PolII
Dicer
M M
AA Dicer
AA secondary siRNAs primary siRNAs 21-24nt
AA M
M
AGO TS
priming?
A RI
A IIl
? RdRP pre-RISC
AA Po
AA
AA M
AA
dsRNA RISC
priming?
ISC
AA
AGO
holo-
nR
AA
AA
AA RNAi signal amplification RISC ? AA
AA
S C
AA AAAAAA AGO nRI
AAAAAAA
AAAAA
AA mRNA slicing
AAA nRISC?
AAA
nRISC? A
M.J. Kemppainen and A.G. Pardo
silencing gene families in fungi. The minimum sequence homology needed for
efficient cosilencing is not yet determined for fungi and may vary between species.
While simultaneous silencing can be a benefit, it is also the biggest drawback of the
RNA silencing technology. Unwanted off-target effects originating from short
sequence homologies are possible especially when long dsRNAs are used as
primary RNAi triggers.
These off-target effects were recently studied by Nguyen et al. in M. oryzae.
Genes sharing sequence similarity with an E-value of only about e3 or e4 were
sometimes silenced at similar levels and only 13 nt perfect homology between genes
was enough for simultaneous silencing of an unwanted target (Nguyen et al. 2008)
Also in HeLa cells only a 7 nt sequence complementary between the unwanted target
and a siRNA or a short-hairpin RNA trigger has been reported to launch off-site
effects (Jackson et al. 2006). Special attention should thus be paid in future for
designing RNAi triggers. It seems that RNA silencing is not as sequence specific as it
was previously believed and probable off-target effects cannot be completely
avoided. These can however be reduced by using more discriminatory shorter
RNA triggers. In Coprinopsis cinerea (syn. Coprinus cinereus) as short as 19 bp
dsRNA trigger can initiate a specific RNA silencing response (Costa et al. 2008).
As fungi possess RdRPs the silencing off-target effects are not necessarily linked
only to the primary silencing trigger. The degree of transitive silencing and possible
off-target effects of secondary cascade siRNAs has however not been profoundly
Fig. 9.2 The most common triggers for artificially initiated RNAi in fungi are variable integrative
or auto-replicative promoter-directed DNA elements. Their introduction to cells results in direct
dsRNA production by transcription (hpRNAs), dsRNA formation by base paring of produced
sense and antisense transcripts (convergent promoter expression), or their form dsRNAs by paring
with target mRNAs (antisense expression). Recently also direct dsRNA introduction by feeding or
electroporation has been demonstrated to launch RNAi in fungi. dsRNAs originating from these
different introduced triggers are recognized in cytosol by the fungal RNAi pathway leading to
PTGS. This involves dsRNA Dicer cutting, formation of 21–24 nt primary siRNAs and RISC
assembly. Dicer binding to dsRNA is assisted by dsRNA binding proteins (dsRBP) and RISC
assembly initiates with pre-RISC formation, leads to discard of one of the siRNA strands and
results in formation of mature holo-RISC. RISC targets mRNAs homologous to siRNAs. A perfect
base pairing leads to slicing of target by Argonaute (AGO) and reduction of mRNA concentration
(gene knock-down). However, imperfect mRNA–RISC pairing which leads to translational arrest
or mRNA decay in plants and animals might also occur in fungi even though it has not yet been
demonstrated. Cut mRNAs (or intact mRNAs) are potential targets for signal amplification by
RdRP. These new dsRNAs can be detected by Dicer and processed into secondary siRNAs. The
RNAi signal amplification step is not well characterized in filamentous fungi. Short dsRNAs or
siRNAs may act as primers at this step. Neither is the extent of this transitive silencing effect clear
in the fungal kingdom in general. RNAi is linked to epigenetic marks in different eukaryotes and
also in fungi. These nuclear effects can potentially affect the silencing triggering locus and/or the
target endogene by DNA methylations and histone modifications via mechanisms such as RNA-
induced transcriptional silencing complex RITS. If these epigenetic modifications can also be
internuclear, reaching untransformed nuclei in multinucleate fungal cells is not clear. A cytosol-
loaded nuclear siRISC (nRISC), detected in animals and apparently also present in plants, could
mediate such sequence specific effects between different nuclei through fungal cytoplasm
studied yet in this group of eukaryotes. Transitive silencing has been demonstrated
in the zygomycete Mucor circinelloides (Nicolás et al. 2003) but its existence in
filamentous ascomycetes is not clear. At least no silencing spreading has been
detected in A. nidulans toward the 30 end on target mRNA transcripts (Barton and
Prade 2008). The fungal RNAi protein machineries are however highly variable
between different fungal taxa and the biological role of especially the RdRP-gene
family expansion in filamentous homobasidiomycetes (Nakayashiki and Kadotani
2006) is intriguing. The possible role of these proteins in spreading the silencing
effect should be investigated in future. Even though miRNAs have not been
detected in fungi the possibility that secondary siRNAs could act via miRNA-
type translational arrest on homologous mRNAs is not an excluded possibility.
9.15 Use of RNAi as a Reverse Genetic Tool in Fungi
RNA silencing does not completely abolish the target gene expression but its
reduction has been shown to result in phenotypes equivalent or close to null-
mutants. The percentage of functional phenotypes is generally much higher than
with gene knock-out making gene function studies remarkably faster to perform.
The power of this approach on modifying endogene expression has already been
demonstrated in several studies where strongly silenced fungal strains have clarified
the role of target genes in processes such as biosynthetic pathways, meiosis, asexual
sporulation or fungal virulence control (Rappleye et al. 2004; McDonald et al.
2005; Namekawa et al. 2005; Cardoza et al. 2006; Bohse and Woods 2007; Nguyen
et al. 2008; Nicolás et al. 2008; Cooper and Woods 2009; Panepinto et al. 2009).
RNA silencing can be also used for confirming gene knock-out phenotypes
obtained by other means (Gong et al. 2007). The true RNAi era on fungal research
has started with the release of the pSilent-1 cloning vector (Nakayashiki et al. 2005)
and therefore numerous reports on the RNAi technology in resolving biological
fungal functions are expected to be released in the following years.
Although it is already successfully used in reverse genetics of several species,
the basic cellular mechanisms behind RNAi in filamentous fungi are still poorly
understood and studied. The detected high species level variability in RNAi protein
machinery and the differences especially between ascomycetes and basidiomycetes
suggest that the true flexibility and limitations of this gene knock-down technique
must be evaluated on a species basis.
9.16 Gene Silencing in Ectomycorrhizal Fungi
Laccaria bicolor is a homobasidiomycete and ectomycorrhizal (ECM) fungus

which forms symbiosis with several boreal and temperate forest trees such as
birch, pine and poplar. Laccaria, the first symbiotic fungus with its genome
sequenced (Martin et al. 2008), is also susceptible to genetic modification via

Agrobacterium-mediated transformation (AMT) (Kemppainen et al. 2005, 2008;
this book, Chap. 6). The availability of the full genomic sequence and the possibil-
ity to genetically modify this fungus has rapidly turned Laccaria into a true ECM
model species. However, gene knock-outs have turned out to be difficult to obtain
in this fungus. As the symbiotic phase of Laccaria is dikaryotic and interrupting one
of the gene copies does not necessarily produce strong haploinsufficiency pheno-
types (Kemppainen et al. 2008) RNA silencing represented an alternative and
highly attractive way for altering expression of Laccaria genes involved in ECM.
Gene knock-down had not been used before for studying plant–fungus symbiotic
interactions. Neither had the functionality of RNA silencing pathway been demon-
strated in ECM fungi.
A preliminary study of Laccaria full genome sequence revealed that it encodes
for a minimum set of genes with predicted protein products shown to be essential
for RNA silencing. Two putative Dicer-like, and several Argonaute and RNA-
dependent RNA polymerase (RdRP) proteins were detected (Kemppainen et al.
2009). Especially high number of putative RdRPs (6) and Argonautes (6) in
Laccaria reflects the homobasidiomycete specific gene expansion of the RNA
silencing machinery (Nakayashiki and Kadotani 2006). The bioinformatical evi-
dence of the existence of Laccaria RNAi machinery suggested the presence of a
functional siRNA-dependent silencing pathway in this fungus. This was assayed
with nitrate reductase as a test endogene target.
Laccaria nitrate reductase encoding gene (Lbnr) can be considered an optimal
test target for RNA silencing. This is a single genomic gene and not a member of a
multigene-family, thus reducing possible silencing off-target effects. Silencing of
Lbnr would also allow a fast growth phenotype screening on nitrate medium.
Moreover, as nitrate metabolism is generally repressed in fungi and other eukar-
yotes by primary N sources, growth of Lbnr-silenced strains would be expected to
be unaffected on ammonium. As N is one of the main mobile nutrients in ECM,
fungal N metabolism genes are therefore of special interest.
As with Laccaria the vast majority of ectomycorrhizal fungi studied this far can
support growth on nitrate indicating that this metabolic trait is evolutionarily
conserved (Nygren et al. 2008). The true role of nitrate as N source for ectomycor-
rhizal fungi in forest soil is however under current debate. Even though traditionally
considered of minor importance for total N pool in forest soil other studies are
proposing that this inorganic N form may have marked temporal and spatial
concentration gradients and be more available for mycorrhizal fungi than previ-
ously believed (Stark and Hart 1997; Laverman et al. 2000). Moreover, the activity
and differential expression of fungal and plant nitrate utilization genes has been
connected to functional ectomycorrhizal symbiosis and enhanced host plant N
nutrition. The nitrate reductases of plant hosts become down-regulated both in
endo- and ectomycorrhizal roots and this repression has been directly linked to
the activity of fungal N metabolism in ectomycorrhiza (Kaldorf et al. 1998;
Guescini et al. 2003; Bailly et al. 2007). This proposes that the mycorrhized hosts
can be highly dependent on the N uptake and utilization pathway of the fungal
partner under nitrate feeding. The fungal response to symbiosis can however
vary between different ectomycorrhizal species. While in the basidiomycete
H. cylindrosporum nitrate reductase expression is lower in mycorrhizal structures
than in extraradical mycelium, in ectomycorrhiza formed by the ascomycete
T. borchii fungal nitrate utilization genes are induced in symbiotic structures
(Bailly et al. 2007; Guescini et al. 2003, 2007, 2009). Despite the observed species
specific variation, increasing data suggest that fungal nitrate metabolism genes can
play an important role in the establishment and/or the function of ectomycorrhizal
interactions in nature. Therefore, knocking-down of Lbnr could also offer informa-
tion on how this mutualistic association is regulated.
The functionality of the Laccaria RNA silencing pathway was tested by trans-
forming the dikaryotic fungus with a promoter-directed inverted repeated sequence
of a partial coding sequence of Lbnr (Kemppainen et al. 2009). RNA silencing was
accomplished in L. bicolor by AMT (Kemppainen et al. 2005, 2008; this book,
Chap. 6). Promoter-directed expression of dsRNA resulted in fungal transgenic
strains strongly affected in growth with nitrate as N source (Kemppainen et al.
2009). The phenotype correlated with a clear reduction of the target gene mRNA
level and this effect was not caused by homologous recombination of the T-DNA in
the nitrate reductase locus. Transformation with the hairpin sequence resulted in
specific but moderate CpG methylation of both the silencing triggering transgene
construct and the nitrate reductase encoding gene demonstrating that epigenetic
modifications accompany RNA silencing in Laccaria like in other eukaryotes. The
methylation in the target gene was restricted to the silencing trigger sequence and
did not represent the entire genomic DNA in the dikaryon suggesting that the
epigenetic changes accompanying RNA silencing affected only the transformed
nucleus. This strongly proposes that the Lbnr silencing phenotypes were predomi-
nantly a result of siRNA-dependent cytosolic PTGS mechanisms (Kemppainen
et al. 2009). Moreover, the silencing strength variation (SSV) between different
transformed strains did not correlate with the transgene copy number but it seems to
be linked to the nature of genomic integration sites of transgenes. Integrations in
euchromatin zones, especially within the coding sequence of active genes results in
strongly silenced fungal strains. These sites most probably allow the maximal
dsRNA-trigger production (Kemppainen et al. 2009; Kemppainen and Pardo 2010).
Mycorrhization experiments of Populus with strongly Lbnr-silenced fungal
strains revealed a systematic inhibition of symbiosis with nitrate as N source
compared to the wild type. This inhibition of mycorrhization was reversed by an
organic N source efficiently utilized by the fungus (i.e. L-asparagine). These
observations strongly suggest that the plant is able to sense the nutritional status
of a potential fungal symbiont avoiding the establishment of an unsatisfactory
interaction. A control mechanism conducted by the plant would inhibit symbiosis
when the metabolic profile of the fungal partner is not proper and mutual benefit
from the symbiotic structure cannot be assured. These results are the first direct
genetic proof showing that the alteration of expression of a fungal gene impairs
mycorrhization and the first demonstration of the RNA silencing pathway in
mycorrhizal fungi. They also highlight the great potential of RNAi technology for
studying symbiotic interactions (Kemppainen et al. 2009).
However, the efficient use of RNA silencing requires a friendly silencing/
transformation vector. Double-stranded hpRNA expression from stable integrated
transgenes or from auto-replicative elements has been shown to be a widely
efficient trigger in inducing RNA silencing in fungi. Vectors for plant silencing
such as pHANNIBAL and pHELLSGATE (Wesley et al. 2001), and pSTARLING
and pOpOFF (CSIRO) have been available for almost a decade and several silenc-
ing vectors for mammalian cells have been reported in the last few years (Wadhwa
et al. 2004; Gou et al. 2007). RNA silencing vectors for fungi however did not exist
before the launch of pSilent-1 (Nakayashiki et al. 2005) which has led to the
successful use of RNA silencing as a genetic tool in filamentous ascomycetes.
While an increasing number of silencing vectors for ascomycetes has been released
during the last few years (Krajaejun et al. 2007; Nguyen et al. 2008; Shafran et al.
2008; Oliveira et al. 2008; Barton and Prade 2008) no silencing vectors adapted to
basidiomycetes have been available.
The successful RNA silencing in Laccaria generated an urgent need for an easy-
to-use RNA silencing/transformation vector compatible with AMT. Unfortunately,
many commonly used ascomycete promoters are weakly recognized in filamentous
basidiomycetes, especially when introduced predominantly as a single copy via
AMT. This makes the use of ascomycete-adapted vectors difficult in basidiomy-
cetes. Neither are these transformation vectors compatible with AMT. To fulfill this
current gap of RNAi tools optimized for homobasidiomycetes we constructed the
pSILBAg silencing vector for efficient RNA silencing triggering in L. bicolor
(Kemppainen and Pardo 2009). This cloning vector carries the Agaricus bisporus
gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reduc-
tase intron and the Aspergillus nidulans trpC terminator. The pSILBAg allows an
easy oriented two-step PCR-cloning of hairpin sequences to be expressed in
basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based
binary vector carrying a hygromycin B resistance cassette, the pHg/pSILBAg
plasmid can be used for AMT (Kemppainen and Pardo 2010). Besides, for RNAi
triggering, the pHg/pSILBAg can also be used for gene expression studies. Due to
the widely recognized heterologous A. bisporus gpdII-promoter, both in the cloning
and the transformants selection cassette, this vector system should also result as
functional in other hygromycin B sensitive homobasidiomycete species.
We have shown that the dsRNA-triggered silencing pathway is functional in
the model ectomycorrhizal fungus L. bicolor (Kemppainen et al. 2009; Kemppai-
nen and Pardo 2010). This together with the availability of a easy-to-use basidio-
mycete-adapted silencing/AMT-vector, opens the possibility for efficient use of
gene knock-down in genetic studies of Laccaria. More importantly, RNA silenc-
ing can result in dikaryotic fungal strains affected in their mycorrhization capac-
ity. This sets the conditions for RNAi studies in ectomycorrhizal symbiosis, the
research field that up today has been hindered by the lack of functional reverse
genetic tools.
9.17 Conclusion
The symbiotic phase in the life-cycle of ectomycorrhizal basidiomycetes is the

dikaryon. Thus, studies on symbiotic fungal gene function would require the
inactivation of both gene copies in the dikaryotic mycelium. Due to the extremely
low homologous recombination rate in this group of fungi, traditional gene knock-
out experiments and particularly double gene interruption/replacement are rather
difficult to achieve. However, an alternative approach, RNA silencing, has shed
some light on the ectomycorrhizal research field.
In many organisms, including fungi, RNA silencing can be artificially triggered
to target and degrade gene transcripts of interests, resulting in the so called gene
knock-down. Most importantly, RNA silencing can act at the cytosolic level
affecting mRNAs originating from several gene copies and different nuclei and it
can thus offer an alternative and fast way for altering gene expression in dikaryotic,
diploid, polyploid, and coenocytic organisms.
Laccaria bicolor, the first symbiotic fungus with its genome sequenced, has
rapidly turned into a model fungus in ectomycorrhizal research. Laccaria possesses
a complete set of genes known to be needed for RNA silencing in eukaryotic cells.
We have demonstrated that RNA silencing is functional in L. bicolor and that it can
be triggered via AMT. Moreover, targeted gene knock-down in dikaryotic myce-
lium can result in functional phenotypes altered in the symbiotic capacity confirm-
ing that RNA silencing is a powerful way to study symbiosis-regulated genes.
These findings have now initiated the RNA silencing era in mycorrhizal research,
a field that has been hindered by the lack of proper genetic tools.
References
Axtell MJ, Jan C, Rajagopalan R, Bartel DP (2006) A two-hit trigger for siRNA biogenesis in
plants. Cell 127:565–577
Bailly J, Debaud J-C, Vegner M-C, Plassard C, Chalot M, Marmeisse R, Fraissinet-Tachet L
(2007) How does a symbiotic fungus modulate expression of its host-plant nitrite reductase?
Barnes SE, Alcocer MJC, Archer DB (2008) siRNA as a molecular tool for use in Aspergillus
niger. Biotechnol Lett 30:885–890
Barton LM, Prade RA (2008) Inducible RNA interference of brlAb in Aspergillus nidulans.
Eukaryot Cell 7:2004–2007
Bernstein E, Caudy AA, Hammond SH, Hannon GJ (2001) Role for a bidentate ribonuclease in the
initiation step of RNA interference. Nature 409:363–366
Berry B, Deddouche S, Kirschner D, Imler JL, Antoniewski C (2009) Viral suppressors of RNA
silencing hinder exogenous and endogenous small RNA pathways in Drosophila. PloS One 4:
e5866
Bleys A, Van Houdt H, Depicker A (2006a) Down-regulation of endogenes mediated by a
transitive silencing signal. RNA 12:1633–1639
Bleys A, Vermeersch L, Van Houdt H, Depicker A (2006b) The frequency and efficiency of
endogene suppression by transitive silencing signals is influenced by the length of sequence
homology. Plant Physiol 142:788–796
Bohse ML, Woods JP (2007) RNA interference-mediated silencing of the YPS3 gene of Hiso-
plasma capsulatum reveals virulence defects. Infect Immun 75:2811–2817
Boutros M, Kiger A, Armknecht S, Kerr K, Hild M, Koch B, Haas S, Paro R, Perrimon N (2004)
Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303:
832–835
B€
uhler M (2009) RNA turnover and chromatin-dependent gene silencing. Chromosoma 118:
141–151
Cardoza RE, Vizcaino JA, Hermosa MR, Sousa S, Gonzalez FJ, Llobell A, Monte E, Gurierrez S
(2006) Cloning and characterization of the erg1 of Trichoderma harzianum: Effect of the erg1
silencing on ergosterol biosynthesis and resistance to terbinafine. Fungal Genet Biol
43:164–178
Caribé dos Santos AC, Sena JA, Santos SC, Dias CV, Pirovani CP, Pungartnik C, Valle RR,
Cascardo JC, Vincentz M (2009) dsRNA-induced gene silencing in Moniliophthora perni-
ciosa, the causal agent of witches’ broom disease of cacao. Fungal Genet Biol 46:825–836
Carmell MA, Hannon GJ (2004) RNase III enzymes and the initiation of gene silencing. Nat Struct
Mol Biol 11:214–218
Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell
136:642–655
Castanotto D, Rossi JJ (2009) The promises and pitfalls of RNA interference-based therapeutics.
Nature 457:426–433
Cecere G, Cogoni C (2009) Quelling targets the rDNA locus and fuctions in rDNA copy number
control. BMC Microbiol 9:44
Chen J, Shi X, Padmanabhan R, Wang Q, Wu Z, Stevenson SC, Hild M, Garza D, Li H (2008)
Identification of novel modulators of mitochondrial function by genome-wide RNAi sreen in
Drosophila melanogaster. Genome Res 18:123–136
Choudhary S, Lee H-C, Maiti M, He Q, Cheng P, Liu Q, Liu Y (2007) A double-stranded-RNA
response program important for RNA interference efficiency. Mol Cell Biol 27:3995–4005
Cogoni C, Macino G (1999) Homology-dependent gene silencing in plants and fungi: a number of
variations on the same theme. Curr Opin Microbiol 2:657–662
Cogoni C, Irelan JT, Schumacher M, Schmidhauser TJ, Selker EU, Macino G (1996) Transgene
silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic
effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J
15:3153–3163
Cooper KG, Woods JP (2009) Secreted dipeptidyl peptidase IV activity in the dimorphic fungal
pathogen Histoplasma capsulatum. Infect Immun 77:2447–2454
Costa AM, Mills PR, Bailey AM, Foste GD, Challen MP (2008) Oligonucleotide sequences
forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis
cinerea genes. J Microbiol Methods 75:205–208
Cronin SJF, Nehme NT, Limmer S, Liegeois S, Pospisilik JA, Schramek D, Leibbrandt A,
de Matos SR, Gruber S, Puc U, Ebersberger I, Zoranovic T, Neely GG, von Haeseler A,
Ferrandon D, Penninger JM (2009) Genome-wide RNAi screen identifies genes involved in
intestinal pathogenic bacterial infection. Science 325:340–343
Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC (2000) An RNA-dependent RNA
polymerase gene in Arabidopsis is required for post-transcriptional gene silencing mediated by
transgene but not by a virus. Cell 101:543–553
de Jong JF, Deelstra HJ, W€ osten HAB, Lugones LG (2006) RNA-mediated gene silencing in
monokaryons and dikaryons of Schizophyllum commune. Appl Environ Mircrobiol 72:1267–1269
Dı́az-Pendón JA, Ding SW (2008) Direct and indirect roles of viral suppressors of RNA silencing
in pathogenesis. Annu Rev Phytopathol 46:303–326
Djupedal I, Ekwall K (2009) Epigenetics: heterchromatin meets RNAi. Cell Res 19:282–295
Erental A, Harel A, Yarden O (2007) Type 2A phosphoprotein phosphatase is required for asexual
development and pathogenesis of Sclerotinia sclerotiorum. Mol Plant Microbe Interact
20:944–954
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic
interference by double-stranded RNA in Caenorhabditidis elegans. Nature 391:806–811
Fitzgerald A, van Kan JAL, Plummer KM (2004) Simultaneous silencing of multiple genes in the
apple scab fungus Venturia inaequalis by expression of RNA with chimeric inverted repeats.
Fungal Genet Biol 41:963–971
Fulci B, Macino G (2007) Quelling: post-transcriptional gene silencing guided by small RNAs in
Neurospora crassa. Curr Opin Microbiol 10:199–203
Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small
RNAs binds mammalian Piwi proteins. Nature 442:199–202
Gitlin L, Andino R (2003) Nuclei acid-based immune system: the antiviral potential of mammalian
RNA silencing. J Virol 77:7159–7165
Gobeil S, Zhu X, Doillon CJ, Green MR (2008) A genome-wide shRNA screen identifies GAS1 as
a novel melanoma metastasis suppressor gene. Genes Dev 22:2932–2940
Golden DE, Gerbasi VR, Sontheimer EJ (2008) An inside job for siRNAs. Mol Cell 31:309–312
Goldoni M, Azzalin G, Macino G, Cogoni C (2004) Efficient gene siliencing by expression of
double stranded RNA in Neurospora crassa. Fungal Genet Biol 41:1016–1024
Gong X, Fu Y, Jiang D, Li G, Yi X, Peng Y (2007) L-arginine is essential for conidiation in the
filamentous fungus Coniothyrium minitans. Fungal Genet Biol 44:1368–1379
Gonzalez S, Pisano DG, Serrano M (2008) Mechanistic principles of chromatin remodeling guided
by siRNAs and miRNAs. Cell Cycle 7:2601–2608
Gorlach JM, McDade HC, Perfect JR, Cox GM (2002) Antisense repression in Cryptococcus
neoformans as a laboratory tool and potential antifungal strategy. Microbiology 128:213–219
Gou D, Weng T, Wang Y, Wang Z, Zhang H, Gao L, Chen Z, Wang P, Liu L (2007) A novel
approach for the construction of multiple shRNA expression vectors. J Gene Med 9:751–763
Guang S, Bochner AF, Pavelec DM, Burkhart KB, Harding S, Lachowiec J, Kennedy S (2008) An
argonaute transports siRNAs from the cytoplasm to the nucleus. Science 321:537–541
Guescini M, Pierloeni R, Palma F, Zeppa S, Vallorani L, Potenza L, Sacconi G, Giomaro G,
Stocchi V (2003) Characterization of the Tuber borchii nitrate reductase gene and its role in
ectomycorrhizae. Mol Genet Genomics 269:807–816
Guescini M, Zeppa S, Pierleoni R, Sisti D, Stocchi L, Stocchi V (2007) The expression profile of
the Tuber borchii nitrite reductase suggests its positive contribution to the host plant nitrogen
nutrition. Curr Genet 51:31–41
Guescini M, Stocchi L, Sisti D, Zeppa S, Polidori E, Ceccaroli P, Satarelli R, Stocchi V (2009)
Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal
fungus Tuber borchii. Curr Genet 55:59–68
Hamada W, Spanu PD (1998) Co-suppression of the hydrophobin gene HCf-1 is correlated with
antisense RNA biosynthesis in Cladosprorum fulvum. Mol Gen Genet 259:630–638
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link
between genetic and biochemical analyses of RNAi. Science 293:1146–1150
Hammond TM, Andrewski MD, Roossinck MJ, Keller NP (2008a) Aspergillus mycoviruses are
targets and suppressors of RNA silencing. Eukaryot Cell 7:350–357
Hammond TM, Bok JW, Andrewski MD, Reyes-Domı́nguez Y, Scazzocchio C, Keller NP
(2008b) RNA silencing gene truncation in the filamentous fungus Aspergillus nidulans.
Hutvagner G, Simard MJ (2008) Argonaute proteins: key players in RNA silencing. Nat Rev Mol
Cell Biol 9:22–32
Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, Lim L, Linsley PS (2006) Widespread
siRNA “off-target” transcript silencing mediated by seed region sequence complementarity.
RNA 12:1179–1187
Jagannath A, Wood MJA (2009) Localization of double-stranded small interfering RNA to

cytoplasmic processing bodies Is Ago2 dependent and results in up-regulation of GW182
and Argonaute-2. Mol Biol Cell 20:521–529
Janus D, Hoff B, Hofmann E, K€ uck U (2007) An efficient fungal RNA-silencing system using the
DsRed reporter gene. Appl Environ Microbiol 73:962–970
J€
ochl C, Rederstorff M, Hertel J, Standler PF, Hofacker IL, Schrettl M, Haas H, H€uttenhofer A
(2008) Small ncRNA transcriptote analisis from Aspergillus fumigatus suggests a novel
mechanism for regulation of protein syntesis. Nucleic Acids Res 36:2677–2689
Jorgensen R (1990) Altered gene expression in plants due to trans interactions between homolo-
gous genes. Trends Biotechnol 8:340–344
Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003) RNA silencing in the phytophatogenic
fungus Magnaporthe oryzae. Mol Plant Microbe Interact 16:769–776
Kalantidis K, Schumacher HT, Alexiadis T, Helm JM (2008) RNA silencing movement in plants.
Biol Cell 100:13–26
Kaldorf M, Schmelzer E, Bothe H (1998) Expression of maize and fungal nitrate reductase genes
in arbuscular mycorrhiza. Mol Plant Microbe Interact 11:439–448
Kamath RS, Fraser AG, Dong Y, Polin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S,
Sohrmann M, Welchman DP, Zipperlen P, Ahringer J (2003) Systemic functional analysis of
the Caenorhabditis elegans genome using RNAi. Nature 421:231–237
Kemppainen M, Pardo AG (2010) pHg/pSILBAg vector system for efficient gene silencing in
homobasidiomycetes: optimization of ihpRNA-triggering in the mycorrhizal fungus Laccaria
bicolor. Microb Biotechnol 3:178–200
Kemppainen M, Circosta A, Tagu D, Martin F, Pardo AG (2005) Agrobacterium–mediated
transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N. Mycorrhiza 16:
19–22
Kemppainen M, Duplessin S, Martin F, Pardo AG (2008) T-DNA insertion, plasmid rescue and
integration analysis in the model mycorrhizal fungus Laccaria bicolor. Microb Biotechnol
1:259–269
Kemppainen M, Duplessin S, Martin F, Pardo AG (2009) RNA silencing in the model mycorrhizal
fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbi-
osis with Populus. Environ Microbiol 11:1878–1896
Khatri M, Rajam MV (2007) Targeting polyamines of Aspergillus nidulans by siRNA specific to
fungal ornithine decarboxylase gene. Med Mycol 45:211–220
Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol
6:376–385
Kim DH, Sætrom P, Snøve O, Rossi JJ (2008) MicroRNA-directed transcriptional gene silencing
in mammalian cells. PNAS 105:16230–16235
Krajaejun T, Gauthier GM, Rappleye GA, Sullivan TD, Klein BS (2007) Development and
application of a green fluorescent protein sentinel system for identification of RNA interference
in Balstomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation.
Krishnan MN, Ng A, Sukumaran B, Gilfoy FD, Uchil PD, Sultana H, Brass AL, Adametz R,
Tsui M, Qian F, Montgomery RR, Lev S, Mason PW, Koski RA, Elledge SJ, Xavier RJ,
Agaisse H, Fikrig E (2008) RNA interference screen for human genes associated with West
Nile virus infection. Nature 455:242–245
Laurie JD, Linning R, Bakkeren G (2008) Hallmarks of RNA silencing are found in the smut
fungus Ustilago hordei but not in its close relative Ustilago maydis. Curr Genet 53:49–58
Laverman AM, Zoomer HR, van Verseveld HW, Verhoef HA (2000) Temporal and spatial
variation of nitrogen transformations in a coniferous forest soil. Soil Biol Biochem 32:
1661–1670
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines,
indicates that thousands of human genes are microRNA targets. Cell 120:15–20
Li WX, Li H, Lu R, Li F, Dus M, Atkinson P, Brydon EW, Johnson KL, Garcı́a-Sastre A, Ball LA,
Palese P, Ding SW (2004) Interferon antagonist proteins of influenza and vaccine virus are
suppressors of RNA silencing. Proc Natl Acad Sci USA 101:1350–1355
Lippman Z, Martienssen R (2004) The role of RNA interference in heterochromatic silencing.
Nature 431:364–370
Liu H, Cottrell T, Pierini LM, Goldman WE, Doering T (2002) RNA interference in the pathogenic
fungus Cryptococcus neoformans. Genetics 160:463–470
Martin F, Aerts A, Ahren D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E,
Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canb€ack B,
Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S,
Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J,
Hoegger PJ, Jain P, Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D,
Montanini B, Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP, Peter M, Quesne-
ville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, K€ues U,
Lucas S, Van de Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR,
Stajich JE, Tunlid A, Tuskan G, Grigoriev IV (2008) The genome of Laccaria bicolor provides
insights into mycorrhizal symbiosis. Nature 452:88–92
Matityahu A, Hadar Y, Dosoretz CG, Belinky PA (2008) Gene silencing by RNA interference in
the white-rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 74:5359–5365
McDonald T, Brown D, Keller NP, Hammond TM (2005) RNA silencing of mycotoxin production
in Aspergillus and Fusarium species. Mol Plant Microbe Interact 18:539–545
Mello CC, Conte D Jr (2004) Revealing the world of RNA interference. Nature 431:338–342
Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a gene
family of rice. Plant Physiol 138:1903–1913
Moazed D (2009) Small RNAs in transcriptional gene silencing and genome defence. Nature
457:413–420
Mochizuki K, Gorovsky MA (2005) A dicer-like protein in Tetrahymena has distinct functions in
genome rearrangement, chromosome segregation, and meiotic prophase. Genes Dev 19:77–89
Moissiard G, Voinnet O (2006) RNA silencing of host transcripts by cauliflower mosaic virus
requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci
USA 103:19593–19598
Moriwaki A, Ueno M, Arase S, Kihara J (2007) RNA-mediated gene silencing in the pythopatho-
genic fungus Bipolaris oryzae. FEMS Microbiol Lett 269:85–89
Mouyna I, Henry C, Doering TL, Latgé J-P (2004) Gene silencing by RNA interference in the
human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 237:317–324
Mummery-Widmer JL, Yamazaki M, Stoeger T, Novatchkova M, Bhalerao S, Chen D, Dietzl G,
Dickson BJ, Knoblich JA (2009) Genome-wide analisis of Notch signallingin Drosophila by
transgenic RNAi. Nature 458:987–992
Murata T, Kadotani N, Yamaguchi M, Tosa Y, Mayama S, Nakayashiki H (2007) siRNA-dependent
and independent post-transcriptional cosuppression of the LTR-retrotransposon MAGGY in the
phytopathogenic fungus Magnaporthe oryzae. Nucleic Acids Res 35:5987–5994
Nakayashiki H, Kadotani NSM (2006) Evolution and diversification of RNA silencing proteins in
fungi. J Mol Evol 63:127–135
Nakayashiki H, Nguyen QB (2008) RNA interference: roles in fungal biology. Curr Opin
Microbiol11:494–502
Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S (2005) RNA silencing as a
tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42:275–283
Namekawa SH, Iwabata K, Sugawara H, Hamada FK, Koshiyama A, Chiku H, Kamada T,
Sakaguchi K (2005) Knockdown of LIM/15DMC1 in the mushroom Coprinus cinereus by
double-stranded RNA-mediated gene silencing. Microbiology 151:3669–3678
Nguyen QB, Kadotani N, Kasahara YT, Mayama S, Nakayashiki H (2008) Systematic functional
analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe
oryzae, using a high-throuhgput RNA-silencing system. J Microbiol 68:1348–1365
Nicolás FE, Torres-Martı́nez S, Ruiz-Vázquez RM (2003) Two classes of small antisense RNAs in
fungal RNA silencing triggered by non-integrative transgenes. EMBO J 22:3983–3991
Nicolás FE, Calo S, Murcia-Flores L, Garre V, Ruiz-Vázquez RM, Torres Martı́nez S (2008) A
RING-finger photocarotenogenic represor involved in asexual sporulation in Mucor circinel-
loides. FEMS Microbiol Lett 280:81–88
Nygren CMR, Eberhardt U, Karlsson M, Parret JL, Lindahl BD, Taylor AFS (2008) Growth on
nitrate and occurrence of nitrate reductase-encoding genes in a phylogenetically diverse range
of ectomycorrhizal fungi. New Phytol 180:875–889
Ohrt T, M€utze J, Staroske W, Weinmann L, H€ ock J, Crell K, Meister G, Schwille P (2008)
Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal
the cytoplasmic origination of loaded nuclear RISC in vivo in human cells. Nucleic Acid Res
36:6439–6449
Oliveira JM, van der Veen D, de Graaff LH, Qui L (2008) Efficient cloning system for construction
of gene silencing vectors in Aspergillus niger. Appl Microbiol Biotechnol 80:917–924
Orban TI, Izaurralde E (2005) Decay of mRNAs targeted by RISC requires XRN1, the Ski
complex, and the exosome. RNA 11:459–469
Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and
other small RNAs. Plant J 53:674–690
Pak J, Fire A (2007) Distinct populations of primary and secondary effectors during RNAi in
C. elegans. Science 315:241–244
Panepinto J, Komperda K, Frases S, Park YD, Djordjevic JT, Casadevall A, Williamson PR (2009)
Sec6-dependent sorting of fangal extracellular exosomes and laccase of Cryptococcus neofor-
mans. Mol Microbiol 71:1165–1176
Petersen BO, Albrechtsen M (2005) Evidence implying only unprimed RdRP activity during
transitive gene silencing in plants. Plant Mol Biol 58:575–583
Petersen CP, Bordeleau ME, Pelletier J, Sharp PA (2006) Short RNAs repress translation after
initiation in mammalian cells. Mol Cell 21:533–542
Pfeffer S, Zavolan M, Gr€asser FA, Chien M, Russo JJ, Ju J, John B, Enright AJ, Marks D, Sanders
C, Tuschl T (2004) Identification of virus-encoded microRNAs. Science 304:734–736
Raponi M, Arndt GM (2003) Double-stranded RNA-mediated gene silencing in fission yeast.
Nucleic Acid Res 31:4481–4489
Rappleye CA, Engle JT, Coldman WE (2004) RNA interference in Hitoplasma capsulatum
demonstrates a role for alpha-(1, 3)-glucan in virulence. Mol Microbiol 53:153–165
Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora
crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353
Ruby JG, Jan C, Plaver C, Axtell MJ, Lee W, Nusbaum C, Ge H, Bartel DP (2006) Large-scale
sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in
C. elegans. Cell 127:1193–1207
Ruiz M, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene
silencing. Plant Cell 10:937–946
Schramke V, Allshire R (2003) Hairpin RNAs and retrotransposons LTRs effect RNAi and
chromatin-based gene silencing. Science 301:1069–1107
Sch€umann J, Hertweck C (2007) Molecular basis of cytochalasin biosynthesis in fungi: gene
cluster analysis and evidence for the involvement of a PKS-NRPS hybrid synthase by RNA
silencing. J Am Chem Soc 129:9564–9565
Schuurs TA, Schaeffer EAM, Wessels JGH (1997) Homology-dependent silencing of the SC3
gene in Schizophyllum commune. Genetics 147:589–596
Segers GC, van Wezel R, Zhang X, Hong Y, Nuss DL (2006) Hypovirus papain-like protease p29
suppresses RNA silencing in the natural fungal host and in a heterologous plant system.
Segers CG, Zhang X, Deng F, Sun Q, Nuss DL (2007) Evidence that RNA silencing functions as
an antiviral defense mechanism in fungi. Proc Natl Acad Sci USA 104:12902–12906
Shafran H, Miyara I, Eshed R, Prusky D, Sherman A (2008) Development of new tools for stydying
gene function in fungi based on the Gateway system. Fungal Genet Biol 45:1147–1154
Sigova A, Rhind N, Zamore P (2004) A single argonaute protein mediates both transcriptional and
posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev 18:2359–2367
Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On
the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476
Sijen T, Steiner FA, Thijssen KL, Plasterk HA (2007) Secondary siRNAs result from unprimed
RNA synthesis and from a distinct class. Science 315:244–247
Stark JM, Hart SC (1997) High rates of nitrification and nitrate turnover in undisturbed coniferous
forests. Nature 385:61–64
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to
interferons. Annu Rev Biochem 67:227–264
Tanguay P, Bozza S, Breui C (2006) Assessing RNAi frequency and efficiensy in Ophiostoma
floccosum and O. piceae. Fungal Genet Biol 43:802–804
Tomari Y, Zamora PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529
Travella S, Keller B (2009) Down-regulation of gene expression by RNA-induced gene silencing.
Methods Mol Biol 478:185–199
Verdel A, Vavasseur A, Le Gorrec M, Touat-Todeschini L (2009) Common themes in siRNA-
mediated epigenetic silencing pathways. Int J Dev Biol 53:245–257
Wadhwa R, Kaul SC, Miyagishi M, Taira K (2004) Vectors for RNA interferente. Curr Opin Mol
Ther 6:367–372
W€alti MA, Villalba C, Buser RM, Gr€ unler A, Aebi M, K€unzler M (2006) Targeted gene silencing
in the model mushroom Coprinus cinerea (Coprinus cinereus) by expression of homologous
hairpin RNAs. Eukaryot Cell 5:732–744
Wassenegger M, Heimes S, Riedel L, Sanger HL (1994) RNA-directed de novo methylation of
genomic sequences in plants. Cell 76:567–576
Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen Z-X, Riggs AD,
Rossi JJ, Morris KV (2006) The antisense strand of small interfering RNAs directs histone
methylation and transcriptional gene silencing in human cells. RNA 12:256–262
Weinmann L, H€ock J, Ivacevic T, Ohrt T, M€ utze J, Schwille P, Kremmer E, Benes V, Urlaub H,
Meister G (2009) Importin 8 is a gene silencing factor that targets Argonaute proteins to
distinct mRNAs. Cell 136:496–507
Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott
D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct
design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590
Wu L, Fan J, Belasco JG (2006) MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad
Sci USA 103:4034–4039
Yu D, Fan B, MacFarlane SA, Chen Z (2003) Analisis of the involvement of an inducible
Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Mol Plant Microbe Inter-
act 16:206–216
Zhang X, Segers GC, Sun Q, Deng F, Nuss DL (2008) Characterization of hypovirus-derived small
RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway.
J Virol 82:2613–2619
Zhou H, Xu M, Huang Q, Gates AT, Zhang XD, Castle JC, Stec E, Ferrer M, Strulovici B, Hazuda
DJ, Espeseth AS (2008) Genome-scale RNAi screen for host factors required for HIV replica-
tion. Cell Host Microbe 2:495–504
.
Part III
Functions and Interactions
Chapter 10
Ectomycoremediation: An Eco-Friendly
Technique for the Remediation of Polluted Sites
Heike B€
ucking
10.1 Introduction
Estimates of the number of sites vary considerably, but approximately 450,000 to 1

million sites in the United States and 750,000 sites in Europe are suspected or
known to be contaminated with a diverse group of pollutants due to previous
anthropogenic impacts (De Sousa 2001; GAO 2004). Fuel hydrocarbons, poly-
cyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlori-
nated aromatic hydrocarbons (CAHs), detergents, and pesticides are the most
dominant organic pollutants in soils. The main inorganic pollutants are heavy
metals, such as cadmium, lead and mercury, nitrates and phosphate, and radio-
nuclides. The United States alone spends annually $6–8 billion and global costs
range between $25 and 50 billion for efforts to remediate contaminated sites (Doty
2008). Costs for the restoration of all contaminated sites in the United States have
been estimated to be approximately $1.7 trillion (Kuiper et al. 2004). Conventional
techniques for the remediation of polluted sites typically include: soil excavation,
transport, washing and extraction, pumping and treating of contaminated water, and
the addition of chemical reactants such as hydrogen peroxide or potassium perman-
ganate, and incineration. However, these techniques are expensive and not always
sufficient, very invasive, and can lead to the release of pollutants into the air or
leaching into the ground water (Kuiper et al. 2004).
H. B€ucking
Biology and Microbiology Department, South Dakota State University, Brookings, SD 57007,
USA
e-mail: heike.bucking@sdstate.edu

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_10,
210 H. B€ucking
10.2 Phytoremediation: An Alternative Method

for the Remediation of Contaminated Sites
Phytoremediation can represent a sustainable and cost-effective alternative to

conventional remediation technologies, and includes all plant-induced bio-
logical, chemical, and physical processes that contribute to the remediation of
contaminated sites (Cunningham et al. 1996). The following processes can be
distinguished:
l Phytostabilization. Containment of pollutants by reducing the dislocation to
other ecosystem compartments by, for example, mechanical stabilization of
the soil, binding to the plant surface, or by reducing the leaching of pollutants
into aquifers.
l Phytoextraction. Removal of pollutants from the soil by uptake from the envi-
ronment and accumulation in the plant biomass, preferably in the above-ground
plant tissue.
l Phytotransformation. Chemical transformation of pollutants through plant
metabolism, resulting in inactivation, degradation (phytodegradation), or immo-
bilization (phytostabilization) of pollutants.
l Phytovolatilization. Uptake of pollutants from the soil and release into the
atmosphere, sometimes after transformation into a more volatile form.
l Phytostimulation. Increase in biodegradation through the stimulation of micro-
bial communities in the rhizosphere. Since most of the processes that lead to the
degradation of pollutants by plants occur in the rhizosphere and not in the whole
plant per se, the term rhizoremediation is also often used (Meharg and Cairney
2000; Kuiper et al. 2004).
Compared to conventional techniques, phytoremediation has several advan-
tages (1) it is much cheaper on a per volume basis (estimated costs of phyto-
remediation depend on site and pollutant but vary between 10 and 50% of the
costs for conventional techniques; Sadowsky 1999); (2) less secondary waste is
generated, and instead a useful product, such as wood, pulp, or bioenergy can be
produced; (3) it retains the top soil and is noninvasive; and (4) it has a high
public acceptance. Phytoremediation is suitable for large contaminated sites
with low-to-moderate pollutant concentration particularly in top layers of the
soil. However, polluted sites are often contaminated with a mixture of various
problematic compounds, and the low nutrient availability and poor soil structure
of, e.g., former industrial sites can prevent a successful establishment of plants
(Meharg and Cairney 2000). Phytoremediation is generally slower than conven-
tional practices, and it is not without environmental implications, since pollu-
tants can get integrated into the wild-life food chains via the plant, and/or toxic
intermediates can be formed by incomplete degradation pathways (Trapp and
Karlson 2000).
10 Ectomycoremediation 211
10.3 Ectomycorrhizal Associations and Their Significance

for Phytoremediation
Numerous studies have shown that plants can successfully be used for the remedia-
tion of soils contaminated with a variety of pollutants, such as salts, agrochemicals,
nitroaromatics, chlorinated compounds, heavy metals, and hydrocarbons (Tsao
2003). Particularly, fast growing trees, such as Populus and Salix, are promising
candidates for phytoremediation or dendroremediation (the remediation of polluted
sites by trees, Komives and Gullner 2006) due to their deep root system, and their
high biomass production and transpiration activity (Tlustoš et al. 2006). Laureysens
et al. (2004), who examined the biomass production of 17 poplar clones on a waste
disposal site, found an annual biomass production of up to 11.4 t ha 1. For both
plant species transpiration rates of approximately 100 l water per day have been
reported (Wullschleger et al. 1998).
However, the majority of tree species that have been considered as potential
candidates for phytoremediation live naturally in symbiosis with ectomycorrhizal
(ECM) and/or arbuscular mycorrhizal (AM) fungi (Smith and Read 2008). For
instance, Populus that has extensively been studied for its suitability in phyto-
remediation also due to its accessibility for genetic engineering (e.g., Rugh et al.
1998; Doty et al. 2007) forms ECM interactions with more than 60 different fungal
species, and some of these fungi have been shown to increase the growth of poplar
seedlings by more than 400% (Cripps 2003). Under natural conditions, more than
60–80% of the root systems of poplar and willow, respectively, are colonized with
ECM fungi. Additionally, approximately 10% of the root system is colonized with
AM fungi (van der Heijden and Vosatka 1999; Kaldorf et al. 2002; Becerra et al.
2009).
Mycorrhizal fungi play a key role in nutrient cycling and ecosystem functioning
and have a profound effect on the composition of plant and microbial communities
and thereby also on the capability of these communities to degrade anthropogenic
pollutants. ECM fungi enhance the uptake of phosphate (P) and nitrogen (N), and
can also contribute to the supply of the host with trace elements, such as copper
(Cu) and zinc (Zn) (Smith and Read 2008). Furthermore, mycorrhizal plants have a
higher resistance against abiotic (e.g., drought, heavy metals) and biotic (plant
pathogens) stresses (Smith and Read 2008). In return, for these beneficial effects on
nutrient uptake and stress resistance, the plant transfers between 10% and 20% of its
photosynthetically fixed carbon to the fungus (Finlay and S€oderstr€om 1992).
However, despite the widely acknowledged significance of ECM fungi for plant
growth, fitness and community composition particularly in difficult environments,
their contribution to phytoremediation processes has so far often been ignored.
The capability of tree species, such as Populus and Salix, to remediate contami-
nated soils has extensively been studied (for review see, e.g., Pulford and Watson
2003; Tlustoš et al. 2006), but the contribution of their ECM communities to the
observed degradation processes has only rarely been examined (e.g., Sell et al.
2005). ECM fungi have been shown to degrade a variety of environmentally
212 H. B€ucking
important organic pollutants, such as 2,4-dichlorophenol (Meharg et al. 1997b),

2,4,6-trinitrotoluene (Scheibner et al. 1997), PCB (Donnelly and Fletcher 1995),
4-fluorobiphenyl (Green et al. 1999), and some PAHs (Braun-L€ullemann et al.
1999) and the capability to degrade organic pollutants seems to be relatively
common for this group of fungi. Meharg and Cairney (2000), for example, screened
different ECM fungal species for their capability to degrade various classes of
organic pollutants (e.g., PAHs, PCB) and found that 33 out of 42 species were able
to degrade one or more classes of these contaminants. However, most of these
studies were performed under pure culture conditions (e.g., Meharg et al. 1997b;
Gramss et al. 1999) that do not necessarily reflect the capability of the fungus to
degrade recalcitrant compounds in the symbiotic stage under field conditions
(Meharg et al. 1997a; Dittmann et al. 2002). However, it is crucial to consider
both, plant and fungus, as one unit, and to assess the capability of plants and fungi to
degrade xenobiotics in the symbiotic stage to use both partners to their full potential
for the remediation of polluted sites. This is important for the following reasons:
l Mycorrhizal fungi are ubiquitous in the soil and plants are under natural condi-
tions normally associated with mycorrhizal fungi. However, the colonization
with ECM fungi can have a synergistic (Meharg et al. 1997a; Sell et al. 2005),
but also an antagonistic effect (Genney et al. 2004; Koivula et al. 2004; Joner
et al. 2006; Gunderson et al. 2007) on the capability of the plant to remediate
polluted sites.
l ECM roots represent the major portion of the nutrient-absorbing surface area in
tree roots (Taylor and Peterson 2000), and thereby also of the root system that is
potentially able to absorb and to extract pollutants from the soil. The fungal
sheath of ECM roots represents for ions a significant apoplastic barrier (B€ucking
et al. 2002), and can be expected to restrict the movement of larger molecules,
such as PAHs, into the root cortex. In the symbiotic stage, the nutrient uptake by
the host is mainly controlled and regulated by the mycorrhizal fungus (Smith and
Read 2008), and this can increase or reduce the bioavailability of the pollutant
for the host (Sell et al. 2005; Gunderson et al. 2007).
l The interaction of a host plant with a mycorrhizal fungus has a much stronger
impact on the whole plant physiology than previously suggested. Additionally,
to the positive effects on nutrient uptake and biotic and abiotic stress resistance,
the symbiosis affects, for example, the photosynthetic activity, transpiration,
stomatal conductance, carbon allocation, and N metabolism of the host (Smith
and Read 2008).
l The symbiosis with a host plant changes the physiology of the mycorrhizal
fungus and could, for example, affect the expression of degrading enzymes
(Meharg and Cairney 2000). In the symbiosis the fungus receives carbon from
its host, and this could potentially improve the degradation rate by an increase in
the fungal biomass, but could also reduce the degradation rate, because the more
readily available substrates are generally preferred (Joner and Leyval 2003).
l Approximately 8,000 plant species and 7,000–10,000 fungal species form ECM
associations (Taylor and Alexander 2005), but only a small fraction of potential
plant/fungal interactions has been tested so far. However, there is a considerable

physiological diversity between individual plant and fungal species and also
between different ECM plant/fungal associations (e.g., Sell et al. 2005), and the
potential degradation rate of these systems is also dependent on the pollutant, its
concentration, and the environmental conditions (e.g., soil characteristics, nutri-
ent availability).
l So far, it has been difficult to translate remediation strategies that have been
proven to be successful under laboratory or greenhouse conditions to the field
and commercialization. Gerhardt et al. (2009) suggested that this is due to
additional stress factors for the plant, or to inadequate methods to analyze
remediation under field conditions. Discrepancies between the degradation
rates under laboratory and under field conditions can be the result of many
different factors, for example, soil characteristics that affect the bioavailability
or weathering of soil pollutants. However, it is also possible that mycorrhizal
communities play a much greater role, and that experiments with nonmycor-
rhizal plants or plants that are only colonized with one ECM partner do not
reflect root systems under field conditions that are colonized on a very small
spatial scale with different taxa and species of ECM fungi. Goodman and
Trofymow (1998) identified, for example, 35 different ECM morphotypes in
3 l of soil from a mature spruce stand.
l ECM fungi influence both plant and microbial communities in the soil (Finlay
2008), and this will also have an effect on the capability of these communities to
remediate polluted soils. Both symbiotic partners interact in the soil with a
variety of microorganisms, and it has been suggested that the diverse microbial
communities in the rhizosphere or mycorrhizosphere have a much greater
impact on biodegradation of organic pollutants than the plant (Heinonsalo
et al. 2000).
For the successful application of ECM plants for the phytoremediation of pol-
luted sites, several interrelated factors need to be considered, including the proper-
ties, concentration, distribution, bioavailability, and toxicity of the pollutant; soil
characteristics such as water and nutrient availability; the effect of the ECM system
on the activity of microbial communities in rhizosphere and mycorrhizosphere; and
the activity of both partners to degrade pollutants in the symbiotic stage.
10.3.1 The Contribution of Rhizosphere

and Ectomycorrhizosphere to Bioremediation
The root rhizosphere with its stimulating effect on microbial activities has often
been described as the main contributor to the phytoremediation of contaminated
sites. Wang et al. (2008), for example, reported that in the rhizosphere three to four
times more petroleum hydrocarbon (PHC) was degraded than in unplanted soil, and
similar effects have also been observed for other pollutants (e.g., Gunderson et al.
214 H. B€ucking
2007). However, the mechanisms that cause a stimulation of degradation in the

rhizosphere are complex and only poorly understood. Putative explanations include
(1) the direct degradation by plant-derived enzymes; (2) the stimulation of micro-
bial activity and changes in the microbial community composition due to root
exudates; (3) the release of metabolic precursors, for example, phenolics, that
induce the activity of enzymes that degrade pollutants; and (4) the establishment
of environmental conditions that stimulate degradation (e.g., pH, O2/CO2 concen-
tration, and osmotic potential) (Joner and Leyval 2003).
Plant roots release approximately 10–20% of their photosynthetically fixed
carbon into the rhizosphere (Bais et al. 2006; Haichar et al. 2008), and the secreted
organic compounds, such as sugars, sugar alcohols, amino acids, fatty acids, and
proteins represent a significant carbon source and promote microbial proliferation
and diversity in the rhizosphere. Root exudates have also been shown to shape the
microbial community composition and its capability to degrade recalcitrant com-
pounds. For instance, phenolic compounds in root exudates stimulate the microbial
degradation of PCBs in the rhizosphere by serving as growth substrates for PCB-
degrading bacteria and/or as inducer of the PCB-degradation pathway (Donnelly
et al. 1994; Gilbert and Crowley 1997). Similar effects were also shown for the
biodegradation of PAHs, such as naphthalene. The expression of naphthalene
dioxygenase in Pseudomonas fluorescens was differentially regulated in response
to different root exudate compounds, and the higher overall expression when root
exudates were available resulted in increased naphthalene-degradation rates in the
root rhizosphere (Kamath et al. 2004).
The plant rhizosphere has also been discussed as a potential way to introduce and
to stabilize communities of genetically engineered microorganisms in the soil that
could carry or transfer degradation genes to indigenous microbial communities
(“bioaugmentation”; Sarand et al. 1998, 1999). Without the positive effect of the
root rhizosphere, the number of introduced degrading microorganisms often
decreases shortly after inoculation, due to competition with indigenous microbial
communities for limited nutrient resources, antagonistic interactions, and other
stresses (Gentry et al. 2004).
However, the mycorrhizosphere represents in most soils an even larger surface
for the establishment of microbial communities than the rhizosphere. The extra-
radical mycelium acts as an extension of the root system and creates a large
interface between hyphae and soil, the mycorrhizosphere that can be defined as
“the zone of soil where the physical, chemical and microbiological processes are
influenced by plant roots and their associated mycorrhizal fungi” (Giri et al. 2005).
Rousseau et al. (1994) estimated, for example, that the extraradical mycelium of
Pisolithus tinctorius represents 99% of the nutrient-absorbing surface length of pine
roots. Mycorrhizal fungi and their extraradical mycelium represent a main part of
the microbial biomass particularly in the top soil, and the fungal biomass in the soil
can reach 700–900 kg ha 1 (Wallander et al. 2001) and accounts for at least 32% of
the total microbial biomass (H€ ogberg and H€ ogberg 2002).
Ectomycorrhizal fungi are part of complex microbial communities in the soil,
and the interactions between the diverse members of this community can be
synergistic, competitive, or antagonistic depending on the species and the soil

conditions. The presence of an ECM fungus leads to quantitative and qualitative
changes in the microbial community composition (Heinonsalo et al. 2004; Assi-
betse et al. 2005) and can result in a shift towards catabolic microbial communities,
and this could also result in changes in the capability of these communities to
degrade pollutants in the soil. ECM roots and the extraradical mycelium are major
sinks for photosynthetically fixed carbon in mycorrhizal root systems and it has
been shown that the quantity and quality of root exudates is altered in response to
ECM colonization. Rygiewicz and Anderson (1994) found that mycorrhizal plants
transfer 23% more carbon from the shoot into the root and that more carbon is
allocated into pools with a higher turnover rate.
Fungal hyphae and the extraradical mycelium release energy-rich compounds
into the mycorrhizosphere and can act as conduit and provide carbon sources to
heterotrophic bacterial and microbial communities and their metabolic activities in
larger distance from the root (Heinonsalo et al. 2004; Finlay 2008). Olsson and
Wallander (1998) reported that the microbial activity in the soil near to mycorrhizal
roots and also in further distance from the root is higher than in noncolonized root
systems. Heinonsalo et al. (2000) examined the bacterial density in different
compartments in microcosms with a petroleum-contaminated soil and found the
lowest number of bacteria in the bulk soil. The rhizosphere and mycorrhizosphere
supported a higher number of bacterial cells and the bacterial density was 4–5 times
higher in the mycorrhizosphere than in the rhizosphere. The mycorrhizosphere
provides an ecological niche and nutritionally favorable conditions for diverse
microbial communities, and transmission electron microscopy of ECM hyphae
from PHC-contaminated soils revealed a microbial biofilm at the soil/fungal inter-
face. Isolates from these communities were able to grow on m-toluate and m-xylene
(two intermediates of BTEX degradation) as sole carbon source and contained
plasmids with marker genes involved in the degradation of mono-aromatics (Sarand
et al. 1998). Sarand et al. (1999) were able to establish in the mycorrhizosphere of
Scots pine and the ECM fungus Suillus bovinus, communities of a Pseudomonas
fluorescens isolate carrying the TOL plasmid pWW0 that encodes enzymes for the
degradation of toluene, p- and m-xylenes, and m-toluate. Plant and fungus alone
were not able to degrade m-toluate, but the bacteria with the catabolic plasmid were
able to degrade this pollutant in the mycorrhizosphere. Free-living microbial com-
munities may act in concert with ECM fungi and facilitate the degradation of
recalcitrant soil pollutants (Sarand et al. 1999; Meharg and Cairney 2000). The
partial degradation of persistent aromatic pollutants by ECM fungi could also
provide intermediate products that can be further broken down by microbial com-
munities in the mycorrhizosphere (Cairney and Meharg 2002).
The ECM colonization can lead not only to quantitative but also qualitative
changes in the bacterial community composition and the competition for limited
soil nutrients, or the biosynthesis of antibiotics by ECM fungi, can also result
in a decrease of the microbial activity in the mycorrhizosphere (Olsson et al.
1996). Assibetse et al. (2005) examined the impact of the ECM fungus Pisolithus
albus on the bacterial community composition in the mycorrhizosphere of Acacia
216 H. B€ucking
auriculiformis and found that the number of actinomycetes was reduced in ECM
root systems. Such antagonistic effects of ECM systems on microbial diversity in
the mycorrhizosphere could potentially also reduce the capability of these commu-
nities to degrade recalcitrant compounds. Saprophytic actinomycetes are involved
in the breakdown of complex biopolymers, such as lignin, hemicelluloses, pectin,
and chitin and have also been shown to be able to degrade recalcitrant pollutants
such as pesticides (De Schrijver and De Mot 1999), hydrocarbons (McCarthy and
Williams 1992), and 1,4-dioxane (Mahendra and Alvarez-Cohen 2005).
10.3.2 Ectomycoremediation of Organic Xenobiotics
White rot fungi (WRF), such as Phanerochaete chrysosporium or Trametes versi-

color, or other saprophytic fungi have often been studied for their potential to
degrade recalcitrant organic pollutants in soils. These saprophytic basidiomycetes
are characterized by their production of extracellular lignin- and humic acid-
degrading enzymes, such as lignin peroxidase, manganese peroxidase, and laccase,
that lack specificity for particular substrates and have been shown to attack a variety
of other compounds, including xenobiotics, such as lindane, benzo(a)-pyrene, and
dichlorodiphenyltrichloroethane (DDT) (Majcherczyk et al. 1993).
ECM fungi play a key role for nutrient cycling in boreal and temperate forest
ecosystems and are also able to utilize complex organic molecules as a source for
reduced carbon (Durall et al. 1994) or other nutrients such as nitrogen or phosphate
(Perez-Moreno and Read 2000). Similar to WRF, ECM fungi also produce a variety
of ligninolytic or cell wall-degrading enzymes (for review see Read and Perez-
Moreno 2003) that could also enable them to metabolize recalcitrant organic
pollutants with structural similarities to lignin, such as PAHs, PCBs, 2,4,6-trinitro-
toluene (TNT), or DDT.
The genes encoding a lignin peroxidase and manganese peroxidase have already
been identified in different orders of ECM fungi, and also a partial laccase sequence
has been described (Chen et al. 2001; Ramesh et al. 2008). This seems to indicate
that the evolutionary relationship between ECM fungi and other saprophytic fungi
is much closer and that a clear distinction into the two different functional groups of
symbiotic or saprophytic fungi is not as clear as previously been suggested (Chen
et al. 2001). However, the activity of ligninolytic enzymes or other phenol oxidiz-
ing enzymes, such as tyrosinase, catechol oxidase, or ascorbate oxidase in ECM
roots or mycelium has mainly been detected in nonsterile soils (e.g., Bending and
Read 1997; Timonen and Sen 1998) and could also have been the result of the
microbial activity of the rhizosphere or mycorrhizosphere.
The ligninolytic and facultative saprophytic lifestyle of ECM fungi is currently
under debate (Baldrian 2009) and it is not clear whether these enzymes will also be
expressed in the symbiotic stage, when the ECM fungus is supplied with carbon by
its host. WRF or ericoid fungi are generally more effective in degrading lignin or
phenolic compounds (Haselwandter et al. 1990; Bending and Read 1997), and
facultative ECM fungi have been shown to have a higher activity than obligate
ECM fungi (Haselwandter et al. 1990). Recent results of Cullings et al. (2008)
indicate that the enzymatic activity of ECM fungi will be affected in the symbiotic
stage. They found that the ECM fungus Suillus granulatus expressed D-glucosidase,
laccase, manganese peroxidase, lignin peroxidase, and protease in symbiosis with
Pinus contorta, and that the activity increased when the photosynthetic capacity of
the host was reduced by partial defoliation. Similar effects of the carbon supply on
the enzymatic activity have also been reported during tree bud break, when the
carbon flux to the mycorrhizal fungus is reduced (Courty et al. 2007).
However, even if ECM fungi have in the symbiotic stage a lower activity of
ligninolytic or phenol oxidizing enzymes than WRF or other wood and straw-
degrading basidiomyetes, the activity of these enzymes may still enable ECM
fungi to successfully degrade organic pollutants in the soil. The sustainability of
ECM fungi in soils may still favor their use in bioremediation over WRF, which are
generally nonedaphic living organisms and require exogenous carbon sources to
facilitate remediation.
10.3.2.1 Petroleum Hydrocarbons
Petroleum hydrocarbons (PHCs) are complex mixtures of numerous alicyclic,

aliphatic, and aromatic compounds and mainly contain paraffins, aromatics,
naphthenics, asphaltenes, and heavy metals in different proportions. Although the
toxicity of individual compounds is known, it is extremely difficult to assess the
toxicity of PHCs, because the chemical composition varies considerably, and our
knowledge about additive, synergistic, or antagonistic effects of the various com-
pounds is limited (Robertson et al. 2007). The inconsistent composition also makes
general statements about potential strategies to remediate contaminated soils
extremely difficult and may have also been the reason why the described effects
of ECM fungi on PHC degradation are inconsistent (see below). An incomplete
biotransformation of PHCs can also lead to potentially toxic metabolites that further
limit the capability of plants, fungi, and other microorganisms to degrade PHCs and
its refined products (Robertson et al. 2007).
PHC contaminations in the soil often cause a decrease in the number of indige-
nous bacteria, followed later by an increase in the number of bacterial species that
can use PHCs as carbon source. Fungi are generally more tolerant to PHCs than
bacteria (Robertson et al. 2007), but ECM fungi differ in their sensitivity. In liquid
cultures, the growth of some ECM fungi is reduced or completely inhibited by
increasing crude oil concentrations, while the growth of other species is stimulated
(Nicolotti and Egli 1998). Crude oil up to a concentration of 50 g kg 1 did not have
an effect on the ECM colonization of Populus and only slightly reduced the ECM
colonization of spruce after a longer exposure (Nicolotti and Egli 1998). Diesel oil
also did not affect the ECM colonization of Populus (Gunderson et al. 2007).
However, changes in the ECM community composition after exposure to PHCs
have been observed (Nicolotti and Egli 1998).
218 H. B€ucking
By contrast, PHCs can significantly reduce the germination and growth of plants
(Nicolotti and Egli 1998; Choi et al. 2005), but the colonization with ECM fungi
can increase the resistance of plants against PHCs. Gunderson et al. (2007) sug-
gested that the positive effect of ECM associations on plant resistance is mainly due
to a better supply with P and N, because the nutrient uptake is often reduced in soils
that are contaminated with hydrocarbons. Despite the positive effect of ECM fungi
on plant growth, the ECM colonization can reduce the capability of plants to
remediate soils that are contaminated with PHCs. The presence of nonmycorrhizal
hybrid poplars led to a significant degradation of diesel oil, but the effect of
mycorrhizal plants was significantly lower (Gunderson et al. 2007). Instead, three
times more PHC was accumulated in mycorrhizal roots, and it has been suggested
that PHCs could get absorbed by hydrophobins in the fungal sheath (Gunderson
et al. 2007). Hydrophobins are fungal cell wall proteins that are involved in cell-to-
cell-surface contact in fungal mycelia and can reduce the apoplastic permeability of
the fungal sheath (B€ ucking et al. 2002), and thereby could also reduce the bioavail-
ability of PHCs for the mycorrhizal host plant. Antagonistic effects of ECM pine
and beech seedlings on the degradation of PHCs have also been described by
Vavrek et al. (2001) and could have also been caused by the competition of ECM
plants and microbial communities for limited soil nutrients (the bioavailability
of phosphate, potassium, calcium, and magnesium in the used soil was low). By
contrast, in forest humus ECM Scots pine seedlings were able to stimulate the
degradation of PHCs. The higher degradation was correlated to increases in the
microbial population and changes in the carbon source utilization patterns of
microbial communities in the mycorrhizosphere (Heinonsalo et al. 2000). The
mycorrhizosphere in PHC-contaminated soil has been shown to support morpho-
logically diverse bacterial populations and microbial biofilms. Isolated bacteria of
these biofilms were able to use m-toluate and m-xylene as sole carbon source and
expressed marker genes that are involved in the degradation of mono-aromatics
(Sarand et al. 1998).
10.3.2.2 Polycyclic Aromatic Hydrocarbons
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that represent

an important risk to the environment and human health (Kuiper et al. 2004; Koivula
et al. 2004). PAHs consist of a diverse group of organic molecules with a wide
range of chemical properties (number of aromatic rings, molecular weight, and
structural configuration), water solubility, and volatility. Particularly, higher-ringed
PAHs are recalcitrant to degradation and include toxic, mutagenic, teratogenic, and
carcinogenic compounds. PAH contaminations of the soil are mainly the result of
leakage from old storage tanks or natural oil reservoirs, oil spills, road surfaces,
domestic waste, incomplete fossil fuel combustion, former gas plant facilities, and
tanker accidents (Morgan and Watkinson 1989).
A wide variety of bacteria, fungi, and algae can degrade and utilize PAHs as a
catabolic carbon source through ring fission. Gramss et al. (1999) tested the ability
of 58 fungi, including 22 ECM fungi and other wood- and straw-degrading,

terricolous, and mitosporic fungi to degrade the PAHs phenanthrene, anthracene,
fluronthene, pyrene, and perylene in liquid cultures. The capability of ECM fungi to
degrade PAHs was generally lower than for wood- and straw-degrading basidio-
mycetes, but the majority of ECM fungi was able to degrade at least four or all of
the tested PAHs to a certain degree. Hebeloma crustuliniforme, H. hienale, and
Lactarius deliciosus showed the highest removal rates of the examined ECM fungi,
and their degradation rates were comparable to rates that have been observed for
wood- and straw-degrading fungi (Gramss et al. 1999). This also confirms the
results of Braun-L€ ullemann et al. (1999), who tested the capability of 27 ECM
strains from 16 different species to degrade benzo(a)pyrene, phenanthrene, pyrene,
and chrysene and found that the majority of ECM fungal species were able to
degrade these pollutants (70% of the fungal species degraded phenanthrene, 65% of
the species pyrene). There were species- and strain-specific differences in the
capability of different ECM fungi, but the degradation potential was independent
of the condensation grade and the number of aromatic rings of the compound. The
degradation, however, was dependent on the nutrient supply. A strain of Suillus
grevillei metabolized more than 50% of the supplied benzo(a)pyrene, phenan-
threne, and pyrene in a nitrogen-depleted medium, but showed no degradation in
a nitrogen-supplied medium (Braun-L€ ullemann et al. 1999).
The described activities, however, were measured under controlled conditions in
pure culture experiments (Braun-L€ ullemann et al. 1999; Gramss et al. 1999), but the
degradation rates in the soil will vary considerably from those in liquid cultures due
to the adsorption to the soil matrix and the low water solubility particularly of PAHs
with a high number of fused rings (Kuiper et al. 2004). Koivula et al. (2004) found
no positive effect of Scots pine and the ECM fungus Paxillus involutus on the
mineralization of pyrene in microcosms with forest humus, and the degradation was
rather reduced by the plant and the ECM fungus. The same fungal species was able
to degrade pyrene under pure culture conditions (Gramss et al. 1999). A reduction
in the degradation of PAHs by plants and their associated ECM communities has
also been observed by Genney et al. (2004) and Joner et al. (2006). Genney et al.
(2004) reported that ECM pine seedlings had no effect on the mineralization of
naphthalene, but reduced the degradation of fluorene by 35% compared to non-
planted microcosms. Instead, both ECM fungi metabolized fluorine to the dead-end
products, 9-fluorenone and 9-hydroxyfluorene, which can themselves cause pro-
blems and could be responsible for the overall lower mineralization rate of fluorene
in the microcosms with ECM plants. A reduced degradation of anthracene, anthra-
quinone, chrysene, and dibenz (a,b)anthracene was also found in the ectomycor-
rhizosphere of Suillus bovinus and may have been the result of a reduced microbial
activity due to the competition for limited nutrients. The microbial degradation of
PAHs has been shown to be dependent on the nutrient supply conditions (Joner
et al. 2002). Also the capability of plants to extract PAHs from the soil can be
affected by a mycorrhizal association. Binet et al. (2000) demonstrated that the
colonization of ryegrass with the AM fungus Glomus mosseae reduced the transport
of PAHs to the shoot, but increased the adsorption to the root system.
220 H. B€ucking
10.3.2.3 Nitro-aromatics
Former production and assembly sites for explosives and ammunition during World
War II are still heavily contaminated with nitro-aromatics such as 2,4,6-trinitrotol-
uene (TNT). The contamination is often heterogeneously distributed in the soil
(Scheibner et al. 1997; Koehler et al. 2002) and the removal of the recalcitrant
xenobiotic TNT has been declared by environmental agencies to an urgent priority
due to its toxicity and its mutagenic impacts. Several ECM fungi have been
tested for their potential to degrade TNT, and the capability to reduce TNT to
aminodinitrotoluenes seems to be a ubiquitous pathway among all ecological and
taxonomic groups of fungi. Based on their observation that especially litter and
wood-degrading fungi showed a high potential to mineralize TNT, Scheibner
et al. (1997) suggested that ligninolytic enzymes (see above) could play a key
role for the mineralization process. By contrast, Meharg et al. (1997b) assumed that
ligninolytic enzymes are not involved in the initial stages of TNT degradation and
that TNT could be reduced via the redox potential at the ECM fungal plasma
membrane. The capability of plants to remediate TNT-contaminated soils depends
on the plant species (Koehler et al. 2002; Schoenmuth and Pestemer 2004), but it
has been shown that the capability of Populus can be increased by the colonization
with an unidentified ECM fungus (presumably Hebeloma sp.) and that the mineral-
ization rate of these ECM systems was higher than for WRF (Dobner 2003). When
14
C-labeled TNT was supplied to the root system, only a small proportion was
transferred to the shoot, and the accumulation into the root biomass did not differ
between mycorrhizal and nonmycorrhizal roots (Dobner 2003). This indicates that
in these microcosm experiments, ECM fungi were able to facilitate the mineraliza-
tion of TNT. However, a similar stimulation in TNT degradation by ECM plants
could not be confirmed by in situ experiments at a former TNT production
site, presumably because the TNT was heterogeneously distributed in the soil
(Dobner 2003).
10.3.2.4 Chlorinated Aromatic Hydrocarbons
Chlorinated aromatic hydrocarbons (CAHs) are highly toxic and represent a major
group of chemicals responsible for the pollution of soils. CAHs, such as chloro-
phenols, chlorobenzenes, chloronitrobenzenes, chloroaniline, and PCBs, are highly
resistant to degradation and can bioaccumulate in human and animal tissue. Many
CAHs have commonly been used as herbicides (e.g., atrazine, 2,4-dichlorophenoxy-
acetic acid or 2,4-D), or as dielectric or hydraulic fluids (PCBs) due to their
chemical and thermal stability. PCBs are considered the most widespread pollutant
on the planet, and were banned in most countries in 1979 (Mackova et al. 2006).
Plants, ECM fungi, ericoid mycorrhizal fungi, and saprophytic wood-degrading
fungi have been shown to degrade a variety of different CAHs, such as atrazine or
2,4-D. The degradation rate depended on the compound, its concentration, the
nutrient availability, and the plant and fungal species, but there was no difference
between saprophytic or symbiotic fungi (Donnelly et al. 1993; Dittmann et al. 2002;
Mackova et al. 2006). Generally, ECM basidiomycetes have a lower capability to
degrade CAHs than WRF or ericoid mycorrhizal fungi (Donnelly et al. 1993;
Dittmann et al. 2002), but the degradation rate of ECM fungi for certain compounds
and concentration ranges is comparable to that of WRF. Donnelly and Fletcher
(1995) screened 21 ECM fungi for their capability to degrade various PCB con-
geners and found that 14 ECM fungi were able to metabolize various PCBs and that
the lower chlorinated congoners were more easily degradable than the higher
chlorinated congoners. The ECM fungus Radiigera atrogleba degraded seven out
of 19 PCB congeners, whereas Phanerochaete chrysosporium, a model organism
for xenobiotic biodegradation studies, only metabolized three out of the 19 con-
geners (Donnelly and Fletcher 1995). The capability of ECM fungi to degrade
CAHs seems to increase when the ECM fungus is in symbiosis with its host
plant. Dittmann et al. (2002) found that Suillus bovinus was not able to degrade
3-chlorobenzoic acid (3-CBA) in pure cultures, but that mycorrhizal pines had a
limited potential to degrade 3-CBA in microcosm experiments. Since the mycor-
rhizal plants were not continuously kept under sterile conditions, the reported
increased degradation in the symbiotic stage could also be the result of microbial
activity in the ectomycorrhizosphere. However, similar effects have also been
reported by Meharg et al. (1997a), who found that the mineralization of 2,4-D by
Paxillus involutus and Suillus variegatus was significantly higher in symbiosis than
under pure culture conditions (these mycorrhizal systems were cultured under
sterile conditions).
A large percentage of the removal of CAHs from the soil matrix by mycorrhizal
systems is not the result of mineralization but also of incorporation into the fungal
and plant biomass (Donnelly et al. 1993; Meharg et al. 1997a; Schnabel and White
2001; Huang et al. 2007). For instance, Meharg et al. (1997a) found that 40% of the
supplied 2,4-D was incorporated into the fungal biomass and not mineralized.
However, incorporation into the fungal biomass would still contribute to a phyto-
stabilization of CAHs in the ectomycorrhizosphere and would thereby also make
CAHs accessible for further microbial degradation. Dittmann et al. (2002) showed
that 3-CBA is not only incorporated into the ECM fungal sheath, but also taken up
and transferred via the stele to the needles. The transport to the above-ground plant
part could contribute to CAH removal from the soil by phytoextraction.
10.3.3 Ectomycoremediation of Heavy Metals
The contamination of soils with the nonradioactive metals arsenic (As), cadmium
(Cd), copper (Cu), mercury (Hg), lead (Pb), and zinc (Zn) and the radioactive
metals strontium (Sr), caesium (Cs), and uranium (U) represents a major environ-
mental and human health problem (Raskin et al. 1997).
Suitable plant species for the phytoremediation of these sites should be able to
extract and to tolerate high heavy metal concentrations and should accumulate these
222 H. B€ucking
metals mainly in their shoot (phytoextraction). However, plants often accumulate

heavy metals in their root system to protect the shoot from toxic heavy metal
concentrations (B€ucking and Heyser 1994). This is the reason why in the past,
studies on the phytoremediation of heavy metal-contaminated soils were mainly
conducted with hyperaccumulating plants, such as Thlaspi caerulescens, which are
able to tolerate and to concentrate high heavy metal concentrations in the shoot
(Robinson et al. 1998). However, the small biomass development of these species
significantly reduces their potential to extract significant amounts of heavy metals
from contaminated soils. Fast growing trees, such as Populus and Salix, could
potentially be used for the phytoremediation of these sites, because (1) both are
known to naturally colonize areas with high metal soil concentrations such as active
and inactive smelter sites (Cripps 2003), (2) they are genetically transformable
(Doty 2008), and (3) the high biomass development can compensate for the
moderate heavy metal concentrations in the shoot (Pulford and Watson 2003;
Tlustoš et al. 2006).
Depending on the conditions, the utilization of mycorrhizal systems can assist
the phytoremediation of heavy metal polluted soils by the positive effect of ECM
fungi on plant tolerance, but could also limit the plants ability to extract heavy
metals from the soil by reducing the uptake and the transfer into the shoot. The
colonization of tree species with ECM fungi is often essential for the reforestation
of old mine sites, because ECM fungi are able to ameliorate the toxicity of heavy
metals (Marx 1975). On the other hand, ECM fungi have been shown to enhance the
uptake of metals by plants particularly when the exogenous supply is low (Colpaert
and Van Assche 1992), but can also reduce the uptake into the plant and the
transport into the shoot when the external supply is high (B€ucking and Heyser
1994; Krznaric et al. 2009). Several mechanisms have been described to be
involved in the reduced heavy metal uptake of ECM plants (1) the larger
cell wall surface that can bind heavy metals, (2) the filter function of the fungal
sheath that restricts the apoplastic movement of heavy metals into the root
cortex, (3) the extracellular precipitation of heavy metals, and (4) the intracellular
chelation and compartmentation with, e.g., polyphosphates in the fungal vacuole
(Hartley et al. 1997).
Whether ECM fungi are able to contribute to the phytoextraction of heavy
metals from the soil despite their widely accepted effect on plant tolerance and
uptake depends on the heavy metal, and the fungal and plant species involved. For
instance, Sell et al. (2005) showed that by the ECM colonization with Paxillus
involutus, the capability of Populus canadensis to extract Cd was increased, but that
the same fungus had no effect on the Cd uptake by Salix viminalis. The effect of P.
involutus on metal uptake and distribution in Salix varied depending on the fungal
strain and its adaptation to heavy metals (Baum et al. 2006). However, the twofold
increase in the capability of mycorrhizal Populus to extract Cd from the soil would
substantially increase the potential of the plant to remove this metal from polluted
soils (Sell et al. 2005).
Zimmer et al. (2009) showed that an ECM fungus can simultaneously increase
the heavy metal tolerance of the plant and the accumulation of heavy metals in the
plant biomass. An ECM colonization of Salix viminalis caprea with Hebeloma

crustuliniforme resulted in an increase in the plant biomass under metal stress, and
increased the Cd and Zn accumulation in the stems. Interestingly, a dual inoculation
of the root system with the bacterial strains Micrococcus luteus and Sphingomonas
sp. 23L can further increase the effect of the ECM fungus on heavy metal accumu-
lation (Zimmer et al. 2009). The increase by a factor of 4.7 and 3.4 for the Cd and
Zn accumulation in the stem, respectively, suggests that a combination of ECM
fungi and associated bacteria may represent a promising approach to increase the
capability of plants to extract heavy metals from the soil. The bacteria could
facilitate the ECM colonization (mycorrhiza helper bacteria) or could increase the
bioavailability of heavy metals for the mycorrhizal plant.
Two thousand fungal strains belonging to 98 genera of fungi have been isolated
from the Chernobyl Atomic Energy Station after its nuclear accident in 1986. Some
isolates showed a growth promotion when exposed to ionizing radiation, and the
hyphal growth was directed towards the radiation source (Zhdanova et al. 2004). By
contrast, control isolates were inhibited or showed no response when exposed to
ionizing radiation (Tugay et al. 2006). Particularly ECM basidiomycetes accumu-
late or hyperaccumulate radionuclides in their fruitbodies, and a directed growth
would allow them to absorb radionuclides from radioactive hotspots in the soil.
Fruitbodies are mainly formed when the fungus is in symbiosis with its host
plant and could be collected from the site to extract radionuclides from the soil.
However, the potential consumption of these fruitbodies by animals or humans also
poses a substantial risk and could transfer these radionuclides into the food chain
(Gray 1998).
10.4 Conclusions
So far, studies about the role of ECM fungi in phytoremediation have mainly
focused on their effect on plant tolerance against environmental stresses and on
recultivation of polluted sites to prevent soil erosion. The impact of ECM commu-
nities on the capability of plants to remediate contaminated sites has only rarely
been studied, and our current understanding of the potential of ECM fungi to
degrade recalcitrant xenobiotics and to facilitate ectomycoremediation is mainly
based on the results of pure culture or microcosm experiments that do not neces-
sarily reflect the potential of ECM communities to degrade pollutants. ECM fungi
can play a key role in the application of woody plant species for the remediation of
polluted sites, based on their ubiquitous nature in soils, their positive effect on
abiotic stress resistance, their impact on microbial and plant communities, and their
capability to degrade recalcitrant xenobiotics. ECM fungi can stimulate the ability
of plants and of microbial communities in the mycorrhizosphere to degrade organic
pollutants (Meharg et al. 1997a; Sell et al. 2005), but can also have an antagonistic
effect on soil remediation (Genney et al. 2004; Joner et al. 2006). And while for
certain pollutants, consistently higher degradation rates by ECM systems were
224 H. B€ucking
found, the results for other contaminants were inconsistent and varied depending on
the plant and fungal species, the nutrient availability in the soil, and its effect on the
microbial community composition. A better understanding of the physiology,
biochemistry, molecular genetics, and regulation of potential degradation pathways
in ECM fungi and mycorrhizal plants and of microbial communities in the mycor-
rhizosphere is crucial for a successful application of these associations for the
phytoremediation of contaminated sites. The potential of plants and their ECM
communities for the remediation of polluted sites depends on several interrelated
factors, such as properties, concentration, toxicity and bioavailability of the pollut-
ant, soil characteristics, such as nutrient and water availability, and the interactions
between ECM plants and indigenous microbial communities in rhizosphere and
mycorrhizosphere and their capability to facilitate remediation. However, the
multifaceted effects of ECM fungi on the capability of plants to remediate soils
and their ubiquitous nature in soils should make them a prime target for further
research to identify efficient and sustainable strategies for the remediation of
contaminated sites by plants and their microbial communities.
References
Assibetse K, Gueye M, Thioulouse J, Duponnois R (2005) Soil bacterial diversity responses to root
colonization by an ectomycorrhizal fungus are not root-growth dependent. Microb Ecol
50:350–359
Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in
rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol 57:233–266
Baldrian P (2009) Ectomycorrhizal fungi and their enzymes in soils: is there enough evidence for
their role as facultative soil saprotrophs? Oecologia 161:657–660
Baum C, Hrynkiewicz K, Leinweber P, Meissner R (2006) Heavy metal mobilization and uptake
by mycorrhizal and non-mycorrhizal willows (Salix dasyclados). J Plant Nutr Soil Sci
169:516–522
Becerra AG, Nouhra ER, Silva MP, McKay D (2009) Ectomycorrhizae, arbuscular mycorrhizae,
and dark-septate fungi on Salix humboldtiana in two riparian populations from central Argen-
tina. Mycoscience 50:343–352
Bending GD, Read DJ (1997) Lignin and soluble phenolic degradation by ectomycorrhizal and
ericoid mycorrhizal fungi. Mycol Res 101:1348–1354
Binet P, Portal JM, Leyval C (2000) Fate of polycyclic aromatic hydrocarbons (PAH) in the
rhizosphere and mycorrhizosphere of ryegrass. Plant Soil 277:207–213
Braun-L€ullemann A, H€ uttermann A, Majchercyzk A (1999) Screening for ectomycorrhizal fungi
for degradation of polycyclic aromatic hydrocarbons. Appl Microbiol Biotechnol 53:127–132
B€
ucking H, Heyser W (1994) The effect of ectomycorrhizal fungi on Zn uptake and distribution in
seedlings of Pinus sylvestris L. Plant Soil 167:203–212
B€
ucking H, Kuhn AJ, Schr€ oder WH, Heyser W (2002) The fungal sheath of ectomycorrhizal roots:
an apoplastic barrier for the entry of calcium, magnesium and potassium into the root cortex.
J Exp Bot 53:1659–1669
Cairney JWG, Meharg AA (2002) Interactions between ectomycorrhizal fungi and soil sapro-
trophs: implications for decomposition of organic matter in soils and degradation of organic
pollutants in the rhizosphere. Can J Bot 80:803–809
Chen DM, Taylor AFS, Burke RM, Cairney JWG (2001) Identification of genes for lignin
peroxidases and manganese peroxidases in ectomycorrhizal fungi. New Phytol 152:151–158
Choi W-J, Chang SX, Hao X (2005) White spruce response to co-composted hydrocarbon-
contaminated drilling waste: effects of compost age and nitrogen fertilization. J Environ
Qual 34:1319–1327
Colpaert JV, Van Assche JA (1992) Zinc toxicity in ectomycorrhizal Pinus sylvestris. Plant Soil
143:201–211
Courty P-E, Bréda N, Garbaye J (2007) Relation between oak tree phenology and the secretion of
organic matter degrading enzymes by Lactarius quietus ectomycorrhizas before and during
bud break. Soil Biol Biochem 39:1655–1663
Cripps CL (2003) Native mycorrhizal fungi with aspen on smelter-impacted sites in the Northern
Rocky Mountains: occurrence and potential use in reclamation. In: Proceedings of the 2003
National meeting of the American Society of Mining and Reclamation and the 9th Billings
Land Reclamation Symposium. American Society for Mining and Reclamation, Lexington,
USA
Cullings K, Ishkhanova G, Henson J (2008) Defoliation effects on enzyme activities of the
ectomycorrhizal fungus Suillus granulatus in a Pinus contorta (lodgepole pine) stand in
Yellowstone National Park. Oecologia 158:77–83
Cunningham SD, Anderson TA, Schwab AP, Hsu FC (1996) Phytoremediation of soils contami-
nated with organic pollutants. Adv Agron 56:55–114
De Schrijver A, De Mot R (1999) Degradation of pesticides by actinomycetes. Crit Rev Microbiol
25:85–119
De Sousa C (2001) Contaminated sites: the Canadian situation in an international context.
J Environ Manage 62:131–154
Dittmann J, Heyser W, B€ ucking H (2002) Biodegradation of aromatic compounds by white rot and
ectomycorrhizal fungal species and the accumulation of chlorinated benzoic acid in ectomy-
corrhizal pine seedlings. Chemosphere 49:297–306
Dobner I (2003) Der Einsatz mykorrhizierter Geh€ olze in biologischen Sanierungsverfahren unter
dem Aspekt TNT-belasteter B€ oden. Ph.D. thesis, University of Bremen, Germany. http://elib.
suub.uni-bremen.de/publications/dissertations/E-Diss696.dobner.pdf. Accessed 30 Jan 2010
Donnelly PK, Fletcher JS (1995) PCB metabolism by ectomycorrhizal fungi. Bull Environ Contam
Toxicol 54:507–513
Donnelly PK, Entry JA, Crawford DL (1993) Degradation of atrazine and 2, 4-dichlorophenox-
yacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro. Appl Environ
Donnelly PK, Hegde RS, Fletcher JS (1994) Growth of PCB-degrading bacteria on compounds
from photosynthetic plants. Chemosphere 28:981–988
Doty SL (2008) Enhancing phytoremediation through the use of transgenics and endophytes. New
Phytol 179:319–333
Doty SL, James CA, Moore AL, Vajzovic A, Singleton GL, Ma C, Khan Z, Xin G, Kang JW, Park
JY, Meilan R, Strauss SH, Wilkerson J, Farin F, Strand SE (2007) Enhanced phytoremediation
of volatile environmental pollutants with transgenic trees. PNAS 104:16816–16821
Durall DM, Todd AW, Trappe JM (1994) Decomposition of 14C-labelled substrates by ectomy-
corrhizal fungi in association with Douglas fir. New Phytol 127:725–729
functional diversity of interactions involving the extraradical mycelium. J Exp Bot
59:1115–1126
Finlay R, S€oderstr€om B (1992) Mycorrhiza and carbon flow to the soil. In: Allen MJ (ed)
Mycorrhizal functioning. Chapman and Hall, New York, pp 134–160
GAO (2004) Brownfield redevelopment. United States Government Accountability Office GAO-
05-94. http://www.gao.gov/new.items/d0594.pdf. Accessed 15 Dec 2009
226 H. B€ucking
Genney DR, Alexander IJ, Killham K, Meharg AA (2004) Degradation of the polycyclic aromatic
hydrocarbon (PAH) fluorine is retarded in a Scots pine ectomycorrhizosphere. New Phytol
163:641–649
Gentry TJ, Rensing C, Pepper IL (2004) New approaches for bioaugmentation as a remediation
technology. Crit Rev Environ Sci Technol 34:447–494
Gerhardt KE, Huang X-D, Glick BR, Greenberg BM (2009) Phytoremediation and rhizoremedia-
tion of organic soil contaminants: potential and challenges. Plant Sci 176:20–30
Gilbert ES, Crowley DE (1997) Plant compounds that induce polychlorinated biphenyl biodegra-
dation by Arthrobacter sp. strain B1B. Appl Environ Microbiol 63:1933–1938
Giri B, Giang PH, Kumari R, Prasad R, Sachdev M, Garg AP, Oelm€uller R, Varma A (2005)
Mycorrhizosphere: strategies and functions. In: Buscot F, Varma A (eds) Soil biology, vol 3,
Microorganisms in soils: roles in genesis and functions. Springer, Berlin, pp 213–252
Goodman DM, Trofymow JA (1998) Comparison of communities of ectomycorrhizal fungi in old-
growth and mature stands of Douglas-fir at two sites on southern Vancouver Island. Can J For
Res 28:574–581
Gramss G, Kirsche B, Voigt K-D, G€ unther T, Fritsche W (1999) Conversion rates of five
polycyclic aromatic hydrocarbons in liquid cultures of fifty-eight fungi and the concomitant
production of oxidative enzymes. Mycol Res 103:1009–1018
Gray SN (1998) Fungi as potential bioremediation agents in soil contaminated with heavy or
radioactive metals. Biochem Soc Trans 26:666–670
Green NA, Meharg AA, Till C, Troke J, Nicholson JK (1999) Degradation of 4-fluorobiphenyl by
mycorrhizal fungi as determined by 19F nuclear magnetic resonance spectroscopy and 14C
radiolabelling analysis. Appl Environ Microbiol 65:4021–4027
Gunderson JJ, Knight JD, Van Rees KCJ (2007) Impact of ectomycorrhizal colonization of hybrid
poplar on the remediation of diesel-contaminated soil. J Environ Qual 36:927–934
Haichar FeZ, Marol C, Berge O, Rangel-Castro JI, Prosser JI, Balesdent J, Heulin T, Achouak W
(2008) Plant host habitat and root exudates shape soil bacterial community structure. ISME J
2:1221–1230
Hartley J, Cairney JWG, Meharg AA (1997) Do ectomycorrhizal fungi exhibit adaptive tolerance
to potentially toxic metals in the environment? Plant Soil 189:303–319
Haselwandter K, Bobleter O, Read DJ (1990) Degradation of 14C-labelled lignin and dehydropo-
lymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi. Arch Microbiol 153:352–354
Heinonsalo J, Jørgensen KS, Haahtela K, Sen R (2000) Effects of Pinus sylvestris root growth and
mycorrhizosphere development on bacterial carbon source utilization and hydrocarbon oxida-
tion in forest and petroleum-contaminated soils. Can J Microbiol 46:451–464
Heinonsalo J, Frey-Klett P, Pierrat J-C, Churin J-L, Vairelles D, Garbaye J (2004) Fate, tree
growth effect and potential impact on soil microbial communities of mycorrhizal and bacterial
inoculation in a forest plantation. Soil Biol Biochem 36:211–216
H€ogberg MN, H€ogberg P (2002) Extramatrical ectomycorrhizal mycelium contributes one-third of
microbial biomass and produces, together with associated roots, half the dissolved organic
carbon in a forest soil. New Phytol 154:791–795
Huang Y, Zhao X, Luan S (2007) Uptake and biodegradation of DDT by 4 ectomycorrhizal fungi.
Sci Total Environ 385:235–241
Joner EJ, Leyval C (2003) Phytoremediation of organic pollutants using mycorrhizal plants: a new
aspect of rhizosphere interactions. Agronomy 23:495–502
Joner EJ, Corgié SC, Amellal N, Leyval C (2002) Nutritional constraints to degradation of
polycyclic aromatic hydrocarbons in a simulated rhizosphere. Soil Biol Biochem 34:859–864
Joner EJ, Leyval C, van Colpaert J (2006) Ectomycorrhizas impede phytoremediation of polycy-
clic aromatic hydrocarbons (PAHs) both within and beyond the rhizosphere. Environ Pollut
142:34–38
Kaldorf M, Fladung M, Muhs H-J, Buscot F (2002) Mycorrhizal colonization of transgenic aspen
in a field trial. Planta 214:653–660
Kamath R, Schnoor JL, Alvarez PJJ (2004) Effect of root-derived substrates on the expression of
nah-lux genes in Pseudomonas fluorescens HK44: implications for PAH biodegradation in the
rhizosphere. Environ Sci Technol 38:1740–1745
Koehler H, Warrelmann J, Frische T, Behrend P, Walter U (2002) In-situ phytoremediation of
TNT-contaminated soil. Acta Biotechnol 22:67–80
Koivula TT, Salkinoja-Salonen M, Peltola R, Romantschuk M (2004) Bioremediation and bio-
degradation. Pyrene degradation in forest humus microcosms with our without pine and its
mycorrhizal fungus. J Environ Qual 33:45–53
Komives T, Gullner G (2006) Dendroremediation: the use of trees in cleaning up polluted soils. In:
Machova M, Dowling D, Macek T (eds) Phytoremediation and rhizoremediation. Springer,
Dordrecht, pp 23–31
Krznaric E, Verbruggen N, Wevers JHL, Carleer R, Vangronsveld J, Colpaert JV (2009) Cd-
tolerant Suillus luteus: a fungal insurance for pines exposed to Cd. Environ Pollut
157:1581–1588
Kuiper I, Lagendijk EL, Bloemberg GV, Lugtenberg BJJ (2004) Rhizomediation: a beneficial
plant-microbe interaction. MPMI 17:6–15
Laureysens I, Bogaert J, Blust R, Ceulemans R (2004) Biomass production of 17 poplar clones in a
short-rotation coppice culture on a waste disposal site and its relation to soil characteristics. For
Ecol Manage 187:295–309
Mackova M, Barriault D, Francova K, Sylvestre M, M€ oder M, Vrchotova B, Lovecka P, Najma-
nová J (2006) Phytoremediation of polychlorinated biphenyls. In: Mackova M, Dowling D,
Macek T (eds) Phytoremediation and rhizoremediation. Springer, Dordrecht, pp 143–167
Mahendra S, Alvarez-Cohen L (2005) Pseudonocardia dioxanivorans sp. nov., a novel actinomy-
cete that grows on 1, 4-dioxane. Int J Syst Evol Microbiol 55:593–598
Majcherczyk A, Zeddel A, Kelschebach M, Loske D, H€uttermann A (1993) Abbau von poly-
zyklischen aromatischen Kohlenwasserstoffen durch Weißf€aulepilze. Bioengineering 2:27–31
Marx DH (1975) Mycorrhizae and establishment of trees on strip-mined land. Ohio J Sci
75:288–297
McCarthy AJ, Williams ST (1992) Actinomycetes as agents of biodegradation in the environment
– a review. Gene 115:189–192
Meharg AA, Cairney JWG (2000) Ectomycorrhizas – extending the capabilities of rhizosphere
remediation? Soil Biol Biochem 32:1475–1484
Meharg AA, Cairney JWG, Maguire N (1997a) Mineralization of 2, 4-dichlorophenol by ecto-
mycorrhizal fungi in axenic culture and in symbiosis with pine. Chemosphere 34:2495–2504
Meharg AA, Dennis GR, Cairney JWG (1997b) Biotransformation of 2, 4, 6-trinitrotoluene (TNT)
by ectomycorrhizal basidiomycetes. Chemosphere 35:513–521
Morgan P, Watkinson PJ (1989) Hydrocarbon degradation in soils and methods for soil treatment.
Crit Rev Biotechnol 8:305–353
Nicolotti G, Egli S (1998) Soil contamination by crude oil: impact on the mycorhizosphere and on
revegetation potential of forest trees. Environ Pollut 99:37–43
Olsson PA, Wallander H (1998) Interactions between ectomycorrhizal fungi and the bacterial
community in soils amended with various primary minerals. FEMS Microbiol Ecol
27:195–205
Olsson PA, Chalot M, Bååth E, Finlay RD, S€ oderstr€
om B (1996) Ectomycorrhizal mycelia reduce
bacterial activity in a sandy soil. FEMS Microbiol Ecol 21:77–86
Perez-Moreno J, Read DJ (2000) Mobilization and transfer of nutrients from litter to tree seedlings
via the vegetative mycelium of ectomycorrhizal plants. New Phytol 145:301–309
Pulford ID, Watson C (2003) Phytoremediation of heavy metal-contaminated land by trees – a
review. Environ Int 29:529–540
Ramesh G, Sweera R, Sudhakara Reddy M (2008) Enhancement of laccase in ectomycorrhizal
fungus Hebeloma cylindrosporum in presence of different substrates. Adv Environ Biol
2:115–120
228 H. B€ucking
Raskin I, Smith RD, Salt DE (1997) Phytoremediation of metals: using plants to remove pollutants
from the environment. Curr Opin Biotechnol 8:221–226
Read DJ, Perez-Moreno J (2003) Mycorrhizas and nutrient cycling in ecosystems – a journey
towards relevance? New Phytol 157:475–492
Robertson SJ, McGill WB, Massicotte HB, Rutherford PM (2007) Petroleum hydrocarbon con-
tamination in boreal forest soils: a mycorrhizal ecosystems perspective. Biol Rev 82:213–240
Robinson B, Leblanc M, Petit D, Brooks RR, Kirkman JH, Gregg PEH (1998) The potential of
Thlaspi caerulescens for phytoremediation of contaminated soils. Plant Soil 203:47–56
Rousseau JVD, Sylvia DM, Fox AJ (1994) Contribution of ectomycorrhiza to the potential
nutrient-absorbing surface of pine. New Phytol 128:639–644
Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of transgenic yellow poplar
for mercury phytoremediation. Nat Biotechnol 16:925–928
Rygiewicz PT, Anderson CP (1994) Mycorrhizae alter quality and quantity of carbon allocated
below ground. Nature 369:58–60
Sadowsky MJ (1999) Phytoremediation: past promises and future practices. In: Bell CR, Brylinsky
M, Johnson-Green P (eds) Microbial biosystems: new frontiers. Proceedings of the 8th
International Symposium on Microbial Ecology, Atlantic Canada Society for Microbial Ecol-
ogy, Halifax
Sarand I, Timonen S, Nurmiaho-Lassila E-L, Koivula T, Haahtela K, Romantschuk M, Sen R
(1998) Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudo-
monads in Scots pine mycorrhizospheres developed on petroleum contaminated soil. FEMS
Microbiol Ecol 27:115–126
Sarand I, Timonen S, Koivula T, Peltola R, Haahtela K, Sen R, Romantschuk M (1999) Tolerance
and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudo-
monads individually and under associative conditions. J Appl Microbiol 86:817–826
Scheibner K, Hofrichter M, Herre A, Michels J, Fritsche W (1997) Screening for fungi intensively
mineralizing 2, 4, 6-trinitrotoluene. Appl Microbiol Biotechnol 47:452–457
Schnabel WE, White DM (2001) The effect of mycorrhizal fungi on the fate of PCBs in two
vegetated systems. Int J Phytoremed 3:203–220
Schoenmuth BW, Pestemer W (2004) Dendroremediation of trinitrotoluene (TNT). Part 2: fate of
radio-labelled TNT in trees. Environ Sci Pollut Res 11:331–339
Sell J, Kayser A, Schulin R, Brunner I (2005) Contribution of ectomycorrhizal fungi to cadmium
uptake of poplars and willows from a heavily polluted soil. Plant Soil 277:245–253
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic, Amsterdam
Taylor AFS, Alexander I (2005) The ectomycorrhizal symbiosis: life in the real world. Mycologist
19:102–112
Taylor JH, Peterson CA (2000) Morphometric analysis of Pinus banksiana Lamb. root anatomy
during a 3-month field study. Trees 14:239–247
Timonen S, Sen R (1998) Heterogeneity of fungal and plant enzyme expression in intact Scots
pine-Suillus bovinus and –Paxillus involutus mycorrhizospheres developed in natural forest
humus. New Phytol 138:355–366
Tlustoš P, Pavlı́ková D, Száková J, Fischerová Z, Balı́k J (2006) Exploitation of fast growing trees
in metal remediation. In: Mackova M, Dowling D, Macek T (eds) Phytoremediation and
rhizoremediation. Springer, Dordrecht, pp 83–102
Trapp S, Karlson U (2000) Phytoremediation organischer Schadstoffe. Z Umweltchem Ökotox
12:245–255
Tsao DT (2003) Overview of phytotechnologies. Adv Biochem Eng Biotechnol 78:1–50
Tugay T, Zhdanova NN, Zheltonozhsky V, Sadovnikov L, Dighton J (2006) The influence of
ionizing radiation on spore germination and emergent hyphal growth response reactions in
microfungi. Mycologia 98:521–527
van der Heijden EW, Vosatka M (1999) Mycorrhizal associations of Salix repens L. communities
in succession of dune ecosystems. II. Mycorrhizal dynamics and interactions of ectomycor-
rhizal and arbuscular mycorrhizal fungi. Can J Bot 77:1833–1841
Vavrek MC, Colgan W III, Campbell WJ (2001) The role of plant-bacteria-fungal interaction in
remediation of oak-hickory-pine systems. Louisiana Applied Oil Spill Research and Develop-
ment Program, OSRADP Technical Report Series, Louisiana
Wallander H, Nilsson LO, Hagerberg D, Bååth E (2001) Estimation of the biomass and seasonal
growth of external mycelium of ectomycorrhizal fungi in the field. New Phytol 151:753–760
Wang J, Zhang Z, Su Y, He W, He F, Song H (2008) Phytoremediation of petroleum polluted soils.
Petrol Sci 5:167–171
Wullschleger S, Meinzer F, Vertessy RA (1998) A review of whole-plant water use studies in
trees. Tree Physiol 18:499–512
Zhdanova NN, Tugay T, Dighton J, Zheltonozhsky V, McDermott P (2004) Ionizing radiation
attracts soil fungi. Mycol Res 108:1089–1096
Zimmer D, Baum C, Leinweber P, Hrynkiewicz K, Meissner R (2009) Associated bacteria
increase the phytoextraction of cadmium and zinc from a metal-contaminated soil by mycor-
rhizal willows. Int J Phytoremed 11:200–213
.
Chapter 11
Metal Elements and the Diversity and Function
of Ectomycorrhizal Communities
Alexander Urban
11.1 Introduction
A few metal elements, Al, Fe, Ca, Na, Mg and K, are major components of the
continental earth crust, while the majority of metals are present in traces only
(Wedepohl 1995). Certain metal elements are required as essential nutrients (e.g.
Na, K, Ca, Mg, Fe, Zn, Cu) while others have no known biological function (e.g. Al,
Pb, Hg). All trace metals can be toxic, depending on concentration and speciation.
Human activities have resulted in the release of trace metals into the environment
far above natural levels. Metal and metalloid pollution is highly persistent and
constitutes a major ecotoxicological concern, particularly in case of highly toxic
elements such as As, Pb, Cd, Cu, Ni and Zn. The natural biogeochemical cycling of
metals, that is the transformation and translocation of metal elements in the
biosphere, has increasingly turned into a recycling of anthropogenic pollutants.
Soils are major sinks of metals emitted by human activities (Berthelsen et al.
1995). The largest quantities of metals are released in the northern hemisphere
(Europe, North America, and, increasingly, Asia; Pacyna and Pacyna 2001). Large
areas of metal-contaminated terrestrial environments are actually or potentially
vegetated with ectomycorrhizal (ECM) forests. Metal-contaminated environments
are dynamic systems, the speciation and compartmentation of metal elements
depends on interactions of physico-chemical soil properties and biotic activities
(Fomina et al. 2007). Recently, free-living and symbiotic fungi have been recog-
nized as important agents in metal biogeochemistry, since they are involved in
metal mobilization, transformation and immobilization (Gadd 2007). Ectomycor-
rhizal fungi (ECMF) are the dominant microbial component of ECM forest soils
and key players in metal biogeochemical cycles in these environments, due to their
important biomass and due to their manifold interactions with the organic and
inorganic soil matrix. Increased interest in ECM–metal interactions was triggered
A. Urban
Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 1030
Vienna, Austria
e-mail: alexander.urban@univie.ac.at

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_11,
232 A. Urban
by the potential applications of ECMF in phytoremediation of polluted sites. The

evolution of metal tolerance in ECMF and potential host protection by ECMF and
the role of ECM fungi in metal biogeochemical cycles (Gadd 2007) were covered
by several reviews (Hartley et al. 1997; Leyval et al. 1997; Godbold et al. 1998;
Jentschke and Godbold 2000; Meharg 2003; Bellion et al. 2006). Here I want to
focus on studies that link various aspects of trace metal toxicity to population
biology, community composition, biodiversity of ECMF and on the integration of
ECMF into food webs.
11.2 Metal Element Cycling in ECM Forests
11.2.1 Biogeochemical Transformations of Metals in ECM

Forest Soil
Transformations and transport of metals are part of natural biogeochemical cycles.

Changes of metal speciation in biochemical cycles have important effects on solubility,
bioavailability and toxicity. The soil pools of trace elements are distributed across
different phases and are associated with different matrices: soil solution, living biomass,
organic matter, organomineral complexes, clay minerals, and hydrous oxides of Al, Fe
and Mn. Main processes that control the distribution, speciation and availability of
metals are adsorption–desorption, precipitation–dissolution, complexation, redox reac-
tions and volatilization (Gadd 2007; Huang 2008). Adsorption to minerals, organic
matter and microbial or plant cell walls limits the transport in aqueous soil solution,
reducing leaching to deeper soil horizons and water bodies. Cu, Pb and Cr are strongly
retained by the forest floor, while Zn and Cd are regarded as more mobile. Fungi can
bind metals to cell walls or extracellular polysaccharides. Precipitation of metals in the
soil solution may occur under the influence of sulphide in reduced environments or by
biotic oxalic acid exudation. Complexation reactions are highly diverse and they may
involve specialized chelates such as siderophores or diverse organic acids (Gadd 2007;
Huang 2008).
11.2.2 Weathering and Mineralization
Weathering of the parental rock matrix is an essential source of most plant nutri-
ents, including biogenic metal elements (Landeweert et al. 2001). Weathering is
important in replacing element losses that may occur naturally or anthropogenically
through leaky nutrient cycles, leaching, removal of biomass, or as a result of forest
fires and subsequent discharge of ashes.
The discovery of micrometer-scale tunnels inside mineral grains (feldspar and
hornblende, which contain K, Ca and Mg) in the elution horizons of podzols led to
11 Metal Elements and the Diversity and Function of Ectomycorrhizal Communities 233
the hypothesis that ECM fungi can actively penetrate and dissolve minerals and
transport nutrients directly from minerals to ECM host plants (Jongmans et al. 1997;
Landeweert et al. 2001). The direct link between rock and trees was thought to
improve tree growth by protecting trees from nutrient deficiency and Al toxicity. It
was demonstrated that certain ECM fungi are able to mobilize P or cationic nutrients
from minerals such as apatite, biotite, goethite and muscovite. Fungal mobilization
and translocation of K, Ca, Mg via ECM hyphae appeared to be driven by reduced
bioavailability of these elements (van Sch€ oll et al. 2008). Pot experiments with
muscovite as only K source demonstrated an increase of weathering rates by tree
seedlings by a factor 1.7, inoculation with Paxillus involutus increased weathering to
a factor of 3.3, compared to abiotic weathering. Inoculation with Suillus bovinus or
Piloderma croceum did not change weathering rates (van Sch€oll et al. 2006). Fungi
target hyphal growth and weathering agents (organic acids, particularly oxalic acid,
and siderophores) at minerals containing nutrient elements (P, K, Ca, Mg). Fungi can
dissolve minerals and metal compounds by acidolysis, complexolysis, redoxolysis
and metal accumulation. Combined acidolysis and complexolysis by organic acids
serving as a source of both protons and ligands was found more effective than
protonation alone (Gadd 2007). The process of heterotrophic leaching had been
first studied from a biotechnological perspective (Burgstaller and Schinner 1993),
before it was recognized as an important ecosystem process. Many minerals contain
both nutrients and potentially toxic metal elements. Weathering, whether mediated
by ECMF or not, is likely to increase the bioavailability of toxic metals. Al, the most
abundant metal element in the earth crust, is toxic when present as free ion. Acidity
increases Al availability but may reduce Al toxicity. Dicarboxylic and tricarboxylic
acids such as oxalic acid and citric acid are involved in both weathering and
detoxification. Complexation with citric acid (Tahara et al. 2005) and precipitation
with oxalic acid are effective means of Al detoxification (Gadd 2007).
(Fomina et al. 2005) inferred metal tolerance of ECM fungi from growth rates in
metal-spiked growth media and found that tolerant strains solubilized metal-con-
taining minerals more effectively. Opposing results were obtained in an experiment
with the ericoid mycorrhizal fungus Oidiodendron maius. Metal-tolerant strains
from polluted soils did hardly respond to ZnO and Zn3(PO4)2 by ligand excretion,
while strains from unpolluted habitats increased ligand excretion and Zn
solubilization (Martino et al. 2003). The lack of responsiveness to Zn minerals in
metal-tolerant Oidiodendron is possibly a strategy to save organic carbon, since
low-molecular weight organic acid (LMWOA) excretion can constitute a major
carbon sink (van Hees et al. 2005). Gibson and Mitchell (2004) confirmed the
Glucose dependency of Zn phosphate solubilization in Hymenoscyphus ericae.
11.2.3 Litter Decomposition
Litter is a major source of nutrients in forest ecosystems and litter decomposi-

tion is essential in nutrient cycling, particularly in ecosystems that are less
234 A. Urban
herbivore-driven. Potentially toxic elements may accumulate in tree foliage and

litter in several ways: through uptake from the soil solution via the xylem mass
flow, through atmospheric deposition onto the tree canopies, or via translocation of
elements from the soil solution to the decomposing litter. Atmospheric deposition is
the main route for anthropogenically mobilized metal elements to enter forest
ecosystems, with tree canopies being important scavengers of pollutants. The
process of litter decomposition involving saprotrophic fungi, bacteria and soil
arthropods results in changes of metal speciation, availability and concentration.
ECM fungi potentially compete with saprotrophic fungi for nutrients released from
decomposed litter and have been reported to partially inhibit litter decomposition
(Gadgil and Gadgil 1971; Koide and Wu 2003). Lindahl et al. (2001) analysed the
transfer of 32P-labelled P in interactions between saprotrophic and ECM mycelia.
The direction and quantity of P transfer was influenced by resource availability.
Recent studies by Lindahl et al. (2007) demonstrated a spatial separation of litter
decomposition and ECM nitrogen uptake. Saprotrophic fungi were essentially
confined to more recently (<4 years) shed litter. Organic carbon was mineralized
during decomposition, while N was retained, most likely within fungal mycelia.
The dwindling source of organic C as expressed by a decreasing C:N ratio was
hypothesized to favour the succession of ECM fungi.
Litter decomposition can be a long-lasting process, particularly in temperature-
and nutrient-limited environments. Brun et al. (2008) measured element contents in
conifer litter bags exposed on forest soils for as long as 8 years and recorded
different types of evolution of mass-normalized concentrations in different groups
of elements. Nutrient elements tended to decrease, along with the unessential
or toxic elements Rb, Sc, Sr and Tl. The concentrations of most unessential
and potentially toxic elements increased during decay. The concentrations of
Cd, Hf, Hg, Ta and Zr initially increased, followed by a net decrease. The anthro-
pogenic component of atmospheric deposition resulted in elevated concentrations
of Pb, Cd and Hg.
11.2.4 Metal Element Cycling and Translocation by ECMF
ECMF are considered highly efficient recyclers of mineral nutrients, which mini-
mize element losses through leaching. Very high affinities to certain nutrients can
result in the uptake of similar non-essential elements, for example Rb and Cs that
are accumulated by certain ECMF along with K. High bioconcentration factors
(BCFs) for certain non-essential trace elements are a side effect of high affinity
essential element capture. The efficiency of ECM element recycling can be illu-
strated by the observation that radioactive Cs has hardly been leached from forest
soils more than 20 years after the fall out impact caused by the accident in the
nuclear power plant in Chernobyl (Dighton et al. 2008). The ability of ECM fungi to
capture cationic nutrient elements and to limit losses due to leaching is particularly
important when the retention capacity of the soil matrix is low and when climatic
conditions enhance the leaching potential.
Fungal translocation of metal elements might explain some surprising results
from both weathering and litter decomposition studies. Budget studies in boreal
podzols indicated an upward transport of Al and Fe from mineral soil to the organic
soil layer, a phenomenon that cannot be explained by passive leaching. Smits and
Hoffland (2009) tested the hypothesis that fungal mycelia in the E horizon
connected to ECM roots in the O horizon might be responsible for the uplift of
Al. A two-compartment in vitro experiment demonstrated that Rhizopogon roseolus
and one isolate of Paxillus involus transported Al, while Piloderma croceum,
Hebeloma longicaudum and Suillus bovinus did not. Ga, an element with similar
properties as Al, was used as a tracer in a pot experiment with Pinus sylvestris
seedlings planted into a reconstructed podzol. Ga uptake by Pinus sylvestris seed-
lings was largest when naturally present ECMF mycelia but not the tree roots had
access to the compartment with Ga added. Comparisons between hyphal transfers
of Ga and Al suggested that Ga is a good proxy for Al.
Scheid et al. (2009) compared accumulation and solubility of metals during the
decomposition of leaf litter harvested from trees grown in non-polluted and pol-
luted sites and exposed in non-polluted and polluted soils. Decomposition rates
were not changed by elevated metal contents, suggesting that the function of
decomposer communities was not affected. Leaves from trees grown on polluted
soil had larger initial Zn, Cd and dissolved organic carbon concentrations. Total
metal contents increased during decomposition in polluted soils. The solubility of
metals decreased over time, indicating that litter acted as a temporal sink for metals
from the soil. The sorbed metals were strongly bound in the litter 2 years after
decomposition. The significant rise of metal contents in uncontaminated litter
exposed on polluted soil documents the tight communication of soil and decom-
posing litter in interchanging elements. The agents of decomposition and metal
translocation were not identified in this study, but it is well established that
saprotrophic fungi have a primordial role in the earlier phases of litter decomposi-
tion, while ECMF take over in later phases.
11.2.5 Relevance of Fungal Soil Metal Transformations
Manifold experiments have demonstrated the potential of ECMF to dissolve nutrient-

containing minerals by means of mechanical and chemical attack. Weathering by
ECMF is fuelled by host tree organic carbon, weathering agents such as LMWOAs are
important sinks of reduced carbon (van Hees et al. 2005). The relative proportion of
ECMF weathering is still a matter of controversy. Earlier estimations that ECMF
contributed to mineral weathering in a podzol via tunnelling alone by 50% (van
Breemen et al. 2000) were probably far too high. Smits et al. (2005) quantified the
contribution of ECM mineral tunnelling to total weathering by image analysis of soil
thin sections and concluded that this process accounted for less than 0.5% of total
236 A. Urban
feldspar weathering in the upper 2 cm of the mineral horizon of the investigated soil.
Surface mineral weathering by ECM fungi was suggested to be quantitatively much
more important than tunnelling (Smits et al. 2005). Pot experiments demonstrated that
ECM weathering by exudation of LMWOAs can be upregulated under deficiency
conditions, this regulatory potential might increase the relevance of ECMF weathering
for forest health (van Sch€oll et al. 2008).
Anthropogenical trace metal contaminations are likely to be metabolized by
ECM forest soils in similar ways like naturally occurring metal species, as long as
the impact on the forest ecosystem does not result in disruption of ecosystem
functions. Berthelsen et al. (1995) investigated a forest in southern Norway that
had been submitted to long-term heavy metal emissions. They concluded that nearly
all Cu in the upper humus layer might be contained in ECM structures, for Zn and Cd
the respective proportions were between 30 and 40%. Only 2% of Pb was found
associated with fungal biomass. Importantly, this study included morphotype-based
identifications of the ECMF and linked the contribution of individual ECMF species
to soil trace metal budgets. The considerable biomass and the larger surface: biomass
ratio of ECMF mycelia compared to host root systems may explain the significant
metal-binding capacity of ECMF. Most quantitative information about adsorption of
trace metals to fungal mycelia was obtained in wastewater treatment studies. High-
affinity adsorption of certain trace metals to the mycelia of fast growing saprotrophic
fungi was found by Kapoor and Viraraghavan (1997). In ECMF, the adsorption
potential is potentially limited by the availability of sorption sites and might be
rapidly saturated in metalliferous soils (Godbold et al. 1998). Precipitation of trace
metals on or near fungal hyphae, typically as oxalates, is not limited by hyphal
surface area.
11.3 Metal Tolerance of ECM Associations
11.3.1 Metal Toxicity
Metal toxicity can interfere with the essential physiological and reproductive
processes in fungi (Gadd 1993; Amir and Pineau 1998). Metal toxicity-based
antimycotica illustrates the relevance of fungal metal sensitivity. Cu(II)sulphate,
a fungicide still used in viticulture, is effective in controlling plant parasitic fungi at
concentrations which are not toxic to the host plant.
Metal toxicity depends largely on speciation. Free metal ions, oxyanions and
certain organic metal compounds such as methylated Hg are particularly toxic
(Gadd 2007).
Mechanisms of metal toxicity comprise the elicitation of oxidative stress (even
in non-redox-active metals such as Cd), depletion of antioxidant pools, competitive
inhibition of the uptake of essential elements, denaturation of proteins, interference
with functional groups of proteins by displacement of essential cationic cofactors,
precipitation of P inducing P deficiency and membrane disruption. Cytoplasmatic
metal homeostasis is an essential cellular function given the dual role of many trace
metals as nutrients and potential toxicants.
11.3.2 Mechanisms of Metal Tolerance in ECMF
Metal tolerance may be defined as the genetically conditioned ability to grow and
reproduce in environments with high concentrations of potentially toxic metals
(Hartley et al. 1997). A metal-tolerant organism must be able to maintain metal
homeostasis in the presence of high metal concentrations, controlling the concen-
trations of free metal ions in the cytosol. A multitude of mechanisms were proposed
to contribute to metal tolerance in ECMF (Hartley et al. 1997; Leyval et al. 1997).
Avoidance mechanisms reduce the exposure of ECMF cells to toxic metals and
limit metal entry into the cell (Gadd 1993; Hartley et al. 1997). Avoidance mechan-
isms include the extracellular biochemical transformations of metals discussed
above and the regulation of metal uptake: extracellular ligation of metal ions to
di- and tricarboxylic acids and chelation with siderophores; extracellular metal
immobilization by adsorption to cell walls, pigments and extracellular polysacchar-
ides (fungal slimes); extracellular precipitation as oxalates; restriction of net metal
uptake by reduced influx or increased efflux, through changes in the activity or
specificity of metal transport channels (Bellion et al. 2006).
Cytoplasmatic and vacuolar sequestration of metals reduces the concentration of
free ions in the cytosol. Mechanism of cellular sequestration and detoxification of
metals comprise cytoplasmatic chelation by thiols (metallothioneins, glutathione
and similar oligopeptides) and metal sequestration in the vacuole. Metal coordina-
tion within cells of ECMF was recently analysed by Fomina et al. (2007).
Increased production of cellular redox buffers such as glutathione and of the
enzyme superoxide dismutase protects the cells from metal-induced oxidative
damage, adding another line of defence against metal toxicity (Ott et al. 2002;
Bellion et al. 2006).
The basic mechanisms of cytoplasmatic metal homeostasis used by fungi are
shared with other eukaryotes. Studies in model organisms indicate that more
cellular functions and molecules are involved in fungal metal tolerance. Kennedy
et al. (2008) found many genes involved in Cd tolerance through a screen of
knockout mutants of Schizosaccharomyces pombe. Their results suggested inter
alia an involvement of coenzyme Q10 (ubiquinone) in Cd tolerance.
Most metal tolerance mechanisms imply metabolic costs: oxalate and exopoly-
saccharide production requires organic carbon and increased efflux by metal transport
channels is energy dependent. Intracellular metal chelation with metallothioneins and
glutathione requires considerable amounts of cysteine. Under acute metal stress,
other cysteine-requiring cellular activities such as hydrophobin synthesis are down-
regulated in support of intracellular thiol levels (Bellion et al. 2006).
Metal adsorption to cell walls does not require additional synthetic effort but
might be of limited relevance due to rapid saturation of potential sorption sites in
238 A. Urban
highly metalliferous soils (Jentschke and Godbold 2000). Down-regulation of the

expression of metal influx channels would save metabolic energy and might be the
most economic adaptation to constantly increased substrate metal concentrations.
The evolution of higher specificity of metal influx channels might improve the
discrimination of non-essential and essential elements, but evidence for such a
process in ECMF is not yet available.
Hartley et al. (1997) and Meharg (2003) pointed out that ECM and ERM fungi
share a long evolutionary history of exposition to toxic metal concentrations, since
ECM forests with understoreys of ERM plants cover huge areas with highly acidic
soils, and soil acidity increases the concentration of free ions of Al, Fe and Mn.
Mechanisms of Al detoxification and of Fe and Mn homeostasis were supposed to
confer cotolerance to other metal elements. More particularly, sites with geogeni-
cally elevated levels or toxic trace metals are potential hotspots for the evolution of
tolerance of certain trace metals. Serpentine soils, the most widespread type
of geogenically trace metal enriched substrate, are characterized by elevated levels
of Mn, Ni and Cr (Urban et al. 2008; Moser et al. 2008). Other types of metallifer-
ous outcrops are relatively rare and more restricted in surface.
The weathering of soil minerals by certain ECM fungi is considered important
for the acquisition of essential cationic elements (van Sch€oll et al. 2008). By fungal
attack on rock, potentially toxic metal ions can be liberated too, depending on the
chemical composition of the rock material. Similar mechanisms are involved in
ECM weathering and in the detoxification of metal ions by fungi, most importantly
the exudation of LMWOAs. Citrate is the main ligand of Al3+ in podzolized forest
soils (Landeweert et al. 2001) and oxalic acid is the main component of mycogenic
precipitates of various metals (Gadd 2007). Extracellular ligation and precipitation,
two essential mechanisms of avoidance of metal toxicity, may thus be an evolu-
tionary by-product of the involvement of ECMF in mineral transformations driven
by nutrient foraging.
Hartley et al. (1997) and Meharg (2003) hypothesized that cotolerance against
several metals was likely to occur and would facilitate the evolution of metal
tolerance. This argument may apply at least to very common mechanisms involving
simple molecules, for example the up-regulation of oxalic acid exudation. On the
other hand, it was frequently observed that increased metal tolerance in ECMF is
metal specific, and that specificity in metal tolerance and local metal exposition can
be correlated. Metal tolerance in Suillus spp. was found strain and metal specific
and could be linked to the respective histories of metal exposure (Adriaensen et al.
2005; Krznaric et al. 2009). Recently, it could be demonstrated that metallothio-
neins of the ECMF Paxillus involutus and Hebeloma cylidrosporum and the
regulation of metallothionein gene expression are metal specific (Bellion et al.
2007; Ramesh et al. 2009). More information on specific mechanisms of metal
homeostasis is available for model organisms. A Cd regulated Cd efflux system
based on a P1B-type ATPase (PCA1) was recently reported from Saccharomyces
cerevisiae (Adle et al. 2006). Lin et al. (2008) discovered that a single amino acid
change in the vacuolar Zn transporter ZRC1 changed the substrate specificity of the
transporter from Zn to Fe.
11.3.3 Protection of Host Trees
Schramm (1966) investigated plant colonization of barren coal mine spoils in

Pennsylvania, a highly acidic substrate with toxic metal concentrations. He
observed that both planted and spontaneously established trees (Pinus spp., Quer-
cus borealis, Betula populifolia) were consistently associated with ECMF. Fruit
bodies of Pisolithus tinctorius, Thelephora terrestris, T. caryophyllea, Astraeus
hygrometricus and Inocybe sp. appeared near young trees established on barren
substrate. The majority of spontaneously established pine seedlings were mycor-
rhized, despite apparent limitation of ECM inoculum. The non-mycorrhized seed-
lings were reported as stunted and chlorotic, in contrast to the mycorrhized
seedlings. Ever since it was observed many times that trees mycorrhized spontane-
ously or inoculated with appropriate ECMF species resist much better to extreme
soil conditions, including metal toxicity. Trees were considered to tolerate metal
contamination by means of phenotypic plasticity and ECM symbiosis. It was
hypothesized that the presumably shorter life cycles of ECM fungi would open
up more opportunity for genetic adaptation. Adaptive genetic change in ECM
communities was considered essential for the understanding of the survival of
ECM tree species challenged with metal toxicity (Wilkinson and Dickinson
1995). This hypothesis raised numerous questions, some of them being still under
discussion.
11.3.4 Are ECMF More Resistant to Toxic Metals

Than Their Host Trees?
Hartley et al. (1997) reviewed available data and concluded that a wide range of
metal sensitivity can be found in both trees and ECMF, but upper tolerance limits
appear to be far lower in the tree species. Growth-inhibiting Cd concentrations
range from 0.3 to 30 mM for non-mycorrhized trees and 0.1 to 90 mM for ECM fungi
in liquid media. In case of Pb exposition, the respective values were 0.5–230 mM for
trees and 125–960 mM for ECMF in liquid media. Hartley et al. (1997) attributed
the higher adaptability of ECMF as expressed by higher upper limits of metal
resistance to the higher diversity of ECMF compared to ECM trees, and not to the
presumably shorter fungal generation cycles (Wilkinson and Dickinson 1995).
11.3.5 Can Metal-Resistant ECMF Confer Resistance

to Their Host Trees?
From a co-evolutionary point of view, it seems obvious that a metal-resistant

ECMF which supplies mineral nutrients to its host and which depends on organic
240 A. Urban
carbon obtained from the host would benefit more if it alleviates metal stress in its
host tree, with other words, selection is likely to favour host protection by ECMF,
especially in pioneer populations, where only one tree individual might be available
as carbon source. However, there is some experimental evidence that the most
resistant ECMF species is not necessarily the most protective one (Jones and
Hutchinson 1986). Godbold et al. (1998) concluded that only in a small number
of experiments, amelioration of metal toxicity could be demonstrated, and that this
was the case for specific metals and certain fungi only. The statement that amelio-
ration of metal toxicity is highly species, strain and metal specific is still valid.
However, it has to be considered that many earlier experiments with negative
results had used ECMF from unpolluted sites, while amelioration of metal toxicity
under experimental conditions had been recorded most often when fungi from
metalliferous were used. Later studies provided unequivocal evidence that certain
ECMF protect their host trees highly efficiently against specific metals (Adriaensen
et al. 2004, 2005, 2006; Krznaric et al. 2009). Given recent evidence that ECMF
communities in highly metalliferous soils can be surprisingly diverse, the question
arises, if all those metal-tolerant ECMF have a similarly beneficial effect on their
host trees. (Colpaert and van Assche 1993) inferred from experimental results in a
semi-hydroponic system that species with abundant mycelia have the most benefi-
cial effect. Field observations suggest that species with abundant mycelia such as
Suillus spp. can be highly metal tolerant and beneficial for their host trees (Colpaert
2008). However, if ECM trees are competent of rewarding the most beneficial
ECMF through selective organic carbon allocation, the diversity of metal tolerant
ECMF might be per se beneficial.
ECM-associated microbes are likely to be important, too. Coinoculation with the
ECM-associated bacterium Pseudomonas putida improved the growth promoting
effect of Amanita rubescens on Pinus sylvestris exposed to Cd (Kozdroj et al.
2007).
11.3.6 ECMF and Host Tree Nutrient Status and Metal Uptake
Many metal polluted sites are poor in essential nutrients, and toxic metals can
interfere with the uptake of essential nutrients. It is not easy to disentangle the
beneficial effects of improved access to nutrients and/or reduced metal uptake. If
revegetation of a devastated area is the first goal, this distinction might seem
secondary. If metal cycles and the potential contamination of food webs or applica-
tions such as phytoextraction are considered, the quantity of metal uptake is of high
practical relevance. Experimental results on the transfer of metal elements to trees
via ECM fungi are manifold, but in many cases the toxic element filter hypothesis
(Turnau et al. 1996) might be applicable. ECM fungi can reduce levels of available
metals in soil by precipitation and by binding to organic compounds (Huang 2008),
they can control symplastic metal transfers and cell wall components with high-
metal affinity are likely to reduce apoplastic transport to the fine root. A clear
amelioration of Cd toxicity in Picea abies seedlings by Paxillus involutus was

found (Godbold et al. 1998), but Cd uptake was not decreased. Shoot metal
concentrations are not necessarily reduced due to ECM colonization, effects on
total shoot metal contents can be very variable. Ahonen-Jonnarth and Finlay (2001)
observed a positive growth response of Ni and Cd exposed Pinus sylvestris seedling
upon inoculation with Laccaria bicolor. Shoot metal concentrations were not
affected, resulting in enhanced total metal uptake. In accumulating tree species,
evidence for an ECM filtering effect may be lacking too (Krpata et al. 2009). Again,
the metal exposition histories of both the ECMF and the host tree are essential to
interpret experimental results.
11.3.7 Can ECM Symbiosis Confer Resistance to Sensitive

Host Tree Genotypes?
The role of host sensitivity in the success of ECM associations was rarely investi-
gated. Brown and Wilkins (1985) found increased Zn tolerance due to ECM
inoculation in both tolerant and non-tolerant Betula. The translocation of Zn to
the shoots of Betula was reduced, but Zn accumulated in the ECM. The differences
of tolerance of the trees as expressed in growth rate and the respective limitations of
leaf Zn concentrations were largely maintained. Adaptive tolerance of metal toxic-
ity was suggested to occur in populations of Pinus ponderosa (Wright 2007) and P.
balfouriana (Oline et al. 2000) growing in serpentine soils, but the potential role of
ECMF was not assessed. Kayama et al. (2006) observed significantly reduced ECM
colonization in non-tolerant Picea abies planted into serpentine soil, while ECM
colonization was not decreased in serpentine adapted Picea glehnii. Two alternative
hypotheses are proposed here to be tested in future studies: (a) metal uptake exceeds
a critical threshold despite ECM colonization due to the low tolerance of non-
adapted trees; (b) the naive, non-adapted host tree fails to select the most beneficial,
toxic metal filtering ECMF via selective carbon allocation. In both cases, the metal
sensitive tree will fail to grow normally despite nutrients offered by metal tolerant
ECMF, the consequent reduction of photosynthesis and carbon supply will reduce
colonization intensity and growth of fungal mycelia and destabilize the symbiosis.
11.4 Population Genetics of Adaptive Metal Tolerance

in ECMF
Metal-contaminated soils are an attractive model system to investigate environment-

driven population genetic processes, and important new insights into the microevo-
lution of adaptive metal tolerance were reported for a few ECM model species. In
subpopulations of the ECM basidiomycete Suillus luteus growing in Zn polluted
242 A. Urban
soils, considerable genetic diversity was found and no reduction of genetic diversity
compared to control populations could be detected using AFLP (Muller et al. 2004)
and microsatellite (Muller et al. 2007) population markers. In contrast to a priori
expectations, there was no evidence for clustering of subpopulations from polluted
vs. unpolluted sites, despite significant differences in metal tolerance. It was con-
cluded that metal pollution had a limited effect on the genetic structure of S. luteus
populations, and that extensive gene flow and a high frequency of sexual reproduc-
tion allowed rapid evolution of tolerance while maintaining high levels of genetic
diversity (Muller et al. 2007).
Adaption to Ni toxicity in naturally metalliferous soils was demonstrated in the
ECM ascomycete Cenococcum geophilum (Gonçalves et al. 2009). Mean in vitro
50% growth-inhibiting concentrations of Ni were about seven times higher in
isolates from serpentine (23.4 mg/ml) than in control isolates (3.38 mg/ml). Further-
more, a marginally significant (P ¼ 0.06) trend towards a negative correlation
between Ni tolerance and growth rates in non-toxic conditions was found. This
trade-off had been postulated earlier (Hartley et al. 1997) in order to explain why
tolerance of metal toxicity fails to become a frequent trait in non-exposed popula-
tions. Moderate costs of metal tolerance are compatible with the observation of
considerable variation in metal tolerance in non-exposed populations (Colpaert
2008). In contrast to the results of Gonçalves et al. (2009), Colpaert et al. (2005)
found no reduction of growth rates at low Zn levels linked to reduced Zn uptake in
Zn-tolerant strains of Suillus spp. However, in vitro experiments can at best
partially reproduce selective forces in ECM symbioses in natural environments.
Results concerning the population genetics of serpentine colonizing C. geophi-
lum are rather contradictory. Panaccione et al. (2001) detected genetic divergence
between C. geophilum from serpentine and from control sites, while Gonçalves
et al. (2007) found no differences linked to serpentine, but this result might be due
to a limited sample size. Furthermore, Douhan et al. (2007) reconfirmed that C.
geophilum s.l. is a species complex and recommended caution when conducting
population genetic studies in C. geophilum due to the risk of comparing unrelated
isolates.
The above-mentioned results suggest that in both anthropogenically and geo-
genically metal-contaminated soils, tolerance of very high levels of toxic metals
can be acquired by adaptive evolution, with or without high rates of genetic
exchange with non-exposed populations. It is not clear if adaptive evolution of
metal tolerance is widespread among ECMF. Strains of Paxillus involutus collected
from Zn polluted sites were as Zn sensitive as control strains (Colpaert 2008). In
certain ECMF species, constitutive levels of metal tolerance seemingly suffice to
survive and compete in contaminated sites, while in others the frequently observed
variation of metal tolerance in non-exposed populations may be the base for rapid
selection of highly metal-tolerant genotypes.
Adaptive evolution of metal tolerance might be compatible with different popu-
lation genetic patterns, respectively, reproductive systems and life history traits.
Suillus luteus is a panmictic, sexually reproducing ECMF, C. geophilum is a
complex of cryptic species possibly lacking a sexual state. Population genetics
might rather be conditioned by life cycle traits than by metal stress. The probability
of evolving distinct, specialized genotypes and genetically isolated populations
might rather be determined by the structure of reproductive systems than by the
nature and intensity of environmental selection pressure. At present, there is no
evidence for serpentine driven speciation in ECMF (Urban et al. 2008) and the
genetic structure of metal-tolerant Suillus luteus is similar like in pseudo-metallo-
phytes (Colpaert 2008).
11.5 Distribution of Metal Elements in ECMF
Most studies on the elemental composition of ECMF were based on sporophores,

motivated by the interest in nutritional value and ecotoxicological concerns, since
wild edible fungi are an important component of human diets in many parts of the
world. Earlier studies on metal contents in macrofungi were reviewed by Kalač and
Svoboda (2000). They summarized that BCFs in wild edible mushrooms were found
high for Cd (50–300), which is probably the most problematic element in mush-
rooms, and Hg (30–500) but low for Pb (102–101). Melgar et al. (2009) confirmed
that all fungal species investigated accumulated Hg (BCF > 1). Highest values
were found in ECM Boletus pinophilus and B. aereus and in saprotrophic Agaricus
macrosporus and Lepista nuda (mean BCF between 300 and 450 in the hymeno-
phore). Other ECM species had generally lower BCF values than saproptrophs.
Borovička and Řanda (2007) found Fe accumulation in Hygrophoropsis aur-
antiaca and Zn accumulation in ECM Russula atropurpurea. Generally, lower Se
acumulation was found in checked ECMF compared to saprotrophs, but some of the
highest Se concentrations were recorded in ECMF (Boletus edulis, Boletus pino-
philus, Amanita strobiliformis, Albatrellus pes-caprae).
Metal hyperaccumulation by fungi was rarely reported. Stijve et al. (1990)
measured As hyperaccumulation in Sarcosphaera coronaria (100–7,000 ppm)
and Borovička et al. (2007) reported Ag hyperaccumulation in Amanita strobili-
formis (mostly 200–700 mg/kg, highest value 1,253; mg/kg; BCF 800–2,500). Ag is
microbicidal at low concentrations. Very high BCFs (about 1,000) were reported
for the K analogue Rb in Suillus grevillei (Chudzynski and Falandysz 2008).
Bioavailability, nutritional value and toxicity of metals and metalloids in ECMF
depend on speciation. Slejkovec et al. (1997) found some relation between As
speciation and phylogenetic relationships in mushrooms. Mutanen (1986) reported
low bioavailability of fungal Se. The metalloid Se is of interest due to frequent Se
deficiency of human nutrition. Serafı́n Muñoz et al. (2006) suggested that a major
part of Se in Pleurotus ostreatus is bound to the cell wall component chitin. Serafı́n
Muñoz et al. (2007) demonstrated a speciation-dependent protective role of Se
against oxidative damage induced by Cd and Ag in liquid cultures of Pleurotus
ostreatus.
Krpata et al. (2009) compared Zn and Cd concentrations in fruit bodies of ECM
fungi and in leaves of their host trees, metal-accumulating Populus tremula, in
244 A. Urban
locations highly contaminated by Pb/Zn smelting. BCFs were based on total metal
concentrations in the mor-type organic layer (BCFtot) or on NH4NO3-extractable
metal concentrations in mineral soil (BCFlab). When plotted on log–log scale, a
linear model described well the decrease of BCFs with increasing soil metal
concentrations. A better correlation was found for BCFlab than for BCFtot. The
observation of decreasing BCFs with increasing substrate metal concentrations is
not uncommon in fungi (Gast et al. 1988) and plants (Langer et al. 2009). Differ-
ences between fungal genera were found in Zn-BCFs but not in Cd-BCFs. Tricho-
loma scalpturatum (762 ppm), Scleroderma verrucosum (598–777 ppm) and
Amanita vaginata (403–571 ppm) had highest Zn concentrations, Laccaria laccata
(12.3–93.3 ppm) and Amanita vaginata (10.1–48.5 ppm) had highest Cd values.
Concentrations of Zn were in the range reported for fungal fruit bodies, Cd con-
centrations were highly elevated. Cd levels above 10 ppm are typically found in
contaminated sites (Gast et al. 1988; Svoboda et al. 2006).
Accumulation and BCFs of Zn and Cd in the host trees were of the same order of
magnitude as in the ECM fungi. Studies on metal element concentrations in
sporophores demonstrated differences in metal affinities between various fungal
species and strains and high metal specificity in fungal BCFs. Fungi growing in
substrates with excessive metal concentrations usually have drastically reduced
BCFs, probably a result of physiological control of metal uptake. The relative
contributions of phenotypic plasticity and population genetic factors to the control
of metal uptake rates as expressed by BCFs may vary according to the species
concerned. Colpaert et al. (2005) found reduced Zn uptake in Zn-tolerant strains of
Suillus spp. at low and high Zn concentrations and concluded that partial Zn
exclusion contributed most to Zn tolerance. Zn tolerance as expressed by reduced
Zn accumulation was specific, the concentrations of other micronutrients were not
affected. Despite reduced Zn uptake in tolerant strains, the Zn concentrations in the
mycelia of all treatments were very high, reaching up to 15.57 mg/g, a concentra-
tion representative of plant hyper-accumulators. Turnau et al. (2001) used micro-
proton-induced X-ray emission (PIXE) true elemental maps to quantify metals in
cryo-fixed S. luteus mycorrhizas collected from Zn wastes. They found similarly
elevated Zn concentration in Suillus rhizomorphs, in average 12.83 mg/g. Colpaert
and van Assche (1992) detected high concentrations of Zn in Suillus ECM grown in
Zn-spiked substrate, while transfer to the host plant remained low. Representative
Zn concentrations in Suillus sporophores are 30–150 mg/kg (Kalač and Svoboda
2000), a value about 100 times lower than in Suillus mycelia. Studies on metal
concentrations in environmental samples of ECM mycelia are still scarce, despite
growing awareness of their ecological significance (Finlay 2008). Wallander et al.
(2003) investigated the elemental composition of ECM mycelia grown in contact
with wood ash or apatite in forest soil. They measured high K accumulation by
mycelium of Suillus granulatus and high concentrations of Ca, Ti, Mn and Pb in
Paxillus involutus rhizomorphs. Piloderma croceum appears to accumulate and
translocate Ca, an element that is scarce in podzols (Blum et al. 2002; Hagerberg
et al. 2005).
11.6 Transfer of Trace Metals to Vertebrate Food Webs

via ECMF
ECM fungi are a part of terrestrial food webs and of various pathways of human
exposure to soil borne contaminants. Apart from direct use of fungi as food, the
consumption of partly fungivorous game may be a major source of contaminant
exposure in some populations.
The knowledge about heavy metal transfers via food webs to game is still
fragmentary, despite concerns about elevated heavy metal concentrations in various
species of game.
Pokorny et al. (2004) found a correlation between amounts of fungal spores and
Hg levels in row deer (Capreolus capreolus) faeces collected in a moderately
polluted area in Slovenia. Fungal spores were present in 89% of all faecal samples.
Following fungal genera were reported: Lycoperdon, Calvatia, Hypholoma, Copri-
nus, Russula, Elaphomyces, Xerocomus, Entoloma, Amanita, Cortinarius, Agari-
cus, Inocybe, Boletus, Macrolepiota, Suillus and Pluteus. Analyses of heavy metal
concentrations in fungal sporophores and in important food plants of row deer
showed that Hg and As concentrations were in average by about two to three orders
of magnitude higher in fungi, and Cd concentrations were elevated by about one
order of magnitude. Pb was accumulated in saprotrophic puffballs (Lycoperdon
perlatum, Calvatia utriformis) only, but these species accounted for a major part
of the spores found in row deer faeces. These results explained well the late aestival
and autumnal increase of Hg observed in row deer kidneys. Surprisingly, no
parallel increase of As levels was found. This might indicate lower bioavailability
of fungal As, or higher relevance of other As sources than those covered by this
study.
Cs transfer from soil to roe deer was suggested by Kiefer et al. (1996), based on
circumstantial evidence such as elevated Cs concentrations in Xerocomus badius,
annual variations of roe deer Cs contamination and correlations with precipitation.
Hohmann and Huckschlag (2005) investigated the pathways of 137Cs contamination
in wild boars in Rhineland-Palatinate, Germany. They found deer truffles (Elapho-
myces granulatus) in significantly higher proportions (average weight proportion
15.3%) in highly contaminated stomach contents (345–1,749 Bq/kg) and in lower
proportions (average weight proportion 1.6%) in less contaminated stomach con-
tents (20–199 Bq/kg, median 20 Bq/kg). 137Cs activity concentrations in Ela-
phomyces granulatus were in average 6,030 Bq/kg (800–18,800 Bq/kg). These
results explain why 137Cs concentrations in wild boar meat remain high in many
regions more than 20 years after the fallout of the Chernobyl nuclear accident. ECM
fungi play a paramount role in the long-term recycling and retention of highly
leachable 137Cs in soil organic matter and as a gateway of this radioactive element
to vertebrate food webs. Top predators such as Lynx lynx may accumulate
extremely high concentrations of 137Cs, up to 125,000 Bq/kg, depending on food
choice (Åhman et al. 2004).
246 A. Urban
11.7 Diversity and Structure of ECM Communities

Exposed to Metal Toxicity
Decreases in species diversity of ECMF are a common result of metal pollution,

suggesting a selective elimination of more sensitive fungi (Gadd 1993; Godbold
et al. 1998). Field studies on sporophore production along heavy-metal gradients in
north and south Sweden found strong evidence for decreasing ECM diversity and
productivity at elevated toxic metal concentrations. Most ECM species were found
to decrease with increasing metal (Cu, Zn) concentration, for example Cantharellus
cibarius, Cortinarius spp., Gomphidius spp., Lactarius spp. and Russula spp.
Among the more tolerant taxa were Albatrellus ovinus, Amanita spp., Cantharellus
tubaeformis, Laccaria laccata, Leccinum spp. (R€uhling et al. 1984; R€uhling and
S€oderstr€om 1990). Colpaert (2008) reported that only four morphotypes of Pinus
sylvestris ECM were detected in the most polluted area along the Zn pollution
gradient in northern Limburg, Belgium. A dark ascomycete from the Rhizoscyphus
ericae aggregate was a frequent mycorrhiza former in the most polluted plots only,
confirming earlier observations by Vralstad et al. (2002).
Recent studies provided deeper insights into the below-ground diversity of
ECMF in geogenically and anthropogenically metal enriched soils. Mleczko
(2004) described 14 ECM morphotypes associated with Pinus sylvestris and Betula
pendula growing on Zn wastes in southern Poland. Staudenrausch et al. (2005)
analysed the ECM community of Birch regenerating on the wastes of a uranium
mine in Thuringia, Germany. Twenty-three ECM morphotypes were distinguished,
14 of them were identified by ITS sequence analysis. Total ECM colonization and
ECM diversity were lowest on a mine heap without organic layer, intermediate on
mine wastes with organic layer and highest in a reference site nearby. All ECMF
dominating on the mine heap were present in the reference site, indicating that fungi
with high resistance against U toxicity and other stress factors associated with
barren substrate had been selected from the local ECM community. Urban et al.
(2008) identified 18 different ECMF from eight fungal orders associated with Pinus
sylvestris in a highly metalliferous serpentine soil. Statistic estimators of species
richness indicated the presence of about 30 (Jackknife estimator) to 40 (Michae-
lis–Menten estimator) ECMF species. Moser et al. (2008) differentiated 18 ECM
morphotypes associated with Quercus garryana. Fifteen of these morphotypes were
found in serpentine soil and 13 in non-serpentine soil. Multivariate analysis
detected no differences between the communities. In contrast to the results of
Urban et al. (2008), pezizalean hypogeous fungi were frequent and diverse. Brear-
ley (2006) found higher percentage ECM colonization and higher ECM diversity in
a pot experiment with Dryobalanops lanceolata (Dipterocarpaceae) grown in Ni-
and Mg-rich ultra-mafic soil compared to seedlings grown in tropical ultisol.
Relatively high diversity of ECMF is not limited to naturally metalliferous serpen-
tine. Hrynkiewicz et al. (2007) detected 14 different ECM fungi, mainly Tomentella
spp. and Cortinariaceae associated with Salix caprea at a heavy metal polluted
former ore mining site near Freiburg in Germany. Krpata et al. (2008) differentiated
as many as 54 ECMF associated with Populus tremula in a severely heavy metal-

contaminated (Pb, Zn, Cd) site. The community was rich in Basidiomycota and
dominated by Cenococcum geophilum and tomentelloid fungi. The study site had a
very long pollution history of about 500 years, and the mining sites were not far
away, hence there had been good opportunity for local microevolution of metal
tolerance in many species.
The finding of phylogenetically diverse ECM communities in metal enriched
soils indicates that the potential of metal tolerance is widespread among ECMF. In
plants on the contrary, the potential to evolve increased metal tolerance appears to
be limited to few phylogenetic groups, for example Brassicaceae or Poaceae. Plant
communities on metalliferous soils such as serpentine are typically limited in
diversity but rich in specialized metallophytes. ECMF communities might be
highly diverse, but no metal-specific fungi have been found as yet. Patterns of
ECM diversity do not seem to agree with patterns of vascular plant diversity in
metalliferous soils.
Are there fungal species, genera or larger phylogenetic groups that are excluded
by metal toxicity? The representation of major phylogenetic groups in ECM com-
munities can be very different in similar edaphic conditions. Urban et al. (2008)
found most major ECMF orders associated with pines and oaks on serpentine,
except Pezizales and Gomphales. Moser et al. (2008) found several pezizalean
ECMF associated with oaks on serpentine. Possibly, the potential of various species
to colonize metalliferous soils is conditioned by the local evolutionary history
rather than by genetic predispositions. At present, our knowledge about constitutive
and adaptive mechanisms of tolerance is limited to a few well-studied model
organisms. It is unknown whether different ECMF species use similar or diverse
tolerance mechanisms, and if their population structures are similar. It appears that
metal-tolerant ECMF genotypes are typically derived from local species pools
(Staudenrausch et al. 2005; Colpaert 2008).
11.8 Biodiversity and Conservation
Geogenically metalliferous sites are potential evolutionary hotspots of metal toler-

ance traits, and they use to harbour unique organisms and communities. The
essential genetic adaptations and innovations that confer metal tolerance are likely
to have evolved originally in naturally metal enriched soils (Ernst 2000). The
proximity of natural outcrops of ore and historic mining and smelting sites might
have facilitated colonization of mine spoils by locally adapted metal-tolerant plants
and fungi. Some historic mining and metal processing sites might merit protection,
like naturally metalliferous soils, since they can host specialized communities of
metal-tolerant plants and fungi.
The effects of widespread background contamination of soils with toxic metals
on ECMF biodiversity, community composition and potential species losses are
difficult to estimate. It can be inferred from the relatively rapid recovery of ECMF
248 A. Urban
diversity in historically polluted sites that many species can adapt to high levels of
toxic metals. If generic, constitutive mechanisms of metal tolerance suffice to resist
moderate levels of metal toxicity, the challenge might not be fatal for most ECM
fungal species. Are toxic trace metals involved in the decline of certain rare ECM
species such as stipitate hydnoids and certain Tricholoma species? The enormous
potential of ECMF from different phylogenetic groups to adapt to metalliferous
soils might suggest that this risk is of minor concern, and that other types of
pollution, particularly N emissions, are more important (Arnolds 1991). However,
rare fungi with small population sizes might lack the genetic diversity necessary to
adapt to metal toxicity. Given the high BCFs of certain fungal species challenged
with elements to which they are not adapted, the accumulation of toxic metal
concentrations cannot be excluded. The effects of experimental Ni pollution on
an ECM community were assessed by Markkola et al. (2002), but the analysis of the
ECM community was too coarse to allow conclusions about the effect of low
pollution levels on rare fungi. Nevertheless, this study design hints at an important
question: what is the fate of biodiversity, when pollution starts? Science usually
enters the stage when public awareness and funding are available, for example
when the results of pollution restrictions should be evaluated. Most studies on
ECMF diversity have assessed effects of historic pollution events, the impact of
ongoing pollution due to emerging industrialization in hitherto uncontaminated
areas is rarely documented, but such studies would be required to know more
about the impact of metal pollution on pristine ECMF communities. The study of
sites with a very long or terminated pollution history rather informs about resilience
of ECMF communities, long term impacts on nutrient cycling (Jamnická et al.
2007) and microevolutionary processes (Muller et al. 2007).
11.9 Conclusions
The effects of metal inputs to ecosystems far above natural levels are a priori
unpredictable. The study of naturally or anthropogenically metal enriched sites,
may assist in understanding the fundamental processes of the biogeochemical
cycling of potentially toxic metals in ecosystems and in modelling the potential
pathways of metal pollutants. The ECM association of trees and fungi is successful
in recycling scarce essential metal elements and in colonizing soils with high levels
of toxic metals. Uptake and translocation of metal elements by ECMF account for
major deviations from simple models of soil metal budgets. Despite considerable
conceptual and analytical progress concerning the fundamental biogeochemical and
cellular processes, the role of ECM fungi in metal cycling and budgets has only
started to be recognized by modellers (Rosling et al. 2009). Only a few studies
attempt to link processes to budgets, and the inference of quantitative information
about the role of ECMF in trace metal cycles is still largely based on microcosm
experiments. New technologies for nano-scale element and isotope analysis may
assist in demonstrating the role of ECMF in metal cycles in the field.
ECMF dispose of a variety of extracellular and cellular metal tolerance and

homeostasis mechanisms. Some of these mechanisms can confer cotolerance of
various metals, while others are metal specific. Metal-adapted ECMF generally
provide better host protection than non-adapted strains.
Accumulation of certain essential and non-essential metals and metalloids (e.g.
K, Rb, Cs, As, Se, Zn, Cd, Hg, Ag) is common in ECMF, while other elements tend
to be excluded (e.g. Al, Pb). BCFs are species, strain and metal specific. A negative
correlation between environmental metal concentrations and BCFs appears to be
the rule. Metal hyperaccumulation is rare in fruit bodies of ECMF but appears to be
rather common in ECM mycelia, which may accumulate a major part of the soil
pools of certain trace metals. The role of ECMF as a nutrient source of rare
elements (e.g. Se) and as an important gateway of toxic metals to vertebrate
foodwebs in polluted areas merits further study.
ECM communities in metalliferous soils are surprisingly diverse, and the diver-
sity of ECMF is likely to be important in alleviating metal toxicity in host trees. The
potential to colonize metalliferous soils is widespread in various phylogenetic
groups of ECMF. The microevolution of metal tolerance does not require popula-
tional differentiation. From a population genetic perspective, metal toxicity is just
another environmental gradient exerting selection pressure. Reports on speciation
in ECMF driven by metal tolerance are lacking thus far.
References
Adle DJ, Sinani D, Kim H, Lee J (2006) A Cadmium-transporting P1B-type ATPase in Yeast
Saccharomyces cerevisiae. J Biol Chem 282:947–955
Adriaensen K, van der Lelie D, Van Laere A, Vangronsveld J, Colpaert JV (2004) A zinc-adapted
fungus protects pines from zinc stress. New Phytol 161:549–555
Adriaensen K, Vralstad T, Noben JP, Vangronsveld J, Colpaert JV (2005) Copper-adapted Suillus
luteus, a symbiotic solution for pines colonizing Cu mine spoils. Appl Environ Microbiol
71:7279
Adriaensen K, Vangronsveld J, Colpaert JV (2006) Zinc-tolerant Suillus bovinus improves growth
of Zn-exposed Pinus sylvestris seedlings. Mycorrhiza 16:553–558
Åhman B, Wright SM, Howard BJ (2004) Radiocaesium in lynx in relation to ground deposition
and diet. Radiat Environ Biophys 43:119–126
Ahonen-Jonnarth U, Finlay RD (2001) Effects of elevated nickel and cadmium concentrations on
growth and nutrient uptake of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings.
Amir H, Pineau R (1998) Effects of metals on the germination and growth of fungal isolates from
New Caledonian ultramafic soils. Soil Biol Biochem 30:2043–2054
Arnolds E (1991) Decline of ectomycorrhizal fungi in Europe. Agric Ecosyst Environ 35:209–244
Bellion M, Courbot M, Jacob C, Blaudez D, Chalot M (2006) Extracellular and cellular mechan-
isms sustaining metal tolerance in ectomycorrhizal fungi. FEMS Microbiol Lett 254:173–181
Bellion M, Courbot M, Jacob C, Guinet F, Blaudez D, Chalot M (2007) Metal induction of a
Paxillus involutus metallothionein and its heterologous expression in Hebeloma cylindros-
porum. New Phytol 174:151–158
250 A. Urban
Berthelsen BO, Olsen RA, Steinnes E (1995) Ectomycorrhizal heavy metal accumulation as a
contributing factor to heavy metal levels in organic surface soils. Sci Total Environ
170:141–149
Blum JD, Klaue A, Nezat CA, Driscoll CT, Johnson CE, Siccama TG, Eagar C, Fahey TJ, Likens
GE (2002) Mycorrhizal weathering of apatite as an important calcium source in base-poor
forest ecosystems. Nature 417:729–731
Borovička J, Řanda Z (2007) Distribution of iron, cobalt, zinc and selenium in macrofungi. Mycol
Prog 6:249–259
Borovička J, Řanda Z, Jelı́nek E, Kotrba P, Dunn CE (2007) Hyperaccumulation of silver by
Amanita strobiliformis and related species of the section Lepidella. Mycol Res 111:1339–1344
Brearley FQ (2006) Differences in the growth and ectomycorrhizal community of Dryobalanops
lanceolata (Dipterocarpaceae) seedlings grown in ultramafic and non-ultramafic soils. Soil
Biol Biochem 38:3407–3410
Brown MT, Wilkins DA (1985) Zinc tolerance of mycorrhizal Betula. New Phytol 99:101–106
Brun CB, Åstr€om ME, Peltola P, Johansson M (2008) Trends in major and trace elements in
decomposing needle litters during a long-term experiment in Swedish forests. Plant Soil
306:199–210
Burgstaller W, Schinner F (1993) Leaching of metals with fungi. J Biotechnol 27:91–116
Chudzynski K, Falandysz J (2008) Multivariate analysis of elements content of Larch Bolete
(Suillus grevillei) mushroom. Chemosphere 73:1230–1239
Colpaert JV (2008) Heavy metal pollution and genetic adaptations in ectomycorrhizal fungi.
In: Simon V Avery, Malcolm Stratford, Pieter Van West (eds) Stress in Yeasts and Ilamentous
Fungi (eds) British Mycological Symposia Series. Academic Press, Elsevier Ltd., London
Colpaert JV, van Assche JA (1992) Zinc toxicity in ectomycorrhizal Pinus sylvestris. Plant Soil
143:201–211
Colpaert JV, van Assche JA (1993) The effects of cadmium on ectomycorrhizal Pinus sylvestris L.
Colpaert JV, Adriaensen K, Muller LAH, Lambaerts M, Faes C, Carleer R, Vangronsveld J (2005)
Element profiles and growth in Zn-sensitive and Zn-resistant Suilloid fungi. Mycorrhiza
15:628–634
Dighton J, Tugay T, Zhdanova N (2008) Fungi and ionizing radiation from radionuclides. FEMS
Microbiol Lett 281:109–120
Douhan GW, Huryn KL, Douhan LI (2007) Significant diversity and potential problems associated
with inferring population structure within the Cenococcum geophilum species complex.
Mycologia 99:812–819
Ernst WH (2000) Evolution of metal hyperaccumulation and phytoremediation hype. New Phytol
146:357–358
functional diversity of interactions involving the extraradical mycelium. J Exp Bot 59:1115
Fomina MA, Alexander IJ, Colpaert JV, Gadd GM (2005) Solubilization of toxic metal minerals
and metal tolerance of mycorrhizal fungi. Soil Biol Biochem 37:851–866
Fomina MA, Charnock J, Bowen AD, Gadd GM (2007) X-ray absorption spectroscopy (XAS) of
toxic metal mineral transformations by fungi. Environ Microbiol 9:308–321
Gadd GM (1993) Interactions of fungi with toxic metals. New Phytol 124:25–60
Gadd GM (2007) Geomycology: biogeochemical transformations of rocks, minerals, metals and
radionuclides by fungi, bioweathering and bioremediation. Mycol Res 111:3–49
Gadgil RL, Gadgil PD (1971) Mycorrhiza and litter decomposition. Nature 233:133
Gast CH, Jansen E, Bierling J, Haanstra L (1988) Heavy metals in mushrooms and their relation-
ship with soil characteristics. Chemosphere 17:789–799
Gibson BR, Mitchell DT (2004) Nutritional influences on the solubilization of metal phosphate by
ericoid mycorrhizal fungi. Mycol Res 108:947–954
Godbold DL, Jentschke G, Winter S, Marschner P (1998) Ectomycorrhizas and amelioration of
metal stress in forest trees. Chemosphere 36:757–762
Gonçalves SC, Portugal A, Gonçalves MT, Vieira R, Martins-Loução MA, Freitas H (2007)
Genetic diversity and differential in vitro responses to Ni in Cenococcum geophilum isolates
from serpentine soils in Portugal. Mycorrhiza 17:677–686
Gonçalves SC, Martins-Loução MA, Freitas H (2009) Evidence of adaptive tolerance to nickel in
isolates of Cenococcum geophilum from serpentine soils. Mycorrhiza 19:221–230
Hagerberg D, Pallon J, Wallander H (2005) The elemental content in the mycelium of the
ectomycorrhizal fungus Piloderma sp. during the colonization of hardened wood ash. Mycor-
rhiza 15:387–392
Hartley J, Cairney JW, Meharg AA (1997) Do ectomycorrhizal fungi exhibit adaptive tolerance to
potentially toxic metals in the environment? Plant Soil 189:303–319
Hohmann U, Huckschlag D (2005) Investigations on the radiocaesium contamination of wild boar
(Sus scrofa) meat in Rhineland-Palatinate: a stomach content analysis. Eur J Wildl Res
51:263–270
Hrynkiewicz K, Haug I, Baum C (2007) Ectomycorrhizal community structure under willows at
former ore mining sites. Eur J Soil Biol 44:37–44
Huang PM (2008) Impacts of physicochemical-biological interactions on metal and metalloid
transformations in soils: an overview. In: Violante A, Huang PM, Gadd GM (eds) Biophysico-
chemical processes of heavy metals and metalloids in soil environments. Wiley, Hoboken, NJ,
pp 3–52
Jamnická G, Bučinová K, Havranová I, Urban A (2007) Current state of mineral nutrition and risk
elements in a beech ecosystem situated near the aluminium smelter in Žiar nad Hronom,
Central Slovakia. For Ecol Manage 248:26–35
Jentschke G, Godbold DL (2000) Metal toxicity and ectomycorrhizas. Physiol Plant 109:107–116
Jones MD, Hutchinson TC (1986) The Effect of Mycorrhizal Infection on the Response of Betula
papyrifera to Nickel and Copper. New Phytol 102:429–442
Jongmans AG, van Breemen N, Lundstrom U, van Hees PAW, Finlay RD, Srinivasan M, Unestam
T, Giesler R, Melkerud P, Olsson M (1997) Rock-eating fungi. Nature 389:682–683
Kalač P, Svoboda L (2000) A review of trace element concentrations in edible mushrooms. Food
Chem 69:273–281
Kapoor A, Viraraghavan T (1997) Heavy metal biosorption sites in Aspergillus niger. Bioresour
Technol 61:221–227
Kayama M, Choi D, Tobita H, Utsugi H, Kitao M, Maruyama Y, Nomura M, Koike T (2006)
Comparison of growth characteristics and tolerance to serpentine soil of three ectomycorrhizal
spruce seedlings in northern Japan. Trees 20:430–440
Kennedy PJ, Vashisht AA, Hoe K, Kim D, Park H, Hayles J, Russell P (2008) A genome-wide
screen of genes involved in cadmium tolerance in Schizosaccharomyces pombe. Toxicol Sci
106:124–139
Kiefer P, Pr€ohl G, M€uller H, Lindner G, Drissner J, Zibold G (1996) Factors affecting the transfer
of radiocaesium from soil to roe deer in forest ecosystems of Southern Germany. Sci Total
Environ 192:49–61
Koide RT, Wu T (2003) Ectomycorrhizas and retarded decomposition in a Pinus resinosa
plantation. New Phytol 158:401–407
Kozdroj J, Piotrowska-Seget Z, Krupa P (2007) Mycorrhizal fungi and ectomycorrhiza associated
bacteria isolated from an industrial desert soil protect pine seedlings against Cd (II) impact.
Krpata D, Peintner U, Langer I, Fitz WJ, Schweiger P (2008) Ectomycorrhizal communities
associated with Populus tremula growing on a heavy metal contaminated site. Mycol Res
112:1069–1079
Krpata D, Fitz W, Peintner U, Langer I, Schweiger P (2009) Bioconcentration of zinc and
cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution.
Environ Pollut 157:280–286
Krznaric E, Verbruggen N, Wevers JH, Carleer R, Vangronsveld J, Colpaert JV (2009) Cd-tolerant
Suillus luteus: a fungal insurance for pines exposed to Cd. Environ Pollut 157:1581–1588
252 A. Urban
Landeweert R, Hoffland E, Finlay RD, Kuyper TW, van Breemen N (2001) Linking plants to
rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 16:248–254
Langer I, Krpata D, Fitz WJ, Wenzel WW, Schweiger PF (2009) Zinc accumulation potential and
toxicity threshold determined for a metal-accumulating Populus canescens clone in a dose-
response study. Environ Pollut 157:2871–2877
Leyval C, Turnau K, Haselwandter K (1997) Effect of heavy metal pollution on mycorrhizal
colonization and function: physiological, ecological and applied aspects. Mycorrhiza
7:139–153
Lin H, Kumanovics A, Nelson JM, Warner DE, Ward DM, Kaplan J (2008) A single amino acid
change in the yeast vacuolar metal transporters Zrc1 and Cot1 alters their substrate specificity.
J Biol Chem 283:33865–33873
Lindahl BD, Stenlid J, Finlay R (2001) Effects of resource availability on mycelial interactions and
32P transfer between a saprotrophic and an ectomycorrhizal fungus in soil microcosms. FEMS
Microbiol Ecol 38:43–52
Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, H€ ogberg P, Stenlid J, Finlay RD (2007) Spatial
separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New
Phytol 173:611–620
Markkola AM, Ahonen-Jonnarth U, Roitto M, Str€ ommer R, Hyv€arinen M (2002) Shift in ecto-
mycorrhizal community composition in Scots pine (Pinus sylvestris L.) seedling roots as a
response to nickel deposition and removal of lichen cover. Environ Pollut 120:797–803
Martino E, Perotto S, Parsons R, Gadd GM (2003) Solubilization of insoluble inorganic zinc
compounds by ericoid mycorrhizal fungi derived from heavy metal polluted sites. Soil Biol
Biochem 35:133–141
Meharg A (2003) The mechanistic basis of interactions between mycorrhizal associations and
toxic metal cations. Mycol Res 107:1253–1265
Melgar MJ, Alonso J, Garcia MA (2009) Mercury in edible mushrooms and underlying soil:
bioconcentration factors and toxicological risk. Sci Total Environ 407:5328–5334
Mleczko P (2004) Mycorrhizal and saprobic macrofungi of two zinc wastes in southern Poland.
Acta Biol Crac Ser Bot 46:25–38
Moser AM, Frank JL, D’Allura JA, Southworth D (2008) Ectomycorrhizal communities of
Quercus garryana are similar on serpentine and nonserpentine soils. Plant Soil 315:185–194
Muller LAH, Lambaerts M, Vangronsveld J, Colpaert JV (2004) AFLP-based assessment of the
effects of environmental heavy metal pollution on the genetic structure of pioneer populations
of Suillus luteus. New Phytol 164:297–303
Muller LAH, Vangronsveld J, Colpaert JV (2007) Genetic structure of Suillus luteus populations
in heavy metal polluted and nonpolluted habitats. Mol Ecol 16:4728–4737
Mutanen M (1986) Bioavailability of selenium in mushrooms, Boletus edulis, to young women.
Int J Vitam Nutr Res 56:297–301
Oline DK, Mitton JB, Grant MC (2000) Population and subspecific genetic differentiation in the
Foxtail pine (Pinus balfouriana). Evolution 54:1813–1819
Ott T, Fritz E, Polle A, Sch€
utzend€ubel A (2002) Characterisation of antioxidative systems in the
ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to
cadmium. FEMS Microbiol Ecol 42:359–366
Pacyna JM, Pacyna EG (2001) An assessment of global and regional emissions of trace metals to
the atmosphere from anthropogenic sources worldwide. Environ Rev 9:269–298
Panaccione DG, Sheets NL, Miller SP, Cumming JR (2001) Diversity of Cenococcum geophilum
isolates from serpentine and non-serpentine soils. Mycologia 93:645–652
Pokorny B, Al Sayegh-Petkovšek S, Ribarič-Lasnik C, Vrtačnik J, Doganoc DZ, Adamič M (2004)
Fungi ingestion as an important factor influencing heavy metal intake in roe deer: evidence
from faeces. Sci Total Environ 324:223–234
Ramesh G, Podila GK, Gay G, Marmeisse R, Reddy MS (2009) Different patterns of regulation for
the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindros-
porum. Appl Environ Microbiol 75:2266–2274
Rosling A, Roose T, Herrmann AM, Davidson FA, Finlay RD, Gadd GM (2009) Approaches to
modelling mineral weathering by fungi. Fungal Biol Rev 23:138–144. Available at: http://
linkinghub.elsevier.com/retrieve/pii/S1749461309000207.
R€
uhling Å, S€oderstr€om B (1990) Changes in fruitbody production of mycorrhizal and litter
decomposing macromycetes in heavy metal polluted coniferous forests in North Sweden.
Water Air Soil Pollut 49:375–387
R€
uhling Å, Bååth E, Nordgren A, S€oderstr€om B (1984) Fungi in metal-contaminated soil near the
Gusum Brass Mill, Sweden. Ambio 13:34–36
Scheid S, G€unthardt-Georg MS, Schulin R, Nowack B (2009) Accumulation and solubility of
metals during leaf litter decomposition in non-polluted and polluted soil. Eur J Soil Sci
60:613–621
Schramm JR (1966) Plant Colonization Studies on Black Wastes from Anthracite Mining in
Pennsylvania. American Philosophical Society. Available at: http://www.jstor.org/stable/
1006024. Accessed 3 Dec 2009
Serafı́n Muñoz AH, Kubachka K, Wrobel K, Gutierrez Corona JF, Yathavakilla SKV, Caruso JA,
Wrobel K (2006) Se-enriched mycelia of Pleurotus ostreatus: distribution of selenium in cell
walls and cell membranes/cytosol. J Agric Food Chem 54:3440–3444
Serafı́n Muñoz AH, Wrobel K, Gutierrez Corona JF, Wrobel K (2007) The protective effect of
selenium inorganic forms against cadmium and silver toxicity in mycelia of Pleurotus ostrea-
tus. Mycol Res 111:626–632
Slejkovec Z, Byrne AR, Stijve T, Goessler W, Irgolic KJ (1997) Arsenic compounds in higher
fungi. Appl Organomet Chem 11:673–682
Smits MM, Hoffland E (2009) Possible role of ectomycorrhizal fungi in cycling of aluminium in
podzols. Soil Biol Biochem 41:491–497
Smits MM, Hoffland E, Jongmans AG, van Breemen N (2005) Contribution of mineral tunneling
to total feldspar weathering. Geoderma 125:59–69
Staudenrausch S, Kaldorf M, Renker C, Luis P, Buscot F (2005) Diversity of the ectomycorrhiza
community at a uranium mining heap. Biol Fertil Soils 41:439–446
Stijve T, Vellinga E, Herrmann A (1990) Arsenic accumulation in some higher fungi. Persoonia
14:161–166
Svoboda L, Havlı́cková B, Kalac P (2006) Contents of cadmium, mercury and lead in edible
mushrooms growing in a historical silver-mining area. Food Chem 96:580–585
Tahara K, Norisada M, Tange T, Yagi H, Kojima K (2005) Ectomycorrhizal association enhances
Al tolerance by inducing citrate secretion in Pinus densiflora. Soil Sci Plant Nutr 51:397–403
Turnau K, Kottke I, Dexheimer J (1996) Toxic element filtering in Rhizopogon roseolus/Pinus
sylvestris mycorrhizas collected from calamine dumps. Mycol Res 100:16–22
Turnau K, Przybylowicz WJ, Mesjasz-Przybylowicz J (2001) Heavy metal distribution in Suillus
luteus mycorrhizas – as revealed by micro-PIXE analysis. Nucl Instrum Methods Phys Res
Sect B 181:649–658
Urban A, Puschenreiter M, Strauss J, Gorfer M (2008) Diversity and structure of ectomycorrhizal
and co-associated fungal communities in a serpentine soil. Mycorrhiza 18:339–354
van Breemen N, Finlay R, Lundstr€ om U, Jongmans AG, Giesler R, Olsson M (2000) Mycorrhizal
weathering: a true case of mineral plant nutrition? Biogeochemistry 49:53–67
van Hees PA, Jones DL, Finlay R, Godbold DL, Lundstr€ om US (2005) The carbon we do not see –
the impact of low molecular weight compounds on carbon dynamics and respiration in forest
soils: a review. Soil Biol Biochem 37:1–13
van Sch€oll LV, Smits MM, Hoffland E (2006) Ectomycorrhizal weathering of the soil minerals
muscovite and hornblende. New Phytol 171:805–814
van Sch€oll LV, Kuyper T, Smits M, Landeweert R, Hoffland E, Breemen N (2008) Rock-eating
mycorrhizas: their role in plant nutrition and biogeochemical cycles. Plant Soil 303:35–47
Vralstad T, Myhre E, Schumacher T (2002) Molecular diversity and phylogenetic affinities of
symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats.
254 A. Urban
Wallander H, Mahmood S, Hagerberg D, Johansson L, Pallon J (2003) Elemental composition of

ectomycorrhizal mycelia identified by PCR-RFLP analysis and grown in contact with apatite or
wood ash in forest soil. FEMS Microbiol Ecol 44:57–65
Wedepohl H (1995) The composition of the continental crust. Geochim Cosmochim Acta
59:1217–1232
Wilkinson DM, Dickinson NM (1995) Metal resistance in trees: the role of mycorrhizae. Oikos
72:298–300
Wright J (2007) Local adaptation to serpentine soils in Pinus ponderosa. Plant Soil 293:209–217
Chapter 12
A Conceptual Framework for Up-Scaling
Ecological Processes and Application
to Ectomycorrhizal Fungi
Virgil Iordache, Erika Kothe, Aurora Neagoe, and Felicia Gherghel
12.1 Introduction
Molecular biologists do not attempt to simply up-scale knowledge concerning

macromolecules to the scale of organisms, but use intermediary theories, while
ecologists make intensive efforts to up-scale knowledge concerning the functioning
of tiny organisms to the large scale of ecosystems and landscapes. There is an
implicit recognition of the fact that the first case (up-scaling from macromolecules
to organisms) represents a nested hierarchy of systems, with each hierarchical level
characterized by new, emergent, and in principle irreducible properties (although
reduction attempts are heuristically valuable). In the second case (up-scaling from
tiny organisms to ecosystems), the underlying assumption is that ecological pro-
cesses are basically of the same type over a large range of scales. The problem is to
find a meaningful way to aggregate the processes over different hierarchical levels.
Hence the question: Is the concept “hierarchy of systems” consistent in biological
sciences?
Mycorrhizal fungi, particularly the ectomycorrhizal fungi – (EMF), are well
suited to study up-scaling because of their important role in controlling the func-
tioning of forest ecosystems in view of climate change (see O’Neill et al. 1991).
Recent reviews deal with methodological aspects of the problem (Pickles et al.
2009) and with mechanisms controlling structural changes of the communities over
V. Iordache (*)
Department of Systems Ecology, University of Bucharest, Bucharest, Romania
e-mail: virgil.iordache@g.unibuc.ro
E. Kothe
Microbial Phytopathology, Institute of Microbiology, Friedrich Schiller University, Jena, Germany
A. Neagoe
Research Center for Ecological Services (CESEC), Faculty of Biology, University of Bucharest,
Bucharest, Romania
F. Gherghel
UMR 1136 Interactions Arbres/Microorganismes, INRA 54280, Champenoux, France

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_12,
256 V. Iordache et al.
a wide range of scales (Wolfe et al. 2009). Wolfe et al. (2009) briefly discuss the
functioning of mycorrhizal communities only to underline the need of research in
this area.
In the present chapter, we focus on the functioning of EMF over scales, which,
however, needs a clear concept about the structural aspects. The goal of the text is to
provide an analytical framework for up-scaling processes involving EMF. Such a
framework has not been proposed in literature but can be developed and then
checked for consistency with the existing literature. As the processes involving
EMF are ecological, one cannot look for the existence of an analytical framework
specific only to EMFs. Consequently, we present a general up-scaling framework
and then apply it to EMF.
We start with an overview of approaches to ecological up-scaling (Sect. 12.2),
introduce a framework (Sect. 12.3), and within this framework identify the relevant
scales for EMF functioning (Sect. 12.4.1) from a structural (Sect. 12.4.1.1) and
functional (Sect. 12.4.1.2) point of view. The problems in interpretation of EMF
responses to disturbances and their succession over scales (Sect. 12.4.2), of their
management for ecosystem services (Sect. 12.4.3), and modeling (Sect. 12.4.4)
provide a complementary perspective. Research directions are presented (Sect. 12.5)
and conclusions end the text.
12.2 Addressing the Up-Scaling Problem
The idea to understand the relationship between patterns and processes at various
scales within a hierarchy of nested systems is currently common to all ecological
disciplines, including EMF ecology (Dahlberg 2001). The study of this relationship
can start with the pattern (detection, description, and analyses of the underlying
process) or with the process (description, simulation, and pattern generation)
(Schroder and Seppelt 2006). The second approach is more interesting scientifically
because it allows for mechanistic, theory-based research questions, a recognized
need in the field of EMF ecology (Bruns and Kennedy 2009).
An objective hierarchy of systems has widely been assumed, but some
researchers have arrived at the conclusion that “ecological hierarchy and asso-
ciated scales do not exist per se; rather they represent an instrument constructed by
the observer or modeler” (Lischke et al. 2006), and “hierarchy theory is but one
way of viewing nature” (O’Neill 1996). Indeed, if one looks at the hierarchical
approaches available in the literature, there is no general approach to the scale
problem. The classic hierarchy of increasing scale systems accepted by most
researchers is organism–population–community–ecosystem–landscape. The diver-
sity of views comes when putting into practice this formal pattern. This can be
illustrated by the study of Bailey (1987), looking for objective criteria for deli-
neating large scale landscapes (ecoregions). The spatial scales of the landscape
units are the plant (10–4–101 m2), the patch (100–104 m2), the flowpath
12 A Conceptual Framework for Up-Scaling Ecological Processes 257
(102–105 m2), the landscape (integrative flow system 106–4 106 m2), and the
region (107–1010 m2) (Reynolds and Wu 1999). According to these authors, the
ecosystem concept fits in the patch level (patch ecosystems form a flowpath).
At the ecosystem scale, the search for objective criteria is related to the delineation
of boundaries between ecosystems (e.g., Fortin et al. 2000). Within ecosystems,
food webs “are portrayed as static networks with highly aggregated trophic groups
over broad scales of time and space” (Berg and Bengtsson 2007). Rillig (2004)
uses three hierarchical levels when conceiving the effect of mycorrhizal fungi on
ecosystem processes: the mycorrhizal fungi, the plants (community and composi-
tion), and the ecosystem. Within the soil compartment of terrestrial ecosystems,
Miller and Lodge (1997) use a complex hierarchy of soil ecology systems, starting
with detritusphere, aggregatusphere, and rhizosphere, continuing with drillosphere
and porosphere, and ending with the soil as a whole. When studying the role of
arbuscular mycorrhizal fungi (AMF) in controlling the effects of CO2 on plants
and ecosystems, Rillig and Allen (1999) use a hierarchy with two branches: host
plant–plant population–plant community and host plant–functional group–ecosys-
tem. Ettema and Wardle (2002) identify as relevant for soil ecology four scales:
the fine scale (at root, organic particle and soil structure level), the plot scale,
the field scale, and the large scale (gradients of texture and soil carbon, topogra-
phy, and vegetation). No explicit dimensions in space and time are given for
these scales, but they also associate specific ecological processes to each of these
scales and discuss mechanisms of process’ disturbance at each scale and point
out qualitatively the connection between scales (“spatial distribution of soil organ-
isms influence both plant growth and plant community structure”). Scaling is
also related to the research on species–area relationships (Dengler 2009a), includ-
ing those of microbes (Zhou et al. 2008); these relationships depend both
on grain size and on the type of organism (Rahbek 2005). A review on the
sampling and mathematical issues of this research area (Dengler 2009b) suggests
a stratified approach at large scale (by environmental gradients, vegetation types,
and landscape sectors) coupled with a nested one at small scale with five subscales
(0.01, 0.1, 1, 10, and 100 m2), thus at least six scales in all (five small and one
large).
The limits of such approaches are:
l If the hierarchy does not exist per se, then there is no objective character of the
entities at the hierarchical levels used, and there is no objective knowledge. This
is a conclusion that is difficult to accept within a science.
l If the ecosystems and landscapes include both abiotic and biotic parts, it is
not clear why the classical nested hierarchy includes only biological sys-
tems (organisms) as its elementary subsystems. Not including the abiotic
factors at all levels leads to bizarre conclusions that the elementary units of
the landscapes, such as the “area” proposed by Lepczyk et al. (2008) in a
landscape ontology, refer only to biological systems such as populations and
communities, and the abiotic factors appear only at larger scales (ecosystem
and landscape).
l The aggregated trophic groups in ecosystems, including those of soil (Klironomos

et al. 1999), are characterized by very different biomass turnover rates and
species’ dynamics in time; so it is not appropriate to treat the food web as
characterized by an aggregated space-time scale.
l Stating that there is an “influence” of processes occurring at different scales is
far from being enough because this influence depends also on the plastic
phenotypic answer of organisms to soil heterogeneity (Maestre et al. 2003).
l An explicit statement of the time scale of the pattern is needed as well because
the patterns can change in time in function of cyclic (e.g., seasonal) or long
term driving forces (Wong and Asseng 2006). The evolutionary time scale
should not be forgotten because evaluating the significance of biodiversity
only at the time scale of ecosystems may lead to the unproductive (from
conservation point of view) idea of functional redundancy not only in general
but also in the case of soil (Beare et al. 1995).
Pickles et al. (2009) formulate in the end of their review a number of research
questions, the first one of which is crucial for linking space-time scale with
function: what constitutes a belowground community of EMF, and is it possible
to determine the limits of a given “community”? In the next part, we will try to
answer this question and to produce a theoretical framework surpassing the limits of
existing approaches. Our method follows and generalizes Pahl-Vostl (1995), who
came up with a singular approach for identifying ecosystems and clearly delineat-
ing communities. Besides the functional niche in differentiating the modules, she
proposes the use of biomass turnover rate (inversely related to the length of the
life cycle) and of the location in space-time. The application of these criteria would
lead to a “trophic-dynamic module” (TDM). She defines a TDM as the group of
biological populations having (1) similar rates of biomass cycling (inversely corre-
lated with lifetime of the individuals), (2) the same location in space and time, and
(3) similar roles of the species in the food web. Application of criterion one leads to
dynamic classes of populations, and further, application of criterion two leads to
dynamic modules, which by criterion three are split into TDMs. Pahl-Vostl’s
method of systems identification in this formulation still has two problems (a) it
does not explicitly address the abiotic part of the ecosystem, so productivity can be
described only at large ecosystem scales (or the problem is to exactly up-scale from
smaller scale to ecosystem scale), and (b) some populations can have structural
parts with very different turnover rates, or with different functional niches, and it is
not clear how a population can, in this case, be included into a single TDM.
It is clear that a conceptual framework for integrating biotic and abiotic pro-
cesses at all scales is needed. This is obvious now at least for theoreticians and
modelers who are in a position to integrate the available information for fundamen-
tal or applied purposes. For instance, Seppelt et al. (2009) argue that modeling with
reliable simulations of the human–environmental interactions necessitates linking
modeling and simulation research much more strongly to science fields such as
landscape ecology, community ecology, ecohydrology, etc. Such linking cannot be
done in the absence of a formal cross-scales conceptual framework.
12.3 An Analytic Conceptual Framework for Integration

Across Scales
A thorough conceptual analysis of the terms used in up-scaling research cannot

have a place here for reasons of space, but it is useful to note that even some classic
concepts such as ecosystem are under strong criticism in the current literature.
O’Neill (2001) discusses logical and scientific problems associated with this con-
cept in an article entitled “Is it time to bury the ecosystem concept?” He proposes
that an ecological system is composed of a range of spatial scales from the local
system to the potential dispersal range of all of the species within the local system.
With the same critical attitude, Reynolds and Wu (1999) ask: “Do landscape
structural and functional units exist?”
Looking for the ontological status of ecosystems and landscapes, in the last
decade, we have approached the up-scaling problem in various contexts and pro-
duced step by step, new theoretical elements. Based on the analyses of biodiversity
structure in large rivers, we introduced the concept of emergent TDM, characteriz-
ing each hierarchical ecosystem level (Vadineanu et al. 2001). Then, in the context
of the natural capital management, we discriminated between the concepts of
natural capital and of ecological systems (Iordache and Bodescu 2005). More
recently, we advanced ideas about the integration across scales in the context of
integrated modeling in the biogeochemistry of metals (Iordache et al. 2009a) and for
assessing the effects of disturbance on ectomycorrhiza diversity (Iordache et al.
2009b). Here, we synthesize and further develop these ideas.
12.3.1 Developmental System as the Basic Unit of Ecological

Functioning
In the most general sense, when we declare of an organism that “it functions,” we
mean that it produces biomass, that it reproduces, and that it has biological
productivity. This process conceptually supports both the standard view of ecosys-
tem functions (flow of energy, cycling of matter, self-regulation) and evolution
(recall Darwin’s 1859 “law of growth with reproduction” characterizing the organ-
isms), but at different space and time (ST) scales. However, an organism as an
isolated system cannot “function,” because when the organism grows and repro-
duces, it uses natural resources and services. Darwin did not state this explicitly, but
it is an implicit assumption of the fight for existence introduced in his explanatory
argument for natural selection (the fight for existence cannot be directly deduced
from an empirical “law of growth with reproduction” without scarcity of resources).
Darwin avoided putting growth and reproduction in functional terms (i.e., to say
“the organism uses natural resources and services in order to grow and reproduce”)
because he avoided making use of any teleological principle, being a convinced
Newtonian. But in the current biological thinking, we are used to speaking about
teleonomic behavior of the organisms, the behavior as if they would pursue the
goals to grow and to reproduce. From such a line of argument (developed in more
detail in Iordache 2009b), we first provide a definition for the basic unit of
ecological functioning: a developmental system (DS) is a teleonomic entity within
its environment producing natural resources and services. Two remarks are needed
now (1) if we include the abiotic resources in the developmental system, it is not
clear why we would not include the biotic resources too because the DS productiv-
ity depends also on biotic resources, and (2) if we include entities with positive
value (resources or entities producing services – e.g., dispersal services) for the
biomass production, we should also include entities with negative value because
both of them influence productivity. In definition, a DS is then a teleonomic entity
(TE) and all environmental entities with value (EV) for it (Iordache 2009a). To
avoid confusion, we mention that EVs are not isomorphic with the multidimen-
sional niche in the ecological sense because they refer not only to external entities
but also to internal ones. Another argument for this lack of overlapping is provided
in Sect. 12.4.1.1.4.
DS is a useful theoretical concept because it fits the formal structure of any
biological organism with structural (internal, e.g., genes) EVs and external EVs (as
“perceived” in its environment), fits the formal structure of human organizations
(with organizational leader in the position of TE and organizational goal as goal
function, the organization system as an extended body with internal EVs, and the
organizational environment in place of external EVs), and fits the structure of
management projects (as short-lived organizations).
The fact that it accommodates both natural and human systems provides a
common theoretical basis for describing coupled natural-human systems (socio-
ecological systems), the connection between biological productivity and organiza-
tional productivity, and for modeling such coupled systems.
12.3.2 Epistemic Status of the Developmental Systems
Here, we provide a way to “translate” the conceptual framework introduced in

Sect. 12.3.1 into measurable phenomena and empirically based scientific knowl-
edge. A DS can be modeled by a state space and the “law of growth” can be
formulated in a such state-space. Usually, any system’s “goal” is formulated as an
attractor point or region in the state space of the properties of the systems or as a
maximization or minimization of an index derived from the space parameters (e.g.,
ascendency in ecology). Here, we introduce the goal of ”growth” through an
underived variable (e.g., biomass) or set of variables (accounting for the life
cycle of the organism) in the state space and the laws of growth through mathemat-
ical functions relating the numerical values of goal variable(s) and the numerical
values of other parameters of the state space. We mention without demonstration
that efficiency and effectiveness of the DS can be introduced from the form of these
mathematical functions, and apparent and general fitness can be related to
efficiency and effectiveness, respectively; this is a way to a common formalization

of ecological and evolutionary theory. As far as the “reproduction” part of the
Darwinian law is concerned, it cannot be formulated in the same organismic state
space but actually involves a multiplication of the organismic state space within the
integrating populational system (formally as subspaces). The full “law of growth
with reproduction” presupposes the existence (and, from an epistemic point of
view, the modeling) of a system with at least three hierarchical levels: parts of
DS (TE and EVs), the DS per se, and a population of DS. In order to model the
relationships between the DSs of a population or of a community, one needs to
integrate the state spaces of each DS in a higher dimensional mathematical space.
Part of this mathematical space should describe the “objective” environment
between the DSs and include the mathematical functions showing how the common
use of this environment by different TEs leads to a change in the value of its entities
for other TEs. The integrated model would include two types of models: teleonomic
(subjectivistic) of the DS and objectivistic (without goal parameters) submodels
(Fig. 4 in Iordache et al. (2009a) illustrates this point). Both types of models need
the concept of physical time, but the concept of physical space is requested only by
the objectivistic models. The objectivistic model may be needed in two types of
situations (a) the environment of the organisms is very heterogenous, and the
organisms may deal with different values of the environmental parameters, (b)
the organisms perceive the environment in different ways, the value of the environ-
ment for each organism being different as a result of their perceptual differences. If
one assumes that such situations do not occur, then we arrive to a simplified state
space having only one set of parameters for the environment and several goal
parameters (reflecting the number of organisms in the intra- or interspecific
group). In the case of natural selection modeling, this simplification leads to the
replecement of the DS with the organism as the selection unit. However, it is
currently accepted that the manner in which organisms perceive the environment
is in itself a trait supporting the sorting of organisms by natural selection, and the
role of environmental heterogeneity in the selection process is documented as well.
For theoretical reasons (including compatibility with cultural and economic selec-
tion theories), it is more appropriate to consider the developmental system as the
unit of selection, which boils down, in biology, to organisms as units of selection in
a simplified model of the environmental pressure, as we have seen. Describing the
formal structure of these models and deriving from them the Price equation (Price
1995) is a matter of ongoing research. Other aspects of the epistemic status of DSs
as related to their scale will be tackled in Sect. 12.3.4.
12.3.3 Scale and Productivity
We limit this discussion to biological productivity. The timescale of DS productiv-

ity is short and limited to the life of the systems. One scale effect on biological
productivity is given by the ST scale of the TE (organism here). Another scale
effect is related to the fact that populations of DS of the same species have a larger
ST scale and productivity (due to intraspecific relationships and processes, includ-
ing reproduction). Still another scale effect is related to the communities of DS of
different species, larger in ST scale and having larger productivity as a result of
interspecific relations. One could think further about metapopulations and meta-
communities of DS and so on. All these systems with larger ST scales are ecologi-
cal systems but with various degrees of complexity. This view of ecological
systems is partly convergent with O’Neill’s (2001) view mentioned above, accord-
ing to which an ecological system crosses a range of scales. We underline that we
have not introduced here the concept of “population” and “community” in the
classic sense (of biological individuals) but as populations and communities of
developmental systems, including both organisms and their environment. Also,
there is not a simple ST nesting hierarchy of these systems, and actually there is
no hierarchy at all (see next point for the hierarchy as epistemically conditioned)
because each DS is characterized by its own ST parameters and usually does not
“stay” within the limits of an ecosystem in the standard sense. The more appropriate
representation is that of a network of overlapped basic ecological systems (DSs)
functioning and interacting through objective (nonteleonomic) systems at various
ST scales.
12.3.4 Epistemic Status of Complex Ecological Systems
The fact that the ecological systems more complex than a DS have no definite
boundaries in the Newtonian space is a problem for their description and manage-
ment. Operationally, one can study or manage only structures well defined in a three-
dimensional physical space, not in an n dimensional mathematical space. Even one
DS may spread over many scales, if the EVs from their ET’s developmental system
spread over many scales, namely from scales smaller than ET’s source scale (e.g.,
fungi for plants) to scales much larger than the source scale (large herbivorous
mammals, or seed eating birds, for plants). The solution to this problem is a rough
discretization, the modularization of the system (pointing out its “compartmentalized
architecture” – Moore et al. 2007).
For a modularization in the interest of scientific description, we adapt Pahl-Vostl’s
(1995) trophic-dynamic concept (in line also with Godbold’s 2005 and Satomura
et al. 2006 views) in the following way: a functional dynamic modules (FDM) is a
group of TEs or of parts of TEs having (1) similar rates of TE biomass cycling
(inversely correlated with lifetime of the individuals), (2) the same location in space
and time, and (3) similar functional niches, i.e., relations with TEs of the same or of
different scales. We prefer the term “functional” to ”trophic” because not just the
trophic relations count for the differentiation of functional niches. Another reason is
that we want this concept to be applicable to nonbiological TEs as well. With this
concept, we tackle the TE part of the ecological systems. The abiotic parts (physical
solids, liquids, and gasses) might be tackled by an apparent nested hierarchy
approach at the scales resulting from the TE systems modularization (Iordache et al.
2011). Down-scaling and up-scaling in their case is a problem of physical modeling
in a large sense.
Specific to the scientific modularization is that the ST scale is not constrained by
the manageability of the delineated system. Some populations of ETs can be
included in more than one FDM at the same time because of their internal structural
diversity (Iordache et al. 2009b) For instance, populations of deciduous tree DSs
have parts with very different rates of biomass cycling, like leaves and wood
(criterion 1), as well as parts with different locations in space like below- vs.
aboveground (criterion 2). Thus, the trees will belong to at least three FDMs: two
aboveground and one belowground. The notions of “same order of magnitude,”
“same location in space and time,” and “same role in food web” are to be defined by
the researcher and can be applied more stringently or relaxed. In the most stringent
application, they will lead to a model identical with the “reality” (“isomorphic”
model). If relaxed too much, they will lead to a model too aggregated, having lost
the key characteristics of the real system (simplistic model). Only at an appropriate
intermediate level will they lead to a model simple enough for explanatory value
but keeping the basic characteristic of the system (“homomorphic” model). The
scale of the FDMs varies hugely, which implies that there is not one “true” scale for
ecological processes. Rather, emergence of new structural (e.g., new FDMs) and
functional (e.g., increase in overall biological productivity or changes in the rates of
biogeochemical processes) properties should be defined and used to derive the
mathematical function that links scale and emergence of new properties in different
areas and in different periods of time (“emergence function”).
As for the functional niche, we have to clarify what it can mean in a context
where there are interactions with systems of many scales. Luck et al. (2003)
introduced the concept of a service production unit (SPU) as a subsystem of or a
full biological population directly contributing to the production of a resource or
service perceived as such by humans. The concept of SPU can be generalized from
the perspective of all other species. For instance, roots can be interpreted as an SPU
for fungi, and sporcarps as SPUs for fungivorous mammals. This generalization
allows for a precise delineation of what part of an EV organism provides value for a
TE organism. For EVs with a scale larger than that of the source scale, we answer
this question as follows: one has to either produce a model linking the EV with the
source scale SPU providing direct services (this works for instance for fine roots of
plants as SPUs for EM fungi) or consider the use of source scale TEs by the large
scale entity as a biotic internal control parameter connected to a fungal SPU (this
could work for consumption of fungal sporocarps by mammals, for instance). By
internal control parameters, we mean those describing the influence from inside the
DS model, but from scales different than the source TE scale; by external control
parameters, we mean those describing the factors influencing from outside the DS
model – e.g., large scale physical ones or human action. For entities with a smaller
scale than the fungal source scale (e.g., bacteria, tiny invertebrates), one needs to
produce a model linking the source scale individual with small scale organisms
through the smaller scale SPU providing direct resources and services (e.g., organic
exudates, hyphae) to these smaller scale organisms and then to up-scale the results
of this model to the source scale to form another internal biotic control parameter.
The particular interaction of the source scale individual with each small scale entity
will not count, but the overall pattern resulting from the structural and functional
characteristics of these small scale FDMs will. We can now interpret the functional
niche of a TE as consisting of variables describing the source scale entities with
value for the fungi, the source scale SPUs that are part of larger scale entities
relevant for fungi, and the internal biotic control parameters. This concept of
functional niche is an epistemic one, with no objective reality associated to it. It
will not imply that the developmental system of fungi will not continue in reality to
be spread across scales but that we have to modularize the scale continuum in order
to obtain workable FDMs, which is a strategic part of the epistemic status of the DS.
The DS of a TE (the DS’s “world”) will be modeled not only by the TE’s FDMs but
also by the entire homomorphic model reflecting also the EVs direct relevance for
the TE, i.e., the epistemic status of a DS is related to the production of a structural
homomorphic model and of the associated mathematical models (and of the
hierarchically structured physical abiotic models). Of course, nobody produces
such homomorphic models just for one DS, but for populations of DSs of different
species. The homomorphic model for a single DS is a theoretical case with number
of species one and number of individuals one. This theoretical case is important
when one needs to move the discussion to natural selection, by providing a
conceptual bridge between ecological and evolutionist theories. An important
point is methodological: in an FDM of ecological use, the physical environment
associated with it is considered homogenous and perceived identically not only for
the individuals of the same species but also for all individuals of different species
grouped in that FDM. This leads to the simplification of the state space used in
modeling to make it workable for ecological purposes (where the evolution of the
organisms is not taken into account) and especially for aggregation in view of up-
scaling the ecological processes. Another point is that the space-time windows
associated with the homomorphic models should be chosen, especially in the case
of managerial modularization, such as to be compatible with the human practical
possibilities of action.
The scientific or managerial modularization leads to a nested hierarchy of
ecosystems but not a true one (Fig. 12.1). In this framework, it is not the case that
“holons at level n form the entities at level n þ 1” (Lischke et al. 2006) because
besides the n level entities, there are also new larger scale entities forming the n þ 1
level; each eco-level is characterized by structurally new types of FDMs that are not
found at lower hierarchical levels (Fig. 12.2). Only the representational three
dimensional physical spaces needed for scientific investigation or management are
nested and not the productive systems analyzed within this three dimensional space.
The standard homomorphic model of an ecological system (including compartments
for primary producers, consumers, decomposers, etc.) will be then about interac-
tions of different developmental ecosystems of different scales. What is not seen in
the standard representation is the large number of smaller scale populations of TEs
Biological production and productivity,

Type of individual and population Ind. Pop.
total resources and services

t t
Structural and functional

Prod
emergent properties
z z
y y
x x
ecolevel ecolevel ecol.

1 2 3
a b c d (a) b c d
Scales of observation in space Scale of emergence in space
Fig. 12.1 The relationship between the scale of biological structural elements and processes
(individuals, populations, left graph – left axes, production and productivity, left graph – right
axes) and the hierarchical structure of ecosystems (right graph). At scales of observation from a to
b (corresponding to ecological level 1), one can perceive all types of individuals (and their
populations) from x to y but only some of the individual types from y to z (and not their
populations). The FDMs including populations of type y to z are said to “emerge” at higher
hierarchical ecological level 2. Grey areas on the right graph suggest the multidimensional spaces
characterizing each ecological level in which the processes supporting the productivity of each
level can be conceptualized. Note that the simplistic linear models (emergence functions) from the
left graph can be cut in a different way, leading to alternative hierarchies. The real forms of the
emergence functions are not linear and depend on the starting point of observation in space
Emg_10
Emg_9
Emg_8
Emg_7
Emg_6
Emg_5
Emg_4
Emg_3
Emg_2
Emg_1
1 2 3 4 5 6 7 8 9 10
Fig. 12.2 Theoretical relationships between the number of eco-levels and the number of emergent
FDMs (communities) of each type (Emg_1 to 10) within an eco-level (1 to 10). The color change
from green to red indicates an increase. Biodiversity of the overall biocenoses (system of
communities) is related to the number of FDM types and instances and to the species diversity
inside each FDM
FDMc
,,emergent’’
at scale
n+1
Level ,,Level’’
n+1 n+1
Holon a Holon b FDM a FDM b
Level n ,,Level’’ n
Fig. 12.3 Simplified representation of a true nested hierarchy (left, applicable, for instance, to
molecules – level n, and cells level n + 1) and the apparent hierarchy of systems (right, resulted
from the epistemic modularization of ecological systems). In the apparent hierarchy (abiotic
physical parts not represented), the holon at n + 1 level includes subsystems that are not present
at n level, which is not the case for true hierarchies. In the true hierarchy, the interactions within the
subsystems of a holon are strong and between holons are weak, while in the apparent hierarchy,
they may be very strong between holons (when, for instance, the FDMs include parts of the same
population of DSs with different functional niches, or parts of the same DSs with different location
in space and time, or different turnover rates)
compared to the large scale ones. Many small scale FDMs are coupled at the same
time to a relatively larger FDM (Fig. 12.3).
Usually, the term ecosystem is used both for scientific and for management
purposes. This leads to conceptual confusion and, as we have seen, even to
proposals of rejecting the ecosystem concept with scientific arguments. Here, we
separate the scientific use from the applied, managerial use, by the additional term
natural capital. An ecosystem is natural capital when the modularization of the
emergence function is done for management goals, i.e., of interest is the value of the
ecosystem for humans. Promoting a separation between basic and applied studies is
in line with other opinions from the literature. For instance, in a conceptual analysis
Yarrow and Marin (2007) conclude that the concept of ecological boundary will
find its primary utility within scientific circles, whereas the system-specific transi-
tion zone is quite useful in public discourse and socioeconomic decision-making.
For a modularization in the interest of management, leading to a theoretical
hierarchy of the natural capital, one has to use only scales appropriate for the
coupling of natural DSs with human developmental systems (organizations, man-
agement projects) by natural resources and services relevant to humans. The
theoretical natural capital hierarchy resulting from a managerial modularization
of the emergence function is a hierarchy of the natural capital, with specific natural
resources and services produced at each level (Iordache and Bodescu 2005) and
with specific managerial organizations created for (“emergent at”) each level.
An emergence function for human developmental systems (organizations) can be
constructed and modularized leading to a hierarchy of socioeconomic systems, with
the importance difference being that this modularization is no longer at the latitude
of the researcher but imposed by national and international institutional reality.
Actually, the theoretical natural capital hierarchy may follow the socioeconomic
current hierarchy for reasons of manageability of the natural modularized entities.
An analysis of the socioeconomic systems (the construction of the organizational
emergence function just mentioned) reveals that the real structure of the natural
capital is not the theoretical one, simply because the modularization of the natural
emergence functions is not only a scientific process but also an institutional one
associated with the functioning of a socioeconomic system that includes the
scientists’ organizations. What is interesting in real management is how particular
organizations and projects deal with the ecological system, what they perceive as
valuable, and how they interact when they want to maximize their separately
perceived values (when they are in a conflict of interests, leading eventually to an
environmental crisis). Exactly at this point, the key role of the theoretical concept of
natural capital is revealed as an attractor for the structure of the real natural capital
if one envisages the maximization of the privately and publicly intercepted natural
resources and services. There is an institutional evolution of the real natural capital
(Iordache 2004) and a cultural history of the natural capital as part of the overall
cultural and institutional evolution. The theoretical concept of natural capital is
useful also as a reference for conceiving the deterioration process – as structural
change leading to the decrease of overall (whatever the intercepting organization is)
natural resources and services produced by the natural capital – and for conceiving
the restoration process (the inverse of deterioration).
Another important distinction to make is that between production and manage-
ment of the natural resources and services. The production takes place at all scales
of the ecological emergence function, but the management can occur only at the
human-relevant scales. One problem is how to take into account the contribution of
small scale organisms (like EMF) to the overall natural resources and services
production at the management scale. This can be done through the SPU concept.
From a managerial perspective, the TEs’ populations of the same species (or
fragments of such populations) found in an FDM constitute service productive
units (SPUs). The SPU concept allows the identification of each species’ contribu-
tion to the overall theoretical or real natural capital value, and on this basis, the
design of targeted management measures. All services provided by SPUs are
dependent on the production of biomass, and the material fluxes associated with
it, but they can range from providing a source of carbon to a simple physical support
(e.g., for smaller scale organisms). One needs long term field studies in order
to appropriately assess the biomass production (Staddon et al. 2002), and hence
the importance of long term ecological research. Besides this ecological knowl-
edge, one will need for its application socioeconomic knowledge concerning the
optimal institutional framework. Optimally, this framework has to allow for the
integrated action of the organizations perceiving different values of the natural
capital and the integrated implementation of management projects having different

standpoints.
12.3.5 Up-Scaling the Ecological Processes
Up-scaling the biomass production function from original DSs of scale 1 to a larger
target scale 2 is not a simple physical space-time (ST) procedure because it involves
an increase in the complexity of the state space from the original to the target
system. The scale issues are related to the position in space-time (ST) of the original
TEs as grouped in FDMs of their internal and external EVs, of DSs of larger scale to
which they are directly connected, and to the ST patterns of physical processes of
larger scale controlling the value of abiotic parameters relevant for the original and
large scale TEs.
Up-scaling the biomass production function from the FDMs of source scale to
target scale may involve previous down-scaling of other processes. One of these
situations occurs when the source scale DSs use as EV an organism of larger scale
(for instance as a carbon source). In this case, one needs a model down-scaling the
biomass production of that organism to the specific portion of biomass/carbon made
available for source scale organisms, which is different from the model describing
the overall biomass production of that large scale organism. Another situation is
connected to the pattern (perceived at large scale) of distribution in space of the
biomass of source scale FDMs, a pattern controlling the choice of mathematical
tools for up-scaling (excellently reviewed by Lischke et al. 2006). This pattern may
depend in time on external control parameters that can be modeled in space and
time only at large, target scale. For instance, if one predicts with hydrogeomorpho-
logical models the dispersion of a toxic pollutant with a resolution of 50 50 m,
this information should be down-scaled if the DSs controlled by this pollutant,
whose up-scaling we are looking for, have a much smaller scale. The particular
distribution at small scale can be influenced by the external control parameters, by
changing the species location in space, changing the species location in time, or
changing the turnover rate of the biomass, with overall consequences on the FDMs
structure and associated productivity.
Within the presented analytical framework, the steps for up-scaling the biomass
production (and related biogeochemical processes) from the original (source) DS
systems could be based on structural aspects like (1) ST location and turnover rate
of the source TEs (characterizing biomass turnover and position in ST of the TEs by
one or several modules reflecting their life cycle and morphological properties), (2)
entities providing resources and services to the original TEs (identifying the EVs
relevant for each TE type by module and characterizing their ST position), (3)
entities using resources and services of the original TEs (identifying the use of
source ET modules as EVs by developmental systems centered on types of TE of
larger scales and identifying the position of these large scale DSs in the system of
target scale), (4) external control factors (identifying the large scale abiotic factors,
described as external parameters, influencing the DS of the original scale identified

above) and (5) the homomorphic model of the system of DSs and of its integrating
system (identifying the overall ST scale of the system of FDMs characterizing the
source DS, the relationships of these FDMs with larger scale ones and the external
control parameters). In addition, functional aspects include (6) biomass production
functions at source scale (characterization of the production functions at DS,
populations of DSs, and FDMs of the source TEs at the original scale in the field,
i.e., implicitly taking into account the influence of smaller scale DSs), (7) large
scale DSs’ influence on the production function (characterizing the mathematical
functions describing the influence of large scale DSs on the productivity of the
original DSs), (8) large scale abiotic factor’s influence on the production function
(characterizing the mathematical relationships underlying the influence of large
scale abiotic parameters on DSs at the original scale, the space distribution of the
abiotic parameters in the system of target scale with a resolution of original scale,
and the mathematical functions predicting their space-time dynamic and the abiotic
processes involved at the target scales) and (9) large scale abiotic factor’s influence
on large scale DSs connected to the source scale (the same as in point 6 done for the
large scale FDMs mentioned at point 3 keeping into account only the abiotic
processes influencing all DSs whatever the scale). The integration relies on (10)
stratification (stratifying the system of target scale into strata having the scale of the
system of FDMs characterizing the original DS as identified) , (11) production
function by strata as controlled by large scale DSs and abiotic factors (assessment
of the productivity or other associated processes of interest of original DSs by types
of strata, as modeled by the system of FDMs developed within stratum under
the control of larger scale DSs and abiotic parameters, providing the possibility
to correct for results obtained in step 6 by results from steps 7, 8 and 9, and (12)
model up-scaling (extrapolation of production function to the target area of interest
by types of strata).
12.4 Application to Ectomycorrhizae
It follows from above that the up-scaling of the function of EMF cannot be done
without modeling their function. We are still far from reaching this level of
understanding EMF ecology. Below, we will concentrate on the structural and
functional aspects, and less on the modeling side (touched on in Sect. 12.4.4) and
in, particular, on the following questions:
l How many hierarchical levels do we need to construct and study from the natural
emergence function in order to fully understand the role of EMF DSs in the
ecological productivity of ecosystems and landscapes?
l What are the appropriate modularization scales for understanding the role of
EMFs? (“which are the most relevant scales of analyses for these organisms?”
Lilleskov et al. 2004)
l How many types of FDMs are needed for the structural and functional modeling
of EMF communities?
These questions are directly relevant to the discussion of EMFs biodiversity. We
have shown elsewhere that (Iordache et al. 2009b), for instance, it is not meaningful
to interpret EMF patterns of alpha diversity at an ecosystem scale (104–106 m2), but
that this should be done at a much smaller scale (subtree level differentiated by soil
layer). Here, we will not develop, however, this line of interpretation but only take
the steps of the analytical framework introduced in the previous part.
12.4.1 Scales Relevant for EMF Developmental Systems
12.4.1.1 Structural Issues
The criteria for delineating FDMs are, as we have seen, the biomass turnover rate,
the location in space-time, and the functional niche. The functional niche is
discussed in terms of resources used by EMF and organisms using parts of EMF
as service providing units.
Dimension and Turnover Rate of EMFs
The simple parameters characterizing an organism are difficult to determine in the

case of fungi and even more difficult for EMF. The reasons for this are the complex
structure and life cycle of these organisms and the underground location of most of
their parts. An analysis of the EMF individual should in principle differentiate
between fungal tissue (fruiting body – sporocarp, sclerotium, spore), the plant fungal
interface (ectomycorrhizae (EM), also referred to as “root tips” in the literature and
further below), and the soil fungal interface (extraradical mycelium or extraradical
hyphae, which can differentiate rhizomorphs for long distance transport of nutrients
and water) (Satomura et al. 2006). Anatomical details like the dimensions of hyphae,
cell walls, etc., are provided in an excellent series of studies on ectomycorrhizae
(e.g., Agerer and Weiss 1989 and references therein). The hyphae have diameters of
3–5 mm, smaller than many soil pores (Smith et al. 2010). The importance of each of
these potential parts varies with the species. For instance, relatively few EMF are
known to form sclerotia, but when present, they support resistance to disturbance
and recolonization (M€ unzenberge et al. 2009). Rhizomorphs can be associated with
root tips, with the extraradical hyphae network between trees, or with the sporocarps.
Further complications (many plant fungal interfaces) occur in the case of fungi
linking several plants. Ectomycorrhizae, rhizomorphs, hyphae, and sporocarps
perform different functions, respond differently to environmental conditions, and
their life span has consequences on soil biogeochemical processes (Treseder et al.
2005). Most microbial measurements to date, such as biomass or hyphal length, have
been single time-point measurements (Allen et al. 2007).
The delineation of a fungal individual starting from its various parts can be done
by direct physiological continuity or by genetic identity (Smith et al. 1992). Most of
the reported sizes refer to the genets. This may be a false indicator when a physical
fragmentation of an individual, especially at ERH level, occurs. In the case of
plants, a distinction between genets and ramets (physiological individuals separated
from the clone) is made. For fungi, the term “ramet” is not used since anastomoses
of physically separated units may occur. Lamour et al. (2007) analyze, for instance,
the network of an Armillaria species in two 25 m2 plots of natural soil. They found a
density of 4.3–6.1 rhyzomorphs per mm2. At one site, the network consisted of 169
rhizomorphs as edges and 107 rhizomorphs as nodes. Only two critical rhizomor-
phal bridges would lead to the separation of significant physiologically independent
fragments, so there was “low probability that amputation of a randomly chosen
edge would separate the network into two disconnected components” (Lamour et al.
2007). So the dimension of a genet is a fairly good approximation for the dimension
of the biological individual in the case of fungi.
Smith et al. (1992) reported a 15 ha large, 1,500 year old genet of a parasitic
fungi with an estimated biomass of 10,000 kg. Similar investigations are missing
for EMF. While ectomycorrhiza life span depends to some extent on the life span of
the host tree, interconnections between trees are generally found and may be seen as
indication of similar sizes and life times for EMF. Griffiths et al. (1996) reported
that EMF mats may persist 2 years after their host trees have been cut down. The
patch size of individual EMF mats studied by Agerer and Gőttlein (2003) were
several dm2. Within the mats, some species were positively correlated to N–NH4þ
concentration, to total K, Na, Mg, Fe, Mn concentrations and pH, but other species
distribution revealed no such correlation. The sizes of the genets of three EMF
species studied by Redecker et al. (2001) were 1.5, 9.3, and 1.1 m2. Bruns et al.
(2002b) have mapped and genotyped the fruiting bodies of EMF in a forest in order
to compare the pre- and postdisturbance distributions and identify the causes of
community reestablishment (dispersal or regeneration from local forms of resis-
tance), and the size of the genets ranged from 1 to 102 m2 (estimates from their
map). In reviews, the size of genets is estimated to vary from 101 to 102 m2
(Lilleskov et al. 2004), 1 m2 to more than 102 m2 (Godbold 2005), and between
1 and 300 m2 (Wolfe et al. 2009). Inside a genet, the root tip abundance itself may
be patchy (Pickles et al. 2009).
The size of the genets is related to the physiological and reproduction strategies
of the fungi (Table 12.1). The extent of extraradical mycelium for the same species
Table 12.1 Comparison of early- and late-stage ECM species characteristics (Iordache et al.
2009b). The relation to exploration types is hypothetical; the other relations are documented by the
literature
Species/ Reproduction Genetic Requirement of Exploration type
characteristic diversity C, N, P
Early Primarily by spores Higher Small Mainly medium and
long distance
Late Primarily by clonal Lower Greater Mainly contact and
expansion short distance
may depend on environmental variables (Scattolin et al. 2008), and consequently,

the individuals of the same species may have different locations in space as a
function of abiotic factors.
The information about the life time of a genet is very important for understand-
ing the speciation process, but from a short and average timescale perspective, the
turnover rate of the biomass of a genet’s parts is of interest as well. The turnover
rate may be estimated not only from the life span of the parts but also as a function
of the decomposition of the associated organic matter.
Rygiewicz et al. (1997) found an average median lifetime for mycorrhizal tips
of 139 days (lifespan þ decomposition), ranging from 123 to 185 days for the
eight treatments in the 18 month study period. These authors also review the older
literature, some of which report a much longer life time of individual root tips
(2–4 years). Treseder et al. (2004) screened the literature showing that ages
extended from 1 to 6 years (including errors and sample variability), a range
that overlaps with the 0.25–4 year life spans reported in other studies that have
visually tracked ectomycorrhizae. The fine root turnover rate is dependent on
external, large scale abiotic parameters such as CO2 concentration (Tingey et al.
2000), with consequences on the root tip FDMs. But “ECM turnover need not
precisely mirror root turnover. A number of ecological factors may influence
fungal lifespan independently of roots, including life history of the fungal species,
predation on the fungi, and shifts in the allocation of host plant C to the fungi”
(Treseder et al. 2004).
The hyphal life span is reported to be 5–7 days (Godbold 2005), less than 1
week, although a subset can live for more than 1 month (Staddon et al. 2003). For
rhizomorphs, this may be even longer. A discussion about the turnover rate of ERH
is made in Godbold et al. (2006), mentioning that the turnover time may be longer
than 30 days depending on the methodological details of the estimation. For
comparison, turnover rates of leaves are about once per year and those of fine
roots about three times per year (Godbold et al. 2006 and citations within). The
turnover rate of organic matter derived from dead hyphae is much longer than the
life of the hyphae because of recalcitrant substances like chitin. Due to the high
turnover rate, “the mycorrhizal external mycelium was the dominant pathway,
62%, through which carbon entered the soil organic matter pool, exceeding the
input via leaf litter and fine root turnover” (Godbold et al. 2006). Coutts and Nicoll
(1990) report several months of life for rhizomorphs. In another study, rhizomorphs
lived an average of 11 months in control plots, indicating that many individual
rhizomorphs survive at least part of a nongrowing season (Treseder et al. 2005).
Mushrooms (sporocarps) are short-lived, with an age of 1–2 weeks, and are formed
by labile carbon derived from the ERH mycelium (Treseder et al. 2004). Based on
the above information, we conclude that parts of EMF would fall, based on the
biomass turnover criteria, into two dynamic classes: one with fast turnover rates
(hyphae and sporocarps) and one with smaller turnover rates (ectomycorrhizae and
rhizomorphs).
ST Location of EM Fungi
Locating an EMF individual in soil is a difficult task, taking into consideration its
distribution between the interface with the plant, soil, and sporocarps (Pickles et al.
2009). There is an obvious difference between the location of aboveground spor-
ocarps and belowground structures. For the belowground parts of the fungi, there is
solid knowledge supporting the idea to differentiate between soil horizons, as
documented below.
Vertical location A niche separation of EM species in coarse woody debris and
mineral soil was reported by Tedersoo et al. (2003). The web of ERH not only
colonized mineral soil, but was also abundant in litter and decaying wood (Buée
et al. 2009). ERHs were differently distributed in logs, stumps, forest floors, and
mineral soil (Goodman and Trofymov 1998). The vertical, gradual differentiation
of EMF community structure with depth has been documented (Landeweert et al.
2003; Calvaruso et al. 2007; Courty et al. 2008). Dickie et al. (2002) also report the
vertical niche differentiation of ERH in soil. In a detailed study, Gebhardt et al.
(2009) analyzed only a 3 cm organic layer and cut it into 1 cm slices. Even at this
resolution, they identified two organic sublayers with different EMF communities
(only four species in common). The vertical patchiness of EMFs is related to the
distribution of substrates (Genney et al. 2006) and soil horizon properties (Rosling
et al. 2003; Baier et al. 2006). This vertical partitioning can be interpreted more
generally as niche portioning based on soil chemistry: nitrogen (ammonium)
content, base saturation, carbon age, and soil moisture (Peay et al. 2008). Speciali-
zation of ERH parts of EMF may also occur with respect to organic matter content,
leaf litter type, and litter source (Rillig 2004). Vertical niche partitioning is thought
to be one way by which the high species diversity of mycorrhizal fungi can be
maintained at small spatial scale (Wolfe et al. 2009). The EMF role changes with
depth, not only because some EMF prefer organic or mineral soil layers but also
because the number of root tips and mycorrhized root tips (EV for EMFs) vary with
depth (Scattolin et al. 2008).
From this information, we infer that the aboveground sporocarps should be
included in a separate dynamic module, and the belowground root tips and ERHs
should be split vertically at least into two dynamic modules as a function of the
organic matter content of the soil layer.
Horizontal location The available information concerning the horizontal distri-
bution of EMF refers to individuals (genets), populations, and communities. Addi-
tional information is provided indirectly by niche differentiation as a function of
host species and by the possibility of connecting several hosts. Such information
can be coupled with the hosts location in ST in order to locate the host-specific
EMF.
At the individual level, a cluster of root tips colonized by the same species is
likely to be colonized by the same genet (Godbold 2005). EMF with saprophytic
abilities colonize (by their ERH) the litter layer and discrete patches of organic
nutrients (Graham and Miller 2005). Genet size patterns may be different as a
function of the site, and large genets may have smaller scale structures because of
fragmentation and intense colonization of microsites (Lilleskov et al. 2004).

Another problem in investigating the spatial structure of EMFs can also be the
cryptic nature of some genera like Cenococcum geophyllum (Pickles et al. 2009).
This genus was found to be distributed ubiquitously at a local tree plot scale, but
patchily at microscale (within the 5 5 m, Matsuda et al. 2009), and with a large
genetic diversity even within a single soil core (Douhan and Rizzo 2005). Clearly,
genetic tools have to be applied for characterizing the ST location of such species
(see Sect. 12.5, research directions). In a study of the genet distribution of spor-
ocarps and ectomycorrhizae of Suillus species, Hirose et al. (2004) found in a
20 24 m plot four genets from sporcarps, which coincided with those identified
for EMFs; the spatial distribution of EMFs of each genet were wider than those of
sporocarps, the area occupied by each genet differed considerably within the plot.
Species are likely to differ in spatial colonization patterns because of different
internal genet structure and rates of vegetative expansion (Lilleskov et al. 2004).
These authors report that dominant EMF taxa showed patchiness at a scale of less
than 3 m, with a range from 0 to more than 17 m. Minimal and maximal distances
between cores for stand level EMF characterization are proposed to be 0.25 and
300 m (Lilleskov et al. 2004). Metapopulations of EMFs with epigeous fruiting
bodies are genetically homogeneous over large distances (1 km), while those with
hypogeous fruiting bodies tend to differentiate genetically at much smaller scales
(Wolfe et al. 2009).
Griffiths et al. (1996) use two scales for the investigation of EMF: sampling
within a stand of 2 10 m (to see the effects of forest floor attributes, understory
vegetation, and other species of fungi) and a sampling of stands located in the forest
(to see the effect of succession gradients). They found no correlation with the forest
floor, but proximity between EMF mats, distance from the closest tree, and density
of living trees in a stand had an impact. At scales of <4 m, there is a high
community similarity, while at scales of <20 cm, the community is temporally
dynamic, suggesting a high degree of species turnover probably due to root
senescence (Wolfe et al. 2009). “Late stage fungi” can be found on roots closest
to the trunk of the tree and “early stage fungi” on roots farthest from the base of the
tree (Wolfe et al. 2009, see also Table 12.1), which was interpreted as a succession
in ectomycorrhizal development. These patterns may be indicative of niche differ-
entiation by host tree species and the influence of the neighboring tree of the same
or of different species on the host tree community, as documented below.
The structure of an ectomycorrhizal community depends on the host trees and
host range of the fungi (reviewed by Bruns et al. 2002a; Buée et al. 2009).
Sympatric oak species had different EMF community structures (Morris et al.
2008), partly explainable by extractable phosphorus, but mainly attributable to
the tree species. Host specificity is a niche dimension in itself (Peay et al. 2008).
“Selection pressure for host specificity may not relate as much to interspecific
interactions between trees in later stages of succession but rather to adaptations to
marginal habitats (post disturbance) by the plant and its fungal symbionts” (Horton
et al. 2005). Neighboring tree species identity shaped the EM community structure
of the host, and the effects were specific to host–neighbor combination (Hubert and
Gehring 2008); tree species may serve as reservoirs of EM inoculation to one

another. At the ecotone of a forest, there was an advancing front of EMFs by
dispersal on barren soil, followed by trees, the invasiveness of a tree species being
regulated by the spatial pattern of fungal inoculum in the soil (Thiet and Boerner
2007). Oak seedlings were less infected by EM fungi in a forest dominated by a
different tree species than in oak forests, with consequences to the productivity
(lower dry biomass, Lewis et al. 2008).
Another mechanism supporting the patterns is generated mainly by long range
exploration fungi connecting trees and trees with mycoheterotrophic species. An
implicit idea from Horton and Bruns (2001) is that this networking by fungi can be
made not only by long distance exploration types but also by short distance types
when the roots of the trees spatially overlap. Orchid EMF can simultaneously form
ectomycorrhiza with forest trees (mycoheterotrophy, Bidartondo et al. 2004). The
EMF can interconnect roots of the same or different species (Simard and Durall
2004). The same idea is also supported by Horton and Bruns (2001): dissimilar
plants are associated with many of the same EMF on a small enough spatial scale to
share those fungi. The observed structure at the tree and intertree levels may be
different also because the observation of low abundance fungi as hyphae or tips is
not possible. For instance, Koide et al. (2005) observed in a community some
species as root tip, but not as hyphae, and vice versa. As long as we are interested in
the function of EMF at an ecosystem level on short and average term, these
methodological limits are acceptable.
Based on the above elements (which in the case of intertree location also include
elements related to the functional niche), one has to separate the fungi species into
potentially six dynamic modules in the two dimensional space:
l At tree level (about 4 m around the tree) and at plot level (group of trees and
mycoheterotrophic plants occupying a surface of 400–900 m2)
l Three types of functionally differentiated plots (a) with trees of the same species,
(b) with trees of different species, and (c) intertree-mycoheterotrophic plant
level
l Tree level dynamic modules in function of their position inside the forest: those
at the ecotones between different vegetation patches should be placed in separate
strata
Location in time Some EMF populations build up mostly in winter and others in
summer periods (Courty et al. 2008; Buée et al. 2009 and the references within).
Koide et al. (2007) found an even more complex temporal partioning of niches:
three groups of EMF separated in time over 13 months with respect to their hyphae
in the bulk soil (ERH) and two groups of EMF separated at the same time with
respect to their parts associated to roots. EMF sporocarps may have a specific
location in time. For instance, Nara (2008) reports on the seasonality of sporocarp
formation in a volcanic desert on Mount Fuji, Japan.
Related to the location in time, some authors use the notion of “ectomycorrhiza
turn-over,” referring not to the biomass turnover but to the community change over
time. Izzo et al. (2005), for instance, conclude that “annual occurrence of the
dominant ectomycorrhizal species was constant at larger spatial scales but varied
more across years at a fine spatial scale. Turnover of ectomycorrhizal species
between years was observed frequently at scales <20 cm.” Such information cannot
be used for delineating the location in time of the communities unless some
periodicity of community structure at the multi-year level is observed (and then
the homomorphic model of the study system should be constructed with a multi-
year characteristic timescale).
Based on the existing information, it seems that in some cases, several dynamic
modules separated in time could be differentiated for each FDM separated in space.
However, it is too early to generalize this. The decision in a real situation should be
made after at least 2 years of monitoring the structure of an EMF community.
Resources for Ectomycorrhizal Fungi and Their Space-Time Location
In general terms, fungi are considered to be organisms that strongly influence the
microscopic and the macroscopic world (Peay et al. 2008) and a good model for
experimenting with the coupling between processes of different scales and con-
necting soil microbes with animal populations via the direct effects on plants
(Smith et al. 2010). The EVs with positive value for EMF are soil abiotic mineral
and organic parts, litter, plants, and bacteria.
Many basidiomycetes EMF have retained some of their saprophytic abilities,
and thus have the potential to access organic sources of nitrogen and phosphorus
and to degrade the lignocellulose fraction of dead plant material (Graham and
Miller 2005). A key service provided to fungi by plants is the carbon transfer to
the fungus in the fine roots (Dell 2002), with the extra carbon accumulating at the
edge of the hyphal mat. Fungi also redistribute water from moist layers to upper dry
layers (“hydraulic lift”), which is beneficial to soil microorganisms and increases
the availability of nutrients to plants (Liste and White 2008). In addition, bacteria,
archaea, phages, saprophytic fungi, and soil fauna may interact with EMF (Buée
et al. 2009).
Biological factors influencing the structure of soil microbial communities (Buée
et al. 2009) are: plant developmental stage, plant species, and plant cultivar (genetic
diversity). Mycorrhizae affect the functional diversity of rhizosphere bacteria,
fungi, and other microbes (Buée et al. 2009 and the references within). In turn,
EMF can be supported by rhizosphere bacteria, a phenomenon that lead to the
concept of “mycorrhization helper bacteria,” reviewed in detail by Tarkka and
Frey-Klett (2008). The distribution of “mycorrhization helper bacteria” followed
the vertical EMF stratification both at tree level and between trees (Calvaruso et al.
2007).
A service provided to EMFs by other fungivorous organisms is spore dispersal.
A variety of organisms have been shown to move viable spores of mycorrhizal
fungi at scales from cms to kms (Wolfe et al. 2009). This is an important aspect
because the scale of selection operating on EMF with direct consequences on
genetic divergence (and in time speciation) is related to the maximal dispersal
capability of the fungi. Direct evidence of EMF spore dispersal by mobile animals
was produced and reviewed by many authors (e.g., Johnson 1996; Carrey and
Harrington 2001). In an excellent study, Lilleskov and Bruns (2005) found that
EMF spore densities were high in the guts of arthropod fungivores (mites, springtails,
millipedes, beetles, fly larvae) but present also in arthropod and vertebrate predators
(centipedes and salamanders). A low percent of the spores had intact nuclei in
predators, but most of the spores in the fungivores had intact nuclei and seemed
viable.
Organisms Using Resources and Services Provided by EMF
Organisms using EMF can be interpreted in many cases as EVs with negative value.
In the particular case of trees and fungivorous animals, there is a reciprocal use (þþ
interspecific relationships).
The key organisms benefiting from EMF are trees. EMF assist forest trees in
exploiting the soil, in uptaking nutrients by solubilizing soil minerals with organic
acids (Buée et al. 2009) and in mobilizing organic forms of nutrients by enzymatic
activities (Courty et al. 2005). The variation in EMF perceived by the host plant
may be of a discrete (presence – absence of EMF) rather than continuous nature
(variation in identity or abundance of EMF) (Karst et al. 2008). The ability of EMF
to capture and transport nutrients is believed to be strongly related to the explora-
tion ability and function of ERH. The relevant contribution to nutrient uptake is
estimated by the proportion of root tips colonized (Graham and Miller 2005).
However, root colonization may not be a good predictor for nutrients uptake
(Graham 2008). Instead of this, measurement of lower level mechanisms for
nutrient uptake being needed is preferred; but it is difficult to scale up the informa-
tion thus obtained to the field. Secondary services are also provided. EMF species
differ in the ability to capture nutrients, uptake water, protect against pathogens,
and increase tolerance to heavy metals (Godbold 2005), unfavorable pH, or salinity
(Dell 2002).
EMF can transfer carbon and nutrients between host plants or to mycohetero-
trophic plants (Bidartondo et al. 2004; Buée et al. 2009; Leake and Cameron 2010)
and can facilitate interplant transfer of carbon, nitrogen, phosphorus, and water,
eventually following source-sink gradients between plants (Simard and Durall
2004). Mycoheterotrophic and mixotrophic plants are dependent on the transfer
of carbon by EMF networks, which in turn depend on their host photosynthetic
plants (review in Selosse et al. 2006). Hyphal connections can also maintain
physiological continuity between ramets of plants (Hutchings and Bradbury
1986) or between a tree and seedlings (Simard et al. 1997).
EMF provide an energy supply to the detrital food web as a result of the large
hyphae turnover, benefiting saprophytic microbes and other soil organisms (Dell
2002). The EMF mycelium constitutes the largest part of the biomass of most EMF
species (Godbold 2005). The hyphal mantle mycelium and extraradical hyphae can
have a biomass of 500–700 kg/ha (Godbold 2005). The EMF mycelium supports
the activity of free living decomposers (Buée et al. 2009). EMF also provide food
by mycophagy of the sporocarps (Dell 2002; details in Sect. 12.4.1.1.3 under the
service of spore dispersal).
EMF provide indirect services to many organisms related to the structure of the
soil (increasing the formation of soil aggregates) and to the cycling of nutrients
(replenishment of the available nutrients pool, Dell 2002). Rillig and Mammey
(2006) review how mycorrhizal fungi can influence soil aggregation at various
scales. Many services provided by EMF SPUs might occur by this mechanism, but
their understanding and quantification is still a matter of further research. EMF can
also indirectly affect plants. In a review, Koricheva et al. (2009) analyze indirect
effects of mycorrhizal fungi on insect herbivores. They describe significant effects
on all aspects of insect herbivores performance, including consumption, growth
rate, mass, fecundity, survival, and density. The scale of this influence is dependent
on space and time distribution of the insects.
It is clearly documented that soil invertebrates disrupt the carbon flow through
mycorrhizal networks by feeding on hyphae (e.g., collembola, – Hiol Hiol et al.
2004, orbatid mites – Schneider et al. 2005). But, taking into consideration the high
turnover rate of ERH and its very complex network (Lamour et al. 2007), the
negative influence of this consumption on the connections between plants sug-
gested by several authors is questionable (Moore et al. 1985; Johnson et al. 2005).
However, predation at the rhizosphere level may have an aboveground effect on
plant primary production if such interspecific interactions have an important influ-
ence on the overall pattern occurring at the root FDMs level (Moore et al. 2003).
Earthworms (Szlavecz et al. 2009) may also disrupt the mycelia through soil mixing
and burrowing, and changes in nutrient availability by altering litter quality and
quantity occur, resulting in a shift in the composition of the fungal community.
Humans also use EMF, but we do not discuss this here because the mechanisms
supporting the use of EMF are not biological alone (see Sect. 12.4.3). In light of the
literature reviewed here, the functional niches occupied by the EMF seem to be no
more diverse than those already identified in Sect. 12.4.1.1.2 based on the species of
the host plants and the number of hosts. Before looking at the structure of the
resulting homomorphic model (Sect. 12.4.1.1.6), we will briefly mention the exter-
nal control factors influencing the EMF system.
External Control Factors
External factors should not be confused with the parameters describing them. The
parameters resulting from the effect of the factors can be measured at all scales
(e.g., concentrations of toxic metals), including within the EMF developmental
system, but the action of the factor (pollution with metals by dry deposition) is from
outside the system. From a scale larger than their DSs, EM fungi are affected by
fire, increase of CO2 in air, warming, drought, nitrogen deposition, deposition of
toxic substances, and management practices. There can be direct effects or/ indirect
effects of one factor (e.g., by plants). The effect of coupled external control factors
may be different than the separately considered effects. More information on the
references supporting this statement and about the effects of these factors is
provided in Sect. 12.4.2 under the heading of disturbance mechanisms, because
the external control factors are investigated in fungal research only in the context of
their effects.
The Homomorphic Model for Up-Scaling
The homomorphic model of the community of fungi DSs is presented in Fig. 12.4.
Some of these FDMs could be split into several ones separated in time with seasonal
periodicity (see Sect. 12.4.1.1.2).
The extraradical FDMs, located between trees, and the sporocarps’ FDMs do not
include only the EM fungi but also other fungi if present in the same ST location,
The root FDMs, as well, may not only be limited to the EM fungi present in the
rhizosphere but could also include other fungi (eventually mycorrhizal) along the
mutualism–parasitism continuum (Schulz and Boyle 2005), if present in the same
–1 (min
DS) scale V1
V4a F1a F3a F4a F1’a V4’a V2 V6

Source
scale
V4b F1b F3b F4b F1’b V4’b V3
F5 V5
+1 scale V4c V7 V4’c
+2 (max
V8
DS) scale
Target External factors

scale
Fig. 12.4 Maximal homomorphic model of a community of EM fungi DSs (relationships not
represented for reason of visibility; a connectivity matrix can be easily constructed using the
information presented in the text). The scale refers to scale in space, not in time. The physical part
is not represented. The part of the model within the source scale (within a stratum) is the
homomorphic model for up-scaling under the constraints from FDMs of different scales and
external factors. Legend: the ET is noted with F (from fungi), the EVs with V (from value);
F1a,b ¼ fungi parts (root tips in the case of EMF) in two vertical layers at the roots of tree 1,
F10 a,b ¼ fungi parts in two vertical layers at the roots of tree 10 , F3a,b ¼ hyphae in the extra-
radical mycelium in two vertical layers, F4a,b ¼ rhizomorphs in the extraradical mycelium in two
vertical layers, F5 ¼ sporocarps, V1 ¼ bacteria, V2 ¼ mineral P and N, V3 ¼ organic P and N,
V4 ¼ trees (a,b ¼ roots by layer, c ¼ aboveground parts), V5 ¼ mycoheterotrophic plants, V6
¼ soil microinvertebrates, V7 ¼ fungivorous invertebrates, V8 ¼ fungivorous mammals
ST location. To the extent that interspecific interactions within the fungi species of
a FDM are important, they should be taken into consideration when assessing the
roles of an individual EMF species, or the EMF community as a whole by each
FDM.
We need four (apparent) hierarchical levels for understanding the ecological
functioning of EMF communities, and one more level eventually for management
purposes, if the target scale (of management) is larger than the maximal DS scale
(given by the size of fungivorous mammal populations). One more scale should be
added if one looks also for speciation processes (Sect. 12.4.1.2.2). There is a conver-
gence with the opinion of other authors about the number of needed scales but not
about exactly what those scales are. Wolfe et al. (2009), for instance, “believe four
scales are most relevant for the discussion of spatial pattern and process of mycorrhizal
population and communities (1) across landscapes, (2) within plant communities/
ecosystems, (3) within an individual host root system, and (4) within an individual
mycelium.”
12.4.1.2 Functional Issues
Stratification
According to the review of the literature, the basic unit for stratification of the target
system in the case of EM fungi is a 400–900 m2 tree plot. However, as we have
shown in Sect. 12.4.1.1.2, several classes of tree plots should be differentiated as a
function of the position in the center or at the ecotone of the target system and as a
function of the structure of the vegetation. Other criteria might include management
practices. As the differences between the soil horizons of the EMF community can
be modified by liming, in addition to the tree host species (Rineau and Garbaye
2009), it means that one should stratify the target system both as a function of the
vegetation structure and of (at least in part) the management measures applied. In
general, the principle for stratification should be that one stratum type corresponds
only to one type of homomorphic model of the EMF community, as reflected both
by the fungi per se and by their symbiotic hosts.
Scale-Specific Mechanisms of EMF Productivity
We have touched on these mechanisms in the previous part because researchers

rarely look only at structural issues without approaching the functional aspects as
well. Here, we attempt to systematize the information from the scales and systems
synthesized in Fig. 12.4. Most of the knowledge concerning the productivity
mechanisms of EMF is in a verbal, nonmathematical form.
Organism and population level Function at the organism level is supported by
metabolic processes and ecophysiological processes (in the classic sense).
The scarcity of literature concerning the function of an individual fungi in an
ecological context is probably due not only to experimental difficulties but also to a
lack of concentrated effort on a model species. Fungi seem to not yet be in the focus
of systems biologists. We have not insisted on the exploration of this type of
literature. In a singular study, Ritter et al. (1989) analyzed in detail cytological
aspects of the stages of ectomycorrhizal vitality. Smith et al. (2010) characterized
plant-inducible phosphorus transporters in fungi and mycorrhiza-inducible trans-
porters in plants. Lewis et al. (1994) studied the effect of CO2 on an individual
plant–fungi system; the pattern of results with the same control factor was found to
show interspecific variation between EMF species. A new approach for the study of
in vitro transport of amino acids in hyphae is proposed by Watkinson et al. (2005).
Bonneville et al. (2009) report a first direct observation of the effect of a soil fungus
on the surface of a mineral. A recent review (Rosling 2009) summarizes the current
knowledge on fungal weathering as affected by experimental setup and conditions
(pure or symbiotic growth, nitrogen source, the means of detecting weathering
activity and the species examined).
The extra productivity (compared to the individual level) emerging at the popula-
tion level is in principle related to the intraspecific mechanisms. We have not found
literature about such interactions, and this is probably due to the methodological
difficulties in delineating such individuals.
Tree and plot level We refer here to the fungal FDMs at tree and intertree levels,
within each stratum. Each such FDM should correspond, in our opinion, to a
community in the classical sense (it is the system within which alfa diversity should
be measured). The extra productivity at this level is due to interspecific interactions;
however, the literature does not always make clear what kind of diversity is
discussed with respect to its effects.
A review of the EMF community ecology was done by Dahlberg (2001). Data
aggregation at different scales may lead to different control variables for the
community composition; soil parameters at subplot level, but plant community at
plot level (Fitzsimons et al. 2008). We interpret this as being within FDM diversity
at subplot level and between FDM diversity at plot level. Some EM species were
found significantly more frequently as mycelia than as root tips, while others were
less dominant as mycelia than as root tips (Kjoller 2006). We can interpret this as
being a consequence of the exploration type in the FDMs structure. Kranabetter
et al. (2009) found significant changes in an EMF community along a 25 km
productivity gradient (to nitrogen rich sites). EMF species abundance in relation
to site productivity included parabolic, negative, linear, and exponential curves.
A functional diversity of EMFs was observed, with the specialization of EMF
communities contributing to the successful soil exploitation by a single tree host
species. The phenotypic plasticity of the tree species was much enhanced as a result
of the interaction with the EMF. The diversity described by Kranabetter et al.
(2009) seems to be of at least gamma type (aggregates of communities).
The detailed heterogeneity of abiotic parameters at the FDM level might influ-
ence the functioning of the community. A detailed review of the space-time
heterogeneity of key abiotic parameters at rhizosphere level was done by Hinsinger
et al. (2009). They point out that the integration of such models into root growth and
root architecture models for up-scaling of rhizosphere processes is still a matter of

future research, but no knowledge of fungal distribution at this fine scale (to be
related to the abiotic parameters) is referred to.
The interaction of EMF with lower scale EVs increases their productivity. The
coinoculation of EMF with bacteria increased mineral weathering, plant uptake of
soluble forms of K and Mg, and tree biomass, as compared to simple EMF
inoculation, while the bacteria alone had no effect (Koele et al. 2009). The
relationship between two plants at the same time may increase the productivity
too. Intertree EM linkages can reduce plant competition for resources, promote
forest recovery, and influence the pattern of plant succession (Amaranthus and
Perry 1994). Seedlings were hydraulically linked by the mycorrhizal network to
large trees in a study by Warren et al. (2008).
Some generalizations accepted for AMF could also hold for EMF. The species
composition and richness of AMF was found to be an important contributor to plant
species composition and productivity, with an effect mainly on subdominant plant
species, and with different plant species benefiting from different AMF taxa (van
der Heijden et al. 1998). The external driving factors affect both AMF and EMF.
For instance, Godbold et al. (1997) found changes in the relative proportion of
EMF to AMF as a result of elevated CO2 concentrations. Consequently, the roles of
EMF and AMF in the functioning of the vegetation communities, especially in the
up-scaling context, should probably be approached in an integrated manner. It is
clear that the FDMs should be seen as based on fungi species, not just separately on
EM, AM, or saprophytic fungi, especially in the case of the FDMs associated with
the external mycelium.
Ecosystem level We refer here to systems of FDMs from the plot scale up to the
“ecosystem” scale, to the control of EMF productivity by feedback from the host
plants, and to indirect effects by changing the soil structure. The scale approached here
is that between the stratum and the target scale envisaged in the up-scaling procedure.
The intermediate scales to be tackled will depend on the organisms retained as relevant
from the point of view of direct and indirect interactions with the EMF.
In an excellent review of mycorrhizal–plant–insect interactions, Gehring and
Bennett (2009) summarize the proven effects of mycorrhizal plants as follows:
negative on root herbivores, positive on pollinators, negative, neutral, or positive on
herbivores, and positive or negative on herbivore enemies. Each such group is
characterized by specific scales, and the decision whether or not to include these
interactions in the overall homomorphic model for up-scaling should be done as a
function of the feedback effects of these groups (as influenced by EMFs) on the
primary productivity of the ecosystem and on the dynamic of carbon in the forest floor.
Other complex effects come from the action of external driving factors on
several types of organisms of different scales. In many systems exposed to elevated
CO2, mycorrhizal fungi sequester increased amounts of carbon in living, dead, and
residual hyphal biomass in the soil (Treseder and Allen 2000). When this is coupled
with nitrogen deposition, an increased turnover rate of hyphal biomass can occur.
The two processes are associated with a shift in the EMF community’s composition
as a result of physiological interspecific differences.
The facilitative effects of mycorrhizal fungal networks depend on the seedling

species identity, mycorrhizal identity, plant species combination, and study system,
but seedlings associated with EMF benefited in the majority of reported cases (van
der Heijden and Horton 2009). Southworth et al. (2005) applied the network theory
to mycorrhizal networks from a phytocentric and fungicentric perspective and
concluded that all individual plants are more or less equal in linking fungi, but
from a mycocentric perspective the network is scale free (Barabasi and Albert 1999;
Barabasi and Bonabeau 2003), meaning that certain species of fungi act as hubs
with frequent connections to the other elements of the network.
Productivity may be increased also by changing over time the structure of the
soil. While AMF produce large quantities of glycoproteins in soil (1.45 106 g/ha,
3.2% of the total soil carbon in the 0–10 cm soil layer, Lovelock et al. 2004), EMF
are characterized mainly by the production of extracellular enzymes responsible for
the mobilization of organic carbon and associated nutrients (Maijala et al. 1991;
Courty et al. 2005). Both mycorrhizal fungi types also produce effectors responsible
for colonization (Martin and Nehls 2009). The influence of AMF’s ERH on soil
structure might be of even greater importance to the carbon stock than the influence
of hyphal standing crops (Miller and Kling 2000), and this statement can hold as
well for EMF. This is because, in both cases, there are feedback responses between
the soil and canopy mediated by fungi and supported by mechanisms linked to
nutrient acquisition and the allocation of the tree’s assimilated carbon by mycor-
rhiza or indirectly by litter fall.
EMF may facilitate the dispersal of plants and thus increase the productivity of
the target system. In an interesting article, Thiet and Boerner (2007) studied the
EMF’s role at the ecotones of a forest. The invasion of a pine species on an
unforested area was dependent on the previous dispersals of EMF either as spores
by animals or wind, or as hyphae from the ecotonal trees. The latter mechanism
proved in this case to be the most important for pine seedlings, underlying the need
for a stratified analysis of EMF’s role in the central and peripheral parts of the
forest.
At the ecosystem scale is supported also the stability of the fungal community,
by dispersal between plot scale areas. With regard to the stability issue (and
assuming that diversity is directly related to functional stability), in a study of
tree islands ranging from <10 to >10,000 m 2 (Peay et al. 2007), the species area
slope was similar to slopes for macroorganisms, suggesting that microbes are not
ubiquitous even in suitable habitats (if small); the trade-off between dispersal and
competition played an important role in structuring EMF assemblages.
Speciation of EMF We refer here to systems larger than ecosystems, at the scale
of which speciation of EMF occur. This scale has not been discussed or included in
Fig. 12.4 because the up-scaling is usually only needed for management over a
short time, without taking into account speciation. Underlining of the importance
of evolutionary processes is needed, both for diversity conservation and for the
interpretation of the apparent redundancy of species within the FDMs. The large
number of rare EMF species (as indicated by non saturated rarefaction curves)
indicates a functional redundancy in the relatively short term, but this apparent
redundancy in fact supports the stability of the ecological system in the average
term and the potential for EMF’s evolution in the long term.
High treeless ridgelines are effective barriers to EMF gene flow even at distances
less than 65 km, whereas populations (according to Amend et al. 2010, but probably
metapopulations) located within watersheds are structured at greater distances
(125 km). So the scale at which speciation takes place is much larger than the
ecosystem scale and is the maximal one indicated by Wolfe et al. (2009) to be
included in the study of EMF. It is documented that the structure of the landscape
influences the evolutionary outcome. For instance, Read and Perez-Moreno (2003)
point out that selection has favored EMF systems with well-developed saprophytic
capabilities in those ecosystems characterized by retention of nitrogen and phos-
phorus as organic complexes in the soil.
To sum up the information presented in Sect. 12.4.1.2.2, different productivity
mechanisms involving EMF are distributed across many scales. The information is
scarce at individual level, apparently absent at population level, richer at community
and ecosystem levels, and scarce again at large landscape levels. The production
takes place at all levels, but with maximal intensity and stability in time at the large
landscape scale.
12.5 Disturbance and Succession of Ectomycorrhizal Systems
The change in the relative importance of fungi and bacteria in forest soils with
succession seems to remain uninvestigated, but in many secondary succession
grassland chronosequence studies, the soil microbial community tends to shift
towards a less bacterial and more fungal-dominated food web (Maharning et al.
2009). Hypothetically, this may occur in forests too. A discussion of fungal
succession is much more complicated, however, because it envisages processes at
very different scales. Iordache et al. (2009b) presented and critiqued the early and
late stage species approach for the succession of EMFs and then introduced an
ecosystem approach to fungal succession in an improved framework based on Pahl-
Vostl’s TDM concept. We refer the reader to Iordache et al. (2009b) for aspects
relating to succession. In this present text, we develop the analytical framework
sketched there and apply it to the more general up-scaling problem of EMF
functioning. Another subject discussed in the paper is EMF disturbance due to
heavy metals, for which a data processing and interpretation framework was
proposed. This picture is complemented here by a short review of disturbance
factors relevant to EMF.
Cudlin et al. (2007) review the effects of acidic deposition, nitrogen deposition,
increased ozone levels, elevated CO2, and drought on fine roots and EMF. EMF
colonization was not a suitable parameter for assessing the effects of these driving
factors, but fine root length and biomass could be useful. This does not mean that
the FDMs of EMF have not been affected because they are not delineated in space
at the fine root level but at the plant root/rhizosphere level. The disturbance factors
usually act at a large scale, but the mycorrhizal response is at an FDM scale and
depends on the species composition of the EM community and the relationships of
ectomycorrhizal FDMs with other types of FDMs (the structure of the FDM
network at stratum scale).
Acid rain reduced the number, length, and biomass of lateral tree roots and the
percent and number of EMF (Esher et al. 1992). Some EMF confer drought tolerance
to their host (via influencing the plant’s osmoregulation), while others confer drought
avoidance (by hyphal transport via EMHs, Mudge et al. 1987). The experimental
warming of root-associated fungal communities in an arctic region increased the
density of different genotypes but did not affect the biodiversity within the time frame
of the experiment (Fujimura et al. 2008). Burning either decreases or increases the
colonization of EMF. The increase is attributed to the reduction of substances that
inhibit germination (Cairney and Bastias 2007), and the decrease occurs mostly in
the organic layer, not in the mineral soil. Peay et al. (2009) provide a comparison of
disturbance factors by scale. With regard to fire, they conclude that spore heat
resistance plays an important role in the disturbance-mediated assemblage shift
of EMF. Fire disturbance favors competitively inferior species, keeping diversity of
EMF at landscape scale. Nilsson and Wallander (2003) report a negative influence
of nitrogen fertilization on the external mycelium of EMF, not directly by the soil
nitrogen concentrations but rather by the nitrogen status of the trees.
The effect of enhanced CO2 concentrations mediated by EM communities takes
place through the modification of carbon inputs from plant to soil, with conse-
quences on the biomass, infectivity, and species composition of the symbionts
(Diaz 1996). Godbold and Berntson (1997) reported changes in EMF community
structure as a result of elevated CO2. A review by Staddon et al. (2002) about
temperature and CO2 effects on EM fungi concluded that they should involve the
study at the individual-plant level, multiple species level, and community level. An
interesting finding about the effect of CO2 is that of Pritchard et al. (2008): “CO2
enrichment increased mycorrhizal root tip production in deep soil, but did not
influence it in shallow soil;” also “the rhizomorph turnover was accelerated in
shallow soil, but effects on survivorship in deep soil varied according to diameter.”
These FDM-specific effects open the way to a line of research on how external
control parameters especially influence some of the FDMs from the structure of the
homomorphic model. For now, the experiments for assessing the effect of CO2 have
been performed mostly at small plot level (102 m2) with fewer at “field” level
(102 m2) and especially with monoculture (Staddon et al. 2002).
Miller and Lodge (1997) review the fungal response to disturbances in agricul-
ture and forestry. By disturbance they mean the physical and chemical phenomena
that disrupt communities and ecosystems. Fungi are concluded to be control points
in management practices (tillage and crop rotation, nutrient additions, air pollution,
site preparation, woody debris, opening the canopy, and moisture fluctuations).
Jones et al. (2003) review in detail the dynamics of ectomycorrhizal communities
after clear-cut logging, identifying the amount and type of inoculum, and the
changes in the soil abiotic and biotic environment as the major groups of factors
controlling the succession without discussion of diversity indexes as aggregated
indicators of ecosystem state. Studies of another forest management practice, gap

opening, show a significant reduction in EMF diversity indices and a change in
EMF and fine root dynamics compared to closed stands (Grebenc et al. 2009) but
with some fungi preferring the new conditions.
Colpaert (2008) reviews the effects of metal on fungi and their adaptation. He
concludes that there is true tolerance of EMF to metals. Investigations of EMF
species at the community level have revealed wide inter- and intraspecific varia-
tions in sensitivity to metals (Hartley et al. 1997). The EMF community in a soil
contaminated with metals was rich (not with just a few specialist fungi as expected)
but did not vary with the soil horizon, season, or plot location in the forest (Krpata
et al. 2008). This suggests an interesting hypothesis that the FDM structure of EMF
communities is simplified as a result of toxic pressure compared to uncontaminated
areas. Different metals controlled in a specific way the EMF community, the results
of a multimetal stress being complex at forest levels and reflecting the distribution
of metals at tree scale (Gherghel 2009). Alleviation of metal toxicity in plants
through EMF has been demonstrated (review by Jentschke and Godbold 2000), but
the mechanism remains unclear. Possibilities include immobilization in fungi,
exudation of metal chelating substances in the soil, or nutritional and hormonal
effects in plants mediated by fungi. EMF and associated bacteria protect pine
seedlings against bioavailable forms of Cd, but there are differences in the level
of protection provided by different fungi species (Kozdrój et al. 2007). Bioaccu-
mulation factors for Zn and Cd in fruiting bodies of EMF decreased with increasing
soil concentration, showing that in such a case fungi did not act by accumulation as
an effective barrier against metal uptake by the symbiotic tree (Krpata et al. 2009).
12.5.1 Role of EMF in the Functioning of the Natural Capital
Through some of its EV, the fungal DS reaches the smaller scale directly relevant
for the management of the natural capital (hectares to square kms, here by conven-
tion the ecosystem scale). Griffiths et al. (1996) propose an even larger system for
analyzing the role of EM SPUs, namely the watershed. This is similar to the scale of
EMF speciation. Fungi and their ecosystem services might be in jeopardy if habitat
(tree patch) size is a strong determinant of fungal richness, as it seems to be (Peay
et al. 2007, 2008).
As an example of the explicit connection between basic and applied ecology in
our target domain, Dighton (2003) analyzes the role of fungi in the production of
ecosystem services, including mycorrhizal fungi and their relationships with arthro-
pods, as well as the consequences of human action on the relevant mechanisms.
Recently, Jackson et al. (2008) also reviewed the effects of root processes (includ-
ing the dependence on mycorrhization for nutrient acquisition) on ecosystem
services. The value of EMF to people is related to local consumption and trade of
sporocarps, to their use in medicine, biomonitoring and bioremediation, to their
esthetic value, and to the services provided by EM SPUs to other biological
compartments supporting the ecosystem function – implicitly the production of

other biological resources and services directly relevant to the people (Dell 2002).
Hall et al. (2003) reviews the literature about edible ectomycorrhizal mushrooms.
The EMF SPUs are perceived as useful mainly through their support of the
resources and services associated with forest ecosystems. Management practices
that create intense disturbance and loss of organic matter decrease the ability of
plants to form EMF linkages, but management practices that retain living trees and
shrubs and the input of organic matter facilitate EMF linkages (Amaranthus and
Perry 1994). Helpful forestry management practices targeted to EMF can be taken at
various scales: small ones (retaining refuge plants, mature trees) and larger ones
(retaining old-growth forest, avoiding high intensity broadcast burn, and retaining the
edge to area ratio of harvested areas within certain limits) (Wiensczyk et al. 2002).
Heneghan et al. (2008) review the use of soil ecological knowledge for restora-
tion. From their perspective, when the goal is loosely defined (no specific recovery
trajectory envisaged), less precise knowledge can be useful (for instance, the
concept of “soil quality” is acceptable in this context for management issues), but
when a complex outcome is desired, exact knowledge is needed (we would say,
down to the SPU’s level). Another point in their analysis (and of others, e.g.,
Neagoe et al. 2009) is that a restoration project should be seen as a scientific
experiment and valued as such by the scientist.
In prescribed burning, unburned patches act as an inoculation source. The return
to the preburn state of the EMF community takes place in about 15 years (Cairney
and Bastias 2007). The role of fungi in ecosystem restoration after fire (Claridge
et al. 2009) is related to the stabilization of the soil in the absence of plants (by some
species of fungi), to nutrient acquisition from minerals, and to mycorrhizal function
once the plants start to recover (other species of fungi). It can be said that the fungi
are strongly involved in the secondary succession and restoration management after
such a disturbance is in fact an attempt to control the secondary succession (Neagoe
et al. 2009; Iordache et al. 2009b). Change in soil structure is an important process
during succession and, as we have noticed in Sect. 12.4.1.1.4, the EM SPUs provide
important services by influencing the soil structure.
The use of mycorrhizal fungi can also be targeted to restoration goals related to
the control of toxic substances, metals, or hydrocarbons (Robertson et al. 2007;
Ghergel 2009). Filamentous fungi and their enzymatic system were found to be a
“potent tool to decrease the levels of contaminants in soils by degradation and
stabilization” (review by Mougin et al. 2009). Measures taken in the frame of
restoration projects, such as liming, strongly affect the EMF community (in this
case by pH increase, Kjoller and Clemmensen 2009). The decline of the services
valued by humans is, of course, related not to mycorrhizal fungi alone. For instance,
Gilliam (2006) identifies six types of mechanisms supporting the deterioration of
the herbaceous layer by nitrogen deposition (Fig. 4 in Gilliam 2006): interspecific
competition, herbivory, mechanisms related to mycorrhizal infection, pathogenic
fungal infection, species invasion, and exotic earthworm activity.
We conclude that understanding the effects of EMF-mediated deterioration at a
large scale needs integration in the up-scaling model of all other mechanisms
observable at intermediate scales related to SPUs of EMF. The integration of other

ecological mechanisms, depending on the service and resources identified, will be
needed as well for a full understanding and effective specific goal-directed man-
agement of that natural capital.
12.5.2 Mathematical Modeling
On the mathematical modeling side, Johnson et al. (2006) provide an excellent

review of seven types of mycorrhizal models (that include mycorrhizal parameters
in their structure), varying in their scale of resolution and dynamics and discuss
approaches for integrating these models with each other and with general models of
terrestrial ecosystems. They use the classical hierarchy (individual, populations,
communities, and ecosystems). At individual and population levels, there are
biomass allocation (functional equilibrium) models, economic biological market
models, and integrative agent-based models. At the community scale, there are
community feedback models, and co-evolutionary mosaic models. At the ecosys-
tem scale, there are food web models and pedogenesis models. In the concluding
part, Johnson et al. (2006) mention that “mycorrhizal effects on resource availabil-
ity and biomass allocation patterns have not been included in these models, partly
because of insufficient information but also because of scaling differences [. . .]
mycorrhizal effects on soil properties, disease resistance, and trophic cascades are
not emphasized to the same extent in current models,” and “encourage future efforts
to develop methods for measuring mycorrhizal structure and function at relevant
spatial and organizational scales.”
If one looks into each category of models for relevance of the mathematical
formalism to EMF, the picture is not encouraging. Functional equilibrium models
are exemplified by a conceptual model dedicated to AM fungi, and the primary
source cited does not include mathematical relationships between the mycorrhizal
biomass and the carbon and nutrient resources. Economic models and food-web
models referred also to AM fungi. Agent-based models and pedogenesis models
referred both to AM and EMF; community feedback models and co-evolutionary
mosaic models referred to mycorrhizal fungi in general.
It seems that the state of mathematical models needed for up-scaling the EMF
ecological processes from the stratum scale to the target scale is not yet appropriate,
and that developing such models is an important research direction.
12.6 Research Directions
The proposed framework for conceptualizing the DS’s functioning across scales is
convergent with recent proposals for coupling traditionally small scale targeted
ecophysiology to the functioning of ecosystems under the umbrella of a “macro-
physiology” (Gaston et al. 2009).
For the up-scaling of EM fungi functions, we must (adapted from Lilleskov et al.
2004) identify individuals in tips, soil (as hyphae), and sporocarps, discern patch
change over time, identify endogenous factors (intraspecific such as clonal expan-
sion or high spore rain and interspecific interactions structured by a FDM
approach), identify exogenous factors (patterns of resource availability, disturbance
history, and current external driving factors), then on the resulting dynamic archi-
tecture add the functional information inside each FDM and between FDMs, and
finally aggregate it from the original to the target scale. Doing this is still precluded
by many gaps, both at structural and at functional levels. A structured analytical
approach to the problem (like the one proposed here) might accelerate the knowl-
edge development in the area.
Coupling observations at the molecular scale (plant–fungus gene expression, TE
level, and internal EVs), interfacial scale (TE-external EV level, to quantify ion
uptake by plants), experiments at pot scales (individual fungi–plant or FDM level),
lysimeter scales (FDM level), and plot scales (system of FDMs level) with long
term field ecosystem studies is crucial for obtaining the knowledge needed for
integrating (up-scaling) lower level processes into the management (remediation,
restoration, control of secondary succession) of the natural capital at ecosystem and
landscape scales (Neagoe et al. 2005; Graham and Miller 2005; Meixner et al. 2006;
Neagoe et al. 2009). Hinsinger et al. (2009) underline the need for the integration
of ST models of abiotic parameters in the rhizosphere into root growth and root
architecture models for up-scaling of rhizosphere processes. Graham (2008) states
that “experimental design should either integrate multiple mechanisms of the
landscape scale and include such measures as mycorrhizal influences on net
primary production, evapotranspiration and nutrient cycling, or integrate measures
of [. . .] fungal diversity into assessment of ecosystem function.” We believe that
an FDM approach allows for the structured investigation of functional diversity
and complementarities between EMF species within FDMs (for example, some
fungi may be effective in scavenging organic nitrogen, and others more effective
in phosphorus uptake and transport – Buée et al. 2009), and the functional
complementarities between different types of FDMs. Pool-flux classical ecosystem
type research can be associated with a FDM based homomorphic model, and
theoretical research on the characteristics of the fungal networks are also compati-
ble with this framework.
A major limitation to scaling the mycorrhizal symbioses to higher organizational
levels is knowledge about the fungal biomass in the characteristic FDMs. The ERH
biomass can be assessed by total hyphal length (Graham and Miller 2005), using
biochemical markers (chitin, ergosterol, or a specific fatty acid) or by competitive
PCR (Godbold 2005). The background biomass of saprophytic fungi should also be
determined. Satomura et al. (2006) present direct methods to quantify the fungal
content in EM fine roots. One needs long term field studies in order to appropriately
assess the biomass production (Staddon et al. 2002).
The available methods for investigating the distribution of functional parameters
across scales summarized by Wolfe et al. (2009) include rotated cores (for nutrient
uptake and decomposition), molecular approaches for expression of functional
genes, and the use of natural gradients. To these, one can add extracellular enzymes
profiles (e.g., microplate assays developed by Courty et al. 2005) and the assay
developed by Rineau et al. (2008) for comparing iron chelation, free iron uptake,
and oxalate production of freshly sampled EMs . Graham (2008) mentions addi-
tional methods for AMF, including in situ root observation windows, mesh dividers,
bags and isotopic tracers, and signature fatty acids, the third of which can be applied
to EMFs as well (Buée et al. 2009). A tool-box for mycorrhizal research at the
ecosystem scale is also provided by Rillig (2004).
Scaling-up from sequence data to a whole plant and its functions requires a
genomic-based approach and a systems approach to study the information flow
(Graham and Miller 2005). There is also a need to understand the genetic basis of
tolerance to metals in ECM symbiosis (Hartley et al. 1997). An important review
of the molecular tools in EM ecology is made by Horton and Bruns (2001). An
overview of the molecular techniques available for the analysis of fungal commu-
nities was done by Peay et al. (2008). The use of genomics for EMF ecological
insights is essential (Martin and Nehls 2009). Tools allowing for the production of
extensive data sets needed for models in order to couple the characterization of
EMF with indicators of their functional rates have also started to be available.
Vargas and Allen (2008), for instance, use CO2 microsensors for characterizing
respiration in an EM root system. The use of new sensor technologies is of great
promise for the generation of both small scale intensive data sets (with structural
and function significance – Allen et al. 2007; Hasselquist et al. 2009) and large scale
ones (indicators of ecosystem functions or external control factors – Porter et al.
2005, 2009; Rundel et al. 2009).
Specific research directions/questions are:
l Describing the formal structure of the DS population’s models and deriving
from them the Price equation, if possible. Formulating a decoupling theory for
the apparent ecological hierarchical levels based on the scale specificity of the
types of abiotic and biological processes (Iordache et al. 2011).
l Exploring the potential of new theoretical tools (Barabasi and Albert 1999;
Barabasi and Bonabeau 2003) for the conceptualization of fungal networks
(and of the emergence function relevant for EMF). A theoretical network
approach to the EMF – plant systems (and more generally MF-plant systems)
at stratum level and interstratum level is proposed by Southworth et al. (2005).
A cost benefit approach to the individual members of a mycorrhizal network is
suggested van der by Heijden and Horton (2009).
l Study of taxa area relationships for EMF taxa based on functional genes (gene
area relationships, Zhou et al. 2008).
l Linking proteomics and ecological processes with a focus on soil enzymes as
mediators of decomposition, dissolved organic carbon production, and nitrogen
and phosphorus mineralization (Allison et al. 2007).
l To what extent the driver/passenger hypotheses formulated for AMF (Hart et al.
2001; fungi drive the plant community or are just a by-product of changes in the
plant community) could be relevant also to EMF?
l How the external control factors influence especially some of the FDMs from the
structure of the homomorphic model (fire for the top FDMs and CO2 for the deep
FDMs).
l Is the gene flow affected in landscapes fragmented by contamination with
metals? Is the FDM structure of EMF communities simplified as a result of
toxic substances pressure?
l Is there an optimal form of natural capital’s modularization maximizing the
value produced by SPUs associated to EMF?
12.7 Conclusions
We have developed an analytical framework for up-scaling ecological processes

and applied it to EMF. One has to construct four “hierarchical” levels in order to
understand the ecological role of EMF in the ecological productivity of ecosystems,
and one more if interested in evolutionary processes (gene flow, speciation). The
modularization scales for understanding the role of EMF are those specific to
bacteria, to fungi (FDM occupying surface of tenths of m2, and tree plot of
400–900 m2), to epigeous fungivorous invertebrates, and to fungivorous mammals,
and, for speciation, to small catchments of several hundreds of km2. The analysis
showed that the source scale for up-scaling has to be a plot of 400–900 m2. This plot
has an associated homomorphic model with a maximum number of nine FDM for
the structural and functional modeling of EMF communities. Only one modeling
step is needed for up-scaling from the source scale (plot) to the ecosystem scale, but
the model’s construction involves the previous construction of several up-scaling
and down-scaling models in order to quantify the effects on smaller and larger scale
organisms on fungi.
Acknowledgments We warmly thank Professor Mahendra Rai for providing the opportunity to
prepare this text, and a native English speaker for checking the language. The research leading to
the results presented here was supported by German program DAAD (research visit awarded to the
first author), the Romanian agencies UEFISCSU (project MECOTER code 1006 nr. 291/2007),
CNMP (projects FITORISC 31012/2007, and PECOTOX 31043/2007), and the European program
FP7 (project UMBRELLA nr. 226870 Grant Agreement 090528).
References
Agerer R, Gőttlein A (2003) Correlations between projection area of ectomycorrhizae and H2O
extractable nutrients in organic soil layers. Mycol Prog 2:45–52
Agerer R, Weiss M (1989) Studies on ectomycorrhizae. XX. Mycorrhizae formed by Thelephora
terrestris on Norway spruce. Mycologia 81:444–453
Allen MF, Vargas R, Graham E, Swenson W, Hamilton M, Taggart M, Harmon TC, Rat’ko A,
Rundel P, Fulkerson B, Estrin D (2007) Soil sensor technology: life within a pixel. Bioscience
57:859–867
Allison SD, Garner TB, Holland K, Weintraub M, Sinsabaugh RL (2007) Soil enzymes. Linking
proteomics and ecological processes. In: Hurst CJ et al (eds) Manual of environmental
microbiology, 3rd edn. American Society for Microbiology, Washington D.C., pp 704–711
Amaranthus MP, Perry DA (1994) The functioning of ectomycorrhizal fungi in the field: linkanges
in space and time. Plant Soil 159:133–140
Amend A, Garbelotto M, Fang Z, Keeley S (2010) Isolation by landscape in populations of a
prized edible mushroom Tricholoma matsutake. Conserv Genet 11(3):795–802
Baier R, Ingenhagg J, Blaschke H, Gőttlein A, Agerer R (2006) Vertical distribution of an
ectomycorrhizal community in upper soil horizons of a young Norway spruce (Picea abies
[L.] Karst.) stand of the Bavarian Limestone Alps. Mycorrhiza 16:197–206
Bailey RG (1987) Suggested hierarchy of criteria for multiscale ecosystem mapping. Landsc
Urban Plan 14:313–319
Barabasi AL, Albert R (1999) Emergence of scaling in random network. Science 286:509–512
Barabasi AL, Bonabeau E (2003) Scale-free networks. Sci Am 288:50–59
Beare MH, Coleman DC, Crossley DA Jr, Henddrix PF, Odum EP (1995) A hierarchical approach
to evaluating the significance of soil biodiversity to biogeochemical cycling. Plant Soil 170:5–22
Berg MP, Bengtsson J (2007) Temporal and spatial variability in soil food web structure. Oikos
116:1789–1804
Bidartondo MI, Burghardt B, Gerbauer G, Bruns RD, Read DJ (2004) Changing partners in the
dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and
trees. Proc R Soc Lond B 271:1799–1806
Bonneville S, Smits MM, Brown A, Harringston J, Leake JR, Brydson R, Benning LG (2009)
Plant-driven fungal weathering: early stages of mineral alteration at the nanometer scale.
Geology 37:615–618
Bruns TD, Kennedy PG (2009) Individuals, populations, communities and function: the growing
field of ectomycorrhizal ecology. New Phytol 182:12–14
Bruns TD, Bidartondo MI, Taylor L (2002a) Host specificity in ectomycorrhizal communities:
what do the exceptions tell us? Integr Comp Biol 42:352–359
Bruns TD, Tan J, Bidartondo M, Szaro T, Redecker D (2002b) Survival of Suillus pungens and
Amanita francheti ectomycorrhizal genets was rare or absent after a stand-replacing wildfire.
Buée M, De Boer W, Martin F, van Overbeek L, Jurkevitch E (2009) The rhizosphere zoo: an
overview of plant-associated communities of microorganisms, including phages, bacteria,
archaea, and fungi, and of some of their structuring factors. Plant Soil 321:189–212
Cairney JWG, Bastias BA (2007) Influences of fire on forest soil fungal communities. Can J For
Res 37:207–215
Calvaruso C, Turpault MP, Leclerc E, Frey-Klett P (2007) Impact of ectomycorrhizosphere on the
fnctional diversity od soil bacterial and fungal communities from a forest stand in relation to
nutrient mobilization processes. Microb Ecol 54:567–577
Carrey AB, Harrington CA (2001) Small mammal in young forests: implications for management
and sustainability. For Ecol Manag 154:289–309
Claridge AW, Trappe JM, Hansen K (2009) Do fungi have a role as soil stabilizers and remediators
after forest fire? For Ecol Manag 257:1063–1069
Colpaert JV (2008) Heavy metal pollution and genetic adaptations in ectomycorrhizal fungi. In:
Avery S et al (eds) Stress in yeasts and filamentous fungi. Elsevier, London, pp 157–173
Courty PE, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005) Activity profiling of ectomy-
chorrhiza communities in two forest soil using multiple enzymatic tests. New Phytol
167:309–319
Courty PE, Franc A, Pierrat JC, Garbaye J (2008) Temporal changes in the ectomycorrhizal
community in two soil horizons of a temperate oak forest. Appl Environ Microbiol 74:
5792–5801
Coutts MP, Nicoll BC (1990) Growth and survival of shoots, roots and mycorrhizal mycelium in
clonal Sitka spruce during the first growing season after planting. Can J For Res 20:861–868
Cudlin P, Kieliszewska-Rojucka B, Rudawska M, Grebenc T, Alberton O, Lehto T, Bakker MR,

Borja I, Konopka B, Leski T, Kraigher H, Kuyper TW (2007) Fine roots and ectomycorrhizas
as indicators of environmental change. Plant Biosyst 141:406–425
Dahlberg A (2001) Community ecology of ectomycorrhizal fungi: an advancing interdisciplinary
field. New Phytol 150:555–562
Darwin C (1859) Origin of species. John Murray, London
Dell B (2002) Role of mycorrhizal fungi in ecosystems. CMU J 1:47–60
Dengler J (2009a) A flexible multi-scale approach for standardized of plant species richness
patterns. Ecol Indic 9:1169–1178
Dengler J (2009b) Which function describes the species-area relationship best? A review and
empirical evaluation. J Biogeogr 36:728–744
Diaz S (1996) Effects of elevated [CO2] at the community level mediated by root symbionts. Plant
Soil 187:309–320
Dickie IA, Bing Xu, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in
soil as shown by T-RFLP analysis. New Phytol 156:527–535
Dighton J (2003) Fungi in ecosystem processes. Marcel Dekker, New York
Douhan GW, Rizzo DM (2005) Phylogenetic divergence in a local population of the ectomycor-
rhizal fungus Cenococcum geophilum. New Phytol 166:263–271
Esher RJ, Marx DH, Ursic J, Baker RL, Brown LR, Coleman DC (1992) Simulated acid rain
effects on fine roots, ectomycorrhizae, microorganisms, and invertebrates in pine forests of the
Southern United States. Water Air Soil Pollut 61:269–278
Ettema CH, Wardle DA (2002) Spatial soil ecology. Trends Ecol Evol 17(4):177–183
Fitzsimons MS, Miller RM, Jastrow JD (2008) Scale-dependent niche axes of arbuscular mycor-
rhizal fungi. Oecologia 158:117–127
Fortin MJ, Olsol RJ, Ferson S, Iverson L, Hunsaker C, Edwards G, Levine D, Butera K, Klemas V
(2000) Issues related to the detection of boundaries. Landsc Ecol 15:453–466
Fujimura KE, Egger KN, Henry GH (2008) The effect of experimental warming on the root-
associated fungal community of Salix arctica. ISME J 2:105–114
Gaston KJ, Chown SL, Calosi P, Bernardo J, Bilton DT, Clarke A, Trullas SC, Ghalambo CK,
Konarzewsk M, Peck LS, Porter WP, P€ ortner HO, Rezende EL, Schulte PM, Spicer JI, Stillman
JH, Terblanche JS, van Kleunen M (2009) Macrophysiology: a conceptual reunification. Am
Nat 174:595–612
Gebhardt S, Wőllecke J, Műnzenberger B, Hűttl RF (2009) Microscale spatial distribution patterns
of red oak (Quercus rubra L.) ectomycorrhizae. Mycol Prog 8:245–257
Gehring C, Bennett A (2009) Mycorrhizal fungal-plant-insect interactions: the importance of a
community approach. Environ Entomol 38(1):93–102
Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution of pine ectomycorrhizas
and their extramatrical mycelium. New Phytol 170:381–390
Gherghel F (2009) ldentification and characterization of Quercus robur ectomycorrhiza in relation
to heavy metal contamination. Dissertation, Friedrich Schiller University, Jena, Germany
Gilliam FS (2006) Response of the herbaceous layer of forest ecosystems to excess nitrogen
deposition. J Ecol 94:1176–1191
Godbold DL (2005) Ectomycorrhizal community structure: linking biodiversity to function. Prog
Bot 66:374–391
Godbold DL, Berntson GM (1997) Elevated atmospheric CO2 concentration changes ectomycor-
rhizal morphotype assamblages in Betula papyrifera. Tree Physiol 17:347–350
Godbold DL, Berntson GM, Bazzaz FA (1997) Growth and mycorrhizal colonization of three
North American tree species under elevated atmospheric CO2. New Phytol 137:433–440
Godbold DL, Hoosbeek MR, Lukac M, Cotrufo MF, Janssens IA, Ceulemans R, Andrea P,
Velthorst EJ, Scarascia-Mugnozza G, De Angelis P, Miglietta F, Peressotti A (2006) Mycor-
rhizal hyphal turnover as a dominant process for carbon input into soil organic matter. Plant
Soil 281:15–24
Goodman DM, Trofymov VA (1998) Distribution of ectomycorrhizas in micro-habitats in mature
and old-growth stands of Douglas-fir on southeastern Vancouver Island. Soil Biol Biochem
30:2127–2138
Graham JH (2008) Scaling-up evaluation of field functioning of arbuscular mycorrhizal fungi.

Graham JH, Miller RM (2005) Mycorrhizas: gene to function. Plant Soil 274:79–100
Grebenc T, Christensen M, Vilhar U, Cater M, Martin MP, Simoncic P, Kraingher H (2009)
Response of ectomycorrhizal community structure to gap opening in natural and managed
temperate beech-dominated forests. Can J For Res 39:1375–1386
Griffiths RP, Bradshaw GA, Marks B, Lienkaemper GW (1996) Spatial distribution of ectomycor-
rhizal mats in coniferous forests of the Pacific Northwest, USA. Plant Soil 180:147–158
Hall IR, Yun W, Amicucci A (2003) Cultivation of edible ectomycorrhizal mushrooms. Trends
Biotechnol 21:433–438
Hart MM, Reader RJ, Klironomos JN (2001) Life-history strategies of arbuscular mycorrhizal
fungi in relation to their successional dynamics. Mycologia 93:1186–1194
Hartley J, Cairney JWG, Meharg AA (1997) Do ectomycorrhizal fungi exhibit adaptive tolerance
to potentially toxic metals in the environment? Plant Soil 189:303–319
Hasselquist NJ, Vargas R, Allen MF (2009) Using soil sensing technology to examine interactions
and controls between ectomycorrhizal growth and environmental factors on soil CO2 dynamics.
Plant Soil. doi:10.1007/s11104-009-0183-y
Heneghan L, Miller SP, Baer S, Callaham MA Jr, Montgomery J, Pavao-Zuckerman M, Rhoades
CC, Richardson S (2008) Integrating soil ecological knowledge into restoration management.
Restor Ecol 16:608–617
Hinsinger P, Bengough AG, Vetterlein D, Young IM (2009) Rhizosphere: biophysics, biogeo-
chemistry and ecological relevance. Plant Soil 321:117–152
Hiol Hiol F, Dixon RK, Curl EA (2004) The feeding preference of mycophagous collembola varies
with the ectomycorrhizal symbiont. Mycorrhiza 5:99–103
Hirose D, Kikuchi J, Kanzaki N, Kazuyoshi F (2004) Genet distribution of sporocarps and
ectomycorrhizas of Suillus pictus in a Japanese white plantation. New Phytol 164:527–541
Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into
the black-box. Mol Ecol 10:1855–1871
Horton TR, Molina R, Hood K (2005) Douglas-fir ectomycorrhizae in 40- and 400-year-old stands:
mycobiont availability to late successional western hemlock. Mycorrhiza 15:393–403
Hubert NA, Gehring CA (2008) Neighboring trees affect ectomycorrhizal fungal community
composition in a woodland-forest ecotone. Mycorrhiza 18:363–374
Hutchings MJ, Bradbury IK (1986) Ecological perspectives on clonal perennial herbs. Bioscience
36:178–182
Iordache V (2004) The constitution from the point of view of human ecology (in Romanian), Sfera
Politicii 106:29–32. http://www.sferapoliticii.ro/sfera/pdf/Sfera_106.pdf
Iordache V (2009a) The general concept of evolution. In Staicu L (ed) Rationality and evolution –
philosophical exploration of complexity. Bucharest University Press, Bucharest, pp 83–155 (in
Romanian)
Iordache V (2009b) Darwin’s law of growth with reproduction in the Origin of Species: law of
nature or hidden teleologic principle? Communication at the Conference “Darwin, the evolu-
tion of species and the evolutionary thinking”, 20–22 Nov 2009. University of Bucharest,
Romania
Iordache V, Bodescu F (2005) Emergent properties of the Lower Danube River System: con-
sequences for the integrated monitoring system. Arch Hydrobiol Suppl Large Rivers 158:
95–128
Iordache V, Gherghel F, Kothe E (2009a) (2009b) Assessing the effect of disturbances on diversity
of ectomycorrhiza. Int J Environ Res Public Health 6:414–432
Iordache V, Ion S, Pohoata A (2009b) Integrated modeling of metals biogeochemistry: potential
and limits. Chem Erde-Geochem 69:125–169
Iordache V, Lacatusu R, Scradeanu D, Onete M, Purice D, Cobzaru I (2011) Scale-specific
mechanisms of metals distribution and mobility in contaminated areas. In: Kothe E,
Varma A (eds) Bio-geo-interactions in contaminated soils, Springer, in preparation
Izzo A, Agbowo J, Bruns TD (2005) Detection of plot-level changes in ectomycorrhizal commu-

nities across years in an old-growth mixed-conifer forest. New Phytol 166:619–630
Jackson LE, Burger M, Cavagnaro TR (2008) Roots, nitrogen transformations, and ecosystem
services. Ann Rev Plant Biol 59:341–363
Johnson CN (1996) Interactions between mammals and ectomycorrhizal fungi. Trends Ecol Evol
11:503–507
Johnson D, Krsek M, Wellington EMH, Stott AW, Cole L, Bardgett RD, Read DJ, DJ JR, Leake JR
(2005) Soil invertebrates disrupt carbon flow through fungal networks. Science 309:1047
Johnson CN, Hoeksema JD, Bever JD, Chaudhary VB, Gehring C, Klironomos J, Koide R, Miller
RM, Moore J, Moutoglis P, Schwartz M, Simard S, Swenson W, Umbanhowar J, Wilson G,
Zabinski C (2006) From lilliput to brobdingnag: extending models of mycorrhizal function
across scales. Bioscience 56:889–900
Jones MD, Durall DM, Cairney JWG (2003) Ectomycorrhizal fungal communities in young forest
stands regenerating after clearcut logging. New Phytol 157:399–422
Karst J, Marczak L, Jones MD, Turkington R (2008) The mutual-parasitism continuum in
ectomycorrhizas: a quantitative assessment using meta-analysis. Ecology 89:1032–1042
Kjoller R (2006) Disproportionate abundance between ectomycorrhizal root tips and their asso-
ciated mycelia. Microbiol Ecol 58:214–224
Kjoller R, Clemmensen KE (2009) Belowground ectomycorrhizal fungal community respond to
liming in three southern Swedish coniferous forest stands. For Ecol Manag 257:2217–2225
Klironomos JN, Rillig MC, Allen MF (1999) Designing belowground field experiments with the
help of semi-variance and power analyses. Appl Soil Ecol 12:227–238
Koele N, Turpault MP, Hildebrand EE, Uroz S, Frey-Klett P (2009) Interactions between mycor-
rhizal fungi and mycorrhizosphere bacteria during mineral weathering: budget analysis and
bacterial quantification. Soil Biol Biochem 41:1935–1942
Koide RT, Xu B, Sharda J (2005) Contrasting below-ground views of an ectomycorrhizal fungal
community. New Phytol 166:251–262
Koide RT, Shumway DL, Xu B, Sharda J (2007) On temporal partitioning of a community of
ectomicorrhizal fungi. New Phytol 174:420–429
Koricheva J, Gange AC, Jones T (2009) Effects of mycorrhizal fungi on insect herbivores: a meta-
analysis. Ecology 90:2088–2097
Kozdrój K, Piotrowska-Seget Z, Krupa P (2007) Mycorrhizal fungi and ectomycorrhiza associated
bacteria isolated from an industrial desert soil protect pine seedlings against Cd (II) impact.
Kranabetter JM, Durall DM, MacKenzie WH (2009) Diversity and species distribution of ecto-
mycorrhizal fungi along productivity gradients of a southern boreal forest. Mycorrhiza
19:99–111
Krpata D, Peintner U, Langer I, Fitz WJ, Schweiger P (2008) Ectomycorrhizal communities
associated with Populus tremula growing on a heavy metal contaminated site. Mycol Res
112:1069–1079
Krpata D, Fitz WJ, Peintner U, Langer I, Schweiger P (2009) Bioconcentration of zinc and
cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution.
Lamour A, Termorshuizen AJ, Volker D, Michael JJ (2007) Network formation by rhizomorphs of
Amillaria lutea in natural soil: their description and ecological significance. FEMS Microbiol
Ecol 62:222–232
Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003)
Molecular identification of ectomycorrhizal mycelium in soil horizons. Appl Environ Micro-
biol 69:327–333
Leake JR, Cameron DD (2010) Physiological ecology of mycoheterotrophy. New Phytol
185:601–605
Lepczyk CA, Lortie CJ, Anderson LJ (2008) An ontology for landscapes. Ecol Complex 5:
272–279
Lewis JD, Thomas RB, Strain BR (1994) Effect of elevated CO2 on mycorrhizal colonization of
loblolly pine (Pinus taedaL.) seedlings. Plant Soil 165:81–88
Lewis JD, Licitra J, Tuininga AR, Sirulnik A, Turner GD, Johnson J (2008) Oak seedling growth
and ectomycorrhizal colonization are less in eastern hemlock stands infested with hemlock
woolly adelgid than in adjacent oak stands. Tree Physiol 28:629–636
Lilleskov EA, Bruns TD (2005) Spore dispersal of a resupinate ectomycorrhizal fungal, Tomen-
tella sublilacina, via soil food webs. Mycologia 97:762–769
Lilleskov EA, Bruns TD, Horton TR, Taylor DL, Grogan P (2004) Detection of forest stand-level
spatial structure in ectomicorrhizal fungal communities. FEMS Microbiol Ecol 49:319–332
Lischke L, L€offler TJ, Thornton PE, Zimmermann NE (2006) Model up-scaling in landscape
research. In: Kienast F et al (eds) A changing world. Challenges for landscape research.
Springer, Dordrecht, pp 259–282
Liste HH, White JC (2008) Plant hydraulic lift of soil water-implications for crop production and
land restoration. Plant Soil 313:1–17
Lovelock CE, Wright SF, Clark DA, Ruess RW (2004) Soil stocks of glomalin produced by
arbusclar mycorrhizal fungi across a tropical rain forest landscape. J Ecol 92:278–287
Luck GW, Daily GC, Ehrlich PR (2003) Population diversity and ecosystem services. Trends Ecol
Evol 18:331–336
Maestre FT, Cortina J, Bautista S, Bellot J, Vallejo R (2003) Small-scale environmental heteroge-
neity and spatiotemporal dynamics of seedling establishment in a semiarid degreded ecosystem.
Ecosystems 6:630–643
Maharning AR, Mills AAS, Adl SM (2009) Soil community changes during secondary succession
to naturalized grasslands. Appl Soil Ecol 41:137–147
Maijala P, Fagerstedt KV, Raudaskoski M (1991) Detection of extracellular and proteolytic activity
in ectomycorrhizal fungus Heterobasidion annosum (Fr.) Bref. New Phytol 117:643–648
Martin F, Nehls U (2009) Harnessing ectomycorrhizal genomics for ecological insight. Curr Opin
Plant Biol 12:508–515
Matsuda Y, Hayakawa N, Ito S (2009) Local and microscale distributions of Cenococcum
geophilum in soil of coastal pine forests. Fungal Ecol 2:31–35
Meixner H, Schnauder I, B€ olscher J, Iordache V (2006) Hydraulic, sedimentological and ecologi-
cal problems of multifunctional riparian forest management – RipFor Guidelines for End-
Users. In: Berliner Geographische Abhandlungen, vol 66. ISBN3-88009-067-x. http://www.
cesec.ro/pdf/RipForGL2006_b1.pdf
Miller RM, Kling M (2000) The importance of integration and scale in the arbuscular mycorrhizal
symbiosis. Plant Soil 226:295–309
Miller RM, Lodge DJ (1997) Fungal responses to disturbance: agriculture and forestry. In:
Wicklow DT, S€oderstr€ om B (eds) Environmental and microbial relationships. Springer, Berlin,
pp 65–84
Moore JC, St John TV, Coleman DC (1985) Ingestion of vesicular arbuscular mycorrhizal hyphae
by soil microarthropods. Ecology 66:1979–1981
Moore JC, McCann K, Setala H, De Ruiter PC (2003) Top-down is bottom-up: does predation in
the rhizosphere regulate aboveground dynamics? Ecology 84:846–857
Moore JC, McCann K, de Ruiter PC (2007) Soil rhizosphere food webs, their stability, and
implications for soil processes in ecosystems. In: Cardon ZG, White JL (eds) Rhizosphere.
An ecological perspective. Academic, Burlington, MA, pp 101–126
Morris AH, Smith ME, Rizzo DM, Rejmánek M, Bledsoe CS (2008) Contrasting ectomycorrhizal
fungal community on the roots of co-occurring oaks (Quercus spp.) in a California woodland.
Mougin C, Boukcim H, Jolivalt C (2009) Soil bioremediation strategies based on the use of fungal
enzymes. In: Singh A et al (eds) Advances in applied bioremediation. Springer, Heidelberg,
pp 123–149
Mudge KW, Diebolt KS, Whitlow TH (1987) Ectomycorrhizal effect on host plant response to
drought stress. J Environ Hortic 5:183–187
M€unzenberge B, Bubner B, Wollecke J, Sieber TN, Bauer R, Fladung M, H€uttl RF (2009) The
ectomycorrhizal morphotype Pinirizha sclerotia is formed by Acephalia macrosclerotium sp.
nov., a close relative of Phialocephala fortinii. Mycorrhiza 19:481–492
Nara K (2008) Community developmental patterns and ecological functions of ectomycorrhizal
fungi: implications from primary succession. In: Varma A (ed) Mycorrhiza: state of the art,
genetics and molecular biology, eco-function, biotechnology, eco-physiology, structure and
systematics. Springer, Heidelberg, pp 581–600
Neagoe A, Ebena G, Carlsson E (2005) The effect of soil amendments on plant performance in an
area affected by acid mine drainage. Chem Erde-Geochem 65:115–129
Neagoe A, Merten D, Iordache V, Buechel G (2009) The effect of bioremediation methods
involving different degrees of soil disturbance on the export of metals by leaching and by
plant uptake. Chem Erde-Geochem 69(S2):57–73
Nilsson LO, Wallander H (2003) Production of external mycelium by ectomycorrhizal fungi in a
Norway spruce forest was reduces in response to nitrogen fertilization. New Phytol 158:
409–416
O’Neill RV (1996) Recent developments in ecological theory: hierarchy and scale. In: Scott JM
et al (eds) Gap analysis: a landscape approach to biodiversity planning. American Society for
Photogrammetry Remote Sensing, Bethesda, MD, pp 7–14
O’Neill RV (2001) Is it time to burry the ecosystem concept? Ecology 82:3275–3284
O’Neill EG, O’Neill RV, Norby RJ (1991) Hierarchy theory as a guide to mycorrhizal research on
large-scale problems. Environ Pollut 73:271–284
Pahl-Vostl C (1995) The dynamic nature of ecosystems. Wiley, New York
Peay KG, Bruns TD, Kennedy PG, Bergemann SE, Garbelotto M (2007) A strong species-area
relationship for eukaryotic soil microbes: island size matters for ectomycorrhzal fungi. Ecol
Lett 10:470–480
Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a
molecular master. Bioscience 58:799–810
Peay KG, Garbelotto M, Bruns TD (2009) Spore heat resistence plays an important role in
disturbance-mediated assemblage shift of ectomycorrhizal fungi colonizing Pinus muricata
seedlings. J Ecol 97:537–547
Pickles BJ, Genney D, Anderson IC, Alexander IJ (2009) Spatial ecology of ectomycorrhizas:
analytical strategies. In: Azcón-Aguilar C et al (eds) Mycorrhizas – functional processes and
ecological impact. Springer, Berlin, pp 155–165
Porter J, Arzberger P, Braun HW, Bryant P, Gage S, Hansen T, Hanson P, Lin CC, Lin FP, Kratz T,
Michener W, Shapiro S, Williams T (2005) Wireless sensor networks for ecology. Bioscience
55:561–572
Porter JH, Nagy E, Kratz TT, Hanson P, Scott L, Collins SI, Arzberger P (2009) New eyes on the
world: advanced sensors for ecology. Bioscience 59:385–397
Price GR (1995) The nature of selection. J Theor Biol 175:389–396
Pritchard SG, Strand AE, McCormack ML, Davis MA, Oren R (2008) Mycorrhizal and rhizo-
morph dynamics in a loblolly pine forest during 5 years of free-air-CO2-enrichment. Glob
Chang Biol 14:1252–1264
Rahbek C (2005) The role of spatial scale and the perception of large-scale species-richness
patterns. Ecol Lett 8:224–239
Redecker D, Szaro TM, Rand B, Bruns TD (2001) Small genets of Lactarius xanthogalactus,
Russula cremoricolor and Amanita francheti in late-stage ectomycorrhizal successions. Mol
Ecol 10:1025–1034
Reynolds JF, Wu J (1999) Do landscape structural and functional units exist? In: Tenhunen HD,
Kabat P (eds) Integrating hydrology, ecosystem dynamics, and biogeochemistry in complex
landscapes. Wiley, West Sussex, pp 273–296
Rillig MC (2004) Arbuscular mycorrhizae and terrestrial ecosystem processes. Ecol Lett 7:
740–754
Rillig MC, Allen MF (1999) What is the role of arbuscular mycorrhizal fungi in plant-to-
ecosystem responses to elevated atmospheric CO2? Mycorrhiza 9:1–8
Rillig MC, Mammey DL (2006) Mycorrhizas and soil structure. New Phytol 171:41–53
Rineau F, Garbaye J (2009) Effects of liming on ectomycorrhizal community structure in relation
to soil horizons and tree hosts. Fungal Ecol 2:103–109
Rineau F, Courty PE, Uroz S, Buée M, Garbaye J (2008) Simple microplate assays to measure iron
mobilization and oxalate secretion by ectomycorrhizal tree roots. Soil Biol Biochem 40:
2460–2463
Ritter T, Weber G, Haug I, Kotke I, Oberwinkler F (1989) Interrelationship between vitality of
ectomycorrhizae and occurrence of microfungi. Ann Sci For 46(Suppl):745s–749s
Robertson SJ, McGill WB, Massicotte HB, Rutherford PM (2007) Petroleum hydrocarbon con-
tamination in boreal forest soils: a mycorrhizal ecosystems perspective. Biol Rev 82:213–240
Rosling A (2009) Trees, mycorrhiza and minerals-field relevance of in vitro experiments. Geo-
biomicrobiol J 26:389–401
Rosling A, Landeweert R, Lindahl BD, Larsson KH, Kuyper TW, Taylor AFS, Finlay RD (2003)
Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytol 159:
775–783
Rundel PW, Graham EA, Allen MF, Fisher JC, Harmon TC (2009) Environmental sensor net-
works in ecological research. New Phytol 182:589–607
Rygiewicz PT, Johnson MG, Ganio LM, Tingey DT, Storm MJ (1997) Lifetime and temporal
occurrence of ectomycorrhizae on ponderosa pine (Pinus ponderosa Laws.) seedlings grown
under varied atmospheric CO2 and nitrogen levels. Plant Soil 189:275–287
Satomura T, Hashimoto Y, Kinoshita A, Horikoshi T (2006) Methods to study the role of
ectomycorrhizal fungi in forest carbon cycling 1: introduction to the direct methods to quantify
the fungal content in ectomicorrhizal fine roots. Root Res 15:119–124
Scattolin L, Montecchio L, Mosca E, Agerer R (2008) Vertical distribution of the ectomycorrhizal
community in the top soil of Norway spruce stands. Eur J For Res 127:347–357
Schneider K, Renker C, Maraun M (2005) Oribatid mite (Acari, Oribatida) feeding on ectomycor-
rhizal fungi. Mycorrhiza 16:67–72
Schroder B, Seppelt R (2006) Analysis of pattern–process interactions based on landscape
models – Overview, general concepts, and methodological issues. Ecol Model 199:505–516
Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686
Selosse MA, Richard F, Xand H, Simard SW (2006) Mycorrhizal networks: des liaisons danger-
euses? Trends Ecol Evol 21:621–628
Seppelt R, M€uller F, Schr€ oder B, Martin Volk M (2009) Challenges of simulating complex
environmental systems at the landscape scale: a controversial dialogue between two cups of
espresso. Ecol Model 220:3481–3489
Simard SW, Durall DM (2004) Mycorrhizal networks: a review of their extent, function, and
importance. Can J Bot 82:1140–1165
Simard SW, Durall DM, Jones MD (1997) Carbon allocation and carbon transfer between Betula
papyrifera and Pseudotsuga menziesii seedlings using a 13C pulse-labeling method. Plant Soil
191:41–45
Smith ML, Bruhn JN, Anderson JB (1992) The fungus Armillaria bulbosa is among the largest and
oldest living organisms. Nature 356:428–431
Smith SE, Facelli E, Pope S, Smith FA (2010) Plant performance in stressful environments:
interpreting new and established knowledge of the roles of arbuscular mycorrhizas. Plant
Soil 326:3–20
Southworth D, He XH, Swenson W, Bledsoe CS, Horwath WR (2005) Application of network
theory to potential mycorrhizal networks. Mycorrhiza 15:589–595
Staddon PL, Heinemeyer A, Fitter AH (2002) Mycorrhizas and global environmental change:
research at different scales. Plant Soil 244:253–261
Staddon PL, Ramsey CB, Ostle N, Ineson P, Fitter AH (2003) Rapid turnover of hyphae of
mycorrhizal fungi determined by AMS microanalysis of 14C. Science 300:1138–1140
Szlavecz K, McCormick MK, Xia L, Whigham D (2009) Direct and indirect effects of earthworms
on mycorrhizal fungi. 94th ESA annual meeting, PS 20-177. http://eco.confex.com/eco/2009/
techprogram/P19960.HTM
Tarkka MT, Frey-Klett P (2008) Mycorrhiza helper bacteria. In: Varma A (ed) Mycorrhiza – state
of the art, genetics and molecular biology, eco-function, biotechnology, eco-physiology,
structure and systematics. Springer, Heidelberg, pp 113–134
Tedersoo L, Kőljalg U, Hallenberg N, Larsson KH (2003) Fine scale distribution of ectomycor-
rhizal fungi and roots across substrate layers including coarse woody debris in a mixed forest.
Thiet RK, Boerner REJ (2007) Spatial patterns of ectomycorrhizal fungal inoculum in arbuscular
mycorrhizal barrens communities: implications for controlling invasion by Pinus virginiana.
Tingey DT, Philips DL, Johnson MG (2000) Elevated CO2 and conifer roots: effects of growth, life
span, and turnover. New Phytol 147:87–103
Treseder KK, Allen MF (2000) Mycorrhizal fungi have a potential role in soil carbon storage
under elevated CO2 and nitrogen deposition. New Phytol 147:189–200
Treseder KK, Masiello CA, Lansing JL, Allen MF (2004) Species-specific measurements of
ectomycorrhizal turnover under N-fertilization: combining isotopic and genetic approaches.
Oecologia 138:419–425
Treseder KK, Allen MF, Ruess RW, Pregitzer KS, Hendrick RL (2005) Lifespans of fungal
rhizomorphs under nitrogen fertilization in a pinyon-juniper woodland. Plant Soil 270:249–255
Vadineanu A, Cristofor S, Iordache V (2001) Lower Danube River System biodiversity changes.
In: Gopal B et al (eds) Biodiversity in wetlands: assessment. Function and conservation.
Backhuys, Leiden, pp 29–63
van der Heijden MGA, Horton TR (2009) Socialism in soil? The importance of mycorrhizal fungal
networks for facilitation in natural ecosystems. J Ecol 97:1139–1150
van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T,
Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity,
ecosystem variability and productivity. Nature 396:69–72
Vargas R, Allen MF (2008) Dynamics of fine root, fungal rhizomorphs, and soil respiration in a
mixed temperature forest: integrating sensors and observation. Vadose Zone J 7:1055–1064
Warren JM, Brooks JR, Meinzer FC, Eberhart JL (2008) Hydraulic redistribution of water from
Pinus ponderosa trees to seedlings: evidence for an ectomycorrhizal pathway. New Phytol
178:382–394
Watkinson SC, Boddy L, Burton K, Darrah PR, Eastwood D, Fricker MD, Tlalka M (2005) New
approaches to investigating the function of mycelial networks. Mycologist 19:11–17
Wiensczyk Am, Gamiet S, Durall DM, Jones MD, Simard SW (2002) Ectomycorrhizae and
forestry in British Columbia: a summary of current research and conservation strategies. BC
J Ecosyst Manag 2. http://www.forrex.org/JEM/ISS2/vol2_no1_art6.pdf
Wolfe BE, Parrent JL, Koch AM, Sikes BA, Gardes M, Klironomos JN (2009) Spatial hetero-
geneitiy of mycorrhizal populations and communities: scales and mechanisms. In: Azcón-
Aguilar C et al (eds) Mycorrhizas – functional processes and ecological impact. Springer,
Heidelberg, pp 167–186
Wong MTF, Asseng S (2006) Determining the causes of spatial and temporal variability of wheat
yields at sub-field scale using a new method of upscaling a crop model. Plant Soil 283:203–215
Yarrow MM, Marin VH (2007) Toward conceptual cohesiveness: a historical analyses of the
theory and utility of ecological boundaries and transition zones. Ecosystems 10:462–476
Zhou JZ, Kang S, Schadt CW, Garten CT (2008) Spatial scaling of functional gene diversity across
various microbial taxa. Proc Natl Acad Sci USA 105:7768–7773
.
Chapter 13
Mycobioindication of Stress in Forest
Ecosystems
Hojka Kraigher and Samar Al Sayegh Petkovšek
13.1 Introduction
Indication of the existence of an environmental stress situation is a prerequisite for

its amelioration or adaptation to it. Environmental monitoring based on chemical
and physical parameters can give a measure of a chemical in the environment, or of
temperature in a certain period of time and (micro)location. However, such mea-
surements do not provide any information on the effects of these factors on the
living organisms, and on any synergistic or antagonistic effects on conversion
products or pathways. The state or reaction of a living organism to physical and
chemical environment, its survival, and early diagnosis of any disease can only be
determined by using bioindicators and a biomonitoring approach.
Soil is one of the principal regulatory compartments in forest ecosystems in
which below-ground processes are regulated and mediated to a large extent through
the mycelia of mycorrhizal fungi. Early stress indication through changes in
mycorrhizal structure and function, therefore, also contributes to an understanding
of complex interactions in the mycelial wood-wide web and the functioning of an
entire ecosystem. Since the functional compatibility and stress tolerance of ecto-
mycorrhizal (ECM) types are species specific, information on the ECM community
can not only be applied as an indication of stress and disturbance of forest soils, but
it also adds to the understanding of processes in forest ecosystems.
We present a brief overview of the impacts of stress factors on mycorrhiza and of
mycobioindication approaches developed for monitoring climate change and air
pollution effects on forest ecosystems in Slovenia in the last two decades.
H. Kraigher (*)
Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenia
e-mail: hojka.kraigher@gozdis.si
S. Al Sayegh Petkovšek
ERICo Velenje, Ecological Research & Industrial Cooperation, Koroška 58, 3320 Velenje,
Slovenia
e-mail: samar.petkovsek@erico.si

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_13,
302 H. Kraigher and S. Al Sayegh Petkovšek
13.2 Impact of Stress Factors on Mycorrhiza
Human-induced stress factors can affect ectomycorrhiza directly through reduced

growth of fine roots and decreased uptake of nutrients, and/or indirectly through
foliar damage to the host plant and, consequently, a changed C allocation below-
ground (Kieliszewska-Rokicka et al. 1997; Brunner 2001; Leake 2001; Andersen
2003; Kernaghan 2005; Kraigher et al. 2007; Cudlin et al. 2007). When natural
stress factors (e.g., water shortage, elevated and low temperature (T), and patho-
gens) interact, they can supplement the effects of pollutants or induce antagonistic
effects. The impact of anthropogenic stress factors is most pronounced in effects on
biodiversity of below-ground ECM communities and, consequently, on the sustain-
ability, productivity, and vitality of forests (Kraigher et al. 1995, 2007; Kraigher
1997, 1999, 2001; Read 1998; Simončič et al. 1998; Kovacs et al. 2000; Erland and
Taylor 2002; Cudlin et al. 2007).
13.2.1 Acid Deposition
In spite of the fact that slow acidification of soil is a natural process in most
coniferous forests (Brunner 2001; Erland and Taylor 2002), the percentage of
forests in Europe with soil pH below 4.2 increased toward the end of the millen-
nium, due to the deposition of high atmospheric inputs of acidifying pollutants
[mostly sulfur (S) and nitrogen (N) compounds] (Arnolds 1991). Moreover, the
release of heavy metals, especially Al ions, into the soil solution as a consequence
of acidification was determined (Brunner 2001). In general, acidification did not
reduce the degree of mycorrhization of tree fine roots, which is close to 100% in
most of the forests studied, but the total number of ECM root tips per soil volume
and the number of morphotypes were reduced (Cudlin et al. 2007). A moderate
reduction in pH does not drastically affect the diverse types of ectomycorrhizae,
since ECM communities are mostly adapted to acid soil conditions (Read 1991). A
pronounced acidification effect on ectomycorrhizal fungi (EMF) is species specific
and can result in an increased abundance of selected types of ectomycorrhiza (e.g.,
Russula ochroleuca in Norway spruce plots subjected to acid irrigation), so it can
trigger a shift in the community structure (Kraigher 1991, 1994, 1997; Erland and
Taylor 2002).
13.2.2 Nitrogen Deposition and Fertilization
Nitrogen deposition, which has increased across a large number of temperate

conifer and deciduous ecosystems, affects tree growth and nutrient cycles (Vitousek
et al. 1997). Increased soil N concentrations are often correlated with changes in the
13 Mycobioindication of Stress in Forest Ecosystems 303
number of ECM community attributes, such as decreased sporocarp production,

lower community diversity, and shifts in the relative abundance of ECM commu-
nity members.
The first documented biological effect of nitrogen deposition on the diversity of
EMF was mainly limited to observations made in sporocarp surveys of EMF
(Arnolds 1991). In nitrogen-supplemented plots, a rapid and substantial decrease
in the diversity and production of ECM sporocarps of most species was observed.
This issue was recently thoroughly reviewed by Lilleskov (2005). The response to
fertilization seems to vary among species, with certain taxa declining in abundance
and diversity (Cortinarius, Hydnum, Russula, and Suillus) and some continuing to
fruit at higher N deposition levels (Lactarius rufus, Lactarius theiogalus, Paxillus
involutus, and Laccaria species).
In addition to sporocarp surveys, research based on below-ground ECM obser-
vations has provided new information about richness or diversity in response to N
inputs and revealed the lack of direct correspondence between the two approaches.
Contrasting results between different studies have also appeared. Several reports
have indicated a decrease in fractional colonization by EMF, or total numbers of
ECM roots (Tetreault et al. 1978; Alexander and Fairley 1983; Rudawska 1986;
Taylor and Alexander 1989; Haug et al. 1992) or have shown a decline in the
abundance and diversity of EMF, or a shift in ECM community composition
(Kraigher et al. 1996; Taylor et al. 2000; Peter et al. 2001; Lilleskov et al. 2002;
Parrent et al. 2006). However, the decrease in ECM colonization has sometimes
been fairly short-lived and disappeared a few years after treatment (Menge et al.
1977). The reduction in ECM root tips in soil cores in spruce and beech forests
along transects through Europe, representing gradients in increasing mineral N
deposition, was analyzed by Taylor et al. (2000). A reduction in biodiversity of
ECM types in polluted areas was shown in spruce forests (Kraigher 1997), whereas
in beech forests, no negative effects on biodiversity were determined (Kraigher
et al. 2007). Within spruce forests, the decline in diversity toward the south of the
transect was accompanied by increases in single species dominance. Tylospora sp.
and L. rufus dominated the ECM community of the most polluted site, forming 75%
of mycorrhizal tips. A reverse trend was determined for species from genera
Cortinarius, Tricholoma, and Russula, these species appearing to be particularly
sensitive to nitrogen enrichment (Taylor et al. 2000).
13.2.3 Metal Deposition
Atmospheric pollution leads to soil acidification and elevated concentrations of

trace elements in the soil. Elevated concentrations affect soil microorganisms and
microbial processes, reduce the development of mycorrhiza (Schneider et al. 1989;
Perrin and Estivalet 1990), and decrease the abundance and biodiversity of spor-
ocarps (Arnolds 1991). However, some fungal species tolerate increased metal
levels in soil (e.g., Amanita, Albatrellus ovinus, and Leccinum scabrum) (Erland
and Taylor 2002). Mycorrhizal fungi can accumulate metals in their sporocarps
(Kalač and Svoboda 2000; Falandysz et al. 2001, 2003; Al Sayegh Petkovšek et al.
2002; Johanson et al. 2004; Cocchi et al. 2006; Al Sayegh Petkovšek 2008).
Moreover, they have the capacity to accumulate metals in the external mycelium
(mostly Cu and to a lesser extent Zn and Cd) (Berthelsen et al. 1995). EMF establish
metal tolerance by binding metals to electronegative sites on the cell walls of
hyphae, or by binding to phosphates and sulfhydryl compounds within the cells
(Brunner 2001).
13.2.4 Ozone
Tropospheric ozone (O3) is a secondary atmospheric pollutant, generated from

oxides of nitrogen and volatile organic compounds reacting in the presence of
sunlight. It has been recognized as an increasing and damaging agent to plants
(Karnosky et al. 2005). O3 triggers physiologic changes in leaves, affecting carbon
source strength, i.e., the amount of carbon available for allocation to sink tissues.
Decreased carbon assimilation, increased metabolic costs for repair mechanisms,
and decreased phloem loading all lead to decreased carbon allocation below-
ground, thus affecting roots, root symbionts, rhizodeposition, litter quality and
quantity, and, consequently, the whole soil food web (Andersen 2003). Carbon
source–sink relationships or the functional balance of roots and shoots has been
reported as primary factors in continuous adjustments between root and shoot
growth (Tingey et al. 1976), possibly acting through root to shoot signaling,
including the hormonal regulation of root proliferation. In this context, the effects
of ozone fumigation on the cytokinins (CK) of beech trees (Winwood et al. 2007)
may be related to mycorrhiza-associated changes in cytokinin concentrations in the
host plants (Kraigher et al. 1991, 1993, 2008) and ozone-induced changes in fine
root growth and ECM community structure (Grebenc and Kraigher 2007a).
Sensitivity to ozone has been reported to differ between species (for review, see
Andersen and Rygiewicz 1995; Bortier et al. 1999, 2000; Andersen 2003); between
different clones and populations (Ballach et al. 1992; Coleman et al. 1996; Vanha-
talo et al. 2003; Ottossen et al. 2003), experimental growth conditions, such as
duration of the fumigation, light regime, irrigation, mineral nutrition, and the
combination of various stress factors (McLaughlin and Downing 1995; Roth and
Fahey 1998; Topa et al. 2004; L€ ow et al. 2006; Železnik et al. 2007); and between
age-related physiologic differences within the same species (Matyssek et al. 2007).
Ozone-caused reduced root growth has been found to alter the functioning of
rhizosphere organisms and make them more susceptible to drought or nutrient
deficiency. A carryover effect has also been established (Andersen and Rygiewicz
1991). The length of fumigation should be considered with a view to the life span of
the fumigated plant (Kraigher et al. 2008).
In adult beech trees, a significant increase in the number of vital ECM root tips,
of nonturgescent short roots, and of types of ectomycorrhiza has been observed.
Species richness appears to be affected only by O3 in a normally humid year and not
under conditions of drought stress (Grebenc and Kraigher 2007a). The same
treatment also demonstrated an impact on individual ECM species abundance. An
increased abundance of Cenococcum geophilum, Russula fellea, and Russula illota
suggested their tolerance to the changed physiology of beech trees under ozone,
while Xerocomus sp., Russula cyanoxantha, R. ochroleuca, and a Lactarius sp.
decreased in abundance under increased ozone treatment or were only found with
control trees (Grebenc and Kraigher 2007b). In terms of the nutrient status of roots,
d15N measurements indicated a reduction in total nitrogen in the fine roots of ozone-
treated adult trees (Haberer et al. 2007), while Mg concentration in seedlings was
reduced under the same conditions (Železnik et al. 2007). In contrast to the results
from adult trees, a significant reduction in number of vital ECM types and number
of ECM root tips was observed in beech seedlings (ibid.).
Through its indirect effects, ozone imposes general stress on the below-ground
community, often occurring in combination with other stresses, so there is no clear
differentiation of ozone-sensitive and ozone-tolerant ECM species, such as have
been proposed in acidic and nitrogen deposition studies (e.g., Kraigher et al. 1996).
Functional groups of ECM types (exploration types as proposed by Agerer 2001)
are perhaps more or less sensitive to changes in carbon allocation below-ground,
and the physiology, and thus bioindication value, of each of the ECM types or
groups, might be studied further.
13.2.5 Elevated CO2
Ectomycorrhizal roots respond positively to elevated CO2 (Alberton et al. 2005).

An increase in root tip abundance under elevated CO2 is consistent with reported
changes in the root:shoot ratio, especially under conditions of severe nutrient
limitation (Poorter and Nagel 2000; Ågren and Franklin 2003). It is important to
note, however, that such an increased carbon allocation to ECM roots does not
automatically translate into an increased uptake of limiting nutrients and hence to
increased forest productivity. ECM forests can already be “saturated” with ECM
(O’Neill 1994) and any further increase in ECM roots and mycelia would only
increase nutrient limitation of the system. This phenomenon has been described as
the progressive nitrogen limitation (PNL) hypothesis (Luo et al. 2004; Johnson
2006; Hu et al. 2006).
13.2.6 Drought
In a meta-analysis of the effects of several stress factors (Cudlin et al. 2007), the
clearest effect found was a decrease in the fine root biomass during drought. A
relative reallocation of growth to below-ground organs at the expense of above-
ground ones during mild drought has often been found, and even absolute root
growth may increase during mild drought (Becker et al. 1987). However, when
water stress continues, the usual response is a reduction in root growth (Joslin et al.
2000). In contrast to fine root biomass, the ECM fractional colonization did not
show a reduction but a slight (insignificant) increase. This may be due to a negative
effect of drought on the total number of root tips. This kind of effect was shown in
Norway spruce by Feil et al. (1988).
Cenococcum geophilum (C. graniforme) is often mentioned as a particularly
drought-tolerant fungus. However, with some exceptions (Worley and Hacskaylo
1959), there are only a few reports based on exactly defined experimental condi-
tions. Pigott (1982) showed that Cenococcum survived better than other mycorrhi-
zal fungi during a drought episode. It has also been proposed that the mycelium of
Cenococcum is more resistant to decomposition than that of some other species
(Meyer 1987). Furthermore, the concepts of taxonomy of this fungus have varied
with time, and morphological identification may have allowed possible confusion
with other fungi. Cenococcum may also be tolerant to other stresses, such as
increased ozone (Grebenc and Kraigher 2007a, b) or salinity (Saleh-Rastin 1976,
cit. in Cudlin et al. 2007). While mycorrhiza formation in other species was strongly
delayed in drought-treated plants, Thelephora terrestris formed abundant mycor-
rhizal systems, irrespective of watering treatment (Lehto 1992). Differences in
community structure in drought conditions can, therefore, also be expected to
occur in forests.
13.3 Mycobioindication in Forest Ecosystems
Forest sustainability, productivity, and vitality depend on the relationship with soil
resources, the interface between soil nutrient pools and tree roots, as uptake organs
to sustain above-ground growth (Bakker 1999). In temperate forests in Europe, the
fine roots of stand-forming forest tree species are predominantly covered with a
fungal sheath of a range of EMF, which take over the function of uptake and
translocation of water and nutrients. As such, mycorrhizae are the main spatial
and temporal linkage between the various constituents of a forest ecosystem
(Dighton and Boddy 1988; Kraigher 1996; Dighton 2003).
The forest decline observed since the early 1980s in both Europe and North
America has increased the interest of environmental research in bioindication
methods (Tausz et al. 1996). Bioindication of environmental stresses provides a
suitable means of evaluating the impact of these variables on biological systems
(Arndt et al. 1987). Reactions that indicate a state of stress make the employment of
sensitive plant, animal, fungal, or other species as bioindicators of environmental
stress, or the use of living organisms or their parts as biomonitors possible.
Bioindicators are organisms or communities of organisms that react to environ-
mental conditions by changing their vital functions and/or their chemical composi-
tion, thus making it possible to draw conclusions about the state of their
environment (Arndt et al. 1987). Plants and fungi are recognized as suitable
bioindicators with respect to forest ecosystems. Bioindicators (Fig. 13.1) can be

differentiated into sensitive (response) and accumulative indicators, and are sub-
divided into three different groups (Arndt et al. 1987) (1) pointer organisms or
ecological indicators provide evidence about the state of entire ecosystems; (2)
tester organisms are used in standardized laboratory procedures; and (3) monitoring
organisms are used to monitor the quality and quantity of harmful substances in the
environment and to detect their effects. These monitoring organisms either already
exist in the ecosystem (passive monitoring) or are introduced into the ecosystem in
a standardized form (active monitoring).
Bioindicators can be obtained from any level of biological organization, from
cell, tissue, organ, organism, and community levels (Fig. 13.2), and several
BIOINDICATORS
ACCUMULATIVE REACTIVE
PHYSIOLOGICAL & ECOLOGICAL TESTER

MONITORING
INDICATORS, POINTER ORGANISMS &
ORGANISMS
ORGANISMS BIOMARKERS
passive bioindication active bioindication
ecosystem level laboratory level
Fig. 13.1 Schematic presentation of bioindicators (modified after Arndt et al. 1987)
Stress Characteristics Response Result Biological complexity

characteristics of an organism in bioindication
Environmental Severity Biochemistry or

Survival
stress & resistance and metabolism of a single
Cell, tissue,
disturbance growth organism
duration organ in
question Individual level
repetition Stage of susceptibility Death Population level
development
combination Community level
Genotype
of stresses Changes in the
ecological network
Fig. 13.2 How the response of an organism can be applied in bioindication of stress and
disturbance at different levels of organization (modified from Buchanan et al. 2000 and
Martı́nez-Crego et al. 2010)
summarizing indices that attempt to integrate relevant ecological information into

an overall expression of biotic integrity are being developed (Martı́nez-Crego et al.
2010). Environmental stress differs in terms of its severity, duration, or number of
exposures and a combination of stresses may occur with synergistic or antagonistic
effects. The response of the organism on which the stress is imposed depends on the
organ or tissue in question, stage of development, and genotype, which all influence
the resistance or susceptibility to stress, and result in survival, modifications in
growth, or death of the organism in question. The relevance of the bioindicator
depends on its relevance to ecological integrity, broad-scale applicability, early
detection capacity, feasibility of implementation, interpretability against reference
conditions, and linking ecosystem degradation to its causative stress agents
(Martı́nez-Crego et al. 2010).
Static and deterministic approaches have been developed as part of phytoindica-
tion, through, e.g., phytocoenological analyses, and “load capacity” estimations
have been applied (Ellenberg 1972, cit. in Fr€anzle 2006). They depend on the
disposition, susceptibility, and regeneration in predefined estimation scales. A
combination of dynamic (reflecting the time evolution of perturbation, sensitivity,
and adaptation) and statistical (defining probability distributions in order to calcu-
late expected values) approaches are being developed (Fr€anzle 2006). Complex
indicator approaches seem to reflect well the dynamics of ecosystem development
and include fluctuations of energy, entropy production of ecosystems, fluctuations
of selected micronutrients, duration of biogeochemical cycles, biomarkers (stress
proteins and phytoalexines), changes in biodiversity of selected assemblages,
dynamics of selected populations, changes in the competitive behavior of function-
ally important species, and modifications of the food web structure.
Decreases in species diversity and in the abundance of sporocarps of macro-
mycetes in Europe were reported in the second half of the twentieth century
(Arnolds 1988, 1991; Jaenike 1991) and the method of mycobioindication of forest
site pollution was soon suggested by Fellner (1989) and Fellner and Peškova
(1995), based on the impoverishment of ECM mycobiocenosis. Arnolds (1991)
suggested an adaptation of Fellner’s qualification of impoverishment, based on the
various sensitivities of different ECM species. However, the occurrence of fruit
bodies depends on a range of climatic factors in different years, while ECM types
are present in the soils at any time of the year. Furthermore, a range of ECM fungi,
such as the group commonly called Fungi Imperfecti (predominantly belonging to
ascomycetes, but without a known anamorph), do not produce any sporocarps. As a
result, more detailed studies of the ECM potential of forest sites by determination of
ECM types were initiated in the 1990s, with a view to applying the results to
bioindication (Fellner and Peškova 1995; Kraigher 1994; Kraigher et al. 1995,
1996; Al Sayegh Petkovšek 1997, 2004, 2005; Al Sayegh Petkovšek and Kraigher
2003; Taylor et al. 2000; Erland and Taylor 2002; Taylor and Alexander 2005).
In Slovenia, a series of studies was carried out by applying types of ECM as tools
for the bioindication of stress in forest sites (Kraigher et al. 1995, 1996, 2007;
Grebenc and Kraigher 2007a; Al Sayegh Petkovšek 2008). The application of ECM
studies in bioindication was based on (1) the diversity of ECM types on Norway
spruce (Picea abies (L) Karst) or beech (Fagus sylvatica L.) in forest stands (in situ
ecological indicators); (2) the determination and quantification of selected pollu-
tion-sensitive or insensitive ECM types (in situ passive monitors); (3) the develop-
ment of ectomycorrhiza on spruce seedlings, planted in studied sites (active
monitors); (4) ECM or root growth parameters on tree seedlings, tested in an
experimental setup (ex situ testers of substrate pollution); and (5) the bioaccumula-
tion of metals in fungal sporocarps (in situ accumulative bioindicators).
13.3.1 Ecological Indicators and Passive Monitors of Stress

in a Forest Ecosystem
13.3.1.1 Responsive In Situ Mycoindicators
Diversity indexes indicate the dynamics of an ecosystem, i.e., its potential to react
to a changing environment (Atlas and Bartha 1981). The species richness index (d)
links the number of species and their importance in the total community. The
Shannon–Weaver index (H) also provides the relative abundance of each species,
which indicates whether there are dominant populations in the sample. Both
indexes are in general lower in populations in stressed environments. A reduction
in the number of species in a community of mycorrhizal fungi can negatively
influence the capacity of populations of mycorrhizal fungi and tree seedlings to
form a functional symbiosis.
Pollution and other anthropogenic stresses have been found to diminish biodi-
versity indices of ECM types on Norway spruce (Kraigher 1999; Taylor et al. 2000;
Peter et al. 2008), oak (Kovacs et al. 2000) and several other tree species (reviewed
by Erland and Taylor 2002). A survey performed in three Norway spruce stands
with different degrees of forest decline due to air pollution revealed that the number
of living trees and their defoliation status may directly impact on the ECM species
composition by affecting the amount of carbon delivered to the symbiotic fungal
partners (Peter et al. 2008). The Shannon–Weaver index of adult trees was signifi-
cantly lower in the heavily damaged site (only 3% of leafing trees); however, adult
tress and seedlings were fully mycorrhizal. The most abundant species in all sites
was Tylospora fibrillosa, especially in the most damaged site (it made up over 60%
of root tips). It was emphasized that atheloids and thelephoroids (Thelephora
terrestris, Tylospora fibrillosa, and Thelephora asterophora) and C. geophilum
might play a crucial role in stressed forest ecosystems (ibid.).
Trends of diminishing ECM biodiversity were also clear in our studies in spruce
stands. However, in European beech, no trend was detected (Table 13.1). Early
studies (Kraigher et al. 1996) of ECM types on Norway spruce showed that several
ECM types disappeared, while others proliferated in polluted sites. Additionally,
some ECM types have been found to be restricted or mainly to occur either in
polluted or in unpolluted sites, even if the identity of the fungus was not determined.
Table 13.1 Comparison of Shannon–Weaver indices (H) in soil samples sampled near different tree species from differenty polluted areas
Species Unpolluted areas Polluted areas and/or presence of stressors References
310
Fagus sylvatica 2.8 (Gribskov, Denmark) 2.3 (Aubure, NE France) Taylor et al. (2000)a
3.2 (Collelongo, Italy)
3.9 (Schacht, Germany)
1.6–1.8 (Val di Sella, Italy; Idrija, Pučko et al. (2004)b
Slovenia; Nizbor, Czech Republic
0.0–1.1 (Snežna jama, Slovenia) Grebenc (2005)c
0.6–1.6 (Rajhenavski Rog, Slovenia) Grebenc and Kraigher (2007a, b)c
0.9–1.3 (Krazberg, Germany) Grebenc et al. (2009)c
1.6–2.6 (Preža, Slovenia) 1.3–2.0 (Zavodnje, Slovenia) Al Sayegh Petkovšek (2008)d
1.9–2.8 (Moravške gredice, Slovenia) 1.6–2.3 (Dobovec, Slovenia)
Picea abies 2.2 (Pokljuka, Slovenia) 2.3 (Zavodnje, Slovenia) Kraigher (1999)
2.2 (Mislinjski graben, Slovenia) Kraigher et al. (2000)e
3.1 (Pokljuka, Slovenia) Vilhar et al. (2004)f
3.5 (Ahden, N Sweden) 2.6 (Waldstein, Germany) Taylor et al. (2000)a
3.3 (Aubure, NE France and Klosterhede, Denmark)
1.1–2.0 (Mumlovska hora, Alzbentinka, Modry dul, Peter et al. (2008)g
Czech republic)
0.7–1.2 (Hudobrežnikov vrh, Slovenia)* Al Sayegh Petkovšek (2008)d
1.0–1.3 (Hudobrežnikov vrh, Slovenia)**
0.2–3.0 (Veliki Vrh, Slovenia)*
0.2–0.7 (Veliki Vrh, Slovenia)**
Quercus spp. 1.2–1.3 (Austria)* Kovacs et al. (2000)h
1.3–1.5 (Austria)**
1.1–1.4 (Spain) de Roman and de Miguel (2005)i
a
Study was performed along north–south transects in Europe regarding anthropogenic N-enrichment
b
Identification of types of ectomycorrhizae on 7 year old seedlings in a beech provenance trial
c
Canopy gaps with no natural regeneration (Snežna jama) and with natural regeneration (Rajhenavski Rog); 2 ambient ozone-treated plot (Krazberg)
d
Research plot exposed to emissions of thermal power plants (air pollution); soil samples sampled near vital trees (*) and declining trees (**)
e
Research plots exposed to emissions from thermal power plants (air pollution)
f
Regeneration center in an autochothon Norway spruce forest on the Pokljuka plateau
g
Damaged spruce stand in the Czech Republic due to air pollution
h
Comparison of two oak stands (Quercus petrarea Liebl. Ouercus robur L.) regarding vital (*) and declining tress (**)
i
Post-fire ectomycorrhizal community in a Quercus ilex L. stand over a 3-years period
H. Kraigher and S. Al Sayegh Petkovšek
This was the case with Piceirhiza terraphila and Piceirhiza inflata in polluted sites
(Kraigher et al. 1996), and Piceirhiza oleiferans (Waller et al. 1993) in unpolluted
plots. Similar to pollution-sensitive or -insensitive ECM species in spruce forests,
we have suggested Hydnum rufescens (sensitive), P. involutus (insensitive), and
Elaphomyces sp. in beech forests (insensitive).
However, since stress in natural conditions is complex, other factors, such as
drought in the upper soil horizons (possibly influencing the high abundance of
C. geophilum in unpolluted site), cannot be ruled out. Jany et al. (2003) determined
that C. geophilum maintains the physiological integrity of beech roots facing
drought stress. In an assessment of air pollution in the emission area of the thermal
power plant Šoštanj (Al Sayegh Petkovšek 2008), C. geophilum was also deter-
mined as the dominant ECM type in soil cores from forest research plots exposed to
air pollution and drought stress (Table 13.2). In addition, a statistically significant
difference in the number of sclerotia of C. geophilum in ozone-fumigated
Table 13.2 List of types of ectomycorrhiza determined on European beech and Norway spruce,
their bioindication applicability as a stress indicator and corresponding references
ECM type Fagus Picea Type of References
sylvatica abies stressa
Cenococcum geophilum þ þ 2, 3 Cudlin et al. (2007), Kraigher et al.
Fr. (2007), Grebenc and Kraigher
(2007a, b), Al Sayegh Petkovšek
(2008)
Dermocybe cinammomea þ þ 1, 2 Al Sayegh Petkovšek (2008)
(L.) W€unsche þ Picea
abies (L.) Karst
Elaphomyces granulatus þ þ 1 Al Sayegh Petkovšek (2008)
Fr. þ Picea abies (L.)
Karst.
Elaphomyces muricatus þ 1 Al Sayegh Petkovšek (2008)
Fr. þ Fagus
sylvatica L.
Elaphomyces sp. 1 þ þ 1 Al Sayegh Petkovšek (2008)
Elaphomyces sp. 2 þ þ 1 Al Sayegh Petkovšek (2008)
Fagirhiza oleifera þ þ 1, 2 Al Sayegh Petkovšek (2008)
Fagirhiza spinulosa þ 1, 2 Al Sayegh Petkovšek (2008)
Paxillus involutus þ 1 Kraigher (1999), Kraigher et al. (2007)
Piceirhiza inflata þ 1 Kraigher (1999), Kraigher et al. (2007)
Piceirhiza terraphila þ 1 Kraigher (1999), Kraigher et al. (2007)
Russula fellea (Fr.: Fr.) Fr. þ 3 Grebenc and Kraigher (2007a, b)
þ Fagus sylvatica L.
Russula illota Romagn. þ þ 3 Grebenc and Kraigher (2007a, b)
Fagus sylvatica L.
Russula sp. 4 þ 1 Kraigher (1999), Kraigher et al. (2007)
Xerocomus badius (Fr.Fr.) þ þ 1 Kraigher (1999), Kraigher et al. (2007)
Gilb. þ Picea abies
(L.) Karst.
a
Different stressors are marked with numbers (1 – polluted air, 2 – drought, and 3 – ozone)
containers when compared to that in nonfumigated controls (description available

at: http://www.casiroz.de), especially at a depth of 2–8 cm, was found (Fig. 13.3).
The average number of short root tips in 274 ml of soil cores ranged from (400)
1,100 to 2,000 (5,600), indicating that the number of active short root tips in the
upper 20 cm of forest soils should be between 3 105 and 5 106 per square
meter. These numbers suggest that even types of ECM occurring in small percen-
tages may form symbiosis on several hundred thousands of roots (i.e., Lactarius
lignyotus on ca 230,000 root tips; Kraigher 1997). Such high numbers of root tips
also indicate that it is impossible to analyze more than a small fraction of the total
ECM fungal community on root tips. The high heterogeneity within forest soils also
interacts with the heterogenous distribution of ECM types in time and space;
therefore, representative sampling is difficult (Kraigher and Agerer 2000; Erland
and Taylor 2002). However, stress-sensitive Norway spruce stands, with a very low
ground vegetation diversity, showed clear shifts in the diversity indices of the very
diverse below-ground ECM community (Kraigher 1999). On the contrary, in
ground vegetation rich beech sites, the same approach failed as a bioindication
method. The lack of changes in the ECM biodiversity indices in beech may reflect
the well-adaptable buffering capacity of the forest soils; the presence of favorable
humus forms, especially in older beech stands; the sustainability of natural beech
forests; their adaptation to N deposits; or the fact that the diversity both above- and
below-ground was too high to allow the detection of any changes (Al Sayegh
Petkovšek 2004, 2008). Our standardized sampling protocols were designed for
Norway spruce stands (Kraigher and Agerer 2000).
13.3.1.2 Accumulative In Situ Passive Monitors
Sporocarps of selected ECM fungi can be used as accumulative bioindicators

(passive monitors) of forest site pollution, since they accumulate high concentrations
Fig. 13.3 Occurrence of sclerotia of Cenococcum geophilum in ozone-fumigated containers (gray

bars) compared to the control (black bars) in the Free Air Ozone Fumigation System in Kranzberg
Forest (for description, see http://www.casiroz.de)
of heavy metals. First of all, they are useful for distinguishing polluted and unpolluted
areas (Kalač and Svoboda 2000; Rudawska and Leski 2005a, b); moreover, a survey
performed in variously polluted areas in Slovenia suggests that several ECM species
are employable as bioindicators of soil polluted with heavy metals (e.g., Boletus
edulis and Laccaria amethystina) (Al Sayegh Petkovšek and Pokorny 2006;
Al Sayegh Petkovšek 2008). In addition, the bioconcentration factor (BCF), calcu-
lated as the quotient between metals in the sporocarp and metal content in soil can be
used as a tool for assessing forest site pollution. BCFs ranged from 50 to 300 (Cd), 30
to 500 (Hg), and 0.1 to 0.2 (Pb) and differ in relation to the metal levels in soil and
ECM fungal species (Kalač and Svoboda 2000; Rudawska and Leski 2005a). It
appears that the bioindicative value of BCF is fairly problematic, since BCF is not
a constant value for a particular species. However, according to Rudawska and Leski
(2005a), BCF factors may be useful for comparing fungal species from different sites
with those from soil with uniform properties.
13.3.1.3 Conclusions on Indicators and Passive Monitors
Conclusions from the application of biodiversity indices and passive monitors in

forest stands can be summarized as follows:
1. ECM species richness (d) for Norway spruce showed higher values in unpolluted
sites than in NOx and SOx polluted ones.
2. ECM species richness for European beech was similar in polluted and unpol-
luted sites, so it cannot be applied as an ecological indicator of soil pollution.
3. Similar H values indicate whether there was no dominant ECM species in
polluted vs. unpolluted sites.
4. Differences in diversity indices in polluted and unpolluted Norway spruce sites
may indicate a potentially fast response toward degradation when a stress factor
is imposed, while beech forest sites may be considered to be more resilient
toward any stresses.
5. Biodiversity indices as ecological indicators of the impact of pollution on forest
soils can be applied in various (but not all) forest ecosystems, show a reasonable
sensitivity to stress, and can provide some insight into changes in below-ground
forest ecosystem processes. However, they are species specific and should be
complemented with other bioindication approaches, cannot be used as a single
indicator of stress, and should be upgraded with functional diversity approaches.
6. Specific persistent structures, such as the sclerotia of C. geophilum, which are
presumed to help this fungus to survive in unfavorable conditions, can be
significantly multiplied in soils exposed to ozone fumigation and/or drought.
They have potential as an applicable bioindicator after the system has been
analyzed under various conditions of stress in comparison with controls – an
active monitoring approach may develop.
7. The application of pollution-sensitive and -insensitive ECM species can reveal

the ecology of a certain ECM species and is, therefore, important for under-
standing the processes in a forest ecosystem.
13.3.2 Active Monitors of Stress in Forest Ecosystems
13.3.2.1 Active monitors: In Situ Exposed Nonmycorrhizal Seedlings
Nonmycorrhizal Norway spruce seedlings were exposed and planted in variously

polluted forest sites as active monitors of stress (Kraigher et al. 2007). In beech
dominated sites, the mycorrhization percentage was significantly lower in the
polluted plot (here identified as Zavodnje B) in two different years when testing
was applied (Fig. 13.3). The results indicated that spruce seedlings and their
mycorrhization can be applied as active monitors of pollution when the seedlings
are planted in polluted and unpolluted stands in broadleaf forests. On the contrary,
the mycorrhization percentage of spruce seedlings planted in a polluted spruce
dominated site (Zavodnje A) was significantly higher than that of the unpolluted
spruce stand (Pokljuka). The opposite result, i.e., a higher mycorrhization in
Pokljuka, was discussed as being due to the presence of the same macrobiont in
the plot for several centuries. Our results might, therefore, indicate less favorable
growing conditions resulting from the high altitude of the plot (Pokljuka, 1,250 m/a.
s.l.) and the relatively short vegetation period when compared to all other plots
(Kraigher et al. 2000). In the within-treatment comparison (between 6 and
12 months of exposure), the average mycorrhization percentage was higher for all
plots after longer exposure. The dry weight of needles was in close linear correla-
tion with mycorrhization in all treatments. Seedlings with low mycorrhization also
had less needle biomass and vice versa. As with the mycorrhization %, the
calculated ratio was significantly higher in unpolluted broadleaf stands but not in
spruce stands. The results indicate that spruce seedlings might serve as a good
active monitoring indicator of pollution if planted in beech stands in which no
specific ECM indicator types have been determined.
13.3.2.2 Ex Situ Testers: Mycorrhizal Inoculum Potential of Differently

Polluted Soil Substrates
Mycorrhization inoculum potential is a method developed by Kropaček et al.

(1989). It is based on naturally present mycorrhizal inoculum, existing in sieved
soil substrate taken from a forest site and applied in a pot experiment with seedlings
of a chosen forest tree species in a growth chamber under controlled environmental
conditions.
Two differently polluted forest sites in the emission area of Thermal Power Plant
Šoštanj were submitted to studies of mycorrhizal inoculum potential in different
years, before and after the installation of cleaning blocks in the Thermal Power
Plant. Emissions were reduced from 80.516 t SO2 in 1994 to 44.253 t in 2000, and
the tests were done in 1992–1993 and in 2002–2003. The percentage of mycorrhizal
short roots of potted seedlings from Zavodnje (polluted substrate) was significantly
lower (p < 0.05) in comparison with that of Pohorje (unpolluted), suggesting that
the mycorrhizal potential of the more polluted area was lower (Fig. 13.4). Other
growth parameters of the seedlings were also different: the fresh weights of roots,
stems, and needles were higher in Pohorje (Al Sayegh Petkovšek and Kraigher
2003). Pollution influenced the mycorrhizal potential of forest soils and this nega-
tive impact was still present more than 5 years after the reduction of the emissions.
We conclude that further analyses of mycorrhizal potential are recommended in
order to monitor the changes in ECM composition continuously after the reduction
of the emissions from Thermal Power Plant Šoštanj. These might also include
testing the application of mycorrhizal fungi in bioremediation processes, as
reviewed by Gadd (2005), for remediation of the thermal power plant wastes.
Polluted area (Zavodnje), 1993 Unpolluted area (Pohorje), 1993

7%
21%
79% unmycorrhizal roots unmycorrhizal roots

mycorrhizal roots 93% mycorrhizal roots
Polluted area (Zavodnje), 2002 Unpolluted area (Pohorje), 2002

17% 4%
83% unmycorrhizal roots unmycorrhizal roots

96%
mycorrhizal roots mycorrhizal roots
Fig. 13.4 Mycorrhization (%) of Norway spruce seedlings (n ¼ 25) from the mycorrhizal
inoculum potential of forest soil from 1993 to 2002. The differences among sites were significant
(Fisher LSD, p < 0,001), differences among years showed trends to higher mycorrhization (Fisher
LSD, p ¼ 0.077)
13.3.2.3 Conclusions on Active Indicators in Mycoindication
Our conclusions from mycorrhizal inoculum potential tests can be summarized as

follows:
1. Mycorrhizal potential as a bioassay for soil pollution (N and S deposit) is an
acceptable mycoindication method.
2. The differences between the two sites were statistically significant, although the
pollution effects were not highly destructive with respect to mycorrhizal soil
inoculum potential.
3. Even though emissions from the thermal power plant were reduced in the last
decade, the revitalization of soil substrates is a slow process, it might, therefore,
take several decades before the mycorrhizal potential of the polluted and the
unpolluted sites reaches the same level.
4. The types of ECM are of primary importance for the functioning of a forest
ecosystem, although the simplified situation in pot studies of mycorrhizal
potential supports the primary importance of the percentage of mycorrhizal
infection.
13.4 Final remarks
Bioindicators have been proposed as “environmental fever thermometers” (Fr€anzle

2006), since they should respond to early stages of either exposure or effects
without disclosing cause–effect relationships. As a follow-up action, preventive
or corrective measures need to be initiated. Their qualities, demands, and limita-
tions can be presented as summarized by Fr€anzle (2006), with respect to mycoindi-
cation approaches:
l Environmental observation techniques face a large number of preselected phys-
ical or chemical stress factors, with multiple synergistic and antagonistic effects
on conversion products and pathways. Biomonitoring techniques provide a
timely, widely applicable, low-cost, feasible way of observing deleterious
effects. They involve passive and active approaches on different scales, from
microcoenoses to ecosystems. Their main task is a general determination of
physiological effects rather than measurement of concentrations of stress fac-
tors. The lack of specificity in early recognition perspective might be considered
to be an advantage inducive of a subsequent systematic search for quantification
of causal interrelationships. The disadvantage is the highly variable susceptibil-
ity of the multitude of species exposed to stress factors, resulting in the need for
fuzzy logic approaches.
l The field of biomarkers has evolved rapidly in recent decades; a biomarker may
be considered to be a biological response to chemical(s) that might provide its
quantification and measure of toxicity. A parallel exposure to ambient stress
factors and a control set would be ideal to estimate the ecological significance of
a slight increase or decrease in a measured parameter. In a mycobioindication,

this would imply analysis of a natural mycorrhizal community structure with
special emphasis on resistant and sensitive species, exposure of nonmycorrhizal
seedlings in sites with a gradually increasing stress, and establishment of a
controlled experimental system using the mycorrhizal inoculum potential of a
systematically increased level of stress.
l The interpretation of the results of studies in complex ecosystems such as forests
is problematic. Their functional redundancy might interfere with clear indication
of stress, since the loss of functional capacity by one organism will immediately
be compensated by increased activity of another. An intersystemic comparison
of biotic reactions must also take into account natural successions, and spatial
and temporal variability of ecosystems, whose communities are organized by
competition and collaboration, and predation and disturbances. All these sys-
tems are therefore dynamic; there is no single stability, susceptibility, or vulner-
ability measure for a community or a whole ecosystem. Active and passive
biomonitoring techniques demand representative sampling over temporal and
spatial scales, and their interpretation through geostatistical requirements and
kriging, both at the single species and the community level.
l However, each bioindication method contributes to an understanding of ecosys-
tem functioning, community interactions, and single species physiology and
ecology. A combination of approaches provides a timely and feasible interpre-
tation of stresses, and their application leads to step-wise revelations of pro-
cesses in highly complex below-ground processes.
Acknowledgments The study was part of the research program P4-0107 financed by the Slove-
nian Research Agency, summarizing the results of a number of national and international projects,
with numerous collaborators contributing to obtain the data. Among these, we would like to
acknowledge the collaboration of Dr. Tine Grebenc, Barbara Štupar, Melita Hrenko, and Jana
Janša from the Slovenian Forestry Institute; Dr. Boštjan Pokorny from ERICo Ecological Research
Institute; the coordinator of the EU project CASIROZ Prof. Dr. Rainer Matyssek; and his team.
We would also like to acknowledge the early suggestions on mycorrhizal inoculum potential from
Dr. Pavel Cudlin, instructions on identification of ectomycorrhizae from Prof. Dr. Reinhard
Agerer, on the development of bioindication methods from Prof. Dr. Franc Batič and Prof. Dr.
Dieter Grill, and Martin Creegen for final language corrections.
References
Agerer R (2001) Exploration types of ectomycorrhizae. A proposal to classify ectomycorrhizal

mycelial systems according to their patterns of differentiation and putative ecological impor-
tance. Mycorrhiza 11:107–114
Ågren GI, Franklin O (2003) Root: shoot ratios, optimization and nitrogen productivity. Ann Bot
92:795–800
Al Sayegh Petkovšek S (1997) Mycorrhizal potential of two differently polluted forest sites in the
emission region of the Thermal Power Plant Šoštanj. Zb GL 52:323–350
Al Sayegh Petkovšek S (2004) Biodiversity of types of ectomycorrhizae in fagus stands in
differently polluted forest research plots. Zb GL 75:5–19
Al Sayegh Petkovšek S (2005) Belowground ectomycorrhizal fungal communities at fagus stands

in differently polluted forest research plots. Zb GL 76:5–38
Al Sayegh Petkovšek S (2008) Fungi as sensitive and accumulative bioindicators of forest site
pollution in the Šalek Valley. Doctoral dissertation, University of Ljubljana
Al Sayegh Petkovšek S, Kraigher H (2003) Mycorrhizal potential of two forest research plots with
respect to reduction of the emissions from the Thermal Power Plant Šoštanj. Acta Biol Slov
46:9–16
Al Sayegh Petkovšek S, Pokorny B (2006) Fungi as sensitive and accumulative bioindicators of
forest site pollutions in the Šalek Valley. Zb GL 81:61–71
Al Sayegh Petkovšek S, Pokorny B, Ribarič Lasnik C, Vrtačnik J (2002) Cd, Pb, Hg and As in
fruiting bodies of higher fungi from the forest landscape of the Šalek Valley. Zb GL 67:5–46
Alberton O, Kuyper TW, Gorissen A (2005) Taking mycocentrism seriously: mycorrhizal fungal
and plant responses to elevated CO2. New Phytol 167:859–868
Alexander IJ, Fairley RI (1983) Effects of N fertilisation on populations of fine roots and
mycorrhizas in spruce humus. Plant Soil 71:49–53
Andersen CP (2003) Source–sink balance and carbon allocation below ground in plants exposed to
ozone (Tansley review). New Phytol 157:213–228
Andersen CP, Rygiewicz PT (1991) Stress interactions and mycorrhizal plant response: under-
standing carbon allocation priorities. Environ Pollut 73:217–244
Andersen CP, Rygiewicz PT (1995) Effects of ozone on temporal allocation of carbon in
mycorrhizal Pinus ponderosa seedlings. New Phytol 131:471–480
Arndt U, Nobel W, Schweizer B (1987) Bioindikatoren – M€oglichkeiten, Grenzen und neue
Erkentnisse. Ulmer, Stuttgart
Arnolds E (1988) The changing macromycete flora of the Netherlands. Trans Br Mycol Soc
90:391–406
Atlas R, Bartha R (1981) Introduction to microbiology. Addison-Wesley, Reading
Bakker MR (1999) Fine root parameters as indicators of sustainability of forest ecosystems. For
Ecol Manage 122:7–16
Ballach HJ, Oppenheimer S, Mooi J (1992) Reactions of cloned poplars to air pollution: premature
leaf loss and investigations of the nitrogen metabolism. Z Naturforsch 47c:109–119
Becker CA, Mroz CD, Fuller LG (1987) The effects of plant moisture stress on red pine (Pinus
resinosa) seedling growth and establishment. Can J For Res 17:813–820
Berthelsen BO, Olsen RA, Steinnes E (1995) Ectomycorrhizal heavy metal accumulation as a
contributing factor to heavy metal levels in organic surface soil. Sci Total Environ
170:141–149
Bortier K, Ceulemans R, De Temmerman L (1999) Effects of tropospheric ozone on woody plants.
In: Agrawal SB, Agrawal M (eds) Environmental pollution and plant response. Lewis, Boca
Raton, pp 153–182
Bortier K, De Temmerman L, Ceulemans R (2000) Effects of ozone exposure in open-top
chambers on poplar (Populus nigra) and beech (Fagus sylvatica): a comparison. Environ Pollut
109:509–516
Brunner I (2001) Ectomycorrhizas: their role in forest ecosystems under the impact of acidifying
pollutants. Perspect Plant Ecol Evol Syst 4(1):13–27
Buchanan B, Gruissens W, Jones R (2000) Biochemistry and molecular biology of plants.
American Society of Plant Physiologists, Rockville, MD
Cocchi L, Vescovi L, Petrini LE, Petrinini O (2006) Heavy metals in edible mushrooms in Italy.
Food Chem 98:277–284
Coleman MD, Dickson RE, Isebrands JG, Karnosky DF (1996) Root growth and physiology of potted
and field-grown trembling aspen exposed to tropospheric ozone. Tree Physiol 16:145–152
Cudlin P, Kieliszewska-Rokicka B, Rudawska M, Grebenc T, Alberton O, Lehto T, Bakker MR,
B€orja I, Konopka B, Leski T, Kraigher H, Kuyper TW (2007) Fine roots and ectomycorrhizas
as indicators of environmental change. Plant Biosyst 141(3):406–525
de Roman M, de Miguel AM (2005) Post-fire, seasonal and annual dynamics of the ectomycor-
rhizal community in a Quercus ilex L. over 3-year period. Mycorrhizae 15:471–482
Dighton J (2003) Fungi in Ecosystem Processes. Dekker, New York
Dighton J, Boddy L (1988) Role of fungi in nitrogen, phosphorus and sulphur cycling in temperate
forest ecosystems. In: Boddy L, Marchant R, Read DJ (eds) Nitrogen, phosphorus and sulphur
utilization by fungi. Symposium of the BMS, Cambridge University Press, Cambridge, pp 269–299
Erland S, Taylor AFS (2002) Diversity of ectomycorrhizal fungal communities in relation to the
abiotic environment. In: van der Heijden M, Sanders T (eds) The ecology of ectomycorrhizas.
Ecological studies Series, Vol 157. Chapter 7, Springer, pp 163–193
Falandysz J, Szymczyk K, Ichihashi H, Bielawski L, Gucia M, Frankowska A, Yamasaki S (2001)
ICP/MS and ICP/AES elemental analyses (38 elements) of edible wild mushrooms growing in
Poland. Food Addit Contam 18:503–513
Falandysz J, Kawano M, Swieczkowski A, Brzostowski A, Dadej M (2003) Total mercury in wild-
growing higher mushrooms and underlying soil from Wdzydze Landscape Park, Northern
Poland. Food Chem 81:21–26
Feil W, Kottke I, Oberwinkler F (1988) The effect of drought on mycorrhizal production and very
fine root system development of Norway spruce under natural and experimental conditions.
Fellner R (1989) Mycorrhiza-forming fungi as bioindicators of air pollution. Agric Ecosyst
Environ 28:115–120
Fellner R, Peškova V (1995) Effects of industrial pollutants on ectomycorrhizal relationships in
temperate forest. Can J Bot 73(Suppl 1):1310–1315
Fr€anzle O (2006) Complex bioindication and environmental stress assessment. Ecol Indic
6:114–136
Gadd GM (2005) Microorganisms in toxic metal-polluted soils. In: Buscot F, Varma A (eds) Micro-
organisms in soils: roles in genesis and functions, vol 3, Soil biology. Springer, Berlin, pp 325–356
Grebenc T (2005) Tipi ektomikorize na bukvi (Fagus sylvatica L.) v naravnem in gospodarskem
gozdu: doktorska disertacija. Univerza v Ljubljani, Biotehniška fakulteta, Interdisciplinaren
podiplomski študij biotehnologije. Ljubljana
Grebenc T, Kraigher H (2007a) Types of ectomycorrhiza of mature beech and spruce at ozone-
fumigated and control forest plots. Environ Monit Assess 128:47–59
Grebenc T, Kraigher H (2007b) Changes in the community of ectomycorrhizal fungi and increased
fine root number under adult beech trees chronically fumigated with double ambient ozone
concentration. Plant Biol (Stuttg) 9(2):279–287
Grebenc T, Christensen M, Vilhar U, Čater M, Martin MP, Simončič P, Kraigher H (2009)
Response of ectomycorrhizal community structure to gap opening in natural and managed
temperate beech-dominated forests. Can J For Res 39(7):1375–1386
Haberer K, Grebenc T, Alexou M, Gessler A, Kraigher H, Rennenberg H (2007) Effects of long-
term free-air ozone fumigation on d15N and total N in Fagus sylvatica and associated
mycorrhizal fungi. Plant Biol 9:242–252
Haug I, Pritsch K, Oberwinkler F (1992) Der Einfluss der D€ungung auf Feinwurzeln und Mykor-
rhizen im Kulturversuch und im Freiland. KfK-PEF 97:1–159
Hu S, Tu C, Chen X, Gruver JB (2006) Progressive N limitation of plant response to elevated CO2:
a microbiological perspective. Plant Soil 289:47–58
Jaenike J (1991) Mass extinction of European fungi. Tree 6:174–175
Jany JL, Martin F, Garbaye J (2003) Respiration activity of ectomycorrhizas from Cenoccocum
geophilum and Lactarius spec.: in relation to soil water potential in five beech forests. Plant
Soil 225:487–494
Johanson KJ, Nikolova I, Taylor AFS (2004) Uptake of elements by fungi in the Forsmark area.
Technical Report TR-04-26, Swedish Nuclear Fuel and Waste Management
Johnson DW (2006) Progressive limitation in forests: review and implications for long-term
responses to elevated CO2. Ecology 84:4–75
Joslin JD, Wolfe MH, Hanson PJ (2000) Effects of altered water regimes on forest root systems.
Kalač P, Svoboda L (2000) A review of trace element concentrations in edible mushrooms. Food
Chem 69:273–281
Karnosky DF, Pregitzer KS, Zak DR, Kubiske M, Hendrey GR, Weinstein D, Nosal M, Percy KE
(2005) Scaling ozone responses of forest trees to the ecosystem level in a changing climate.
Plant Cell Environ 28:965–981
Kernaghan G (2005) Mycorrhizal diversity: cause and effect? Pedobiologia 49:511–520
Kieliszewska-Rokicka B, Rudawska M, Leski T (1997) Ectomycorrhizae of young and mature
scots pine trees in industrial regions in Poland. Environ Pollut 98(3):315–324
Kovacs G, Pausch M, Urban A (2000) Diversity of ectomycorhizal morphotypes and oak decline.
Phyton (Horn, Austria) 40(4):109–116
Kraigher H (1991) Mineral nutrients in mycorrhizal Norway spruce trees on Pohorje. MSc Thesis,
University of Ljubljana, Ljubljana
Kraigher H (1994) Cytokinins and types of ectomycorrhiza in Norway spruce (Picea abies (L.)
Karst) seedlings as indicators of forest site pollution. PhD Thesis, University of Ljubljana,
Ljubljana
Kraigher H (1996) Tipi ektomikorize – taksonomija, pomen in aplikacije (Types of ectomycor-
rhizae – their taxonomy, role and application). ZbGL 49:33–66
Kraigher H (1997) Mycobioindication of pollution of two forest sites. In: Robek R (ed) Prouče-
vanje propadanja gozdov v Sloveniji v obdobju 1985–1995. ZbGL 52:279–322
Kraigher H (1999) Diversity of types of ectomycorrhizae on Norway spruce in Slovenia. Phyton
39(3):199–202
Kraigher H (2001) Mycorrhizal fungi. In: Hlad B, Skoberne P (eds) Overview of biodiversity in
Slovenia, 1st edn. Ministry for environment and spatial planning, Environmental Agency od
RS, Ljubljana
Kraigher H, Agerer R (2000). Identification and characterisation of types of ectomycorrhizae. V:
Methods in root–soil interactions research: protocols compiled for the Technical Workshop of
the COST E6 Action Eurosilva. In: Martin MP (ed) Forest Tree Physiology Research held in
Gozd Martuljek, Slovenia. Slovenian Forestry Institute, Ljubljana, 8 Sep 1999, pp 29–34
Kraigher H, Grayling A, Wang TL, Hanke DE (1991) Cytokinin production by two ectomycor-
rhizal fungi in liquid culture. Phytochemistry 30(7):2249–2254
Kraigher H, Strnad M, Hanke DE, Batič F (1993) Cytokiningehalte von Fichtennadeln (Picea abies
[L.] Karst) nach Inokulation mit zwei St€ammen des Mykorrhizapilzes Thelephora terrestris
(Ehrh.) Fr. Forstwiss Cbl 112:107–111
Kraigher H, Agerer R, Javornik B (1995) Ectomycorrhiza of Lactarius lignyotus on Norway
spruce, characterised by anatomical and molecular tools. Mycorrhiza 5:175–180
Kraigher H, Batič F, Agerer R (1996) Types of ectomycorrhizae and mycobioindication of forest
site pollution. Phyton 36(3):115–120
Kraigher H, Simončič P, Smolej I, Diaci J (2000) Conclusions, applications and perspectives of
research. In: Kraigher H, Smolej I (eds) The rhizosphere: studies of forest soils and the
rhizosphere and their influences on chosen physiological parameters of forest trees in selected
forest ecosystems, forest types and developmental phases of the forest. Strokovna in znanst-
vena dela, vol 118. Gozdarski inštitut Slovenije, Ljubljana, pp 246–262
Kraigher H, Al Sayegh-Petkovšek S, Grebenc T, Simončič P (2007) Types of ectomycorrhiza as
pollution stress indicators: case studies in Slovenia. Environ Monit Assess 128:31–45
Kraigher H, Grebenc T, Hanke DE (2008) Ozone stress and ectomycorrhizal root-shoot signaling.
In: Varma A (ed) Mycorrhiza: state of the art, genetics and molecular biology, eco-function,
biotechnology, eco-physiology, structure and systematics, 3rd edn. Springer, Berlin,
pp 337–357
Kropaček K, Kristinova M, Chemelikova E, Cudlin P (1989) The mycorrhizal inoculation
potential of forest soils exposed to different pollutions stress. Agric Ecosyst Environ
28:217–277
Leake JR (2001) Is diversity of ECM important for ecosystem function? New Phytol 152:1–3
Lehto T (1992) Mycorrhizas and drought resistance of Picea sitchensis. II. In conditions of
adequate nutrition. New Phytol 122:669–673
Lilleskov EA (2005) How do composition, structure, and function of mycorrhizal fungal commu-
nities respond to nitrogen deposition and ozone exposure? In: Dighton J, Oudemans P, White J
(eds) The fungal community: its organization and role in the ecosystem, 3rd edn. Dekker,
New York, pp 769–801
Lilleskov EA, Fahey TJ, Horton TR, Lovett GM (2002) Belowground ectomycorrhizal fungal
community change over a nitrogen deposition gradient in Alaska. Ecology 83:104–115
L€
ow M, Herbinger K, Nunn AJ, H€aberle K-H, Leuchner M, Heerdt C, Werner H, Wipfler P,
Pretzsch H, Tausz M, Matyssek R (2006) Extraordinary drought of 2003 overrules ozone
impact on adult beech trees (Fagus sylvatica). Trees Struct Funct 20:539–548
Luo Y, Su B, Currie WS, Dukes JS, Finzi A, Hartwig U, Hungate B, McMurtrie RS, Oren R,
Parton WJ, Pataki DE, Shaw R, Zak DR, Field CB (2004) Progressive nitrogen limitation of
ecosystem responses to rising atmospheric carbon dioxide. Bioscience 54:731–739
Martı́nez-Crego B, Alcoverro T, Romero J (2010) Biotic indices for assessing the status of coastal
waters: a review of strengths and weaknesses. J Environ Monit 12:1013–1028
Matyssek R, Bahnweg G, Ceulemans R, Fabian W, Grill D, Hanke DE, Kraigher H, Oßwald W,
Rennenberg H, Sandermann H, Tausz M, Wieser G (2007) Synopsis of the CASIROZ Case
Study: carbon sink strength of Fagus sylvatica L. in a changing environment – experimental
risk assessment of mitigation by chronic ozone impact. Plant Biol 9:163–180
McLaughlin SB, Downing DJ (1995) Interactive effects of ambient ozone measured on mature
forest trees. Nature 374:252–257
Menge JA, Grand LF, Haines LW (1977) The effect of fertilization on growth and mycorrhizae
numbers in 11-year old loblolly pine plantations. For Sci 23:37–44
Meyer FH (1987) Der Verzweigungsindex, ein Indikator f€ur Sch€aden am Feinwurzelsystem.
Forstwiss Cbl 106:84–92
O’Neill EG (1994) Responses of soil biota to elevated atmospheric carbon dioxide. Plant Soil
165:55–65
Ottossen S, Wallin G, Sk€arby L, Karlsson P-E, Medin E-L, R€antfors M, Pleijel H, Selldén G
(2003) Four years of ozone exposure at high or low phosphorus reduced biomass in Norway
spruce. Trees 17:299–307
Parrent JL, Morris WF, Vilgalys R (2006) CO2 enrichment and nutrient availability alter ectomy-
corrhizal fungal communities. Ecology 87(9):2278–2287
Perrin R, Estivalet D (1990) Mycorrhizal association and forest decline (yellowing of spruce).
Agric Ecosyst Environ 28:381–387
Peter M, Ayer F, Egli S (2001) Nitrogen addition in a Norway spruce stand altered macromycete
sporocarp production and below-ground ectomycorrhizal species composition. New Phytol
149:311–325
Peter M, Ayer F, Cudlin P, Egli S (2008) Belowground ectomycorrhizal communities in three
Norway spruce stands with different degrees of decline in the Czech Republic. Mycorrhizae
18:157–169
Pigott CD (1982) Survival of mycorrhiza formed by Cenococcum geophilum Fr. in dry soils. New
Phytol 92:513–517
Poorter H, Nagel O (2000) The role of biomass allocation in the growth response of plants to
different levels of light, CO2, nutrients and water: a quantitative review. Aust J Plant Physiol
27:595–607
Pučko M, Grebenc T, Božič G, Brus R, Kraigher H (2004) Identifikacija tipov ektomikorize na
sadikah v bukovem provenienčnem poskusu. Zb GL 75:87–104
Read DJ (1991) Mycorrhizas in ecosystems. Experientia 47(4):376–390
Read DJ (1998) Plants on the web. Nature 396:22–23
Roth DR, Fahey TJ (1998) The effects of acid precipitation and ozone on the ectomycorrhizae of
red spruce saplings. Water Air Soil Pollut 103:263–276
Rudawska M (1986) Sugar metabolism of ectomycorrhizal Scots pine seedlings as influenced by

different nitrogen forms and levels. In: Gianinazzi-Pearson V, Gianinazzi S (eds) Physiological
and genetical aspects of Mycorrhizae. Proceedings of the 1st European Symposium on
Mycorrhizae. INRA, Paris, pp:389–394
Rudawska M, Leski T (2005a) Trace elements in fruiting bodies of ectomycorrhizal fungi growing
in Scots pine (Pinus sylvestris L.) stands in Poland. Sci Total Environ 339:103–115
Rudawska M, Leski T (2005b) Macro- and microelement contents in fruiting bodies of wild
mushrooms from the Notecka forest in west-central Poland. Food Chem 92:499–506
Schneider BU, Meyer J, Schulze ED, Zech W (1989) Root and mycorrhizal development in
healthy and declining Norway spruce stands. Forest decline air pollution, vol 77. Berlin,
Springer, pp 370–391
Simončič P, Smolej I, Rupel M, Urbančič M, Kalan P, Kraigher H (1998) Kroženje hranil in
pestrost ektomikorize v smrekovem gozdu na Pokljuki. In: Zbornik referatov “Gorski gozd”.
XIX. Gozdarski študijski dnevi, Logarska dolina, pp 207–221
Tausz M, Batič F, Grill D (1996) Bioindication at forest sites – concepts, practice and outlook.
Phyton 36:7–14
Taylor AFS, Alexander IJ (1989) Demography and population dynamics of ectomycorrhizas of
Sitka spruce fertilized with N. Agric Ecosyst Environ 28:493–496
Taylor AFS, Alexander I (2005) The ectomycorrhizal symbiosis: life in the real world. Mycologist
19:102–112
Taylor AFS, Martin F, Read DJ (2000) Fungal diversity in ectomycorrhizal communities of
Norway spruce (Picea abies [L.] Karst.) and beech (Fagus sylvatca L.) along North-South
transects in Europe. In: Schulze ED (ed) Carbon and nitrogen cycling in European forest
ecosystems, vol 142, Ecological studies series. Springer, Heidelberg, pp 344–365
Tetreault JP, Bernier B, Fortin JA (1978) Nitrogen fertilization and mycorrhizae of balsam fir
seedlings in natural stands. Naturaliste Can 105:461–466
Tingey DT, Wilhour RG, Standley C (1976) The effect of chronic ozone exposure on the
metabolite content of ponderosa pine seedling. For Sci 22:234–241
Topa MA, McDermitt DJ, Yun SC, King PS (2004) Do elevated ozone and variable light alter
carbon transport to roots in sugar maple? New Phytol 162(1):173–186
Vanhatalo M, B€ack J, Huttunen S (2003) Differential impacts of long-term (CO2) and O3 exposure
on growth of northern conifer and deciduous tree species. Oecologia 17:211–220
Vilhar U, Smolej I, Trošt T, Kutnar L, Kraigher H (2004) Pestrost tipov ektomikorize v smreko-
vem sestoju na Pokljuki. Zb GL 75:71–85
Vitousek P, Aber J, Howarth R, Likens G, Matson P, Schindler D, Schlesinger W, Tilman D (1997)
Human alteration of the global nitrogen cycle: sources and consequences. Ecol Appl
7:737–750
Waller K, Agerer R, Brand F, Taylor AFS, Wanner G (1993) Piceirhiza oleiferans, eine neue
Ektomykorrhizen-Art an Picea abies. Sendtnera 1:11–22
Winwood J, Pate AE, Price AJ, Hanke DE (2007) Effects of long-term, free-air ozone fumigation
on the cytokinin content of mature Beech trees. Plant Biol 9:265–278
Worley JF, Hacskaylo E (1959) The effect of the available soil moisture on the mycorrhizal
association of Virginia pine. For Sci 5:267–268
Železnik P, Hrenko M, Then Ch, Koch N, Grebenc T, Levanič T, Kraigher H (2007) CASIROZ:
root parameters and types of ectomycorrhiza of young beech plants exposed to different ozone
and light regimes. Plant Biol 9:298–308
Chapter 14
Effects of Pesticides on the Growth
of Ectomycorrhizal Fungi and Ectomycorrhiza
Formation
Miguel Marin
14.1 Introduction
In all reforestation programs, the quality of the plant to be used is of vital

importance. This quality is determined by the origin of plant material used and
the management during the production in nursery phase. Often, the criteria for
determining the quality of a plant had been limited to assessing the status and size of
the shoot. In the recent years, the nurseries were also given sufficient attention to the
quality of the root system, in recognition of their tremendous importance in the
uptake of water and nutrients. However, the quality of the seedlings is not always
satisfactory and the mortality of pine seedlings after planting in the forest site can
be very high (Heiskanen and Rikala 2003). A term “transplant shock” has been used
to describe a suite of symptoms in seedlings, including yellowing, short needles,
and reduced growth, arising upon relocation from nursery to forest soil (Reitveld
1989). The formation of ectomycorrhizae is the natural state of almost all of the
trees in forests, and therefore part of their root systems and a factor to take into
account when assessing their quality.
Seedlings are nowadays produced in large commercial tree nurseries mainly as
container seedlings. The proportion of bare-rooted seedlings has decreased over the
last 20 years. In Finland, container seedlings represent 98% of Scots pine, 86% of
Norway spruce, and 89% of silver birch production (Peltola 2001). The production
takes place in tree nurseries where large quantities of seedlings are produced in
controlled conditions using artificial growth substrate, irrigation, fertilization, pest
control, and sometimes day–night shift treatments to promote seedling growth
(Landis 1989). Normally in the production of seedlings, the seedlings spend
between 1 and 3 years of cultivation in the nursery until they are transplanted to
the field. Plant diseases may be the biggest problem in nurseries (Landis 1989) and
their prevention in the large monoculture seedling beds has traditionally been
M. Marin
Department of Physiology, Genetics and Microbiology, University of Alicante, Carretera San
Vicente del Raspeig s/n, E-03690, San Vicente del Raspeig (Alicante), Spain
e-mail: mmarinz@ua.es

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_14,
324 M. Marin
achieved using fungicides, insecticides, and other chemical products. Maintenance

of nursery hygiene is also of major importance. The opportunistic plant control is
done by herbicides. Under nursery conditions, ectomycorrhizal fungi (EMF) live in
an artificial environment and are affected by pest control, watering, and fertilization
(Marin 2009). Generally, all these factors are unfavorable to most EMF, but certain
types are commonly found in the nurseries, e.g., Thelephora spp. and ectendomy-
corrhizal species (Laiho 1965; Landis 1989). Unfortunately, most of the so-called
nursery fungi are not represented as dominant members in forest mycorrhizal
communities, which suggest a poor competitive capacity after transplanting.
In 1984, Trappe et al. wrote a paper which includes everything related to the
interaction between the pesticide and the mycorrhizae. This article discusses the
effect of pesticides by type of mycorrhiza (ectomycorrhiza, endomycorrhiza, and
ectendomicorriza), with in vitro and in vivo conditions, as the major groups of
pesticides (general pesticides, fungicides, herbicides and insecticides). In this
paper, the authors reviewed over 150 articles on every possible combination, of
which over 60 are related with EMF. The present work covers articles published
from 1984 to present. About 30 articles that have relation only with EMF/ectomy-
corrhizae and pesticides have been reviewed. It shows that the questions are still
unresolved, apart from studying the effects of new pesticides on ectomycorrhiza-
tion. It must be said that in recent years the study on the effect of pesticides has
focused on the endomycorrhizae for their application in agriculture.
14.2 Forest Tree Mycorrhization
The first experimental work in the field of physiology of ectomycorrhizae carried out
in the first half of the twentieth century, have already shown that mycorrhizal plants
grow better than non-mycorrhizal and also contained the highest amounts of major
nutrients per unit mass (Hatch 1937). Since then, Hatch was devoted to research of
mycorrhizae and many of his works haven tested in different scenarios to explain the
role of ectomycorrhizal symbiosis in the absorption of water and soil major nutrient
(phosphorus and nitrogen) (Tarkka et al. 2005), the role of these fungi in the vitamin
uptake of substances or plant growth regulators (Barker and Tagu 2000), relationships
with other microorganisms in the rhizosphere (Perry et al. 1987), their role in
biological control of certain root pathogens (Whipps 2004), and its role in the
whole forest ecosystem, where EMF are involved in the renovation of biomass and
nutrient cycling and in competitive relations or cooperation established between the
plants for water and nutrient uptake (Van der Heijden and Sanders 2002).
14.2.1 Controlled Mycorrhization
Controlled mycorrhization has been used in some countries with the aim of
introducing growth-promoting EMF into nurseries, and modifying seedling
14 Effects of Pesticides on the Growth of Ectomycorrhizal Fungi 325
production procedures respectively, in order to achieve improved growth responses

and rapid post-transplantation adaptation in the reforestation site (Marin 2009). In
North America, the research and practical applications have been established for
decades (Castellano and Molina 1989), while in Europe the procedure has been in
commercial use since the last decade of last century (Le Tacon et al. 1997).
The potential advantages of controlled mycorrhization in the nursery are not
only the positive growth response of the seedlings, but also improvement of nutrient
stress under deficiency conditions; EMF can increase nutrient uptake (Jones et al.
1991) and under toxic conditions as with heavy metals, mycorrhizal fungi pull
metals out of the soil and sequester them (Jones and Hutchinson 1986). EMF
stimulates root production and considerably increases the volume of soil the plant
can explore, and enhancement of rooting, and reduction of transplant shock (Teste
et al. 2004). The increased root volume is able to take up more water; the mycor-
rhizal fungi also enhance the host’s osmotic adjusting capabilities, allowing some
plants to continue to extract water from soils as they become drier (Garbaye 2000).
Mycorrhizae reduce directly and/or indirectly, antagonizing disease organisms,
increase the number of biocontrol agents around the roots, occupy potential infec-
tion sites on the root, and increase host plant vigor to the extent that it can survive
disease (Datnoff et al. 1995). Also, mycorrhizal fungi increase beneficial interac-
tions with other microbes (Garbaye 1994); it also increases phosphate uptake to the
plant by phosphate solubilizing bacteria.
Seedling performance in the field, after ectomycorrhizal inoculation, has been
variable in European field studies. In the best cases, 6 years after out planting, an
increase in tree volume of 90% has been observed, while in other conditions
inoculated seedlings performed worse in the field compared to control ones (Le
Tacon et al. 1992). Variable environmental growth conditions and differences in
local mycoflora obviously explain part of the variation.
It is important that the planting of seedlings that have been inoculated with
specific mycorrhizal strains, typically of exotic origin, into nature should not cause
any negative impacts on the local, indigenous microflora. Ecological knowledge on
the interactions of existing community and introduced strains should therefore be
improved to prevent the risk of adverse effects. Understanding the survival and
competitive abilities of selected strains under field conditions is of high priority in
the risk management of controlled mycorrhization. As already mentioned, and we
shall see, the use of pesticides in the nursery may play an important role in the
mycorrhization of seedlings with desired EMF, creating added value and better
marketing.
14.3 Pesticides
Since the 1960s, industrial chemistry has increased number of sophisticated che-
micals for agricultural pest control. At that time, the future of these chemicals
among chemists, agronomists, and resource managers seem to have been overly
326 M. Marin
optimistic about the promise of agricultural chemicals as simple solutions to

complex pest problems during the early part of the new era in agriculture. It is
now apparent that pesticides can have unexpected effects on nontarget organisms
and can thereby influence crop productivity as deeply or even more so than do the
pests they are intended to control (Trappe et al. 1984). Wilde (1958) was one of the
first to emphasize the deleterious effects of pesticides on the productivity of nursery
soils. Today, persistence of many pesticides in the soil, their interactions within the
soil, and the resulting effects on the growth and development of seedlings is of great
concern.
14.3.1 Classification of Pesticides
Pesticide, or biocide, is a common name for compounds, which are used for
protecting cultivated plants or for elimination of parasitic plants, fungi, insects, or
other pests. Plant growth regulators (i.e. phytohormones) are also classified as
pesticides. Pesticides are usually grouped according to their mode of action or the
purpose of their use (e.g., fungicides are used against fungal disease, herbicides
against weeds, and insecticides against insect pests). For instance, protectant is a
fungicide that will shield healthy tissue from fungal invasion, whereas an eradicant
will kill fungi that have already invaded the plant. Systematic pesticide spreads
within the plant, thus compound applied onto foliage (e.g., leaf-action herbicide)
will be transported to stem and root, or vice versa (e.g., soil applied herbicide).
Pesticide can be specific when it affects selectively, only one species or group, or
wide-spectral when it can affect a large number of species or groups of organisms.
Formulated pesticide products contain one or more active ingredients (abbr. a.i.)
and some other inactive ingredients like solvents, diluents, carrier substances, non-
forming, or acidity control chemicals, binding agents or pigments, which increase the
effectiveness of the active ingredient. Many of these inactive ingredients may also be
used as pesticide (Environmental Protection Agency of USA). Active ingredient can
be naturally occurring compounds, like pyrethrin and antibiotics, or synthetic inor-
ganic or organic compounds, or mixture of compounds or ligands. Some pesticides
will be converted to active form after application by soil microbes, e.g., sesone to
2,4-D herbicide (Metcalf 1971). Inorganic pesticides have typically simple chemical
structure like fungicides, e.g., copper oxychloride (Cu(OH)Cl) or so-called Bordeaux
mixture ([Cu(OH)2]·CaSO4). Majority of modern pesticides are, or have been various
kinds of organic compounds (MetCalf 1971). More than 1,100 common names for
pesticides have been assigned by International Organization of Standardization (ISO).
14.3.2 Effects of Pesticides on Soil Microorganisms
The repeated and long-term use of pesticides has raised the concern of accumula-
tion of pesticides in soils and the side effects of pesticides on soil microorganisms
and other biotypes (Smith et al. 2000). Simultaneous use of several pesticides may
lead to synergetic or potential, or antagonistic effects of pesticides on various
organisms. Pesticides may also have influence on the competitive position of soil
microbes in the community by destroying or preventing the growth of some
microbe groups, and thus enhancing the growth of other microbes (Sch€uepp and
Bodmer 1991).
Most of the information about side effects of the pesticide has been obtained
from the studies in agricultural fields, and only few studies have been performed
also in forest nurseries and sapling stands, some of which are presented below. In
general, tests with soils have shown that fungicides can have deleterious effects not
only on fungi, but also on bacteria, and also herbicides and insecticides can affect
bacteria and fungi. Commonly, this depression of microbial growth and activity has
been reversible after some duration (Tu 1978; Ingham and Coleman 1984; Colinas
et al. 1994a).
Tu (1993) studied the effects of two fungicides, captanol and chlorothalonil, and
eight herbicides, including linuron, on microbial and enzymatic activities in soils.
The pesticide application rate was 10 ppm in soil. Fungicides initially diminished
both bacterial and fungal populations, but the recovery was rapid. This caused
increase in oxygen consumption. Both fungicides suppressed invertase and amylase
activities for 1 day, and captanol dehydrogenase activity for 4 days, but all of these
were recovered equal to that of control. Some herbicides had effect on activities of
bacteria and fungi for a week, but populations returned to levels similar to controls.
After several herbicide treatments there was a slight depression in nitrification. In
experiments with 11 insecticides, some pesticides diminished the populations of
bacteria and fungi, but they were recovered after couple of weeks (Tu 1991).
Bacteria seemed to be more sensitive to various insecticides. On the contrary,
cypermethrin, chlordane, and chlorpyrifos stimulated fungal growth. Chakravarty
and Chatarpaul (1990a) found herbicides glyphosate and hexazinone to have a
significant short-time (2 months) decrease in soil microbial activity, which was
recovered in 6 months.
Chen and Edwards (2001) studied the effects of three broad-spectrum fungi-
cides, benomyl, captan, and chlorothalonil, on soil microbial activities and biomass.
Benomyl and chlorothalonil had relatively transient effects, while captan had a
greater and long-lasting overall influence on soil microbial activities. In a test with
forest soil (Colinas et al. 1994b), fungicide captan reduced 90% of active fungal
length compared to control, sampled during 5 days of incubation, although total
hyphal length was not affected. Besides that, captan reduced 50% of the number of
active bacteria and 30% of bacteria-feeding nematodes.
14.3.3 Pesticide Persistence in the Soil
Many pesticides are quite persistent in the environment and this is one explains, why
they can be detected in surface waters (Burgoa and Wauchope 1995), groundwater
328 M. Marin
(D€orfler et al. 1997), in soils and sediments (Rostad 1997), and in precipitation or
air samples (Dubus et al. 2000) for months or even years, after the last treatment.
Besides its functional toxicity, a pesticide can be toxic or has other impacts to
humans (Betarbet et al. 2000), animals (Elbetieha et al. 2001), or other organisms
(DeLorenzo et al. 2002). Agricultural and forest pesticides are applied in the fields
where they can also affect organisms other than the protected plants (McLaughlin
and Mineau 1995).
Persistence of a pesticide is derived from its bioaccumulation that is related to
degrability (half life time) and physicochemical properties (Gramatica and Di
Guarno 2002). Climate and weather are also important factors, which affect the
persistence of the pesticide and its degradation rates (Russell 1995). Soil structure
(density and porosity), composition (organic C content), chemistry (pH) and
microbiology, as well as the uptake of the pesticide by plants and soil animals
also have a remarkable influence on pesticide persistence in the environment
(Bergstr€om and Stenstr€ om 1998).
Some persistent pesticides may accumulate in the soil from repeated application.
Volatilization, leaching from the soil, and degradation by soil microorganisms may
account for loss of a major part of some pesticides that disappear rapidly. The
longer a pesticide persists in the soil, the greater the probability that several
processes will become involved in its inactivation and disappearance. Some organic
pesticides that persist for several months create a long period for the recolonization
of soil microorganisms. Pesticides, such methyl bromide, may disappear within 2–4
days. The herbicide Eptam (s-ethyl dipropylthiocarbamate) disappears in 3–10
weeks, while others such as the fungicide benzene hexachloride and the insecticide
chlordane may remain for a year or more. Many herbicides that persist from one
season to the next can injure sensitive plants. The triazine herbicides (atrazine and
simazine) applied as pre-emergent herbicides for selective weed control in corn,
sometimes persist and injure sensitive crops the next year. Additionally, they can be
spread as sprays, or volatilised and transported in air, or run off, or leached out
from the application area. Occasionally, amounts of pesticides in surface waters
(Gerecke et al. 2002) or in ground water (Gaus 2000) have been close to or exceed
the levels known to have toxic impact on the aquatic flora or fauna (Kreuger 1998),
or has been set for limits for drinking water.
Modern pesticides are usually more economic to use than the old ones, because
the dosage of specific pesticides is much smaller, even one-hundredth of that, which
was in old compounds (Chrispeels and Sadava 1994). However, this does not
necessarily reduce environmental and public hazards of pesticides. In general,
most modern pesticides are more than ten times as toxic to organisms than those
used in 1950 (Pimentel et al. 1998). Increasing concern of the harmful side effects
to non-target organisms, caused by more and more complex and environmentally
stable synthetic compounds used as pesticides, have put pressure on manufacturers
to develop pesticides, which are more specific, less toxic, and biologically degrad-
able in nature.
It has been estimated that the concentration of pesticides after one application
would leach into a soil top-layer of 5 cm depth in mineral and in organic soil. In
most cases, the pesticide concentrations exceed 1 ppm (Flykt et al. 2008). Pesticide
concentration may be even higher on the surface of plants roots, because the
pesticides are generally sprayed on seeds, or seedlings (fungicides and insecti-
cides), or on weeds (herbicides); seedlings may even be dipped into pesticide
solution (insecticide). Pesticide solution or residues can leach into the container
or soil either during the application or afterwards due to irrigation or rainwater.
As discussed above, the depths to which a pesticide can leach into the soil is
dependent on the organic matter content of soil and the physico-chemical proper-
ties of the pesticide. In peat-pots, pesticides can be bound to the peat medium and
only a small fraction may be detectable in leaching water. For example, in a trial
with peat-pots seedlings, less than 1% of applied chlorothalonil, but almost 30%
of applied propiconazole leached through the peat medium (Juntunen and Kitunen
2003). Soil microbial activity can release soil-bound pesticides back to undergo
environmental interactions (Levanon et al. 1994), and thus, the mycorrhiza and
mycelium, which are mainly present in this organic top-layer of the soil (Smith
and Read 1997) can be exposed to pesticides. On pines treated with triadimefon in
foliar sprays, Marx observed that this chemical was translocated downward from
the needles and also was leached by water into the root zone, so a considerable
amount of triadimefon is sprayed on the soil surface during its application on
small pine seedlings.
However, Sidhu and Chakravarty (1990) performed a comparison of the results
of mycorrhization of Pinus contorta var. latifolia and Picea glauca with Suillus
tomentosus in the laboratory, greenhouse, and field conditions with herbicides
hexazinone, glyphosate, and tryclopyr. The results showed that seedling growth
and ectomycorrhizal formation was affected significantly in the laboratory and
greenhouse, but not in field except at higher rates. The lower levels of the herbicides
available to the seedlings in field at the time of planting probably accounted for the
lower sensitivity of pine and spruce seedlings in the field.
14.4 Pesticides and EMF
As discussed at the beginning of this work, Trappe et al. (1984) conducted a first
review study of the effect of pesticides on mycorrhizal fungi. This actual work
covers articles published from 1984 to present, the last 25 years, and reviews about
30 articles that have a relation only between EMF and pesticides. Most of these
works were performed between 1980s and 1990s, but even today new studies are
conducted with the emergence of new pesticides (Laatikainen and Heinonen-
Tanski 2002) and the high diversity of EMF and pesticides (Hutchison 1990).
Tables 14.1–14.6 are a summary of articles concerning studies on the effect of
pesticides on the ectomycorrhizal mycelium and the formation of ectomycorrhizae
in forest plants. Research has been conducted both in vitro and in vivo concerning
fungicides, herbicides, and insecticides (and other pesticides).
330 M. Marin
Table 14.1 Fungicide effects on ectomycorrhizal fungi in vitro culture

Fungicide Concentrations Number of Effects on References
(mg/ml) species growtha
Benomyl 10–10,000 1 0 Chakravarty et al. (1990)
10 96 , 0 Hutchison (1990)
2–3 7 , 0 Niini and Raudaskoski (1993)
10 105 , 0 Dickinson and Hutchison (1997)
1–10 15 , 0, + Laatikainen and Heinonen-
Tanski (2002)
1–1,000 2 0 Dı́az et al. (2003)
Captan 1–1,000 2 0 Dı́az et al. (2003)
Carbendazim 10–100 2 Zambonelli and Iotti (2001)
Chlorothalonilb 1–10 15 Laatikainen and Heinonen-
Tanski (2002)
Copper 1–10 15 , 0 Laatikainen and Heinonen-
oxychloride Tanski (2002)
Cyproconazoleb 0.1–10 7 Desprez-Loustau et al. (1992)
Flusilazole 0.1–10 7 , 0 Desprez-Loustau et al. (1992)
Flutriafol 0.1–10 7 , 0 Desprez-Loustau et al. (1992)
Hymexazol 1–1,000 2 Dı́az et al. (2003)
Iprodione 1–1,000 2 Dı́az et al. (2003)
Mancozeb 5–15 2 Reddy and Natarajan (1995)
10–100 2 , 0 Zambonelli and Iotti (2001)
Maneb 1–10 15 , 0 Laatikainen and Heinonen-
Tanski (2002)
Oxine 10–10,000 1 0 Chakravarty et al. (1990)
benzoateb
Oxycarboxin 10–100 2 , 0 Zambonelli and Iotti (2001)
Propamocarb 1–1,000 2 0 Dı́az et al. (2003)
hydroc.
Propiconazole 10–100 2 Zambonelli and Iotti (2001)
0.1–10 15 , + Laatikainen and Heinonen-
Tanski (2002)
0.0015 1 Lekounougou et al. (2008)
Tebuconazole 0.1–10 7 Desprez-Loustau et al. (1992)
Tolclofos- 10–100 2 , 0 Zambonelli and Iotti (2001)
methyl
Thiram 1–1,000 2 0 Dı́az et al. (2003)
Triadimenol 0.1–10 2 Marx et al. (1986)
0.1–10 7 Desprez-Loustau et al. (1992)
Triadimefonb 0.1–10 2 Marx et al. (1986)
0.1–10 7 , 0 Desprez-Loustau et al. (1992)
a
Fungicide effects to low concentrations (<10 mg/ml). 0: no effect; +: growth increased; : growth
decreased
b
Chlorothalonil: not listed; Cyproconazole: expires 30/06/11; Oxine benzoate: not defended their
inclusion; Triadimefon: not defended their inclusion (Directive 91/414/CEE, October 2009). The
rest of pesticides are accepted
There have been many studies on the in vitro effects of pesticides on the EMF;
in vitro tests for mycelial growth can be used when estimating pesticide effects on
mycorrhiza, but for example, Unestam et al. (1989) did not recommend using them
when deciding pesticide usefulness in forest nurseries or plantations. In some
Table 14.2 Herbicide effects on ectomycorrhizal fungi in vitro culture

Herbicide Concentrations Number of Effects on References
(mg/ml) species growtha
2,4-D 1–10,000 3 , 0 Estok et al. (1989)
200–800 2 , 0 Donnelly et al. (1993)
Atrazineb 200–800 2 0 Donnelly et al. (1993)
Chlorthiamidb 1 15 0 Laatikainen and Heinonen-Tanski
(2002)
Glyphosate 0.1–1,000 5 , 0 Chakravarty and Sidhu (1987)
0.1–1,000 5 , 0 Chakravarty and Chatarpaul
(1990a)
1–10,000 3 , 0 Estok et al. (1989)
1 15 , 0, + Laatikainen and Heinonen-Tanski
(2002)
1–1,000 2 0 Dı́az et al. (2003)
Hexazinone 0.1–1,000 5 , 0 Chakravarty and Sidhu (1987)
0.1–1,000 5 , 0 Chakravarty and Chatarpaul (1989)
1–10,000 3 , 0, + Estok et al. (1989)
1 15 0, + Laatikainen and Heinonen-Tanski
(2002)
1–1,000 2 0 Dı́az et al. (2003)
Linuron 1 15 , 0 Laatikainen and Heinonen-Tanski
(2002)
Simazine b 1–1,000 2 0 Dı́az et al. (2003)
Terbuthylazineb 1 15 , 0, + Laatikainen and Heinonen-Tanski
(2002)
Triclopyr 0.1–1,000 5 Chakravarty and Sidhu (1987)
1–10,000 3 , + Estok et al. (1989)
a
Herbicide effects to low concentrations (<10 mg/ml). 0: no effect; +: growth increased; : growth
decreased
b
Chlorthiamid: not listed; Atrazine: not defended their inclusion; Simazine: excluded; Terbuthy-
lazine: expires 30/06/11 (Directive 91/414/CEE, October 2009). The rest of pesticides are
accepted
Table 14.3 Insecticides effects on ectomycorrhizal fungi in vitro culture

Insecticide Concentrations Number Effects References
(mg/ml) of species on growtha
Cypermethrin 1–10 15 , 0 Laatikainen and Heinonen-
Tanski (2002)
Fenvalerate 0.25–1.5 2 Reddy and Natarajan (1994)
Nocodazoleb 2 7 Niini and Raudaskoski (1993)
a
Insecticide effects to low concentrations (<10 mg/ml). 0: no effect; þ: growth increased;
: growth decreased
b
Nocodazole: not listed (Directive 91/414/CEE, October 2009). The rest of pesticides are accepted
studies, pesticide toxicity to EMF in vitro has been confirmed with experiment
in vivo (Marx et al. 1986; Chakravarty and Sidhu 1987; Chakravarty and Chatarpaul
1988). However, the influence of pesticide on the growth of EMF can be different
when pure culture mycelium in vitro is tested compared to tests by using inoculated
332 M. Marin
Table 14.4 Fungicide effects on ectomycorrhizal formation

Fungicide Application Host Effects on References
speciesa mycorrhizaeb
Benomyl Commercial labels Pire 0, + Chakravarty et al. (1990)
1–100 ppm Pist + De la Bastide and Kendrick (1990)
500 ppm/pot Thpl Cade-Menun and Berch (1997)
20–150 kg/ha Piel 0 Pedersen and Sylvia (1997)
Not known Pisi O’Neill and Mitchell (2000)
Commercial labels Piab Flykt et al. (2008)
Captan 25 ppm dry soil Psme 0, + Colinas et al. (1994b)
Not known Pisi + O’Neill and Mitchell (2000)
Chlorothalonil 2,000 g ai/ha Pisy 0 Laatikainen et al. (2000)
Not known Pisy 0 Aleksandrowicz-Trzcinska (2007)
Copper oxychloride 588 ppm Pisy 0 Manninen et al. (1998)
Ferbam Unspecific Piel, Pita 0 Marx et al. (1986)
Fenhexamid Commercial labels Piab Flykt et al. (2008)
Iprodione 1 g/Kg Quro Garbaye et al. (1992)
Mancozeb 800–2,400 ppm Pipa Reddy and Natarajan (1995)
Oxine benzoate Commercial labels Pire Chakravarty et al. (1990)
Oxycarboxinc 100 mg ai/l sprayed Qupu , 0 Zambonelli and Iotti (2001)
Propiconazole 250 ppm Pisy Manninen et al. (1998)
125 g ai/ha Pisy 0 Laatikainen et al. (2000)
125–375 ppm Psme Teste et al. (2006)
25 mg ai/m2 Bepe Nerg et al. (2008)
Pyrimethanil Commercial labels Piab Flykt et al. (2008)
spi + teb + trid Not known Pisy 0 Aleksandrowicz-Trzcinska (2007)
Tolylfluanid Commercial labels Piab Flykt et al. (2008)
Thiophanate- 350–1,050 ppm Psme Teste et al. (2006)
methyl Commercial labels Piab Flykt et al. (2008)
Triadimefon 0.56 Kg ai/ha Piel, Pita Marx et al. (1986)
6, 12 and 24 oz/ac Pita , 0 Kelley (1987)
Not known Pipo Page-Dumroese et al. (1996)
a
Betua pendula: Bepe; Picea abies: Piab; Picea sitchensis: Pisi; Pinus elliotti: Piel; Pinus patula:
Pipa; Pinus ponderosa: Pipo; Pinus resinosa: Pire; Pinus strobus: Pist; Pinus sylvestris: Pisy;
Pinus tadea: Pita; Pseudotsuga menziesii: Psme; Quercus pubescens: Qucu; Quercus robur: Quro;
Thuja plicata: Thpl
b
0: no effect; þ: mycorrhiza formation increased; : mycorrhiza formation decreased
c
Oxycarboxin: not defended their inclusion (Directive 91/414/CEE, October 2009). The rest of
pesticides are accepted
d
Spiroxamine þ tebuconazole þ triadimenol
seedlings. Marx and Rowan (1981) studied the influence of four fungicides on two
EMF, Pisolithus tinctorius and Thelephora terrestris, by infesting the nursery
soil with these fungi and growing Pinus tadea seedlings in this soil. Ectomycor-
rhizal development of P. tinctorius was depressed by benomyl and captan, but
enhanced by benodanil, whereas ectomycorrhizal development of T. terrestris was
the greatest in plots with benomyl and captan applications. In the laboratory test,
benomyl had slight inhibitory and captan no effect on the mycelial growth of both
P. tinctorius and T. terrestris.
Table 14.5 Herbicides effects on ectomycorrhizal formation

Herbicide Application Host speciesa Effects on References
mycorrhizaeb
Clopyralid Commercial Piab Flykt et al. (2008)
labels
Glyphosate 0.1–100 ppm Pico, Pigl , 0 Sidhu and Chakravarty
(1990)
Hexazinone 1.0–4.0 kg ai/ha Pico, Pigl , 0 Sidhu and Chakravarty
drench (1990)
Imazapyr 1.1–2.2 kg ai/ha Abco, Pipo, Psme 0 Busse et al. (2004)
Phosphinothricinc Not known Pira 0 Gonzalez-Moro et al.
(2000)
Sulfometuron 0.14–0.28 kg Abco, Pipo, Psme 0 Busse et al. (2004)
methylc ai/ha
Triazinec Commercial Piab Flykt et al. (2008)
labels
Triclopyr 0.1–100 ppm Pico, Pigl , 0 Sidhu and Chakravarty
(1990)
4.5–9.0 kg ai/ha Abco, Pipo, Psme 0 Busse et al. (2004)
a
Abies concolor: Abco; Picea abies: Piab; Picea glauca: Pigl; Pinus contorta: Pico; Pinus
ponderosa: Pipo; Pinus radiata: Pira; Pseudotsuga menziesii: Psme
b
0: no effect; +: mycorrhiza formation increased; : mycorrhiza formation decreased
c
Phosphinothricin: not listed; Sulfometuron methyl: not listed; Triazine: not listed (Directive
91/414/CEE, October 2009). The rest of pesticides are accepted
Table 14.6 Insecticide and others chemicals (O) effects on ectomycorrhizal formation
Chemical Application Host Effects on Reference
speciesa mycorrhizaeb
Chloropicrin (O)c 0.66–3.3 ml/kg Psme 0 Massicote et al. (1998)
stump
Deltamethrin Commercial labels Piab 0 Flykt et al. (2008)
Dimethoate 200 ppm dry soil Psme , 0 Colinas et al. (1994b)
Commercial labels Piab 0 Flykt et al. (2008)
Fenvalerate Commercial labels Pipa Reddy and Natarajan
(1994)
Fumagillin (O)c 10 ppm dry soil Psme 0, + Colinas et al. (1994b)
Oxydemeton-methylc Commercial labels Piab 0 Flykt et al. (2008)
Quinoclamine (O) Commercial labels Piab 0 Flykt et al. (2008)
Permethrin Commercial labels Piab 0 Flykt et al. (2008)
Vapam (O) Not known Pipo Page-Dumroese et al.
(1996)
a
Picea abies: Piab; Pinus patula: Pipa; Pinus ponderosa: Pipo; Pseudotsuga menziesii: Psme
b
0: no effect; +: mycorrhiza formation increased; : mycorrhiza formation decreased
c
Chloropicrin: expires 30/06/11; Fumagillin: not listed; Oxydemeton-methyl: excluded (Directive
91/414/CEE, October 2009). The rest of pesticides are accepted
Pesticides may influence the growth of mycorrhizal plants by affecting the

nutrient uptake and allocation by suppressing the mycorrhizal symbiosis (Schweiger
and Jakobsen 1998). The detrimental effect on ectomycorrhiza can be seen as reduced
growth in seedlings (Reddy and Natarajan 1995).
334 M. Marin
14.4.1 Fungicides
Generally, the fungicides proved to be toxic to EMF, presumably due to their

general mode of actions, and such fungicides can inhibit fungal cell division
(benomyl), impair ergosterol biosynthesis (propiconazole), inactivate fungal cell
thiols (chlorothalonil), cause protein damage (copper oxychloride), or bind to cell
copper compounds (maneb). Thus, chlorothalonil is viewed as an effective fungi-
cide against a broad spectrum of plant pathogens. Propiconazole, which is a
systemic fungicide, belonging to the group called sterol synthesis inhibitors also
has a broad range of activity.
Fungicides can be useful in studies of controlled mycorrhization with different
inoculated EMF because not all fungi are equally sensitive to fungicides: zygomy-
cetes are particularly susceptible to most of benzimidazole fungicides (Trappe et al.
1984). Benomyl is generally known to inhibit the growth of ascomycetes fungi like
Cenoccocum geophilum, Tuber spp., and ectendomycorrhizae, and ascomycetes
tend to be more sensitive in low concentrations than basidiomycetes (Niini and
Raudaskoski 1993). Similarly, in axenic culture, some EMF respond differently to
fungicides than others (Hutchison 1990); the differential sensitivity of EMF to
fungicides can be a valuable tool in controlling mycorrhizal formation in the
nursery and in the field (Marx and Rowan 1981; Trappe et al. 1984). This genetical
distance from the other EMF could account for its different response from all other
identified fungi when tested with the pesticides. It has been observed that in field
conditions, benomyl increases the development of ectomycorrhizae that are nurs-
ery-grown (Trappe et al. 1984; Chakravarty et al. 1990; De la Bastide and Kendrick
1990) and associated to decrease the rhizosphere competition. However, O’Neill
and Mitchell (2000) observed that benomyl has deleterious effect on root length and
mycorrhizal colonization of Picea sitchensis.
Edible EMF is cultivated by establishing plantations with inoculated plants
(Hall and Wang 1998). A major problem during the cultivation process is the
replacement of the inoculated fungus by more aggressive competing fungi. Selec-
tive fungicides might therefore be used to control competitive EMF during the
production of edible EMF, Tuber spp. infected plants, and later in plantations of
mycorrhizated trees. For example, Tuber melanosporum can be replaced by Sclero-
derma spp. (Hall and Wang 1998), T. brumale (Chevalier et al. 1982; Etayo and
De Miguel 1998), or Hebeloma spp. (Granetti and Angelini 1992). Similar pro-
blems have been detected during the cultivation of T. magnatum and T. uncinatum
(Capecchi et al. 1999).
There is every reason to suspect that the same problems will occur during the
cultivation of other EMF such as Tuber borchii, which was cultivated by Zambonelli
et al. (2000). In greenhouse experiments, Hebeloma sinapizans actively competed
with T. borchii not only during the initial phases of ectomycorrhizal formation,
but also when H. sinapizans was introduced after infection had been established
(Zambonelli and Govi 1991). Zambonelli and Iotti (2001) demonstrated that fungi-
cides such as oxycarboxin, which have relatively little effect on T. borchii, can be
used to selectively suppress ectomycorrhizal development of contaminant basidio-

mycetes such as H. sinapizans. This fungicide is commonly used to control plant
pathogenic basidiomycetes. These results support and extend Unestam’s findings
that oxycarboxin also suppresses the development of ectomycorrhizal basidio-
mycetes (Unestam et al. 1989). This opens up the possibility of using selective
fungicides, such as oxycarboxin, during the commercial production of plants
infected with T. borchii and other species of Tuber. Although it is difficult to predict
the field competition of greenhouse contaminant fungi, many authors (see Govi et al.
1997) agree that their presence should be avoided in Tuber infected plants.
In a field experiment, propiconazole and copper oxychloride reduced ectomy-
corrhizal growth of Scots pine seedlings growing in sand-filled-pots (Manninen
et al. 1998). In the field experiment with Loblolly and Slash pine seedlings, the
ectomycorrhizal development with P. tinctorius and naturally occurring fungi were
both suppressed by triadimefon treatments, which confirmed the results from
in vitro tests. Reddy and Natarajan (1995) examined the effect of fungicide man-
cozeb on Laccaria laccata and Thelephora terrestris both in vitro and inoculated
with Pinus patula seedlings. Fungicide inhibited the growth of mycelia of both
fungi, as well as, mycorrhization of seedlings roots was clearly reduced. Coloniza-
tion with L. laccata was totally inhibited and colonization with T. terrestris reduced
more than 50% at the recommended fungicide dose. In a greenhouse experiment
with container-grown Pinus palustris seedlings, benomyl stimulated the ectomy-
corrhizal development with tested fungi, P. tinctorius and T. terrestris (Pawuk et al.
1980). Theodorou and Skinner (1976) found seed dressing with fungicides captan,
zineb, and thiram to inhibit mycorrhizal development on Pinus radiata seedlings
with various inoculated mycorrhizal fungi, while natural mycorrhization in soil
were not affected. However, studies in Spain with Pinus halepensis (Aleppo pine)
and the use of fungicides (benomyl, captan, and thiram) in the nursery, necessary
for its cultivation , given its sensitivity to fungal pathogens at this stage of
development does not adversely affect the process of controlled mycorrhization
(Carrillo 2000).
14.4.2 Herbicides
Herbicides such as glyphosate and hexazinone have been tested with various
EMF in pure culture tests, e.g., Hebeloma crustuliforme, L. laccata, and S. tomen-
tosus, and they inhibited all of the tested fungi at concentrations above 10 ppm
(Chakravarty and Sidhu 1987), and in a second experiment they had inhibitory
effects on C. geophilum, Hebeloma longicaudum and P. tintorius at concentrations
below 100 ppm (Estok et al. 1989), though such high concentrations may not be
particularly relevant to the situation in forest nurseries.
The growth stimulation of some EMF strains was caused mainly by herbicides
glyphosate, terbuthylazine, and hexazinone (Suillus species), but in a few rare
336 M. Marin
cases also by the insecticides, cypermethrin, and all of the fungicides, except
chlorothalonil. The growth stimulation of EMF might indicate that these fungi are
able to degrade pesticides, but this stimulation noted in the laboratory may be less
likely to occur in field conditions if there is a lack of nutrients such as potassium or
phosphorus in the soil (DaSilva et al. 1977). In some cases pesticide molecule is not
mineralized, but can become incorporated into the fungal tissue (Donnelly et al.
1993). Ectomycorrhizae having the potential to degrade and mineralize pesticides
and other persistent organic pollutants could also be used in bioremediation
(Meharg et al. 1997a). For example, Amanita species, P. involutus, and Suillus
species are known to degrade several organic pollutants (Meharg and Cairney
2000), and C. geophilim can immobilize, for example, hexazinone (Donnelly and
Fletcher 1994). The growth of EMF in symbiosis with Pinus sylvestris has stimu-
lated even greater pollutant mineralization than in pure cultures (Meharg et al.
1997b). The ability to degrade some aromatic herbicides appears to be dependent
on the specific EMF and the herbicide. When the EMF is growing with the host
plant, it has an ample supply of carbohydrates provided by the host plant for its
growth. It is know that extracellular enzymatic activity of the fungus dramatically
increases when the EMF is growing with the host plant vs. growing in pure culture
(Donnelly et al. 1993).
Estok et al. (1989) have studied the effects of herbicides 2,4-D, glyphosate,
hexazinone, and triclopyr on the growth of C. geophilum, Pisolithus tinctorius and
Hebeloma longicaudum. C. geophilum was the least sensitive to tested herbicides
but the growth of other two tested fungi was slightly reduced already at the
concentration of 1 ppm. Generally, the low concentrations of herbicides could
promote the growth, whereas concentrations of 10–1,000 ppm strongly inhibited
the growth of all five EMF. The order of decreasing sensitivity of fungi to tested
herbicides was Suillus tomentosus, Thelephora americana, Hebeloma crustulini-
forme, Laccaria laccata, and finally, Thelephora terrestris. Later, hexazinone at the
recommended field rates of applications (4–18 ppm) was found to reduce the
mycorrhizal development of inoculated L. laccata with Pinus resinosa, as well
as inhibit naturally occurring mycorrhizal fungi in the field (Chakravarty and
Chatarpaul 1988).
During the other experiment, Chakravarty and Chatarpaul (1990a) examined
in vitro the effects of glyphosate and hexazinone on the growth of five EMF,
Cenococcum graniforme, Hebeloma crustuliniforme, Laccaria laccata, Suillus
tormentosus, and Paxillus involutus, and all were significantly reduced at the
concentrations of 50 ppm. Glyphosate was also tested in the field experiment with
Pinus resinosa seedlings inoculated with Paxillus involutus (Chakravarty and
Chatarpaul 1990b). Neither seedling growth nor ectomycorrhizal development
was affected by glyphosate treatment. In the field experiment, twofold higher
recommended dose of copper oxychloride and propiconazole reduced ectomycor-
rhizal development and growth of Scot pine (Pinus sylvestris L.) seedlings
(Manninen et al. 1998), and propiconazole, as in the case of some fungicides,
selectively killed ascomycete symbionts, while basidiomycete symbionts were
less affected.
14.4.3 Insecticides and Other Pesticides
There were not very many studies made with insecticides and EMF. The cases
studied show the inhibitory effect of insecticides on mycelial growth at low con-
centrations, contradicting what is observed by Trappe et al (1984). This fact is
consistent with the observation mentioned before, in that new pesticides can affect
EMF despite the use of small doses of the product.
As in the case of fungicides, insecticides and other pesticides listed in Table 14.6,
when applied in forest plant nursery on mycorrhized plants, their effect on mycor-
rhizal colonization is not significant or very low.
14.5 Pesticides and Lactarius spp.
Here, a special case of in vitro effect of some fungicides and herbicides on various
species of Lactarius and strains of Lactarius deliciosus is shown. The basidiomy-
cete L. deliciosus is an ectomycorrhizal fungus principally on Pinus spp. roots in the
Mediterranean forests (Sánchez et al. 1994). This species is socioeconomically
important in Spain and others countries of the world, because it is a popular edible
wild mushroom that gives a new value to our forest ecosystems (Singer 1986).
L. deliciosus has been used in mycorrhization of pine seedlings in nursery (Guerin-
Laguette et al. 2000).
Tables 14.7 and 14.8 shows the effect of some pesticides on mycelial growth on
species of the genus Lactarius at different concentrations. At the same time, the
fungistatic effect of each of the pesticide on the fungus was observed by transfer-
ring the piece of agar with mycelium (not grown on culture medium with pesticide)
to a new culture medium without pesticide, and noting the presence or absence of
mycelial growth, thus showing that the fungus was not dead. The effects of each
pesticide on the growth of different Lactarius spp. in axenic culture were similar,
but differences between the species studied and even among isolates of L. deliciosus
were also observed.
14.5.1 Fungicides
Benomyl, a common systemic fungicide in agricultural practices (Torstensson and

Wessén 1984) has been extensively studied in axenic culture on several EMF (see
Trappe et al. 1984, and Table 14.1). Lactarius isolates had a strong inhibition of
growth to low concentrations of fungicide (Table 14.7). Hutchison (1990) found
between the Lactarii, a similar sensitive response to benomyl. In addition, this
author found that species of Lactarius of section Dapetes (Singer 1986) where
carpophore exudes bright-colored latex when cut, exhibited tolerance to benomyl.
Certainly, more resistance is found in some isolates of L. deliciosus and the isolate
338 M. Marin
Table 14.7 Fungicide effects on mycelial diameter growth (d) of Lactarius isolates on fungicide-
amended BAF for 6 weeks
Ld Ld Ld Ls Lh Lc
CBS 334.65 DAOM 197163 UAMH 5547 CBS 409.75 Baar CECT 20239
BEN (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 38.2 18.0 23.0** 15.8 23.8 10.3
10 (33.3)a 18.3 ng 6.2* (100) (100)
50 ng (100) ng (100) (66.7) (100)
100 ng (100) ng (100) (33.3) (100)
500 ng (100) ng (100) ng ng
1,000 ng ng ng ng ng ng
FAL (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 38.7 22.7 15.0 25.3** 15.3 11.0
10 41.3** 18.2 24.7 17.0 10.0* 1.5*
50 21.8 18.2 13.5 17.5 13.0* 2.5*
100 30.8 17.8 (66.7) 15.7 8.8* (100)
500 (33.3) 6.7* ng 7.7* (100) (100)
1,000 ng (66.7) ng (100) ng ng
PRO (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 35.0 23.5 20.0 17.8 17.3 11.3
10 30.5 23.8 24.2 17.0 33.2** 15.2
50 37.8 27.3 26.5 19.2 33.5** 11.5
100 33.0 32.3** 29.0 19.0 20.8 10.0
500 21.2* 28.8 23.3 15.2 25.5 6.8*
1,000 ng 27.8 23.5 13.5 16.0 3.2*
THI (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 40.0** 23.3 18.8 18.3** 23.8 10.5
10 ng 10.3* ng 7.2* ng ng
50 ng (100) ng (66.7) ng ng
100 ng (33.3) ng (33.3) ng ng
500 ng ng ng ng ng ng
Significant differences compared to control (0 mg/ml fungicide-medium) are indicated with “*” for
low values and “**” for high values (P < 0.05 Tukey or KW)
CBS Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; CECT Spanish Collection
of Type Cultures, València, Spain; DAOM Canadian Collection of Fungus Cultures, Ottawa,
Canada; UAMH University of Alberta Microfungus Collection, Alberta, Canada; Baar provide
by J. Baar, originating from a nitrogen-enriched Scots pine
ng not growing
BEN benomyl; FAL fosetyl-aluminum; PRO procymidone; THI thiram
a
Percentage of samples that recovered growth after reharvesting in new media BAF without biocides
of L. sanguifluus, but this characteristic is not general in the section Dapetes of

Lactarii (Hutchison 1990).
Fosetyl-Al is a systemic fungicide active against oomycetes. Jalai-Hare and
Kendrick (1987) found that fosetyl-Al stimulated the growth of Glomus intrara-
dices in allium roots. This fungicide had only fungitoxic effect on Lactarius spp. at
Table 14.8 Herbicide effects on mycelial diameter growth (d) of Lactarius isolates on herbicide-
amended BAF for 6 weeks
Ld Ld Ld Ls Lh Lc
CBS 334.65 DAOM 197163 UAMH 5547 CBS 409.75 Baar CECT 20239
GLU (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 35.8** 16.0* 12.8* 18.0** 25.8 5.8*
10 ng ng 7.8* (100) 6.8* 3.0*
50 ng ng (100)a (100) (66.7) (100)
100 ng ng ng ng ng (100)
GLY (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 39.3 18.5 16.3* 18.2 24.3 16.7
10 38.2 25.5 17.0* 5.8* 8.8* 9.2
50 40.2 20.5 13.0* 4.5* 9.3* 10.5
100 41.0 20.8 10.5* 5.3* 7.3* 9.0
500 15.0 4.5* (100) ng (100) 5.5*
1,000 ng ng (100) ng (100) 2.0*
MCPA (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 37.7 16.8* 19.3 16.2 17.0 11.2
10 36.3 17.3 18.7 17.3 11.3* 10.3
50 35.0 20.5 10.5* 16.5 4.3* 8.3*
100 27.2 13.5* 10.5* 10.5 ng 8.0*
OXY (mg/ml) d (mm)
0 28.5 22.0 19.8 15.3 21.7 17.3
1 28.2 12.8* 19.3 20.7** 23.3 9.0
10 24.8 16.2* (100) 8.2* (100) 5.0*
50 4.5* ng (100) (66.7) (100) ng
100 (100) ng ng ng ng ng
Significant differences compared to control (0 mg/ml herbicide-medium) are indicated with “*” for
low values and “**” for high values (P < 0.05 Tukey or KW)
CBS Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; CECT Spanish Collection
of Type Cultures, València, Spain; DAOM Canadian Collection of Fungus Cultures, Ottawa,
Canada; UAMH University of Alberta Microfungus Collection, Alberta, Canada; Baar provide
by J. Baar, originating from a nitrogen-enriched Scots pine
GLU glufosinate; GLY glyfosate; MCPA MCPA; OXY oxyfluorfen
ng not growing
a
Percentage of samples that recovered growth after reharvested in new media BAF without biocides
high concentrations of the product. The fungicide effects of fosetyl-Al have not
been shown previously in EMF.
Procymidone is an example of pesticide that can be utilized as energy sources for
some fungi (Altman 1969). Four of the six strains tested had not accepted at
concentrations of 1,000 ppm. Procymidone is a systemic fungicide used on lupins,
340 M. Marin
grapes, stone fruit, strawberries, and some vegetables. It is widely used in horticul-
ture, either as a seed dressing, pre-harvest spray, or post-harvest dip. As well,
procymidone has not been shown previously in direct fungitoxicity on EMF.
Thiram is a preventive fungicide of large spectrum, and similarly in the studied
strains of Lactarius. A high fungitoxicity is observed to low concentrations in EMF
in axenic culture (Trappe et al. 1984; Dı́az et al. 2003). In addition, negative effects
have been found in mycorrhiza formation (Trappe et al. 1984).
14.5.2 Herbicides
Glufosinate (Table 14.8) is a non-selective herbicide that inhibits the glutamine

synthetase activity, produce accumulation of ammonia, and inhibit formation of
glutamine amino acid (Montanini et al. 2003). Lactarius isolates are affected
strongly by this herbicide. However, glufosinate has low residual effect after
reharvesting in control media, except over some L. deliciosus. Direct fungitoxicity
on EMF has not been shown previously.
Glyphosate is an herbicide that is very commonly used in forest and agricultural
activity (Sidhu and Chakravarty 1990). Table 14.2 shows that the effect in axenic
culture at low concentrations of this herbicide on mycelial growth of EMF was
different according to several studies. Dı́az et al (2003) observed in L. deliciosus not
effect, perhaps, stimulating mycelial growth. In this experience, inhibition or
reduction of mycelial growth in medium or high concentrations was found.
MCPA is a phenoxyacetic herbicide, as 2,4-D, aromatic herbicides. MCPA is
similar to 2,4-D, except in-group chloride on position 2. Donnelly et al. (1993)
found that 2,4-D was degraded by incorporation of herbicide carbon into tissue.
Donnelly and Fletcher (1995) found that the degradation capacity of these herbi-
cides depends of the number and the position of chloride groups in the aromatic
molecule. Dasilva et al. (1977) found strong stimulation of growth to low con-
centrations of MCPA on two Boletus spp., while with 2,4-D, the stimulation was
lower. Similarly, the growth of Lactarius isolates was also stimulated. In principle,
it would be prudent to replace 2,4-D by MCPA in agriculture and forestry practices
when the plant is to be mycorrhized with EMF.
Oxyfluorfen produced the rupture of cellular membrane in presence of light as
all the diphenyl ethers (Choi et al. 1999). This group of herbicides produced in EMF
a strong inhibition of growth in axenic conditions (Kelley and South 1980) like we
have observed in Lactarius spp.
14.6 Conclusions
In recent years, some interest among scientists have been lost in studying the effects
of pesticides on the EMF, and consequently on the formation of ectomycorrhizae in
forest plants. Recent studies have developed in the production of mycorrhized
forest plant, especially with container seedlings. However, most of the forest
productions still need the use of pesticide. However, at present, the use of pesticides
is being reduced in general agricultural practices and particularly in forest produc-
tions, but this will be still difficult to occur in all situations and nurseries. The study
of Trappe et al. (1984) showed the irrationality of the research on the effect of
pesticides on mycorrhiza, and that these would increase as new substances are used
as pesticides. This situation continued to occur over recent years, and the variety
and diversity of studies makes it difficult to rationalize the observations found.
Based on the variety of forest plants, soils, and climates under pesticides are used, it
is difficult to propose one type of pesticide for each of the multiple combinations
EMF/plant.
There are a number of parameters that are observed among all studies, and more
important as expected is that fungicides have a greater effect on EMF than other
pesticides. Knowledge on the capabilities of EMF to tolerance of pesticides might
be useful in deciding which pesticides would be less harmful to mycorrhizal fungi
when used in forest nurseries and in afforested fields. Pesticides may affect directly
the seedling roots, and thus pure culture tests alone are not recommended when
estimating the effects of pesticides on mycorrhizae and deciding the usefulness of
different pesticides in the nursery or new plantations.
It should be noted that production of container seedlings is more similar to
horticultural production than to agricultural production. Seedlings are started in
greenhouses, and depending on seedling type, they are raised in greenhouses from
2 to 6 months with peat as the growing media. When container seedlings are
sprayed with pesticides, most of the pesticides are sprayed on very densely growing
seedlings. In production of container seedlings the pesticides leaching to the ground
include both pesticides leaching from the growth medium and pesticides applied
directly to aisles and other empty space around the container blocks. For production
of bareroot seedlings, the situation is similar to that on agricultural fields. On
bareroot fields, seedlings grow at much lower density than in containers, and
there are empty areas between seedling rows and beds. Much of the pesticide
suspension is applied directly on the ground than in container production.
A better integration of systems of mycorrhized plant production and use of
pesticides can be carried out by a better development of Integrated Pest Manage-
ment (IPM), a system that combines cultural, biological, and chemical technologies
to reduce insect, fungal, and weed populations. Finally, pesticides cause many
complex reactions of all organisms in the forest plant production, and generaliza-
tion should be made with caution. General biocides, fungicides, herbicides, and
insecticides cause complex reactions to be studied in each case on a small scale.
References
Aleksandrowicz-Trzcinska M (2007) The effect of fungicides used in the protection of natural

regenerations of pine against Lophodermium needle cast on mycorrhizae and seedling growth.
Sylwan 151:27–34
Altman J (1969) Predisposition of sugarbeets to Rhizoctonia damping-off with herbicides. Phyto-
phathology 59:1015
342 M. Marin
Barker SJ, Tagu D (2000) The role of auxins and cytokinines in mycorrhizal symbioses. J Plant
Growth Regul 19:144–154
Bergstr€om L, Stenstr€om J (1998) Environmental fate of chemicals in soil. Ambio 27:16–23
Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT (2000)
Chronic systematic pesticide exposure reproduces features of Parkinson’s disease. Nat Neu-
rosci 3:1301–1306
Burgoa B, Wauchope RD (1995) Pesticides in run-off and surface waters. In: Roberts TR, Kearney
PC (eds) Environmental behaviour of agrochemicals. Wiley, Chichester, pp 131–184
Busse MD, Fiddler GO, Ratcliff AW (2004) Ectomycorrhizal formation in herbicide-treated soils
of differing clay and organic matter content. Water Air Soil Pollut 152:23–34
Cade-Menun BJ, Berch SM (1997) Response of mycorrhizal western red cedar to organic
phosphorus sources and benomyl. Can J Bot 75:1226–1235
Capecchi M, Vecchiati P, Zambonelli A (1999) Dinamica della micorrizazione in due tartufaie
sperimentali di Tuber magnatum Pico in provincia di Modena. Micol Ital 17:9–18
Carrillo C (2000) Producción de inóculo de hongos ectomicorrı́cicos y micorrización controlada
de Pinus halepensis Miller en vivero. (Production of ectomycorrhizal fungal inoculum and
controlled mycorrhization of Pinus halepensis Miller in nursery). Ph.D. thesis, University of
Murcia, Spain, 241 pp
Castellano MA, Molina R (1989) Mycorrhizae. In: Landis TD, Tinus RW, McDonald SE, Barnett
JP (eds) The container tree nursery manual, vol 5, Agricultural handbook 674. Department of
Agriculture, Forest Service, Washington, DC, pp 101–167
Chakravarty P, Chatarpaul L (1988) The effects of Velpar L. (hexazinone) on seedling growth and
ectomycorrhizal symbiosis of Pinus resinosa. Can J For Res 18:917–921
Chakravarty P, Chatarpaul L (1990a) Non-target effect of herbicides: I. Effect of glyphosate and
hexazinone on soil microbial activity. Microbial population, and in vitro growth of ectomycor-
rhizal fungi. Pestic Sci 28:233–241
Chakravarty P, Chatarpaul L (1990b) Non-target effect of herbicides: II. The influence of glypho-
sate on ectomycorrhizal symbiosis of red pine (Pinus resinosa) under greenhouse and field
conditions. Pestic Sci 28:243–247
Chakravarty P, Sidhu SS (1987) Effect of glyphosate, hexazinone and triclopyr on in vitro growth
of five species of ectomycorrhizal fungi. Eur J For Pathol 17:204–210
Chakravarty P, Peterson RL, Ellis BE (1990) Integrated control of Fusarium damping-off in red
pine seedlings with the ectomycorrhizal fungus Paxillus involutus and fungicides. Can J For
Res 20:1283–1288
Chen SK, Edwards CA (2001) A microcosm approach to assess the effects of fungicides on soil
ecological processed and plant growth: comparisons of two soil types. Soil Biol Biochem
33:1981–1991
Chevalier G, Giraud M, Bardet MC (1982) Interactions entre les mycorhizes de Tuber melanos-
porum et celles d’autres champignons ectomycorhiziens en sols favorables à la truffe. In:
INRA (ed) Les Mycorhizes: biologie et utilisation, vol 13. Les Colloques de l’INRA, Paris, pp
313–321
Choi JS, Hee HJ, Hwang IT, Pyon JY, Cho KY (1999) Differential susceptibilities of wheat and
barley to diphenyl ether herbicide oxyfluorfen. Pestic Biochem Phys 65:62–67
Chrispeels MJ, Sadava DE (1994) Plants, genes and agriculture. Jones and Bartlett, Boston, p 478
Colinas C, Ingham E, Molina R (1994a) Population responses of target and non-target forest soil
organisms to selective biocides. Soil Biol Biochem 26:41–47
Colinas C, Molina R, Trappe J, Perry D (1994b) Ectomycorrhizas and rhizosphere microorganisms
of seedlings of Pseudotsuga menziesii (Mirb.) Franco planted on a degraded site and inoculated
with forest soils pretreated with selective biocides. New Phytol 127:529–537
DaSilva EJ, Henriksson LE, Urdis M (1977) Growth responses of mycorrhizal Boletus and
Rhizopogon species to pesticides. Trans Br Mycol Soc 68:434–437
Datnoff LE, Nemec S, Pernezny K (1995) Biological control of fusarium crown and root rot of
tomato in Florida using Trichoderma harzianum and Glomus intraradices. Biol Control
5:427–431
De la Bastide PY, Kendrick B (1990) The in vitro effects of benomyl on disease tolerance,
ectomycorrhiza formation, and growth of white pine (Pinus strobus) seedlings. Can J Bot
68:444–448
DeLorenzo ME, Taylor LA, Lund SA, Pennington PL, Strozier ED, Fulton MH (2002) Toxicity
and bioconcentration potential of the agricultural pesticide endosulfan in phytoplankton
zooplakton. Arch Environ Contam Toxicol 42:173–181
Desprez-Loustau M-L, Dupuis F, Viguié A (1992) Evaluation of single annual applications of
sterol-inhibiting fungicides for control of pine twisting rust. Plant Dis 76:376–382
Dı́az G, Carrillo C, Honrubia M (2003) Differential responses of ectomycorrhizal fungi to
pesticides in pure culture. Cryptogam Mycol 24:199–211
Dickinson TA, Hutchison LJ (1997) Numerical taxonomic methods cultural characters, and the
systematics of ectomycorrhizal agarics, boletes and gastermycetes. Mycol Res 101:477–492
Donnelly PK, Fletcher JS (1995) PCB metabolism by ectomycorrhizal fungi. Bull Environ Contam
Toxicol 54:507–513
Donnelly PK, Entry JA, Crawford DL (1993) Degradation of atrazine and 2, 4-dochlorophenox-
yacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro. Appl Environ
Donnelly PK, Fletcher JS (1994) Potential use of mycorrhizal fungi as bioremediation agents. In:
Anderson TA, Coats JR (eds), Bioremediation Through Rhizosphere Technology, American
Chemical Society, Washington, pp 93–99
D€orfler U, Feicht EA, Scheunert I (1997) S-triazine residues in groundwater. Chemosphere
35:99–106
Dubus IG, Hollis JM, Brown CD (2000) Pesticides in rainfall in Europe. Environ Pollut
110:331–344
Elbetieha A, Da’as SI, Khamas W, Darmani H (2001) Evaluation of the toxic potencials of
cypermethrin pesticide on some reproductive and fertility parameters in the male rats. Arch
Environ Contam Toxicol 41:522–528
Estok D, Freedman B, Boyle D (1989) Effects of the herbicides 2, 4-D, glyphosate, hexazinone,
and triclopyr on the growth of three species of ectomycorrhizal fungi. Bull Environ Contam
Toxicol 42:835–839
Etayo ML, De Miguel AM (1998) Estudio de las ectomycorrizas en una trufera cultivada situada
en Olóriz (Navarra). Publicaciones de Biologia de la Universidad de Navarra. Serie Botanica
11:55–114
Flykt E, Timonen S, Pennanen T (2008) Variation of ectomycorrhizal colonisation in Norway
spruce seedlings in Finnish forest nurseries. Silva Fenn 42:571–585
Garbaye J (1994) Helper bacteria – a new dimension to the mycorrhizal symbiosis. New Phytol
128:197–210
Garbaye J (2000) The role of ectomycorrhizal symbiosis in the resistance of forests to water stress.
Outlook Agric 29:63–69
Garbaye J, Churin J-L, Duponnois R (1992) Effects of substrate sterilization, fungicide treatment
and mycorrhization helper bacteria on ectomycorrhizal formation of pedunculate oak (Quercus
robur) inoculated wtih Laccaria laccata in two peat bare-root nurseries. Biol Fertil Soils
13:55–57
Gaus I (2000) Effects of water extraction in a vulnerable phreatic aquifer: consequences for
groundwater contamination by pesticides, Sint-Jansteen area, The Netherlands. Hydrogeol J
8:218–229
Gerecke AC, Sch€arer M, Singer HP, M€ uller SR, Schwarzenbach RP, S€agesser M, Ochsenbein U,
Popow G (2002) Sources of pesticides in surface waters in Switzerland: pesticide load through
waste water treatment plants – current situation and reduction potential. Chemosphere
48:307–315
Gonzalez-Moro MB, Iribeei N, Duñabeitia MK, Loureiro-Beldarrain I, González-Murua C (2000)
Effect of phosphinothricin herbicide on nitrogen metabolism in Pinus radiata and Laccaria
bicolor. Phyton (Special Issue Root-soil interactions) 40:71–77
344 M. Marin
Govi G, Bencivenga M, Granetti B, Pacioni G, Palenzona M, Tocci A, Zambonelli A (1997)

Metodo basato sulla caratterizzazione morfologica delle micorrize. In: Toscana R (ed) Il
Tartufo. Florence, Centro Stampa Giunta Regionale, pp 49–62
Gramatica P, Di Guarno A (2002) Screening of pesticides for environmental partitioning tendency.
Chemosphere 47:947–956
Granetti B, Angelini P (1992) Competizione tra alcuni funghi ectomicorrizici e T. melanosporum
in una tartufaia coltivata. Micol Vegetazione Mediterr 7:173–188
Guerin-Laguette A, Plassard C, Mousain D (2000) Effects of experimental conditions on mycor-
rhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-
body formation of the Saffron milk cap under controlled soilless conditions. Can J Microbiol
46:790–799
Hall IR, Wang Y (1998) Methods for cultivating edible ectomycorrhizal mushrooms. In: Varma A
(ed) Mycorrhiza manual. Springer, Berlin, pp 99–114
Hatch AB (1937) The physiological basis of mycotrophy in the genus Pinus. Black Rock For Bull
6:168
Heiskanen J, Rikala R (2003) Effect of peat-based container media on establishment of Scots pine,
Norway spruce and silver birch seedlings. Tree Planter’s Notes 50:28–33
Hutchison LJ (1990) Studies on the systematics of ectomycorrhizal fungi in axenic culture. IV. The
effect of some selected fungitoxic compounds upon linear growth. Can J Bot 68:2172–2178
Ingham ER, Coleman DC (1984) Effects of streptomycin, cycloheximide, fungizone, captan,
carbofuran, cygon, and PCNB on soil microorganisms. Microb Ecol 10:345–358
Jalai-Hare SH, Kendrick WB (1987) Response of an endomycorrhizal fungus in Allium porrum L.
to different concentrations of the systemic fungicides, metalaxyl (Ridomil®) and fosetyl-Al
(Aliette®). Soil Biol Biochem 19:95–99
Jones MD, Hutchinson TC (1986) The effect of mycorrhizal infection on the response of Betula
papyerifera to nickel and copper. New Phytol 102:429–442
Jones MD, Durall DM, Tinker PB (1991) Fluxes of carbon and phosphorus between symbionts in
willow ectomycorrhizas and their changes with time. New Phytol 118:99–106
Juntunen ML, Kitunen V (2003) Leaching of propiconazole and chlorothalonil during protection
of Pinus sylvestris seedlings in containers. Scand J For Res 18:45–53
Kelley WD, South DB (1980) Effects of herbicides on in vitro growth of mycorrhizae of Pine
(Pinus spp.). Weed Sci 28:599–602
Kelley WD (1987) Effect of triadimefon on development of mycorrhizae from natural inoculum
in loblolly pine nursery beds. Southern J Appl Forestry 11:49–52
Kreuger J (1998) Pesticides in stream water within an agricultural catchment in southern Sweden,
1990–1996. Sci Total Environ 216:227–251
Laatikainen T, Juntunen M-L, Heinonen-Tanski H (2000) The effect of nursery applied fungicides
on the growth and mycorrhiza abundance of Scots pine seedlings. In: Lilja A, Sutherland JR
(eds) Proceedings of the 4th Meeting of IUFRO Working Party 7.03.04 – Diseases and Insects
in Forest Nurseries. The Finnish Forest Research Institute, Research Papers 781:177–187
Laatikainen T, Heinonen-Tanski H (2002) Mycorrhizal growth in pure cultures in the presence of
pesticides. Microbiol Res 157:127–137
Laiho O (1965) Further studies on the ectendotrophic mycorrhizae. Acta For Fenn 79:1–34
Landis TD (1989) Mineral nutrients and fertilization. In: Landis TD, Tinus RW, McDonald SE,
Barnett JP (eds) The container tree nursery manual, vol 4. Department of Agriculture, Forest
Service, Washington DC, pp 1–67
Le Tacon F, Alvarez IF, Bouchard D, Henrion B, Jackson RM, Luff S, Parlade JI, Pera J,
Stenstr€om E, Villeneuve N, Walker C (1992) Variations in field response of forest trees to
nursery ectomycorrhizal inoculation in Europe. In: Read DJ, Lewis DH, Fitter AH, Alexander
IJ (eds) Mycorrhizas in ecosystems. CAB International, Wallingford, pp 119–132
Le Tacon F, Mousan D, Garbaye J, Bouchard D, Churin J-L, Argillier C, Amirault J-M, Généré R
(1997) Mycorrhizes, pépinières et plantations forestières en France (Summary in English).
Revue Forestière Française 49:131–154
Lekounougou S, Jacquot JP, Gérardin P, Gelhaye E (2008) Effects of propiconazole on extra-

cellular enzymes involved in nutrient mobilization during Trametes versicolor wood coloniza-
tion. Wood Sci Technol 42:169–177
Levanon D, Meisinger JJ, Codling EE, Starr JL (1994) Impact of tillage activity and the fate of
pesticides in the upper soil. Water Air Soil Pollut 72:179–189
Manninen AM, Laatikainen T, Holopainen T (1998) Condition of Scots pine fine roots and
mycorrhiza after fungicide application and low-level ozone exposure in a 2-year field experi-
ment. Trees 12:347–355
Marin M (2009) Ectomycorrhizal fungi and its application in forest nursery. In: Rai M (ed)
Advances in fungal biotechnology. IK International, New Delhi, pp 379–408
Marx DH, Rowan SJ (1981) Fungicides influence growth and development of specific ectomycor-
rhizae on Loblolly pine seedlings. Forest Sci 27:167–176
Marx DH, Cordell CH, France RC (1986) Effects of triadmefon on grown and ectomycorrhizal
development on loblolly and slash pines in nurseries. Phytopathology 76:824–831
Massicote HB, Takaberry LE, Ingham ER, Thies WG (1998) Ectomycorrhizae establishment on
Douglas-fir seedlings following chloropicrin treatment to control laminated-root rot disease:
assessment of 4 and 5 years after outplanting. Appl Soil Ecol 10:117–126
McLaughlin A, Mineau P (1995) The impact of agricultural practices on biodiversity. Agric
Ecosyst Environ 55:201–212
Meharg AA, Cairney JWG (2000) Ectomycorrhizas - extending the capabilities of rhizosphere
remediation? Rev Soil Biol Biochem 32:1475–1484
Meharg AA, Cairney JWG, Maguire N (1997a) Mineralization of 2, 4-dichlorophenol by ecto-
mycorrhizal fungi in axenic culture and in symbiosis with pine. Chemosphere 34:2495–2504
Meharg AA, Denis GR, Cairney JWG (1997b) Biotransformation of 2, 4, 6-trinitrotoluene (TNT)
by ectomycorrhizal basidiomycetes. Chemosphere 35:513–521
Metcalf RL (1971) The chemistry and biology of pesticides. In: White-Stevens R (ed) Pesticides in
the environment, Part 1, vol 1. Marcel Dekker, New York, pp 1–144
Montanini B, Marco Betti M, Márquez AJ, Balestrini R, Bonfante P, Ottonello S (2003) Distinc-
tive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.
Biochem J 373:357–368
Nerg A-M, Kasurinen A, Holopainen T, Julkunen-Tiitto R, Neuvonen S, Holopainen JK (2008)
The significance of ectomycorrhizas in chemical quality of silver birch foliage and above-
ground insect herbivore performance. J Chem Ecol 34:1322–1330
Niini SS, Raudaskoski M (1993) Response of ectomycorrhizal fungi to benomyl and nocodazole:
growth inhibition and microtubule depolymeration. Mycorrhiza 3:83–91
O’Neill JJM, Mitchell DT (2000) Effects of benomyl and captan on growth and mycorrhizal
colonization of Sitka-spruce (Picea sitchensis) and ash (Fraxinus excelsior) in Irish nursery
soil. For Pathol 30:165–174
Page-Dumroese DS, Harvey AE, Jurgensen MF, Larsen MJ (1996) Ponderosa pine seedling
response to planting-site soil fumigation and fungicide application. Northwest Sci 70:139–146
Pawuk WH, Ruehle IE, Marx DH (1980) Fungicide drenches affect ectomycorrhizal development
of container-grown Pinus palustris seedlings. Can J For Res 10:61–64
Pedersen CT, Sylvia DM (1997) Limitations to use benomyl in evaluating mycorrhizal function-
ing. Biol Fertil Soils 25:163–168
Peltola A (2001) Finnish statistical yearbook of forestry 2000. Jyv€askyl€a, Agriculture, Forestry
and Fishery, vol 52. p 374 (in Finnish with English summary)
Perry DA, Molina R, Amaranthus MP (1987) Mycorrhizae, mycorrhizospheres, and reforestation:
current knowledge and research needs. Can J For Res 17:929–940
Pimentel D, Tort M, D’Anna L, Krawic A, Berger J, Rossman J, Mugo F, Doon N, Shriberg M,
Howard E, Lee S, Talbot J (1998) Ecology of increasing disease. Bioscience 48:817–826
Reddy MS, Natarajan K (1994) Effect of a synthetic pyrethroid on the growth of ectomycorrhizal
fungi and mycorrhiza formation in Pinus patula. Mycorrhiza 5:115–117
Reddy MS, Natarajan K (1995) Effects of the fungicide dithane M-45 on the growth and
mycorrhizal formation of Pinus patula seedlings. Soil Biol Biochem 27:1503–1504
346 M. Marin
Reitveld WJ (1989) Transplanting stress in bareroot conifer seedlings: its development and
progression to establishment. North J Appl For 6:99–107
Rostad CE (1997) Concentration and transport of chlordaner and nonachlor associated with
suspended sediments in the Missisippi river, May 1988 to June 1990. Arch Environ Contam
Toxicol 33:369–377
Russell MH (1995) Recommend approaches to assess pesticide mobility in soil. In: Roberts TR,
Kearney PC (eds) Environmental behaviour of agrochemicals. Wiley, Chichester, pp 57–129
Sánchez F, Honrubia M, Torres P, Dı́az G, Garcı́a G, Pérez P (1994) Biodiversity and ecological
distribution of ectomycorrhizal fungi in mediterranean forests of the Sistema Ibérico Mountains.
In: Azcon-Aguilar C, Barea JM (eds) Mycorrhizas in integrated systems from genes to plant
development. Proceedings of the fourth European symposium on mycorrhizas, Granada, Spain
Sch€uepp H, Bodmer M (1991) Complex response of VA-mycorrhizae to xenobiotic substances.
Toxicol Environ Chem 30:193–199
Schweiger PF, Jakobsen I (1998) Dose–response relationships between four pesticides and
phosphorus uptake by hyphae of arbuscular mycorrhizas. Soil Biol Biochem 30:1425–1432
Sidhu SS, Chakravarty P (1990) Effect of select forestry herbicides on ectomycorrhizal develop-
ment and seedling growth of lodgepole pine and white spruce under controlled and field
enviroment. Eur J For Pathol 20:77–94
Singer R (1986) The Agaricales in modern taxonomy, 4th edn. Koeltz Scientific Books, Koenig-
stein, Germany, pp 829–839
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn. Academic, London
Smith MD, Hartnett DC, Rice CW (2000) Effects of long-term fungicide application on microbial
properties in tallgrass prairie soil. Soil Biol Biochem 32:935–946
Tarkka M, Nehls V, Hampp R (2005) Physiology of ectomycorrhiza (ECM). Prog Bot 66:247–276
Teste FP, Schmidt MG, Berch SM, Bulmer C, Egger KN (2004) Effects of ectomycorrhizal
inoculants on survival and growth of interior Douglas-fir seedlings on reforestation sites and
partially rehabilitated landings. Can J For Res 34:2074–2088
Teste FP, Karst J, Jones MD, Simard SW, Durall DM (2006) Methods to control ectomycorrhizal
colonization: effectiveness of chemical and physical barriers. Mycorrhiza 17:51–65
Theodorou C, Skinner MF (1976) Effects of fungicides on seed inocula of basidiospores of
mycorrhizal fungi. Aust For Res 7:53–58
Torstensson L, Wessén B (1984) Interactions between the fungicide benomyl and soil microrgan-
isms. Soil Biol Biochem 16:445–452
Trappe JM, Molina R, Castellano M (1984) Reactions of mycorrhizal fungi and mycorrhiza
formation to pesticides. Ann Rev Phytopathol 22:331–359
Tu CM (1978) Effect of some pesticides on acetylene reduction and microorganisms in sandy
loam. Soil Biol Biochem 10:451–456
Tu CM (1991) Effect of some technical and formulated insecticides on microbial activities in soil.
J Environ Sci Health B 26:557–573
Tu CM (1993) Effect of fungicides, captafol and chlorothalonil, on microbial and enzymatic
activities in mineral soil. J Environ Sci Health B 28:67–80
Unestam T, Chakravarty P, Damm E (1989) Fungicides: in vitro tests not useful for evaluating
effects on ectomycorrhizae. Agric Ecosyst Environ 28:535–538
Van der Heijden MGA, Sanders IR (2002) Mycorrhizal ecology. Ecological studies 157. Springer,
Berlin, p 469
Whipps JM (2004) Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Can
J Bot 82:1198–1227
Wilde SA (1958) Forest soils-their properties and relation to silviculture. Ronald, New York, p 537
Zambonelli A, Govi G (1991) Competizione fra Tuber albidum ed altri funghi. 3 contributo.
Micol Ital 10:5–12
Zambonelli A, Iotti M (2001) Effects on fungicide on Tuber borchii and Hebeloma sinapizans
ectomycorrhizas. Mycol Res 105:611–614
Zambonelli A, Iotti M, Rossi I, Hall I (2000) Interaction between Tuber borchii and other
ectomycorrhizal fungi in a field plantation. Mycol Res 104:698–702
Chapter 15
Metal-Chelating Agents from Ectomycorrhizal
Fungi and Their Biotechnological Potential
Ángela Machuca
15.1 Introduction
Ectomycorrhizal (ECM) fungi have been investigated for many years concerning
the inoculation of plants for the recovery of degraded terrestrial ecosystems.
Inocula production and mycorrhization techniques have been enhanced over time
due to the proven importance of symbiotic associations on the growth and develop-
ment of plants. Furthermore, ECM fungi that exhibit an unusual capacity to regulate
the bioavailability of metal ions in their natural environments by either increasing
or decreasing them are of particular interest in the revegetation of disturbed
ecosystems, poor in mineral nutrients or contaminated with metal ions (O’Dell
et al. 1993; Haselwandter and Bowen 1996; Leyval et al. 1997; Prasad and Freitas
1999; Brunner 2001).
Both the ECM fungi and their host plants can interact with metal ions, essential
(e.g., Ca, Fe, Cu, Zn) or nonessential (e.g., Cd, Pb, Hg) for growth, through physical
or chemical mechanisms and/or transport systems of various specificities, either to
acquire ions for their nutrition or to avoid the toxic effects that high concentrations
of certain ions can cause, depending on the environmental conditions (Jentschke
and Godbold 2000; Hall 2002; Meharg 2003). ECM fungi can benefit the growth of
the host plant, promoting the uptake of mineral nutrients or increasing the plant’s
tolerance to certain metal ions in soils. The beneficial effects, however, depend on
the fungal species, the plant species, the metal involved, and the soil conditions that
surround the mycorrhizal association and that affect the bioavailability of the metal
ions (Galli et al. 1994; Prasad and Freitas 1999, Meharg 2003). Despite the
numerous investigations in the area of the amelioration of metal toxicity by ECM
associations, the results are contradictory regarding the protective role of ECM
Á. Machuca
Department of Plant Science and Technology, Universidad de Concepción, Campus Los Ángeles,
J.A.Coloma 0201 Los Ángeles, Chile
e-mail: angmachu@udec.cl

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_15,
348 Á. Machuca
fungi on their host plants. Some studies clearly describe a protective effect of
ECM fungi in contaminated environments, while others describe an increase in
metal ion uptake in the mycorrhizal roots, thereby increasing the toxic effect on the
plants (Godbold et al. 1998; Jentschke and Godbold 2000). It is likely that the
limited amount of knowledge that exists regarding the mechanisms involved in the
modification of the bioavailability of metal ions by ECM fungi in association with
their host plants is responsible for the contradictory results.
The mechanisms used by living organisms to mobilize essential metal ions from
cultures or natural environments to the interior of the cells have been very well
described for most bacterial, plant, and some fungal species, but the studies are
limited to only a few species when it comes to ECM fungi. Something similar
occurs when trying to determine the mechanisms that allow ECM fungi to deal with
elevated, potentially toxic concentrations of certain essential or nonessential metal
ions, metalloids, or radionuclides. In this way, a fungus is able to use a combination
of various strategies to take up or detoxify metal ions, through mobilization or
immobilization, extracellularly or intracellularly, depending on culture conditions
or environment. Independently of the fungus using these mechanisms to take up or
avoid the potentially toxic effect of metal ions, most of these mechanisms are based
on chelation capacity. Chelation occurs when a bi- or multidentate ligand molecule,
organic in nature, binds or sequesters a metal ion by forming multiple coordinated
bonds. The ligands are also known as chelators or chelating agents and the complex
formed as a chelate; the ions of most transition metals form stable chelates. A very
important group of chelating agents with different affinities for metal ions includes
the siderophores and low molecular weight organic acids (citric, oxalic, malic, etc.),
widely distributed in a variety of organisms (Gadd 1999; Renshaw et al. 2002;
Bellion et al. 2006). Another important group is formed by the peptides metal-
lothioneins (MTs), phytochelatins (PCs), and reduced glutathione (GSH), some of
which are restricted to certain organisms (Gadd 1993; Mejáre and B€ulow 2001;
Bellion et al. 2006). Although metal binding to the fungal cell wall (adsorption) is
among the mechanisms that many ECM fungi use for metal detoxification (Gadd
1993; Bellion et al. 2006), this will not be treated here since this chapter focuses on
those chelation mechanisms that depend on the metabolic activity of the fungi.
15.2 Specific Metal-Chelating Agents: Siderophores
Despite the abundance of iron in the Earth’s crust (5% by weight), in aerobic
environments it exists mainly as iron oxyhydroxides (e.g., goethite) of very low
solubility at neutral pH (~1038 M), which causes a bioavailability problem for
most living organisms whose metabolism depends on this element. Therefore, most
plants, fungi, and bacteria produce an efficient and highly specific system for iron
acquisition, comprised of siderophores (Greek “iron carriers”) that act as iron
scavengers or chelators of high affinity (Hider 1984; Neilands 1995; Renshaw
et al. 2002; Boukhalfa and Crumbliss 2002; Kraemer 2004). However, other
15 Metal-Chelating Agents from Ectomycorrhizal Fungi 349
mechanisms such as reductive assimilation and protonation are also used by some
organisms with the aim of solubilizing iron from their environments (Guerinot
1994; Kosman 2003). Although plants (grasses) produce these compounds (phyto-
siderophores), the chemical structures and chelation mechanisms differ from those
of fungal and bacterial origin (Sugiura and Nomoto 1984; Kraemer et al. 2006;
Crowley 2006).
Siderophores are iron-chelating compounds of low molecular weight (500–
1,500 Da), with the ability to form stable complexes of high affinity constants
(Kf > 1030) with the ferric form of iron (Fe3+), but not with the ferrous form (Fe2+).
These compounds are excreted into the environment by microorganisms and after
chelating the Fe3+ they return to the cell (Hider 1984; Guerinot 1994; Renshaw et al.
2002). The synthesis of siderophores and the proteins involved in their recognition
and, in some cases, in their transport to the interior of the cells, is strictly regulated
at the molecular level by the concentration of iron perceived by the cell, causing an
induction of these systems only under iron stress. Depending on the organism,
concentrations higher than 1 mM of Fe3+ can dramatically reduce or completely
repress siderophore biosynthesis (van der Helm and Winkelmann 1994).
Siderophores are synthesized by cells as metal-free ligands (desferrisidero-
phores), which are excreted into the extracellular environment where they solubi-
lize the iron by chelation and return to the cell as ferrisiderophores, releasing the
iron through different mechanisms (Hider 1984; Guerinot 1994; Neilands 1995;
Renshaw et al. 2002; Kraemer 2004). At least four different mechanisms have been
proposed for the transport of ferrisiderophores to the cell, mostly based on the
recognition of ferric complexes by specific transport systems present in the cell
membrane. Once inside the cell, the Fe3+ is reduced to Fe2+, which is released from
the siderophore. A different mechanism performs the reduction of siderophore-
transported Fe3+ by a reductase enzyme present in the membrane. The Fe2+ and not
the siderophore is subsequently transported to the interior of the cell (van der Helm
and Winkelmann 1994; Renshaw et al. 2002). Despite the existing knowledge on
the ferrisiderophore transport, this has been much more studied in bacteria than in
fungi. Although siderophores are produced by the majority of fungi and bacteria,
there are some exceptions where the presence of these compounds has not been
demonstrated. However, some microorganisms that do not produce siderophores
are capable of using exogenous siderophores (xenosiderophores), produced by
other species of bacteria or fungi (Guerinot 1994; Kosman 2003).
The chemical nature of siderophores varies a great deal, but it is possible to
distinguish three large groups according to the functional groups involved in
chelation: hydroxamates, catecholates (phenolates), and hydroxycarboxylates. In
addition to these groups, characterized most frequently in microorganisms, side-
rophores with other structures or a combination of the structures previously men-
tioned have also been described. In bacteria, the production of siderophores mainly
of the catecholate and hydroxamate types has been reported. In most fungi,
by contrast, it has only been possible to detect and isolate hydroxamate-type side-
rophores of the ferrichrome, fusarinine, and coprogen families and, in a few
cases, hydroxycarboxylates (Hider 1984; Renshaw et al. 2002; Haselwandter
350 Á. Machuca
and Winkelmann 2007). Independent of the chemical nature, the kinetic and
thermodynamically more stable complexes are obtained by the formation of hex-
adentate or six-coordinate siderophores with Fe3þ, which allows them to act as
efficient iron scavengers from the insoluble oxyhydroxides (Hider 1984; Boukhalfa
and Crumbliss 2002; Kraemer 2004). Although catechol-type siderophores can
form complexes with a higher Fe3þ affinity than hydroxamates, they are very
susceptible to oxidation, depending on the pH. By contrast, hydroxamates form
complexes of lower affinity, but very stable over a broad pH range (Hider 1984;
Boukhalfa and Crumbliss 2002).
The siderophore production by mycorrhizal fungi has been demonstrated in a
limited number of investigations and for very few fungal species; however, less
research has been done to illustrate the chemical nature of siderophores isolated
from ECM fungi. Nevertheless, those studies demonstrate that siderophores pro-
duced by ectomycorrhizal, ericoid, orchidaceous, and ectendomycorrhizal fungi are
hydroxamate ligands: mainly of the ferrichrome structural family (Haselwandter
1995; Haselwandter and Winkelmann 2007). The ferrichromes are cyclic peptides
containing a tripeptide of N-acyl-N-hydroxy-ornithine and combinations of the
amino acids glycine, serine, or alanine (Renshaw et al. 2002). The few studies of
some species of ECM fungi (Table 15.1) have detected the presence of siderophores
in axenic cultures using (a) the reagent chrome azurol S (CAS) in liquid or solid
medium (Schwyn and Neilands 1987; Milagres et al. 1999), (b) the chemical assays
to detect hydroxamate structures or the Csáky test (Csáky 1948), and catecholate
structures or the Arnow test (Arnow 1937), or (c) through bioassays using the
Aureobacterium (Arthrobacter) flavescens JG-9 strain, a hydroxamate siderophore
auxotrophic soil organism (Neilands 1984). Cenococcum geophilum (only one
ascomycete in Table 15.1) was the first ECM fungus described, from which side-
rophores were isolated and characterized through HPLC, mass spectrometry, and
NMR spectra (Haselwandter and Winkelmann 2002). This fungus produced mainly
ferricrocin-type hydroxamates; other lower concentration compounds were found
and although their structures were not completely identified, seemed to correspond
to ferrichrome, fusarinine, fusigen, and coprogen.
On the other hand, if the studies showing the production of siderophores by ECM
fungi are few, studies describing siderophore production by ECM fungi in associa-
tion with roots are even fewer. Nevertheless, van Hees et al. (2006), in a careful and
precise study demonstrated the simultaneous release of siderophores and organic
acids by hyphae of the extraradical mycelium of Hebeloma crustuliniforme in
association with Pinus sylvestris seedlings. The siderophores characterized corre-
sponded mainly to ferricrocin; ferrichrome also appeared in the exudates, but in a
much lower concentration. Oxalate in concentrations 10,000 times higher than
ferricrocin was also detected in exudates. The authors suggest that the combination
of hyphal exudates, siderophores and oxalic acid, can significantly alter soil con-
ditions through mineral dissolution (van Hees et al. 2006). Using the CAS assay, the
Csáky and Arnow assays, and HPLC, the presence of iron-chelating compounds
and organic acids was detected in cultures of Suillus luteus, Rhizopogon luteolus
and Scleroderma verrucosum, collected from pine plantations (Machuca et al. 2007).
Table 15.1 Ectomycorrhizal fungi described by production and/or characterization of siderophores
15
Fungal species Fungal growth (solid, liquid, Demonstration of siderophore Purification/characterization of References
symbiosis) production siderophores
Amanita muscaria Pure culture Bioassay with Aureobacterium Only for B. edulis were the compounds Szaniszlo et al. (1981)
Boletus edulis Hagen medium (agar and broth), flavescens JG-9, specific for partially purified and characterized.
Suillus brevipes deferrated, without Fe3+ hydroxamates Czaky assay confirmed
S. lakei addition, or with 50 ng mL1 hydroxamate nature of compounds.
S. punctipes Fe3+ Thin layer chromatography (TLC)
S. granulatus revealed compounds mixture that
S. tomentosus contains ferricrocin
Pisolithus tinctorius
Cenococcum geophilum
P. tinctorius Pure culture No assay was carried out for Siderophores isolated using ion- Leyval and Reid (1991)
Iron-deficient liquid culture (iron siderophore detection exchange resin and TLC.
concentration not specified) Siderophores partially purified
were radiolabeled with 59Fe.
Structural characteristics not
specified
S. granulatus Pure culture Hydroxamate detection by Czaky assay No Watteau and Berthelin
Pachlewsky broth, in presence of in goethite, but not in biotite or (1994)
goethite, biotite and pyrite pyrite presence. Iron solubilization
Metal-Chelating Agents from Ectomycorrhizal Fungi
from goethite was attributed to

siderophore production more than
to organic acids
C. geophilum Pure culture Siderophore detection in culture Siderophore isolation using ion- Haselwandter and
LIM-1 medium (Szaniszlo et al. filtrates by CAS assay exchange resin and size exclusion Winkelmann (2002)
1981) and deferrated medium chromatography. Ferricrocin was
identified by HPLC, mass
spectrometry, and 1H–13C-NMR
spectra
Laccaria laccata Pure culture Siderophore detection by CAS-agar No Gupta and Satyanarayana
P. tinctorius Modified Melin-Norkans (MMN) assay and hydroxamate detection (2002)
Rhizopogon vulgaris broth, with and without Fe3+ by chemical assay
R. luteolus addition
(continued)
351
352
Fungal species Fungal growth (solid, liquid, Demonstration of siderophore Purification/characterization of References
symbiosis) production siderophores
Hebeloma crustuliniforme Fungus in symbiosis with Pinus Release of siderophores and organic Siderophore isolation using ion- van Hees et al. (2006)
sylvestris, using aseptic acids from extraradical mycelium exchange resin. Hydroxamates
multicompartment dishes were purified by HPLC and
identified by mass spectrometry
(ESI-MS/MS), mainly as
ferricrocin, but ferrichrome was
also detected. Organic acids were
also detected, mainly oxalic acid
S. luteus Pure culture Siderophore detection by CAS assay. No Machuca et al. (2007)
R. luteolus MMN broth, without Fe3+ addition Hydroxamate and catecholate
Scleroderma verrucosum and with 35 mmol L1 Fe3+. detection by Czaky and Arnow
assays, respectively
Á. Machuca
The Arnow assay revealed the presence of catecholate-type structures in the three
species in higher concentrations than the hydroxamates; however, these compounds
were not proven to be true siderophores (Machuca et al. 2007). Phenolic com-
pounds with Fe3+-chelating capacity have already been described in a variety of
wood-rotting basidiomycetes (Goodell et al. 1997; Milagres et al. 1999; Machuca
et al. 2001; Arantes and Milagres 2006); but their function as siderophores in the
transport of Fe3+ has not been proven. Moreover, phenolic pigments produced by
these species, responsible for the brown staining of the mycelia and culture
medium, may be responsible for the positive reaction in the Arnow assay. Some
pigments of this type (e.g., melanins) located in the cell wall, extracellular phenolic
polymers, and other phenolic metabolites with metal-chelating properties have been
involved in processes of metal uptake and detoxification by fungi (Gadd 1993;
Fogarty and Tobin 1996; Bellion et al. 2006). Other ECM fungi showed iron-
chelating compound production in liquid and/or solid medium through CAS
assay: S. bellinii, S. granulatus, Lactarius deliciosus, Paxillus filamentosus and
Amanita sp.. Unexpectedly, the ascomycete Tuber borchii was the only species
we tested that has not shown any positive CAS reaction, either in liquid or solid
medium.
The exudation of compounds with the capacity to chelate and mobilize Fe3+ has
also been confirmed in the ECM root tips of Xerocomus sp. and L. subdulcis,
collected in forest soils (Rineau et al. 2008). On the other hand, the presence of
hydroxamate siderophores, mainly ferricrocin and ferrichrome, in forest soil in
concentrations that vary between 2 and 12 nM has also been described (Holmstr€om
et al. 2004; Essén et al. 2006). The origin of these siderophores must be linked to the
microorganisms that inhabit terrestrial ecosystems, among them ECM fungi. This
has considerable ecological implications, since both microorganisms (fungi and
bacteria) and plants could benefit from the production of siderophores by other
species and use them as exogenous siderophores along with their own in order
to increase the uptake of iron from the environment. The ability to utilize exoge-
nous siderophores gives some plants and microorganisms a competitive advantage
in environments deficient in this essential micronutrient (Crowley et al. 1991;
Guerinot 1994; Winkelmann 2007; Johnson 2008).
Furthermore, the role of hydroxamate siderophores in iron uptake from soils
has been shown using Fe3+-minerals (e.g., goethite), from which siderophores
release iron through dissolution of the mineral, acting alone or in combination
with organic acids (Kraemer et al. 1999; Cervini-Silva and Sposito 2002; Borer
et al. 2005; Reichard et al. 2007). The solubilization of goethite by S. granulatus
was attributed more to the production of siderophores than to organic acids; and
most of the Fe3+ mobilized was accumulated in the mycelium (Watteau and
Berthelin 1994). S. granulatus also dissolved biotite and mobilized Fe3þ and
Al3þ, but siderophores were not produced in the presence of this mineral, and
the authors attribute this to Fe3+ being more available in biotite. Peña et al. (2007)
proved the participation of siderophores in the dissolution of a Mn mineral
(hausmannite), through strong chelation of Mn3+. These studies confirm the
importance of siderophores in the biogeochemical cycle of Fe and their connection
354 Á. Machuca
to other cycles like the Mn cycle (Duckworth et al. 2009). Most of the research
in this area has been undertaken using the commercial hydroxamate siderophore
desferrioxamine B, while the studies with ECM fungi producing siderophores
are limited.
Despite the high affinity of siderophores for Fe3+, these compounds can also
form stable complexes with other metals, such as Al, Cd, Cr, Cu, Pb, Mn, Zn, and
some actinides like Pu, Th, and U (Hider 1984; Brainard et al. 1992; Renshaw
et al. 2003; Zou and Boyer 2005). Ferricrocin, the main siderophore produced by
most of the ECM fungi investigated (Table 15.1), which is also present in forest
soils, can form stable mononuclear complexes in aqueous solution with trivalent
ions (Al3+, Cr3+, Ga3+, and Fe3+); and with divalent ions (Cu2+, Zn2+), it can
form multinuclear complexes (Zou and Boyer 2005). Although siderophores can
form complexes with other metal ions besides iron, these complexes often do
not complete their cycle of returning to the cell and delivering the metal ion
because they are not recognized, or because metal ion reduction does not occur
(Hider 1984; Clarke et al. 1987; Zou and Boyer 2005). These results are of great
importance in view of the role that siderophores may play for ECM fungi and their
host plants in metal-contaminated terrestrial ecosystems, where they might regu-
late the transport and mobility of these metal ions as part of detoxification
mechanisms. Nevertheless, research is still necessary to determine the conditions
under which siderophores can prevent the entrance of certain metal ions into the
cell, thereby reducing the toxic effect on the fungus and its host plant, or the
conditions under which the mobilization of metal ions could be increased, pro-
ducing an adverse toxic effect as has been observed in other microorganisms
(Arceneaux et al. 1984). Studies into the effects of ECM fungi on their host plants
growing in metal-contaminated soils have often been contradictory; some illus-
trate the beneficial effect of ECM fungi on the protection of their host plants,
while many others indicate an increase in metal ion uptake by mycorrhized plants
(Godbold et al. 1998; Jentschke and Godbold 2000). Perhaps the contradictory
effects described in the literature are related to the type of siderophore or other
metal-chelating agents being produced by a certain fungal species and to the
characteristics of the metal complexes formed.
From these results, it seems evident that there is a gap in our knowledge
regarding the production and characterization of siderophores by ECM fungi, in
the absence and/or presence of symbiotic association, since to date very few species
have been evaluated (Table 15.1). The role that siderophores play as chelators of
other metal ions besides Fe when EcM fungi are growing in contaminated environ-
ments must be investigated as part of possible detoxification mechanisms. Studies
are also needed in relation to the effect of other metal ions besides Fe on side-
rophore synthesis regulation in EcM fungi. In addition, investigations that con-
tribute to increasing our understanding of the role of fungal siderophores in the
metabolism of iron in plants are necessary to determine, for example, whether ECM
fungal siderophores are used by host plants, under what conditions and what
mechanisms are involved.
15.3 Non-specific Metal-Chelating Agents: Low Molecular

Weight Organic Acids
Low molecular weight (LMW) organic acids, along with siderophores, are among
the main mediators of the biological weathering of soils caused by living organi-
sms, leading to the dissolution of a variety of minerals through a double effect of
metal cation chelation and anion displacement by protonation (Landeweert et al.
2001; Gadd 2007; van Sch€ oll et al. 2008). In soils, organic acids come mainly from
plants that release them into the rhizosphere by exudation from their roots, as well
as from fungi and bacteria, and chemically they are carboxylic acids, with one or
more functional groups per molecule (e.g., formic, malic, oxalic, citric, succinic,
etc.). Those acids that present more than one carboxylic group (di- or tricarboxylic)
are important in the formation of complexes with metal ions and can be considered
as metal-chelating agents. Under the normally existing pH conditions in biological
systems, these acids are found in the form of carboxylate anions (e.g., oxalate,
citrate, etc.).
Citric (tricarboxylic) and oxalic (dicarboxylic) acids have been the most inves-
tigated in plants and fungi, mainly in saprophytes and pathogens, due not only to
their nutritional and physiological importance for these organisms, but also to their
industrial importance. Oxalate can form complexes with a variety of trivalent (e.g.,
Fe3+, Al3+, Cr3+) and divalent (e.g., Ca2+, Cu2+, Mg2+, Zn2+, Pb2+) metal ions and
also with actinides and lanthanides; some oxalates are soluble, like that of Fe3+, but
others can be insoluble like those of Ca2+ and Pb2+. Similarly, the citrate can form
different complexes with a variety of metal ions (e.g., Ca2+, Cd2+, Cu2+, Pb2+, Fe3+)
(Dutton and Evans 1996; Jones 1998; Gadd 1999).
LMW organic acids perform important and varied metabolic roles for living
cells and also participate in the mobilization and uptake of a series of mineral
nutrients (e.g., Fe and P), essential to the metabolism of plants and most micro-
organisms in the soil. LMW organic acids are directly involved in the biogeochem-
ical cycles of a variety of metal elements; they serve as pathogenic agents for
certain fungi and as wood biodeterioration agents for others; and they have been
involved in the detoxification of metal ions in plants, fungi, and bacteria (Dutton
and Evans 1996; Jones 1998; Gadd 1999, 2007; Landeweert et al. 2001). Although
LMW organic acids are important agents for the mobilization of mineral nutrients
from inorganic sources, the strategy most utilized by ECM fungi for the mobiliza-
tion of N and P from organic sources is enzyme production. Both strategies are used
with more or less efficiency depending on the fungal species (Chalot and Brun
1998; Landeweert et al. 2001).
Malate and citrate are the main acids released into the rhizosphere from many
plant roots under conditions of iron or phosphorus deficiency. In iron deficiency, it
has been suggested that citrate may participate in the dissolution and uptake of Fe3+
by forming Fe3+-citrate complexes in some dicotyledons (Jones 1998). In grasses,
phytosiderophores excreted by the roots may be responsible for the solubilization of
Fe3+ (Kraemer et al. 2006; Crowley 2006). On the other hand, oxalate, malate, and
356 Á. Machuca
citrate have been involved in the detoxification of Al in plants through the forma-
tion of Al3+-organic acid complexes, which are of lower toxicity for plants than free
Al3+ (Jones 1998; Sch€ ottelndreier et al. 2001).
LMW organic acids can regulate the speciation and bioavailability of certain
metal ions in soils through mobilization by acidification (protonation), formation of
soluble complexes (complexolysis), or immobilization by formation of insoluble
complexes. These mechanisms are of considerable importance in the acquisition of
essential metal ions or metal detoxification by LMW organic acids. The formation
of complexes, however, depends not only on the concentration of carboxylate
anions, but also on the type and concentration of metal ion, the pH of the medium,
and the stability constants of the complexes formed (Jones 1998; Gadd 1999;
Meharg 2003). Citrate, malate, and oxalate form stable complexes of high affinity
constants with trivalent ions like Fe3+ and Al3+. Oxalate also forms insoluble
complexes with Ca2+ and this is one of the main forms in which it is found in
soils, fungi, plants, and animals (Jones 1998; Gadd 1999). Despite the high affi-
nities for Fe3+, the stability constants of the complexes with organic acids are
inferior to those obtained with hydroxamate or catecholate (Hider 1984; Kraemer
2004), and therefore the role of siderophores in the uptake and transport of Fe3+
must be of higher relevance than chelation by organic acids for living organisms
dependent on these metal-chelating agents.
LMW organic acids have been studied in ECM fungi much more than side-
rophores, mainly in relation to the weathering of minerals. However, as in the case
of siderophores, most detailed studies are limited to a few fungal species. Organic
acid exudation by the hyphae of ECM fungi produces the solubilization and
mobilization of a variety of nutrients (P, K, Ca, and Mg) directly from solid mineral
substrates far from the rhizosphere, insoluble and inaccessible to the roots, thereby
contributing to the nutritional status of the host plants (Landeweert et al. 2001;
Gadd 2007; van Sch€ oll et al. 2008). The formation of tubular pores in certain
minerals has been attributed to organic acid exudation by the hyphae of ECM
fungi inside mineral particles, and for this reason they are called rock-eating fungi.
In this way, the dissolution of minerals by ECM fungi to obtain essential ions is not
limited to an attack on particle surfaces, but inside the mineral particles as well
(Jongmans et al. 1997; Landeweert et al. 2001; van Sch€oll et al. 2008).
The exudation of LMW organic acids, mainly oxalate, by different ECM fungi
has been described in vitro in the absence of symbiotic association (Lapeyrie 1988;
Lapeyrie et al. 1991; Arvieu et al. 2003; Machuca et al. 2007), in the presence of
symbiosis (Ahonen-Jonnarth et al. 2000; Casarin et al. 2003; van Hees et al. 2005,
2006; van Sch€oll et al. 2006a), and also by ECM root tips harvested in forest
ecosystems (Rineau et al. 2008). Some of these studies have shown a greater
production of oxalate by mycorrhized compared to nonmycorrhized plants
cultivated under axenic conditions (Ahonen-Jonnarth et al. 2000; Casarin et al.
2003). P. involutus, one of the most studied species in relation to oxalate produc-
tion, can use bicarbonate (NaHCO3) as a source of C and ammonium (NH4+), or
nitrate (NO3) as N sources to produce the acid and biosynthesis can happen
directly from oxaloacetate or via citrate, isocitrate, and glyoxylate (Lapeyrie 1988;
Lapeyrie et al. 1991). In the presence of NO3, calcium carbonate (CaCO3) and
different concentrations of orthophosphate (Pi), P. involutus, H. cylindrosporum,
R. roseolus and, S. collinitus released oxalate and protons (H+) to the culture
medium (Arvieu et al. 2003). When carboxylates are exuded from the cells, the
negative charges must be balanced by a simultaneous efflux of positive charges
(like H+), leading to a reduction of pH outside the cells (Jones 1998; Casarin et al.
2003). The increase in the excretion of oxalate and H+ in the presence of CaCO3
suggests the importance that these fungal species may have in the mobilization
of nutrients in calcareous soils. In the rhizosphere of the associations between
R. roseolus and H. cylindrosporum with P. pinaster, it was possible to detect the
release of oxalate in presence of R. roseolus with simultaneous rhizosphere acidifi-
cation. However, no excretion of oxalate was obtained with H. cylindrosporum and
an alkalinization of the rhizosphere was observed (Casarin et al. 2003).
The excretion of citrate and succinate, in addition to oxalate, was detected in a
culture medium of R. luteolus, S. verrucosum and S. luteus. S. luteus was the species
that produced the highest concentrations of all the organic acids and the only one
that also produced malonate. A strong pH reduction was also recorded in the
cultures of all the species (Machuca et al. 2007). It must be emphasized that even
though these species were not subjected to a nutrient deficit (P, N, or C) and used
NH4+ as the source of N, they were able to excrete high organic acid concentrations
and H+. Strong acidification was also produced by these species in solid medium in
the presence of high concentrations of Cu2+ and Zn2+, but not in the presence of Cd.
This suggests that the capacity to produce organic acids and acidify culture media is
an intrinsic characteristic of these species. These species were harvested in forest
plantations of P. radiata, where the fruiting bodies were found frequently and in
large quantities in the different regions studied (Machuca et al. 2007). Van Sch€oll
et al. (2008) indicate that species of the genera Rhizopogon and Suillus are phylo-
genetically related to species of the genera belonging to brown rot fungi like
Serpula, Coniophora, and Hygrophoropsis, known to produce large amounts of
oxalate.
LMW organic acids have also been investigated with respect to the solubiliza-
tion of mineral nutrients from a great variety of insoluble substrates and their
connection to plant nutrition via ECM fungi. The mobilization of P and Ca from
the mineral apatite has been described (Wallander 2000a; Blum et al. 2002;
Wallander et al. 2003), as well as K from biotite, microcline, and phlogopite
(Paris et al. 1995, 1996; Wallander and Wickman 1999; Wallander 2000b). Using
pot experiments, P. sylvestris plants mycorrhized with P. involutus, Piloderma
croceum, and H. longicaudum were cultivated with mineral muscovite as the only
source of K and hornblende as the source of Mg (van Sch€oll et al. 2006b). Under
these conditions, P. involutus was the only species able to solubilize the muscovite,
but not the hornblende; mobilizing K for its host plants. The authors emphasize that
ECM fungi can indeed increase the dissolution of minerals in response to a
deficiency in nutrients, but the efficiency of the process is clearly species-specific.
The exudation of LMW organic acids has been related to metal ion detoxifica-
tion mechanisms in ECM fungi (Jentschke and Godbold 2000; Meharg 2003;
358 Á. Machuca
Bellion et al. 2006) and plants (Jones 1998). LMW organic acids may act as
extracellular chelators, forming complexes with metal ions and preventing them
from entering the cell (avoidance mechanisms) in the same way that may occur with
siderophores (Jentschke and Godbold 2000; Bellion et al. 2006). In this respect,
there is a great deal of controversy, given that when LMW organic acids produce
the dissolution of certain minerals in soil, together with increasing the bioavail-
ability of essential metal ions, they may also cause the mobilization of potentially
toxic metal ions. Furthermore, the efflux of H+ that can occur with the exudation of
carboxylate anions may decrease the medium pH, increasing the bioavailability of
certain metal ions, making them toxic to fungi and plants (Gadd 2007).
When P. sylvestris seedlings mycorrhized with S. variegatus, R. roseolus, and
P. involutus were exposed to high concentrations of metals, a significant increase in
oxalate production was observed in the presence of S. variegatus and R. roseolus
exposed to Al. In addition, Cu exposure stimulated the production of oxalate in the
presence of S. variegatus and P. involutus, and, by contrast, Ni and Cd had no effect
on oxalate production (Ahonen-Jonnarth et al. 2000). Using several insoluble
minerals, the solubilization capacity was attributed to the strong decrease in
extracellular pH caused by different species of ECM fungi (Rosling et al. 2004),
and this decrease was also related to metal tolerance (Fomina et al. 2005). Ray and
Adholeya (2009) determined in vitro organic acid exudation by P. tinctorius and
S. verrucosum using coal ash pond. Formic, malic, and succinic acids were pro-
duced by the fungi and a variety of metals (Al, As, Cd, Cr, Ni, and Pb) were
detected in their mycelia. No oxalic acid production was described in this paper.
Large differences were observed between the strains regarding the patterns of
LMW organic acids exuded and metal accumulation. A correlation between organic
acid production and a metal accumulation in fungal mycelia was also demonstrated
(Ray and Adholeya 2009).
The presence of Ca oxalate crystals has been observed in the cultures of ECM
fungi, mycorrhized roots, in the rhizosphere, and around the hyphae of the extra-
radical mycelium (Cromack et al. 1979; Lapeyrie et al. 1990; Allen et al. 1996;
Mahmood et al. 2001; Tuason and Arocena 2009) and it has been suggested that
these crystals constitute a reservoir of Ca in the ecosystems, where they can also
affect phosphate availability (Dutton and Evans 1996; Gadd 1999, 2007). The
formation of these crystals by reprecipitation of solubilized Ca may serve as a
fungal detoxification mechanism when the plants grow in soils with high concen-
trations of Ca; this has also been suggested for other metals that form insoluble
oxalates (Cromack et al. 1977; Dutton and Evans 1996; Gadd 2007).
Even though metal tolerance has been extensively researched in ECM fungi, the
results with respect to the benefits of the fungi on their host plants can be contra-
dictory at times (Godbold et al. 1998; Jentschke and Godbold 2000; Gadd 2007).
This may be related to the lack of experimental evidence clearly showing which
mechanisms are involved and how much they contribute to tolerance in fungi. The
participation of chelating agents such as siderophores and LMW organic acids has
often been suggested among these tolerance mechanisms. However, to date, there
seem to be no conclusive studies that are able to correlate the degree of metal
tolerance with the type and concentration of chelators produced by an ECM fungus
when exposed to high concentrations of one or several metals simultaneously. The
situation is of greater complexity when attempting to analyze the fungus–plant
system in contaminated soils where the mycorrhized roots might be exposed to
more than one metal ion and several other factors that affect the bioavailability
of metals. This knowledge is essential if the possible application of certain ECM
fungi is to be considered in bioremediation projects or degraded environment
reforestation.
15.4 Other Metal-Chelating Agents: Thiol-Peptides
Another group of agents with metal-chelating properties has been described in

some species of ECM fungi, but unlike siderophores and LMW organic acids,
they are more related to detoxification mechanisms than to metal ion nutrition or
mineral solubilization. This group includes MTs, PCs, and GSH, all the thiol
peptides (Gadd 1993; Meharg 2003; Pocsi et al. 2004; Bellion et al. 2006). These
peptides act intracellularly; once the metal ions have entered the cell, they regulate
or prevent the activity of metal ions in the cytosol through complexation. Thus,
unlike siderophores and LMW organic acids that can represent avoidance mechan-
isms in relation to metal detoxification, these peptides represent intracellular
detoxification mechanisms.
MTs are peptides of low molecular weight (6–7 kDa), rich in cysteine (Cys),
highly ubiquitous – given that they have been identified in animals, plants, fungi,
and some bacteria – with the capacity to chelate metals through the thiol (–SH)
groups. Some MTs can contain up to 60 amino acids, of which fewer than half
correspond to Cys; they do not exhibit aromatic amino acids or histidine. MTs can
form clusters with a large amount of divalent or monovalent metal ions through the
domains rich in Cys (Robinson et al. 1993). The biosynthesis of MTs is regulated at
transcriptional level by a variety of factors, among them some metals: Cd, Zn, Hg,
Cu, Au, Ag, Co, Ni, and Bi (K€agi and Sh€affer 1988); and of these Cu, Zn, Cd, Hg,
Bi, Ag, Au, as well as Pb and Pt can be indeed chelated (K€agi 1991). However, the
preference for one or the other metal, both in the regulation of biosynthesis and in
chelation, depends exclusively on the organism (Robinson et al. 1993; Hall 2002).
MTs were described in ECM fungi in 1986 and their production was induced by the
presence of Cd2+ in a P. tinctorius culture medium (Morselt et al. 1986). Later, the
production of MTs by P. involutus and L. laccata was shown, regulated by high
Cd2þ concentrations (Howe et al. 1997). Using HPLC, several thiol-containing
compounds were investigated in P. involutus (Courbot et al. 2004), with GSH,
g-glutamylcysteine, and a compound of low molecular weight being detected that
the authors suggested to correspond to MTs; all of these increased by exposing the
fungus to Cd2+. The genes that codify for MTs were characterized in P. involutus
(Pimt1 gene) (Bellion et al. 2007); the gene expression was regulated by Cu and Cd,
but not by Zn, and the overexpression of Pimt1 in a strain transformed from
360 Á. Machuca
H. cylindrosporum gave it an increased tolerance to Cu. Recently, these genes were

also characterized in H. cylindrosporum and their regulation by Cu and Cd was
shown, but not by Zn, Pb, or Ni (Ramesh et al. 2009).
PCs are peptides rich in Cys, with a general (g-Glu-Cys)n-Gly (n ¼ 2–11)
structure, of low molecular weight, which in fungi does not seem to exceed
2 kDa. Unlike MTs, PCs are not codified in the genome and their presence has
been shown mainly in plants, where they have been related to the detoxification of
Cd (Cobbett 2000; Mejáre and B€ ulow 2001; Hall 2002). They are synthesized from
GSH, through the action of an enzyme (PC synthase), whose activity is regulated by
metal ions such as Hg, Ag, Cu, Ni, Au, Pb, Zn, but mainly by Cd, which is the
strongest inducer of PC synthase (Gadd 1993; Cobbett 2000; Mejáre and B€ulow
2001). Until recently, there were no reports of their presence in ECM fungi, and
even the synthesis of these peptides has been shown in only a very few fungal
species (Bellion et al. 2006). Nevertheless, Collin-Hansen et al. (2007) described
for the first time the presence of PCs in the well known edible wild mushroom
Boletus edulis. In this fungus, metal exposure induced the production of thiol
compounds such as GSH, and an accumulation of Cd in the caps of this macro-
mycete was observed due to the formation of complexes with the PCs. When the
roots of Picea abies mycorrhized with L. laccata and the pure cultures of fungus
were exposed to Cd, an increase was detected in the GSH, but not in the MTs (Galli
et al. 1993). Recently, the role of tripeptide GSH (g-Glu-Cys-Gly) was established
as a key agent in the responses to various stress situations in fungi (Pocsi et al.
2004); and in the case of metal stress, GSH can act as a precursor to the synthesis of
PCs, as a scavenger of reactive oxygen species or as a metal-chelating agent (Ott
et al. 2002; Courbot et al. 2004; Heged€ us et al. 2007).
PCs, like MTs, have not only been linked to metal detoxification, but also to the
regulation of the homeostasis of essential metal ions and other biological processes,
acting as xenobiotic detoxifying agents, or antioxidants (Gadd 1993; Rauser 1995;
Cobbett 2000; Bellion et al. 2006). Furthermore, these thiol peptides have also been
related to the accumulation capacity of metal ions in the fruiting bodies of some
basidiomycetes when they grow in contaminated environments, and this represents
an enormous risk to human health due to the high consumption of edible wild
mushrooms in many countries (Gadd 1993). Although there is a large number of
studies in the literature on the accumulation of metals, metalloids, and radio-
nuclides by several mushrooms (saprophyte and ECM fungi), most aim at deter-
mining the metal concentrations in the caps and stipes of the macrofungi and in
their growth substrates. Nevertheless, the physiological and biochemical mechani-
sms involved in the accumulation are still not well understood, and the participation
of metal-chelating agents like siderophores, MTs, PCs, and GSH have been sug-
gested and in some cases proven, as with B. edulis (Collin-Hansen et al. 2007). On
the other hand, a correlation between the GSH concentration and the Hg2+ and Cd2+
contents was described in the fruiting bodies of various ECM fungi (Kojo and
Lodenius 1989). Recently, Amanita stroboliformes and A. solitaria were described
as hyperaccumulators of Ag, accumulating 2,500 times more Ag in their fruiting
bodies than in the growth soil (Borovicka et al. 2007). Despite Ag accumulation
mechanisms not being investigated in this study, it may be similar to that described
for saprophyte Agaricus bisporus, where the participation of MTs was suggested
(Byrne and Tusek-Znidaric 1990). The accumulation of 137Cs has been observed in
several species of saprophytes and ECM fungi, edible wild mushrooms (Gaso et al.
2000). Among these, the ECM fungus Clavariadelphus truncatus was described as
an accumulator of 137Cs, Rb, and Pb; and this was related to metal-chelating
compounds (siderophore-type) detected in its fruiting bodies (Gaso et al. 2007).
Given the metal-accumulation capacity of some macromycetes, their use as
bioindicators of contaminated environments has been suggested by several authors.
However, the variability in accumulation among fungal species is enormous and
depends on the developmental stage of the fruiting bodies, the type of metal, and a
series of environmental factors (Mejstrik and Lepsova 1993; Gadd 2007). More-
over, some ECM fungi may accumulate large concentrations of metals in their
fruiting bodies with respect to growth soils, even when they are grown in non-
contaminated environments. By contrast, other species exclude or do not accumu-
late metals in their fruiting bodies despite growing in close proximity to mine
tailings. For this reason, the use of some saprophytic or ECM macromycetes as
bioindicators of a metal-contaminated terrestrial ecosystem must be considered
with caution.
Some of the metal-chelating agents or mechanisms described here for ECM
fungi have also been described for arbuscular mycorrhizal fungi (AMF) (Khan et al.
2000; G€ohre and Paszkowski 2006). However, given the symbiotic dependency
required in AMF, the experiments are often of greater complexity. Regarding the
metal-chelating mechanisms, there is a great difference between both groups of
fungi, which is represented by glomalin, described to date exclusively in AMF.
Glomalin is a glycoprotein produced by the hyphae, capable of chelating metals
such as Cu, Pb, and Cd, and because of this some researchers have highlighted the
significant role of the glomalin-producing AMF in the stabilization of the contami-
nated soils and in the protection of their host plants (González-Chávez et al. 2004;
Khan 2006).
15.5 Biotechnological Potential of ECM Fungi Producing

Metal-Chelating Agents
The biotechnological potential of ECM fungi producing metal-chelating agents is

related to the isolated chelators with specific aims, or to the use of the fungi in the
presence or absence of symbiotic association. Due to their extremely efficient
metal-chelating properties, for many years the main application of siderophores
has been in chelation therapy to treat Fe and Al overload in humans; its possible use
in the decorporation of actinides is also being investigated. The main siderophore
used for this purpose is desferrioxamine B, a hydroxamate obtained from Strepto-
myces pilosus (Messenger and Ratledge 1985; Renshaw et al. 2002; Ansoborlo
362 Á. Machuca
et al. 2007). However, siderophores can have other important applications related to
the environment; in the uptake of metals from industrial waste, low-grade ores,
serpentine soils, contaminated terrestrial and aquatic environments, tailings of
abandoned mines, etc. The uptake of metals can serve to remediate an environment
and/or recover metals for recycling. In addition, the capacity to chelate actinides
(Pu, U, Np, and Th) has been demonstrated in siderophores and for this reason their
application has been proposed for the remediation of radioactive waste and the
reprocessing of nuclear fuel (Hernlem et al. 1999; Renshaw et al. 2002, 2003). The
majority of these studies have been conducted with commercial hydroxamate
siderophores like desferrioxamine B and some with siderophore-producing soil
microbes (John et al. 2001; Keith-Roach et al 2005; Mullen et al 2007). Therefore,
this is an area where other fungal hydroxamate siderophores, like those produced by
ECM fungi, could have a great potential for application. Another important possible
application of siderophores is in the treatment of asbestos, a carcinogenic fibrous
mineral with varied industrial applications that is prohibited in many countries.
Although the toxicity mechanisms are not well understood, it is believed that the Fe
present on the surface of asbestos fibers cause cell damage by generating free
radicals. It was recently demonstrated that hydroxamates and soil fungi-producing
siderophores, among them ECM fungi, inactivate fibers by removing the Fe from
the material surfaces (Martino et al. 2003, 2004; Daghino et al. 2008). These fungi
represent a potential tool for the bioremediation of asbestos waste, contaminated
waters and soils, and asbestos abandoned mines. It must be pointed out that due to
their chelating properties, LMW organic acids could have most of the environmen-
tal applications found for siderophores, as previously described.
The identification and selection of new strains of ECM fungi, efficiently produc-
ing siderophores and/or LMW organic acids, is fundamental to the mycorrhization
of plants used in programs to recover degraded or destabilized forest ecosystems,
poor in mineral nutrients either from natural causes or through anthropogenic
action. The production of these chelators by ECM roots must contribute to the
solubilization and uptake of mineral nutrients into the rhizosphere, facilitating
the establishment and development of plants in these ecosystems. In addition, the
modifications in the speciation and bioavailability of metals and nonmetals as a
result of the chelators released by the hyphae beyond the rhizosphere promotes the
restoration in the mineral nutrient balance, favoring not only vegetal species, but
also all microorganisms inhabiting these ecosystems (Gadd 2007; Van Sch€oll et al.
2008).
The revegetation of metal-contaminated soils is another important application of
plants mycorrhized with ECM fungi producing a wide range of chelators: side-
rophores, LMW organic acids, MTs, PC, and GSH. Given that these chelators
contribute to the detoxification of metals through intracellular or extracellular
mechanisms, the fungal species that produce them must protect their host plants
in contaminated environments. This protection consists of excluding metals or
accumulating them in their fruiting bodies, using both ways to prevent their entry
into the mycorrhized roots. The use of plants in the bioremediation of soils
contaminated by metals, aided by synthetic chelators like EDTA and called assisted
phytoremediation, has been described in the literature (Khan et al. 2000; Lasat
2002; Wenzel 2009). Plants inoculated with ECM fungi producing metal-chelating
agents could be used in the same way to aid in the increased uptake of metals by the
roots, and at the same time improve the nutritional status of the plants, and with it
the production of biomass for phytoremediation.
15.6 Conclusions
The results described illustrate the fundamental role that extracellular metal-
chelating agents like siderophores and LMW organic acids play in the acquisition
of essential metal ions for many ECM fungi and their host plants. At the same time,
siderophores and LMW organic acids, together with the intracellular thiol-peptides
metallothioneins, phytochelatins, and glutathione, have been related to detoxifica-
tion mechanisms and metal ion accumulation, when the ECM fungi are growing in
contaminated substrates or natural environments. Despite their biotechnological
potential, very few species of ECM fungi have been investigated in relation to the
production and characterization of metal-chelating agents. Moreover, the simulta-
neous production of more than one type of metal-chelating agent by the same fungal
species has not often been considered, even though the efficiency of the combined
action of these agents in the solubilization of minerals has been shown. Along with
this, the demonstration that the transfer of the genes that codify for the metallothio-
neins provides increased copper tolerance between fungal species indicates the
need to extend our knowledge regarding the genes that codify metal-chelating
agents and the factors that regulate their expression in ECM fungi. Because of
that, the urgent search for new species of ECM fungi producing more than one type
of metal-chelating agent efficiently is necessary, since these species could be
converted into valuable tools for applications in the bioremediation of substrates
or contaminated environments or in the rehabilitation of disturbed forest ecosys-
tems, alone or in association with their host plants. Nevertheless, success in the
application of ECM fungi based on metal-chelating agent production depends on
much-needed prior research that considers which type of metal-chelating agents are
being produced by the ECM fungi, alone or in symbiotic association under axenic
conditions. This is because, depending on the chelators produced, ECM fungi can
promote the mobilization of metals towards the interior of the roots, or they can
promote the immobilization of metals in the rhizosphere or the interior of their
cells, preventing entry into the roots. The results of these studies will make it
possible to select the most appropriate fungal partner for a particular vegetal species
under a certain metal-stress condition (by excess or deficit), before applying the
ECM fungi and/or ectomycorrhized plants in field assays.
Acknowledgments The author wishes to thank Dr. Adriane M.F. Milagres and Dr. Carolin
Córdova for their helpful comments and revisions of the manuscript.
364 Á. Machuca
References
Ahonen-Jonnarth U, Van Hees PAW, Lundstr€ om US, Finlay RD (2000) Organic acids produced
by mycorrhizal Pinus sylvestris exposed to elevated aluminium and heavy metal concentra-
tions. New Phytol 146:557–567
Allen MF, Figueroa C, Weinbaum BS, Barlow SB, Allen EB (1996) Differential production of
oxalate by mycorrhizal fungi in arid ecosystems. Biol Fertil Soils 22:287–292
Ansoborlo E, Amekraz B, Moulin C, Moulin V, Taran F, Bailly T, Burgada R, Hengé-Napoli M-H,
Jeanson A, Den Auwer C, Bonin L, Moisy P (2007) Review of actinide decorporation with
chelating agents. CR Chim 10:1010–1019
Arantes V, Milagres AMF (2006) Evaluation of different carbon sources for production of iron-
reducing compounds by Wolfiporia cocos and Perenniporia medulla-panis. Process Biochem
41:887–891
Arceneaux JEL, Boutwell ME, Byers BR (1984) Enhancement of copper toxicity by siderophores
in Bacillus megaterium. Antimicrob Agents Chemother 25:650–652
Arnow LE (1937) Colorimetric determination of the components of 3, 4-dihydroxy-phenylalanine
tyrosine mixtures. J Biol Chem 118:531–537
Arvieu J-C, Leprince F, Plassard C (2003) Release of oxalate and protons by ectomycorrhizal
fungi in response to P-deficiency and calcium carbonate in nutrient solution. Ann For Sci
60:815–821
Bellion M, Courbot M, Jacob C, Blaudez D, Chalot M (2006) Extracellular and cellular mechan-
isms sustaining metal tolerance in ectomycorrhizal fungi. FEMS Microbiol Lett 254:173–181
Bellion M, Courbot M, Jacob C, Guinet F, Blaudez D, Chalot M (2007) Metal induction of a
Paxillus involutus metallothionein and its heterologous expression in Hebeloma cylindros-
porum. New Phytol 174:151–158
Blum JD, Klaue A, Nezat CA, Driscoll CT, Johnson CE, Siccama TG, Eagar C, Fahey TJ, Linkens
GE (2002) Mycorrhizal dissolution of apatite as an important calcium source in base-poor
forest ecosystems. Nature 417:729–731
Borer PM, Sulzberger B, Reichard PU, Kraemer SM (2005) Effect of siderophores on the light-
induced dissolution of colloidal iron (III) (hydr)oxides. Mar Chem 73:179–193
Borovicka J, Randa Z, Jelı́nek E, Kotrba P, Dunn CE (2007) Hyperaccumulation of silver by
Amanita strobiliformis and related species of the section Lepidella. Mycol Res 111:1339–1344
Boukhalfa H, Crumbliss AL (2002) Chemical aspects of siderophore mediated iron transport.
Biometals 15:325–339
Brainard JR, Strietelmeier BA, Smith PH, Langsten-Unkefer PF, Barr ME, Ryan RR (1992)
Actinide binding and solubilization by microbial siderophores. Radiochim Acta 58
(59):357–363
Brunner I (2001) Ectomycorrhizas: their role in forest ecosystems under the impact of acidifying
pollutants. Perspect Plant Ecol Evol Syst 4:13–27
Byrne AR, Tusek-Znidaric M (1990) Studies of the uptake and binding of trace metals in fungi,
Part I: accumulation and characterisation of mercury and silver in the cultivated mushroom,
Agaricus bisporus. Appl Organomet Chem 4:43–48
Casarin V, Plassard C, Souche G, Arvieu JC (2003) Quantification of oxalate ions and protons
released by ectomycorrhizal fungi in rizosphere soil. Agronomie 23:461–469
Cervini-Silva J, Sposito G (2002) Steady-state dissolution kinetics of aluminum-goethite in the
presence of desferrioxamine-B and oxalate ligands. Environ Sci Technol 36:337–342
Chalot M, Brun A (1998) Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and
ectomycorrhizas. FEMS Microbiol Rev 22:21–44
Clarke SE, Stuart J, Sanders-Loehr J (1987) Induction of siderophore activity in Anabaena spp.
and its moderation of copper toxicity. Appl Environ Microbiol 53:917–922
Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol
123:825–832
Collin-Hansen C, Pedersen SA, Andersen RA, Steinnes E (2007) First report of phytochelatins in a
mushroom: induction of phytochelatins by metal exposure in Boletus edulis. Mycologia
99:161–174
Courbot M, Diez L, Ruotolo R, Chalot M, Leroy P (2004) Cadmium-responsive thiols in the
ectomycorrhizal fungal Paxillus involutus. Appl Environ Microbiol 70:7413–7417
Cromack K, Sollins P, Todd RL, Fogel R, Todd AW, Fender WM, Crossley ME, Crossley DA
(1977) The role of oxalic acid and bicarbonate in calcium cycling by fungi and bacteria: some
possible implications for soil animals. Ecol Bull 25:246–252
Cromack K, Sollins P, Graustein WC, Speidel K, Todd AW, Spycher G, Li CY, Todd RL (1979)
Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hyster-
angium crassum. Soil Biol Biochem 11:463–468
Crowley DE (2006) Microbial siderophores in the plant rhizosphere. In: Barton LL, Abadı́a J
(eds) Iron nutrition in plants and rhizospheric microorganisms. Springer, Dordrecht, The
Netherlands, pp 169–198
Crowley DE, Wang YC, Reid CPP, Szaniszlo PJ (1991) Mechanisms of iron acquisition from
siderophores by microorganisms and plants. Plant Soil 130:179–198
Csáky TZ (1948) On the estimation of bound hydroxylamine in biological materials. Acta Chem
Scand 2:450–454
Daghino S, Martino E, Vurro E, Tomatis M, Girlanda M, Fubini B, Perotto S (2008) Bioweather-
ing of chrysolite by fungi isolated in ophiolitic sites. FEMS Microbiol Lett 285:242–249
Duckworth OW, Bargar JR, Sposito G (2009) Coupled biogeochemical cycling of iron and
manganese as mediated by microbial siderophores. Biometals 22:605–613
Dutton MV, Evans CS (1996) Oxalate production by fungi: its role in pathogenicity and ecology in
the soil environment. Can J Microbiol 42:881–895
Essén SA, Bylund D, Holmstr€ om SJM, Moberg M, Lundstr€om US (2006) Quantification of
hydroxamate siderophores in soil solutions of podzolic soil profiles in Sweden. Biometals
19:269–282
Fogarty RV, Tobin JM (1996) Fungal melanins and their interactions with metals. Enzyme Microb
Technol 19:311–317
Fomina MA, Alexander IJ, Colpaert JV, Gadd GM (2005) Solubilization of toxic metal minerals
and metal tolerance of mycorrhizal fungi. Soil Biol Biochem 37:851–866
Gadd GM (1993) Interactions of fungi with toxic metals. New Phytol 124:25–60
Gadd GM (1999) Fungal production of citric and oxalic acid: importance in metal speciation,
physiology and biogeochemical process. Adv Microb Physiol 41:47–92
Gadd GM (2007) Geomycology: biogeochemical transformations of rocks, minerals, metals and
radionuclides by fungi, bioweathering and bioremediation. Mycol Res 111:3–49
Galli U, Meier M, Brunold C (1993) Effects of cadmium on nonmycorrhizal and mycorrhizal
Norway Spruce seedlings Picea abies (L.) Karst and its ectomycorrhizal fungus Laccaria
laccata (Scop. ex Fr.) Bk. & Br. Sulfate reduction, thiols and distribution of the heavy metal.
Galli U, Schuepp H, Brunold C (1994) Heavy metal binding by mycorrhizal fungi. Physiol Plant
92:364–368
Gaso MI, Segovia N, Morton O, Cervantes ML, Godinez L, Peña P, Acosta E (2000) 137Cs and
relationships with major and trace elements in edible mushrooms from Mexico. Sci Total
Environ 262:73–89
Gaso MI, Segovia N, Morton O, Lopez JL, Machuca A, Hernandez E (2007) Radioactive and
stable bioaccumulation, crystalline compound and siderophore detection in Clavariadelphus
truncatus. J Environ Radioactiv 97:57–69
Godbold DL, Jentschke G, Winter S, Marschner P (1998) Ectomycorrhizas and amelioration of
metal stress in forest trees. Chemosphere 36:757–762
G€ohre V, Paszkowski U (2006) Contribution of the arbuscular mycorrhizal symbiosis to heavy
metal phytoremediation. Planta 223:1115–1122
366 Á. Machuca
González-Chávez MC, Carrillo-González R, Wright SF, Nichols KA (2004) The role of glomalin,
a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements.
Goodell B, Jellison J, Liu J, Daniel G, Paszczynski A, Fekete F, Krishnamurthy S, Jun L, Xu G
(1997) Low molecular weight chelators isolated from wood decay fungi and their role in the
fungal biodegradation of wood. J Biotechnol 53:133–162
Guerinot ML (1994) Microbial iron transport. Annu Rev Microbiol 48:743–772
Gupta V, Satyanarayana T (2002) Production of extracellular siderophores by ectomycorrhizal
fungi. Indian J Microbiol 42:107–110
Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot
53:1–11
Haselwandter K (1995) Mycorrhizal fungi: siderophore production. Crit Rev Biotechnol
15:287–291
Haselwandter K, Bowen GD (1996) Mycorrhizal relations in trees for agroforestry and land
rehabilitation. For Ecol Manag 18:1–17
Haselwandter K, Winkelmann G (2002) Ferricrocin – an ectomycorrhizal siderophore of Cen-
ococcum geophilum. Biometals 15:73–77
Haselwandter K, Winkelmann G (2007) Siderophores of symbiotic fungi. In: Varma A, Chinch-
olkar SB (eds) Soil biology, vol 12. Spriger, Berlin, pp 91–103
Heged€us N, Emri T, Szilágyi J, Karányi Z, Nagy I, Penninckx MJ, Pócsi I (2007) Effect of heavy
metals on the glutathione status in different ectomycorrhizal Paxillus involutus strains. World J
Microbiol Biotechnol 23:1339–1343
Hernlem BJ, Vane LM, Sayles GD (1999) The application of siderophores for metal recovery and
waste remediation: examination of correlations for prediction of metal affinities. Water Res
33:951–960
Hider RC (1984) Siderophore mediated absorption of iron. Struct Bonding 58:25–87
Holmstr€om SJM, Lundstr€ om US, Finlay RD, Van Hees AW (2004) Siderophores in forest soil
solution. Biogeochemistry 71:247–258
Howe R, Levans R, Ketteridge SW (1997) Copper-binding proteins in ectomycorrhizal fungi. New
Phytol 135:123–131
John SG, Ruggiero CE, Hersman LE, Tung C-S, Neu MP (2001) Siderophore mediated plutonium
accumulation by Microbacterium flavescens (JG-9). Environ Sci Technol 35:2942–2948
Johnson L (2008) Iron and siderophores in fungal–host interactions. Mycol Res 112:170–183
Jones DL (1998) Organic acids in the rhizosphere – a critical review. Plant Soil 205:25–44
Jongmans AG, van Breemen N, Lundstr€ om US, van Hees PAW, Finlay RD, Srinivasan M,
Unestam T, Giesler R, Melkerud P-A, Olsson M (1997) Rock-eating fungi. Nature
389:682–683
K€agi JHR (1991) Overview of metallothioneins. Methods Enzymol 205:613–626
K€agi JHR, Sh€affer A (1988) Biochemistry of metallothionein. Biochemistry 27:8509–8515
Keith-Roach MJ, Vetriburatti M, Worsfold PJ (2005) Thorium complexation by hydroxamate
siderophores in perturbed multicomponent systems using flow injection electrospray ionization
mass spectrometry. Anal Chem 77:7335–7341
Khan AG (2006) Mycorrhizoremediation – an enhanced form of phytoremediation. J Zhejiang
Univ Sci B 7:503–514
Khan AG, Kuek C, Chaudhry TM, Khoo CS, Hayes WJ (2000) Role of plants, mycorrhizae and
phytochelators in heavy metal contamined land remediation. Chemosphere 21:197–207
Kojo MR, Lodenius M (1989) Cadmium and mercury in macrofungi-mechanisms of transport and
accumulation. Angew Bot 63:279–292
Kosman DJ (2003) Molecular mechanisms of iron uptake in fungi. Mol Microbiol 47:1185–1197
Kraemer SM (2004) Iron oxide dissolution and solubility in the presence of siderophores. Aquat
Sci 66:3–18
Kraemer SM, Cheah S-F, Zapf R, Xu JD, Raymond KN, Sposito G (1999) Effect of hydroxamate
siderophores on Fe release and Pb(II) adsorption by goethite. Geochim Cosmochim Acta
63:3003–3008
Kraemer SM, Crowley DE, Kretzschmar R (2006) Geochemical aspects of phytosiderophore-
promoted iron acquisition by plants. Adv Agron 91:1–46
Landeweert R, Hoffland E, Finlay RD, Kuyper TW, van Breemen N (2001) Linking plants to
rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 16:248–254
Lapeyrie F (1988) Oxalate synthesis from soil bicarbonate by the mycorrhizal fungus Paxillus
involutus. Plant Soil 110:3–8
Lapeyrie F, Picatto C, Gerard J, Dexheimer J (1990) TEM study of intracellular and extracellular
calcium oxalate accumulation by ectomycorrhizal fungi in pure culture or in association with
Eucalyptus seedlings. Symbiosis 9:163–166
Lapeyrie F, Ranger J, Vairelles D (1991) Phosphate solubilizing activity of ectomycorrhizal fungi
in vitro. Can J Bot 69:342–346
Lasat MM (2002) Phytoextraction of toxic metals: a review of biological mechanisms. J Environ
Qual 31:109–120
Leyval C, Reid CPP (1991) Utilization of microbial siderophores by mycorrhizal and non-
mycorrhizal pine roots. New Phytol 119:93–98
Leyval C, Turnau K, Haselwandter K (1997) Effect of heavy metal pollution on mycorrhizal
colonization and function: physiological, ecological and applied aspects. Mycorrhiza
7:139–153
Machuca A, Napoleao D, Milagres AMF (2001) Detection of metal-chelating compounds from
wood-rotting Trametes versicolor and Wolfiporia cocos. World J Microbiol Biotechnol
17:687–690
Machuca A, Pereira G, Aguiar A, Milagres AMF (2007) Metal-chelating compounds produced by
ectomycorrhizal fungi collected from pine plantations. Lett Appl Microbiol 44:7–12
Mahmood S, Finlay RD, Erland S, Wallander H (2001) Solubilisation and colonisation of wood
ash by ectomycorrhizal fungi isolated from wood ash fertilized spruce forest. FEMS Microbiol
Ecol 35:151–161
Martino E, Prandi L, Fenoglio I, Bonfante P, Perotto S, Fubini B (2003) Soil fungal hyphae bind
and attack asbestos fibers. Angew Chem Int Ed 42:219–222
Martino E, Cerminara S, Prandi L, Fubini B, Perotto S (2004) Physical and biochemical interac-
tions of soil fungi with asbestos fibers. Environ Toxicol Chem 23:938–944
Meharg AA (2003) The mechanistic basis of interactions between mycorrhizal associations and
toxic metal cations. Mycol Res 107:1253–1265
Mejáre M, B€ulow L (2001) Metal-binding proteins and peptides in bioremediation and phytor-
emediation of heavy metals. Trends Biotechnol 19:67–73
Mejstrik V, Lepsova A (1993) Applicability of fungi to the monitoring of environmental pollution
by heavy metals. In: Markert B (ed) Plants as biomonitors. Indicators for heavy metals in the
terrestrial environment. VCH, Weinheim, pp 365–378
Messenger AJM, Ratledge C (1985) Siderophores. In: Moo-Young M (ed) Comprehensive
biotechnology: the principles, applications and regulations of biotechnology in industry,
agriculture and medicine, vol 3. Pergamon, New York, pp 275–295
Milagres AMF, Machuca A, Napoleao D (1999) Detection of siderophores production from
several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J
Microbiol Methods 37:1–6
Morselt AFW, Smits WTM, Limonard T (1986) Histochemical demonstration of heavy metal
tolerance in ectomycorrhizal fungi. Plant Soil 96:417–420
Mullen L, Gong C, Czerwinski K (2007) Complexation of uranium(VI) with the siderophore
desferrioxamine B. J Radioanal Nucl Chem 273:683–688
Neilands JB (1984) Methodology of siderophores. Struct Bonding 58:1–24
Neilands JB (1995) Siderophores: structure and function of microbial iron transport compounds. J
Biol Chem 270:26723–26726
368 Á. Machuca
O’Dell TE, Castellano MA, Trappe JM (1993) Biology and application of ectomycorrhizal fungi.
In: Metting BF (ed) Soil microbial ecology: applications in agriculture and environmental
management. Dekker, New York, pp 379–415
Ott T, Fritz E, Polle A, Schutzendubel A (2002) Characterisation of antioxidative systems in the
ectomycorrhiza-building basidiomycete Paxillus involutus (Barsch)Fr. and its reaction to
cadmium. FEMS Micrbiol Ecol 42:359–366
Paris F, Bonnaud P, Ranger J, Lapeyrie F (1995) In vitro weathering of phlogopite by ectomycor-
rhizal fungi 1. Effect of K+ and Mg2+ deficiency on phyllosilicate evolution. Plant Soil
177:191–201
Paris F, Botton B, Lapeyrie F (1996) In vitro weathering of phlogopite by ectomycorrhizal fungi 2.
Effect of K+ and Mg2+ deficiency and N sources on accumulation of oxalate and H+. Plant Soil
179:141–150
Peña J, Duckworth OW, Bargar JR, Sposito G (2007) Dissolution of hausmannite (Mn3O4) in the
presence of the trihydroxamate siderophore desferrioxamine B. Geochim Cosmochim Acta
71:5661–5671
Pocsi I, Prade RA, Penninckx MJ (2004) Glutathione, altruistic metabolite in fungi. Adv Microb
Physiol 49:1–76
Prasad MNV, Freitas HMO (1999) Feasible biotechnological and bioremediation strategies for
serpentine soils and mine spoils. Electron J Biotechnol 2:35–50
Ramesh G, Podila GK, Gay G, Marmeisse R, Reddy MS (2009) Copper and cadmium metal-
lothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum have different patterns of
regulation. Appl Environ Microbiol 75:2266–2274
Rauser WE (1995) Phytochelatins and related peptides. Plant Physiol 109:1141–1149
Ray P, Adholeya A (2009) Correlation between organic acid exudation and metal uptake by
ectomycorrhizal fungi grown on pond ash in vitro. Biometals 22:275–281
Reichard PU, Kretzschmar R, Kraemer SM (2007) Dissolution mechanisms of goethite in the
presence of siderophores and organic acids. Geochim Cosmochim Acta 71:5635–5650
Renshaw JC, Robson GD, Trinci APJ, Wiebe MG, Livens F, Collison D, Taylor RJ (2002) Fungal
siderophores: structures, functions and applications. Mycol Res 106:1123–1142
Renshaw JC, Halliday V, Robson GD, Trinci APJ, Wiebe MG, Livens FR, Collison D, Taylor RJ
(2003) Development and application of an assay for uranyl complexation by fungal metabo-
lites, including siderophores. Appl Environ Microbiol 69:3600–3606
Rineau F, Courty P-E, Uroz S, Buée M, Garbaye J (2008) Simple microplate assays to measure
iron mobilization and oxalate secretion by ectomycorrhizal tree roots. Soil Biol Biochem
40:2460–2463
Robinson NJ, Tommey AM, Kuske C, Jackson PJ (1993) Plant metallothioneins. Biochem J
295:1–10
Rosling A, Lindahl BJ, Taylor AFS, Finlay RD (2004) Mycelial growth and substrate acidification
of ectomycorrhizal fungi in response to different minerals. FEMS Microbiol Ecol 47:31–37
Sch€ottelndreier M, Norddahl MM, Str€ om L, Falkengren-Grerup U (2001) Organic acid exudation
by wild herbs in response to elevated Al concentrations. Ann Bot 87:769–775
Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of
siderophores. Anal Biochem 160:46–56
Sugiura Y, Nomoto K (1984) Phytosiderophores. Structures and properties of mugineic acids and
their metal complexes. Struct Bonding 58:107–135
Szaniszlo PJ, Powell PE, Reid CPP, Cline GR (1981) Production of hydroxamate siderophore iron
chelators by ectomycorrhizal fungi. Mycologia 73:1158–1174
Tuason MMS, Arocena JM (2009) Calcium oxalate biomineralization by Piloderma fallax in
response to various levels of calcium and phosphorus. Appl Environ Microbiol 75:7079–7085
Van der Helm D, Winkelmann G (1994) Hydroxamates and polycarboxylates as iron transport
agents (siderophores) in fungi. In: Winkelmann G, Winge D (eds) Metal ions in fungi. Dekker,
Van Hees PAW, Jones DL, Jentschke G, Godbold DL (2005) Organic acid concentrations in soil
solution: effects of young coniferous trees and ectomycorrhizal fungi. Soil Biol Biochem
37:771–776
Van Hees PAW, Rosling A, Essén S, Godbold DL, Jones DL, Finlay RD (2006) Oxalate and
ferricrocin exudation by the extramatrical mycelium of an ectomycorrhizal fungus in symbio-
sis with Pinus sylvestris. New Phytol 169:367–378
Van Sch€oll L, Hoffland E, van Breemen N (2006a) Organic anion exudation by ectomycorrhizal
fungi and Pinus sylvestris in response to nutrient deficiencies. New Phytol 170:153–163
Van Sch€oll L, Smits MM, Hoffland E (2006b) Ectomycorrhizal weathering of the soil minerals
muscovite and hornblende. New Phytol 171:805–814
Van Sch€oll L, Kuyper TW, Smits MM, Landeweert R, Hoffland E, van Breemen N (2008) Rock-
eating mycorrhizas: their role in plant nutrition and biogeochemical cycles. Plant Soil
303:35–47
Wallander H (2000a) Uptake of P from apatite by Pinus sylvestris colonized by different ectomy-
corrhizal fungi. Plant Soil 218:249–256
Wallander H (2000b) Use of strontium isotopes and foliar K content to estimate weathering of
biotite induced by pine seedlings colonized by ectomycorrhizal fungi from two different soils.
Wallander H, Wickman T (1999) Biotite and microcline as potassium sources in ectomycorrhizal
and non-mycorrhizal Pinus sylvestris seedlings. Mycorrhiza 9:25–32
Wallander H, Mahmood S, Hagerberg D, Johansson L (2003) Elemental composition of ectomy-
corrhizal mycelia identified by PCR-RFLP analysis and grown in contact with apatite or wood
ash in forest soil. FEMS Microbiol Ecol 44:57–65
Watteau F, Berthelin J (1994) Microbial dissolution of iron and aluminium from soil minerals:
efficiency and specificity of hydroxamate siderophores compared to aliphatic acids. Eur J Soil
Biol 30:1–9
Wenzel WW (2009) Rhizosphere processes and management in plant-assisted bioremediation
(Phytoremediation) of soils. Plant Soil 321:385–408
Winkelmann G (2007) Ecology of siderophores with special reference to the fungi. Biometals
20:379–392
Zou G, Boyer GL (2005) Synthesis and properties of different metal complexes of the siderophore
desferriferricrocin. Biometals 18:63–74
.
Chapter 16
Ectomycorrhiza and Secondary Metabolites
Hanna Dahm and Patrycja Golińska
16.1 Introduction
Approximately 6,000 species of ectomycorrhizal (EM) fungi have been described,

considerably more than arbuscular (AM) fungi. This has led to assumption that EM
fungi are most host specific than AM fungi. Host plant diversity, species composi-
tion, and age do have a role in regulation mycorrhizal communities. Some studies
also suggest that plant secondary metabolites (SM) which are in part under plant
genetic control can affect EM colonization.
The main function of SM is defense against herbivores and microbes; some SM
are signal and attract compounds for seed dispersing animals and some play a role
in the symbiotic relationships with plants and microorganisms.
Early in the twentieth century, it was considered that SM arise either spontaneously
or with the aid of nonspecific enzymes. Now, there is good evidence that biosynthetic
enzymes are highly specific. As a consequence of specific enzymatic synthesis, final
products always have a distinct stereochemistry (Wink 2008). Only the enzymes that
are involved in the degradation of SM (glucosidases, esterases, and other hydrolases)
are less substrate specific.
SM are not functionless waste products, but are important substances for the
symbiotic organisms. Precursor for SM synthesis usually derive from basic metabolic
pathways such as glycolytic, Krebs cycle, and shikimate pathway. These bioprocesses
may lead to synthesis of glucosinates, cyanogenic glucosides, alkaloids, nonprotein
aminoacids, amines, flavonoids, terpenes, quinoline, indole, pyrrolidine, pyrrolizi-
dine, alkaloids, cumarins, mono-, sesqui-, and triterpenes.
Some of the genes that encode biosynthetic enzymes have already been isolated
and characterized. Wink (2008) consider question of when, where, and how the plant
genes evolved that encode enzymes of SM biosynthesis, as well as those of transport,
storage, and turnover.
H. Dahm (*) and P. Golińska

Faculty of Biology and Earth Sciences, Department of Microbiology, Institute of General
and Molecular Biology, University of Nicolaus Copernicus, 87-100 Toruń Gagarina 9, Poland
e-mail: dahm@biol.uni.torun.pl

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_16,
372 H. Dahm and P. Golińska
Theoretically some scenarios can be considered:

l Secondary metabolism could be a young phenomenon and modern plants have
developed their pathways independently.
l Alternatively, secondary metabolism is an old innovation, which was developed
early in the evolution of land plants and was inherited by modern plants.
l Plants could have developed the genes of SM from their own genes of primary
metabolism. Starting with duplication of a gene, the new gene became mutated,
exhibited new metabolic functions and was established by natural selection.
l Plant might have inherited some of the genes in early evolution by horizontal
gene transfer from their bacterial symbionts, which later developed into modern
mitochondria and plastids. Bacteria, especially Actinomyces, Streptomyces, cya-
nobacteria produce a wide diversity of SM, showing similar structures as plant
SM (antraquinones, terpenoids, and alkaloids).
About 80% of modern plants live in symbiosis with fungi (endo-, ectomycor-
hiza). These fungi could directly have supplied its host with SM or might have
transferred (horizontal) the genes to the host’s genome.
Environmental factors (biotic and abiotic) control and regulate the biosynthesis
of SM in plants (Laitinen et al. 2005; Zhi-lin et al. 2007).
As a defense reaction plants evolved bioactive compounds, which repel, deter, or
poison herbivores and which can inhibit growth and development of bacteria, fungi,
and viruses. Some of the defense compounds are constitutive, while others can be
induced under stress conditions. Several SM (phytoalexins) and defense proteins
are synthesized de novo when plant is invaded by microorganisms (Wink 2008).
Type of SM Estimated numbersa

Nitrogen containing SM
Alkaloids 21,000
Nonprotein amino acids (NPAAs) 700
Amines 100
Cyanogenic glycosides 60
Glucosinolates 100
Alkylamides 150
Lectins, peptides, polypeptides 2,000
SM without nitrogen
Monoterpenes (C10)b 2,500
Sesquiterpenes (C15)b 5,000
Diterpenes (C20)b 2,500
Triterpenes, steroids, saponins (C30, C27)b 5,000
Tetraterpenes (C40)b 500
Flavonoids, tannins 5,000
Phenylpropanoids, lignin, coumarins, lignans 2,000
Polyacetylenes, fatty acids, waxes 1,500
Polyketides 750
Carbohydrates, simple acids 400
a
Approximate number of known structures
b
Total number of all terpenoides exceeds 22,000 at present
16 Ectomycorrhiza and Secondary Metabolites 373
In the plant–microbe interaction, coevolution between plants and their microbial

partners are mediated via plant chemical defense. Plant SM usually act as signal
molecules or respond to pathogen and symbiont colonization. Mycorrhizal associa-
tions are the most important mutualist symbiosis which involve three-way interactions
between plants, mycorrhizal fungi, and soil factors. Interactions in the mycorrhizal
associations between macro- and microsymbiont in contrast to plant–pathogen inter-
actions are for both profitable.
In presymbiotic phase, plant and their fungal partner secret signals into soil,
mostly SM, recognized by roots and mycelium, inducing morphological and phys-
iological modifications.
According to some investigators (Baron and Zambryski 1995; Garcia-Garrido
and Ocampo 2002) signal perception and transductions proceed via similar path-
ways between symbiosis and pathogenesis of plants. However, the defense response
in plant-mycorrhizal is probably weak.
The nature of signaling molecules, signal perception, and transduction in mycor-
rhiza are unknown or mistakenly denied (Martin et al. 2001). In the first stage, host
plants release into the rhizosphere metabolites that are able to trigger basidiospore
germination, growth of hyphae toward the roots and the early steps of mycorrhizal
formation.
According to Kottke and Oberwinkler (1987), Horan et al. (1988), Lagrange
et al. (2001), Martin et al. (2001), molecules that control the interactions between
symbionts can be classified as follows:
l Tropism of hyphae for root tissues (rhizospheric signals)
l Attachment and penetration of host tissues by hyphae (adhesions, hydrolases)
l Induction of organogenetic programs in both fungal and root cells (hormones
and secondary signals)
l Facilitating survival of the mycobiont despite plant defense responses
l Coordinating strategies for exchanging carbon and other metabolites for plant
Signals secreted into the rhizosphere can include flavonoids, terpenes, hormones,
and various nutrients. These substances stimulate growth and modified hyphal
morphology.
Some of these substances might be produced and released into rhizosphere by
bacteria, namely mycorrhization helper bacteria (MHBs). Root exudates enhanced
accumulation of fungal molecules such as hypaphorine, the betaine of tryptophan
(Martin et al. 2001). This fungal alkaloid is the major indole compound produced in
larger amounts by some EM fungi (e.g., Pisolithus sp.) during mycorrhiza formation
and development (Béguiristain and Lapeyrie 1997; Martin et al. 2001).
Hypaphorine induces morphological changes in root hairs, which lead to a
decreased rate of elongation and transitory swelling of the apex of the root hair
(Ditengou et al. 2000).
Root hairs are a significant site for microbial interaction in the rhizosphere and it
has been suggested that interaction between the EM fungus and root hairs may play
a role in the symbiosis development. Growth in root hairs is associated with an
apex-high cytosolic free Ca2þ gradient generated by a local Ca2þ influx at the tip.
Some investigators suggest that hypaphorine-induced cytoskeleton changes are

related to interaction with calcium channels, cofilin/actin depolymerizing proteins
and auxin signaling pathways (Ditengou et al. 2000).
The aim of this chapter was to point out a presence of various SM released both
the micro- and macrosymbiont to the mutual interactions zone.
Any such chemical compounds (both the volatile and nonvolatile ones) affect
either positively or negatively the mycorrhiza symbiosis formation and functioning
as well as such chemical compounds interact between themselves. The role of some
metabolites in these processes is better known (auxins), however any importance of
the majority of them is still unknown and requires future, detailed studies.
16.2 Flavonoids
Flavonoids are derived from y-pyrone. They are either 2-phenylbenzopyrone or

3-phenylbenzopyrone. More than 1,300 different flavonoid compounds have been
isolated from plants. Individual flavonoids in a group differ from each other by the
number and position of the hydroxyl, methoxy, and sugar substituents.
O 2-Phenyl-1,4-benzopyrone
Flavonoid compounds occur in plants as glycosides, with hexoses such as glucose,

galactose and rhamnose, and pentoses such as arabinose and xylose as the most
commonly found sugars. The sugars can be attached singly or in combination with
each other.
Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which
the amino acid phenylalanine is used to produce 4-coumaroyl-CoA. This can be
combined with malonyl-CoA to yield of compounds called chalcones, which
contain two phenyl rings. Conjugate ring-closure of chalcones results in the familiar
form of flavonoids, the three-ringed structure of a flavone.
Flavonoids form a large and heterogeneous group of SM having a bioactive role
in the major processes of plants (e.g., attraction of seed disperses, defense reaction
against predators, pathogens and abiotic stress condition [Taylor and Grotewold
2005; Niemi et al. 2007]).
Some authors suggest the role of flavonoids in modulating cell signaling path-
ways including polar auxin transport (Brown et al. 2001; Peer et al. 2004; Kakiuchi
et al. 2006).
Despite increasing evidence the role of flavonoids and other phenolic substances
in plant development, their role in EM symbioses is contradictory. Studies on the
changes in the concentrations flavonoids in Scots pine seedlings during the
establishment of the EM symbiosis with Suillus variegatus showed that in contrast

to shoots, the concentrations of catechin and condensed tannins showed a tendency
to decrease in the roots of both noninoculated and inoculated seedlings and regard-
less of high mycorrhiza frequencies the fungi caused hardly changes in the flavo-
noid concentrations of the roots.
Weiss et al. (1997, 1999) suggested that catechin and epicatechin accumulated in
the inner part of the cortex to prevent the growth of the EM fungus into the inner
cortex.
Epicatechin
In contrast, Beyler and Heyser (1997) reported that reduction of catechin and
epicatechin in the root tips is a prerequisite for rapid mycorrhization. Similarly,
Sch€utzend€ubel and Polle (2004) showed that Scots pine short root tips covered
by the mycelium of Pisolithus tinctorius contained less catechin than nonmycor-
rhizal ones.
16.3 Terpenes
Terpenes are widespread in nature, mainly in plants as constituents of essential oils.

Many terpenes are hydrocarbons, but oxygen-containing compounds such as alco-
hols, aldehydes, or ketones (terpenoids) are also found. Their building block is the
hydrocarbon isoprene, CH2 ¼ C(CH3)–CH ¼ CH2.
Terpene hydrocarbons are classified according to the number of isoprene units:
monoterpenes (2 isoprene units [i.u.]), sesquiterpenes (3 i.u.), diterpenes (4 i.u.),
triterpenes (6 i.u.), tetraterpenes (8 i.u.). Examples of monoterpenes are: pinene,
nerol, citral, camphor, menthol. Examples of sesquiterpenes are: nerolidol, farne-
sol. Examples of diterpenes are: phytol, vitamin A1. Squalene is an example of a
triterpene and carotene is a tetraterpene.
When terpenes are modified chemically, such as by oxidation or rearrangement
of the carbon skeleton, the resulting compounds are generally referred to as
terpenoids. Terpenoids are also known as isoprenoids.
Terpenes are produced by a wide variety of plants, particularly conifers. They
are the major components of resin and of terpentine produced from resin. In
conifers, biosynthesis and infiltration of tissues with resins are involved in defense
system in response to wounding and subsequent inhabitation of the wounds by fungi
and insects (Higuchi 1985; Werner 1993; Napierała-Filipiak et al. 2002). The
response is nonspecific and similar after wounding and infection.
Members of Pinaceae produce two types of resins – oleoresin and parenchyma

resins. Oleoresin is a super saturated solution of resin acids in liquid terpenes and is
located in resin ducts and surrounding epithelial cells. Parenchyma or medullary
resins are composed mainly of fatty acids (Prior 1976; Napierała-Filipiak et al. 2002).
Pine oleoresin contain 60–70% resin acids. The rest is comprised of volatile and
other terpenoids. Among monoterpens, a-pinene, D3-carene, and b-pinene domi-
nate in the volatile fraction (Asiegbu et al. 1998).
The monoterpenes are toxic to wood-fungi, whereas the resin acids display low
toxicity and function mainly as mechanical barrier (Prior 1976; Napierała-Filipiak
et al. 2002). According to Mekin and Krupa (1971) and Krupa et al. (1973) volatile
substances may play a role in the initiation and development of the mycorrhizal
symbiosis, because colonization of pine roots by different mycorrhizal fungi results
in quantitative changes in the concentration of the individual volatiles.
Phenolics and volatiles are significant factor root exudates and influence the
activity of the rhizosphere microorganisms. Due to their volatility, the monoter-
penes and some sesquiterpenes can display significant effect on fungi composition
structure (Smith 1987). They can regulate competitive or antagonistic interactions
and finally create environment stimulating the symbiotic associations.
Forest litter reach in volatile substances may be too a significant factor in the
interactions between microorganisms in soil and rhizosphere. Koide et al. (1998)
found that á-pinene and â-pinene showed differential effects on the growth of
various EM fungi.
Varese et al. (1996) observed enhanced fungal growth due to volatiles; however,
the stimulation was seldom significant as some of the substances, when present in
sufficient concentration, may cause inhibition of the vegetative growth of mycor-
rhizal and pathogenic fungi outside the roots.
Differences in the sensitivity of EM fungi to several volatile substances might
characterize of their ability to induce host reaction and consequently the ability to
initiate symbiosis (Mekin and Krupa 1971; Napierała-Filipiak et al. 2002). Increased
production of volatile and nonvolatile substances might be a mechanism of control over
growth of mycorrhizal fungi in tissues of macrosymbiont (Molina and Trappe 1982).
Although the major volatiles identified in nonmycorrhizal and mycorrhizal roots
of pine, the degree of accumulation of several compounds varied among fungal
treatments. This suggests that each mycorrhizal fungus may elicit a different response
in trees (Napierała-Filipiak et al. 2002). Also in the previous study by Krupa et al.
(1973), the diverse levels of D3-carene and b-phellandrene in roots of Pinus echinata
inoculated with P. tinctorius and Cenococcum graniforme were explained in terms of
the different ability to elicit a specific host response of the two fungi.
16.4 Plant Growth Regulating Substances (phytohormones)
It is assumed that hormones of plant and fungal origin may take part information and
functioning of mycorrhizae (Gogala 1991). Phytohormones, SM synthesis by plant
and EM fungi include auxins, cytokinins, GAs, abscisic acid (ABA), ethylene as well
as alkaloids and phenylglycoside (Gogala 1991). The soil pool of phytohormones

might have partially originated from plants released into the rhizosphere as root
exudates and/or synthesized by soil microorganisms. These biomolecules respond to
exchange of rhizospheric signals between microorganisms and plants. Signal percep-
tion may culminate in the induction of down-stream target gene products whose
expressions are physiological and/or development responses (Martin et al. 2001).
16.4.1 Auxins
Several naturally occurring auxins include indole-3-acetic acid (IAA), its haloge-
nated derivatives (4-Cl-IAA), and indole-3 butyric acid (IBA). On molecular level,
auxins have an aromatic ring and a carboxylic acid group.
Auxins play an essential role in coordination of many growth and behavioral
processes in the plant life cycle. They act in concert with (or opposition) other plant
hormones. For example, the ratio of auxin to cytokinin in certain plant tissues
determines initiation of root vs. shoot buds.
The plant hormones stimulate cell elongation. Auxin induces new root formation
by breaking root apical dominance induced by cytokinins. However, high concen-
tration of auxin inhibits root elongation and instead enhances adventitious root
formation. In low concentration, auxin can inhibit ethylene formation and transport
of precursors in plant; however, high concentration of auxin can induce the synthesis
of ethylene.
Auxin production is widespread among many mycorrhizal fungi (Gay 1986; Gay
and Debaud 1987; Frankenberger and Poth 1987; Kampert and Strzelczyk 1989).
Several studies have demonstrated increased auxin content (hyperauxiny) in
response to mycorrhizal infection, which may indicate a role of auxin in EM
symbiosis. Studies initiated by Slankis (1950) had shown that auxins as well as
cytokinins are necessary for the formation of mycorrhizal structures.
Auxins added to the synthetic media inhibited elongation of pine seedlings
roots. The roots became thicker and dichotomically branched devoid of roots
hairs and caps, structures characteristic for the nonmycorrhizal roots.
O
OH
N
H IAA
Many studies indicate that changes in auxin balance are a prerequisite for
mycorrhiza organogenesis (Gay et al. 1994; Martin et al. 2001). EM fungi enhance
proliferation of short roots and the presence of plant-derived tryptophan in the root
exudates could be sufficient for EM fungi to enhance the biosynthesis of fungal IAA
(Rupp et al. 1989).
Although the structure of ectomycorrhizae in a natural habitat may show consid-

erable variation, common features are a swollen appearance, lack of root hairs, and
variable radial growth of cortical cells within the swollen region (Slankis 1973).
The fact that roots are very sensitive to auxins and that auxins take part in many
physiological and metabolic processes can be expected that the presence of excess
auxin in mycorrhizae would profoundly affect their physiology and metabolism.
EM roots morphology reflect a specific physiological and metabolic state which is
necessary for the functioning of the symbiosis.
According to Slankis (1973), the hyperauxiny in mycorrhizal roots is more
likely to result from the host plant’s endogenous auxins than from the fungus
auxins. However, seedlings of pine inoculated with mutant strain of Hebeloma
crustuliniforme that overproduced IAA generated an increased number of EM
roots (Gay et al. 1994).
16.4.2 Cytokinins
Cytokinins are N6-substituted aminopurines, including ribosides, ribotides, and

glucosides. These are adenine derivatives characterized by their ability to induce
cell division in tissue culture in the presence of auxins. The most common cytokinin
in plants is zeatin, which is converted to other cytokinins. Over 40 cytokinins have
been characterized in plant tissues (McGaw and Burch 1995).
Cytokinins are responsible for the translocation of carbohydrates to the mycor-
rhizal roots. They also act indirectly on the activity of auxins (Gogala 1991).
Several mycorrhizal fungi have been shown to be capable of producing cytoki-
nins in vitro. However, it is unclear whether these fungi that are capable of
producing cytokinins also do so in association with the macrosymbiont (Arshad
and Frankerberger 1998). No direct, unequivocal evidence indicates that cytokinins
are a prerequisite for the formation of mycorrhizae. However, higher cytokinin
levels in mycorrhizal plants have been reported, but the source of increased
cytokinin levels in mycorrhizal plants is somewhat unresolved.
Allen et al. (1980) reported higher cytokinin activity in mycorrhizal plants
compared with noninfected (control) plants. Similarly, Thiagarajan and Ahmad
(1994) reported significantly greater cytokinin content (156%) in mycorrhizal roots
compared to nonmycorrhizal roots.
Several other studies confirmed these findings and provided evidence that
inoculation with mycorrhizal fungi results in increasing the endogenous cytokinin
contents of host plants (Dixon 1989; Danneberg et al. 1992).
In the plant root zone (rhizosphere), there are also plant growth regulators
elaborated by microorganisms accompanying mycorrhizae (Strzelczyk and Pokojska-
Burdziej 1984) and those originating from the root exudates (Gogala 1991).
HO
NH
H
N
N
N N Zeatin
Little is known about the direct effects of these compounds on mycorrhizal fungi.
However, Pokojska et al. (1993) showed differences in the effects of plant regulators
on mycorrhizal fungi (H. crustuliniforme, Laccaria laccata, Rhizopogon vinicolor)
depending upon the kind of hormones, its concentration, and the kind of fungus.
Kinetin inhibited biomass production by L. laccata in a liquid medium but it did
not inhibit the linear growth of this fungus on agar medium. Reverse results were
observed with R. vinicolor.
Auxins did not affect the growth of L. laccata, but some of them exhibited
both inhibitory and stimulatory effects on the growth of H. crustuliniforme and
R. vinicolor depending upon the concentration and type of the medium.
Gogala and Pohleven (1976) have shown that cytokinins promoted the mycelial
growth of S. variegatus and affected the content of K, Ca, P, and Na in the
mycelium of this fungus. In the presence of kinetin, the uptake of Cd, Zn, P by
some EM fungi increased significantly (Stegnar et al. 1978).
The importance of auxins and cytokinins in plant growth and development is
known. The role of these substances in microorganisms is not elucidated as yet.
According to the data obtained from the literature, it can be assumed that auxins and
cytokinins do not play the role of hormonal factor in microorganisms (Gogala and
Pohleven 1976; Pohleven and Gogala 1986; Gogala 1989; Pokojska et al. 1993).
16.4.3 Gibberellins (GAs)
GAs are tetracyclic diterpenoid acids with an ent-gibberellane ring system. Meva-
lonic acid is the primary precursor of GAs biosynthesis in plants.
Although the most widely recognized gibberellin is GA3 (gibberellic acid)
which is a fungal product, the most active GA in plants is GA1 which is primarily
responsible for stem elongation (Arshad and Frankerberger 1998).
O
OH
CO CH2
HO
H3C COOH
Gibberelic acid
Very little work has been conducted on the detection of GAs released by
mycorrhizal fungi. Gogala (1971) detected gibberellin-like substances in culture
medium of the EM fungus Boletus edulis and Ho (1987) in culture of P. tinctorius.
Strzelczyk et al. (1975) found gibberellin-like substances produced by Suillus
bovinus, H. crustuliniforme, and C. graniforme.
16.4.4 Ethylene
Ethylene is synthesized from methionine in many plant tissues, mostly in response

to stress (Arshad and Frankerberger 1998). It is the only hydrocarbon (C2H4) with a
pronounced effect on plants and is involved in developmental processes, from
germination of seeds to senescence of various organs.
H2C¼CH2
Graham and Linderman (1980) found that EM fungi produced ethylene when
grown in medium containing methionine. DeVries et al. (1987) noted an apparent
correlation between C2H4 production and morphological effects, such as stimula-
tion of lateral root formation by mycorrhizal fungi.
16.4.5 Abscisic Acid
ABA is a sesquiterpene, derived from mevalonic acid. ABA appears to act as much
as a promotor (e.g., storage protein), as an inhibitor, and a more open attitude
toward its overall role in plant development is warranted (Davies 1995).
COOH
OH
O
Abscisic acid (ABA)
Production of ABA by mycorrhizal fungi has not been demonstrated as yet;

however, few studies have investigated the alteration in ABA levels in mycorrhizal-
infected plants.
16.5 Sterols
Sterols play an essential role in the physiology of eukaryotic organisms. Sterols are
also known as steroid alcohols. They are a subgroup of steroids with a hydroxyl
group. They are amphipathic lipids synthesized from acetyl-coenzymes.
Ergosterol is a component of fungal cell membranes, serving the same function
that cholesterol serves in animal cells. Ergosterol is used as an indicator of fungal
biomass in soil.
Ergosterol
The composition of fatty acids and sterols in soil lipid fraction is often used as an
indicator for the changes of soil microorganisms.
Laczko et al. (2004) performed an experiment in which seedlings of Pinus
sylvestris and EM fungus P. tinctorius were grown separately or combined to
form ectomycorrhiza. Fatty acids of the neutral lipid fraction (NLFAs) and the
phospholipids fraction (PLFAs) as well as sterol were identified. When grown
separately, the two organisms differed strongly with respect to the sterol composi-
tion. Sterols had a much higher relative abundance in the fungus in comparison with
the plant and the two main fungal sterols, ergosterol and 24-ethyllanosta-8,24(24)-
diene-3 beta, 22 zeta-chiol (Et lano 8.24) as well as six minor fungal sterols were
not found in the plant roots. When the fungus and plant were brought together, there
was a drastic change in the lipid composition of the root.
It was detected that in symbiosis, the fungus transports plant lipids from the
symbiotic interface to the extramatrical mycelium. Concerning sterols, the extra-
matrical mycelium acquired only a small amount of plant-specific sterols. However,
its ergosterol content steadily decreased whereas the content of Et lano 8,24
remained high, causing the ratio of these two sterols to decrease from 1:70 to
1:20, whereas in the EM roots, the opposite phenomenon occurred, so that the ratio
increased to a value of almost 1:1.
These results showed that an EM fungus may display markedly different lipid
composition in its intraradical and extraradical part and highlight a potential role of
plant lipid transfer from the root to the fungus in the functioning the of EM symbiosis.
16.6 Conclusions and Future Perspectives
Production of SM is widespread among plants and rhizosphere microorganisms

including mycorrhizal fungi. However, conditions in the rhizosphere are often quite
variable and many factors, mainly availability of nutrients composition and amount
of root exudates as well as interaction between rhizosphere microorganisms, can
affect the synthesis of SM by plants and microorganisms associated with plant root.
Research on the role of SM in the initiation development function of mycorrhiza
is mainly concerned with two tasks: the determination of their role in the meta-
bolism, growth, and development of the mycorrhizal fungi and the determination
of their role in root morphology in the growth of the entire plant and in causing
metabolic changes in plants.
Widespread ability of mycorrhizal fungi to produce plant hormones in culture

media and induction of mycorrhizal-like changes in response to exogenous applica-
tion of plant hormones favor the speculation that fungal hormones may have a role in
establishment of the symbiotic relation and in the physiology of mycorrhizal plants.
However, physiology of hormones released by the microsymbiont and the role of
these metabolites in the symbiotic association are still poorly understood. Very
little is known about the molecules regulating the interaction between plants and
EM fungi during root colonization.
The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and
questioned, suggesting that, if fungal auxin controls some steps of colonized root
development, its activity might be tightly controlled in time and in space by plant
and/or fungal regulatory mechanisms.
Increase in auxin synthesis or auxin accumulation was noted in most plant–
microbe interactions in plant tissues. However, in some interactions (e.g., P. tinctorius
and Eucalyptus) downregulation of the auxin activity in the host plant was
observed.
It is assumed that hypaphorine (betaine of tryptophan) might be the specific IAA
antagonist.
Despite increasing evidence, the role of flavonoids and other phenolic sub-
stances in EM symbiosis is yet contradictory. The change in the balance of plant
hormones and other SM have yet to be examined for plant development and first
signal for initiation of mutualistic symbiosis. Understanding of these problems
could be of great ecological benefit to the agriculture and forestry industry.
A large number of studies have verified that multiplicity of signals and diversity
of signaling pathways exist during the establishment of mycorrhizal associations
with regulation of symbiosis-specific genes expression.
In presymbiotic phase, plant and their fungal partner secrete signals into soil,
mostly SM inducing morphological and physiological changes. The nature of the
signals released by the EM symbionts and processes triggering the expression of
genes that participate and regulate symbiosis in partner recognition are only the
beginning to be understood. Although many genes have been identified in various
EM association, the product of which play role in recognition and attachment of the
mycobiont on the root surface remains unknown. Many questions concerning the
differentiation of plant and fungal symbiotic structure are also poorly recognized. It
is interesting to be analyzed how is elicitor’s signal achieved depending on the
activation factors and which substances participate in this signaling network.
References
Allen MF, Moore TS, Christensen M (1980) Phytohormone changes in Bouteloua gracilis infected
by vesicular-arbuscular mycorrhizae: I. Cytokinin increases in the host plant. Can J Bot 58:
371–374
Arshad M, Frankerberger WT (1998) Plant growth-regulating substances in the rhizosphere:
microbial production and functions. Advan Agron 62:45–151
Asiegbu FO, Johansson M, Woodward S, H€ utterman A (1998) Biochemistry of the host–parasite

interaction. In: Woodward S, Stenlid J, Karialajnen R, H€utterman A (eds) Heterobasidion
annosum: Biology, ecology, impact and control. University Press, Cambridge, pp 167–193
Baron C, Zambryski PC (1995) The plant response in pathogenesis, symbiosis, and wounding:
variations on a common theme? Ann Rev Genet 29:107–129
Béguiristain T, Lapeyrie F (1997) Host plant stimulates hypaphorine accumulation in Pisolithus
tinctorius hyphae during ectomycorrhizal infection while excreted fungal hypaphorine controls
root hair development. New Phytol 136:525–532
Beyler M, Heyser W (1997) The influence of mycorrhizal colonization on growth in the green-
house and on catechin, epicatechin and procyanidin in roots of Fagus sylvatica L. Mycorhhiza
7:171–177
Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA, Taiz L, Muday GK
(2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant
Physiol 126:524–535
Danneberg G, Latus C, Zimmer W, Hundeshagen B, Schneider-Poetsch H, Bothe H (1992)
Influence of vesicular-arbuscular mycorrhiza on phytohormone balances in maize (Zea mays L.).
J Plant Physiol 141:33–39
Davies PJ (1995) The plant hormones: their nature, occurence, and functions. In: Davies PJ (ed)
Plant hormones: physiology, biochemistry and molecular biology. Kluwer Academic Publish-
ers, Dordrecht, The Netherlands, pp 1–12
DeVries HE, Mudge KW, Lardner JP (1987) Ethylene production by several ectomycorrhizal
fungi and effects on host root morphology. In: Sylvia EM, Hung LL, Graham JH (eds)
Mycorrhizae in the next decade. Institute of Food and Agricultural Sciences, University of
Florida, Gainesville, p 245
Ditengou FA, Béguiristain T, Lapeyrie F (2000) Root hair elongation is inhibited by hypaphorine,
the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by IAA.
Planta 211:722–728
Dixon RK (1989) Cytokinin activity in Citrus jambhiri seedlings colonized by mycorrhizal fungi.
Agric Ecosystems Environ 29:103–106
Frankenberger WT, Poth M (1987) Biosynthesis of indole-3-acetic acid by the pine ectomycor-
rhizal fungus Pisolithus tinctorius. Appl Environ Microbiol 53:2908–2913
Garcia-Garrido JM, Ocampo JA (2002) Regulation of the plant defence response in arbuscular
mycorrhizal symbiosis. J Exp Bot 53:1377–1386
Gay G (1986) Effect of glucose on indole-3-acetic production by the ectomycorrhizal fungus
Hebeloma hiemale in pure culture. Physiol Veg 24:185–192
Gay G, Debaud JC (1987) Genetic study on indole-3-acetic acid production by ectomycorrhizal
Hebeloma species: inter- and intraspecific variability in homo- and dikaryotic mycelia. Appl
Microbiol Biotechnol 26:141–146. doi:10.1007/BF00253898
Gay G, Normand L, Marmeisse R, Sotta B, Debaud JC (1994) Auxin overproducer mutants of
Hebeloma cylindrosporum Romagnési have increased mycorrhizal activity. New Phytol 128:
645–657
Gogala N (1971) Growth substances in mycorrhiza of the fungus Boletus pinicola Vitt. and the
pine-tree Pinus sylvestris L. Razprave 14:123–202
Gogala N (1989) Growth substances in root exudate Pinus sylvestris – their influence on mycor-
rhizal fungi (effect of jasmonic acid). Agric Ecosystems Environ 28:151–154
Gogala N (1991) Regulation of mycorrhizal infection by hormonal factors produced by hosts and
fungi. Experimentia 47:331–339
Gogala N, Pohleven F (1976) The effect of cytokinins and auxins on the growth of mycorrhizal
fungus Suillus variegatus. Acta Bot Croat 35:129–134
Graham JH, Linderman RG (1980) Ethylene production by ectomycorrhizal fungi, Fusarium
oxysporum f. sp. pini, and by aseptically synthesized ectomycorrhizae and Fusarium-infected
Douglas fir roots. Can J Microbiol 26:1340–1347
Higuchi T (1985) Biosynthesis and biodegradation of wood components. Academic Press,

Orlando, p 679
Ho I (1987) Comparison of eight Pisolithus tinctorius isolates for growth rate, enzyme activity,
and phytohormone production. Can J For Res 17:31–35
Horan DP, Chilvers GA, Lapeyrie F (1988) Time sequence of the infection process in eucalypt
Kakiuchi Y, Gàlis I, Tamogami S, Wabiko H (2006) Reduction of polar auxin transport in tobacco
by the tumorigenic Agrobacterium tumefaciens AK-6b gene. Planta 223:237–242
Kampert M, Strzelczyk E (1989) Effect of amino acids on cytokinin-like substances production by
micorrhizal fungi of pine (Pinus sylvestris L.). Agric Ecosyst Environ 28:219–228
Koide RT, Suomi L, Stevens CM, McCormick L (1998) Interactions between needles of Pinus
resinosa and ectomycorrhizal fungi. New Phytol 140:539–547
Kottke I, Oberwinkler F (1987) The cellular structure of the Hartig net: coenocytic and transfer
cell-like organization. Nordic J Bot 7:85–95
Krupa S, Andersson J, Marx DH (1973) Studies on ectomicorrhizae of pine IV. Volatile organic
constituents in micorrhizal and nonmycorrhizal root systems of Pinus echinata Mill. Eur J For
Pathol 3:194–200
Laczko E, Boller T, Wiemken V (2004) Lipids in roots of Pinus sylvestris seedlings and in mycelia
of Pisolithus tinctorius during ectomycorrhiza formation: changes in fatty acid and sterol
composition. Plant Cell Environ 27:27–40
Lagrange H, Jay-Allemand C, Lapeyrie F (2001) Rutin, the phenolglycoside from Eucalyptus root
exudates, stimulates Pisolithus hyphal growth at picomolar concentrations. New Phytol
150:349–355
Laitinen ML, Julkunen-Tiitto R, Tahvanainen J, Heinonen J, Rousi M (2005) Variation in birch
(Betula pendula) shoot secondary chemistry due to genotype, environment, and ontogeny.
J Chem Ecol 31:697–717
Martin F, Duplessis S, Ditengou F, Lagrange H, Voiblet C, Lapeyrie F (2001) Developmental
cross talking in the ectomycorrhizal symbiosis: signals and communication genes. New Phytol
151:145–154
McGaw BA, Burch LR (1995) Cytokinin biosynthesis and metabolism. In: Davies PJ (ed) Plant
hormones: physiology, biochemistry and molecular biology. Kluwer Academic Publishers,
Dordrecht, The Netherlands, pp 98–117
Mekin E, Krupa S (1971) Studies on ectomicorrhizae of pine. II. Growth inhibition of mycorrhizal
fungi by volatile organic constituents of Pinus silvestis L. (Scots pine) roots. Physiol Plantarum
25:337–340
Molina E, Trappe JM (1982) Patterns of ectomycorrhizal host specificity and potential among
pacific northwest conifers and fungi. Forest Sci 28:423–458
Napierała-Filipiak A, Werner A, Mardarowicz M, Gawdzik J (2002) Concentrations of terpenes in
micorrhizal roots of Scots pine (Pinus sylvestris L.) seedlings grown in vitro. Acta Physiol
Plant 24:137–143
Niemi K, Julkunen-Tiitto R, H€aggman H, Sarjala T (2007) Suillus variegatus causes significant
changes in the content of individual polyamines and flavonoids in Scots pine seedlings during
mycorrhiza formation in vitro. J Exp Bot 58:391–401. doi:10.1093/jxb/erl209
Peer WA, Bandyopadhyay A, Blakeslee AA, Makam SN, Chen RJ, Masson PH, Murphy AS
(2004) Variation in expression and protein localization of the PIN family of auxin efflux
facilitator proteins in flavonoid mutatnts with altered auxin transport in Arabidopsis thaliana.
Plant Cell 16:1898–1911
Pohleven F, Gogala N (1986) The influence of natural cytokinins on the content of K, P, Ca and Na
in the mycelium of the mycorrhizal fungus Suillus variegatus. Biol Vestn 34:79–88
Pokojska A, Strzelczyk E, Li CY, Rozycki H, Szablewska M (1993) Effect of plant growth
regulators on growth of ectomycorrhizal fungi. Cryptogam Bot 4:8–13
Prior C (1976) Resistance by Corsican pine to Fomes annosus. Ann Bot 40:261–279
Rupp LA, Mudge KW, Negm FB (1989) Involvement of ethylene in ectomycorrhiza formation
and dichotomous branching of roots of mugo pine seedlings. Can J Bot 67:477–482
Sch€utzend€ubel A, Polle A (2004) Plant responses to abiotic stress: heavy metal-induced oxidative
stress and protection by mycorrhization. J Exp Bot 53:1351–1365
Slankis V (1950) Effect of a napthaleneacetic acid on dichotomus branching of isolated roots of
Pinus silvestris. Physiol Plantarum 3:40
Slankis V (1973) Hormonal relationships in mycorrhizal development. In: Marks GC, Kozlowski
TT (eds) Ectomycorrhizae: their ecology and physiology. Academic Press, New York,
pp 231–298
Smith WH (1987) The atmosphere and the rhizosphere: linkages with potential significance for
forest tree health. In: Blasser RO (ed) Technical Bulletin of national council of the paper
industry for air and stream improvements, vol. 527. New York, pp 30–94
Stegnar P, Gogala N, Pohleven F (1978) The uptake of cadmium, zinc, phosphorus, and plant
hormone kinetin by ectomycorrhizal fungi. Acta Bot Croat 37:67–73
Strzelczyk E, Pokojska-Burdziej A (1984) Production of auxins and gibberellin-like substances
production by mycorrhizal fungi, bacteria and actinomycetes isolated from soil and mycor-
rhizosphere of pine (Pinus sylvestris L.). Plant Soil 81:185–194
Strzelczyk E, Sitek JM, Kowalski S (1975) Production of gibberelin-like substances by fungi
isolated from mycorrhizae of pine (Pinus silvestris L.). Acta Microbiol Pol 7:145–153
Taylor LP, Grotewold E (2005) Flavoniods as developmental regulators. Curr Opinion Plant Biol
8:317–323
Thiagarajan TR, Ahmad MH (1994) Phosphatase activity and citokinin content in cowpeas (Vagna
unguiculata) inoculated with a vesicular-arbuscular mycorrhizal fungus. Biol Fertil Soils
17:51–56
Varese TC, Portinaro S, Trotta A, Scannerini S, Luppi-Mosca AM, Martinotti MG (1996) Bacteria
associated with Suillus grevillei sporocarps and ectendomycorrhizae and their effects on
in vitro growth of the mycobiont. Symbiosis-Rehovot 21:113–129
Weiss M, Mikolajewski S, Peipp H, Schmitt U, Schmidt J, Wray V, Strack D (1997) Tissue-
specific and developmental-dependent accumulation of phenylpropanoids in larch mycorrhi-
zas. Plant Physiol 114:15–27
Weiss M, Schmidt J, Neumann D, Wray V, Crist R, Strack D (1999) Phenylpropanoids in
mycorrhizas of the Pinaceae. Planta 208:491–502
Werner A (1993) Pathological anatomy of thin woody roots of Scots pine invaded by Hetero-
basidion annosum (Fr.) Bref. Arboretum Kórnickie 38:113–129
Wink M (2008) Plant secondary metabolism: diversity, function and its evolution. Nat Prod
Communicat 3:1205–1216
Zhi-lin Y, Chuan-chao D, Lian-qing C (2007) Regulation and accumulation of secondary
metabolites in plant-fungus symbiotic system. Afr J Biotechnol 6:1266–1271
.
Chapter 17
C:N Interactions and the Cost:Benefit Balance
in Ectomycorrhizae
Ana Corrêa and Maria-Amélia Martins-Loução
17.1 Introduction
Mycorrhizal (M) symbioses are known to improve plant nutrient uptake due to the
fine exploration of the substrate by the fungal hyphae, while in return the plant
supplies the fungus with carbohydrates that are essential for the completion of its
life cycle.
The fungal hyphae constitute an extension of the nutrient absorption surface
since they penetrate the soil more extensively than the root. The nutrients can be
translocated through the depletion zones surrounding the roots, and inaccessible
sources of nutrients can be mobilized. Due to their high surface-to-volume ratio and
capacity for rapid exponential growth, fungal hyphae are superior to roots in the
acquisition of immobile nutrient ions from the low and both spatially and tempo-
rally variable nutrient concentrations in natural soils. Fungi may also have access to
other nutrient sources than plant roots, namely through production of degradative
extracellular enzymes or organic acids (Leake and Read 1990; Zhu et al. 1994).
In particular, the role of ECM in improving the nitrogen (N) nutrition of the host
plant is of central importance in this association, as ECM fungi evolved, and ECM
interactions predominate, in N-limiting ecosystems (Martin and Botton 1993; Read
and Pérez-Moreno 2003).
At the other end of the mycorrhizal partnership, the fungus is also dependent on
the carbon (C) supply by the plant. A substantial part of the C fixed through
A. Corrêa (*)
Departamento de Microbiologı́a del Suelo y Sistemas Simbióticos, Estación Experimental del
Zaidı́n, CSIC, Profesor Albareda 1, 18008 Granada, Spain
and
Universidade de Lisboa, Faculdade de Ciências, Centro de Biologia Ambiental, Campo Grande,
1749-016 Lisboa, Portugal
e-mail: ambcorrea@gmail.com
M.-A. Martins-Loução
Universidade de Lisboa, Faculdade de Ciências, Centro de Biologia Ambiental, Campo Grande,
1749-016 Lisboa, Portugal

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_17,
388 A. Corrêa and M.-A. Martins-Loução
photosynthesis is allocated to the fungus, for formation, maintenance, and function-

ing of mycorrhizal structures (Jones et al. 1991; Wu et al. 2002; Heinemeyer et al.
2006). Host-derived carbohydrates are necessary for the mycorrhizal fungus as
precursors for the synthesis of fungus-specific carbohydrates and sugar-alcohols
like trehalose, glycogen or mannitol (Fajardo-López et al. 2007; Deveau et al.
2008), and fruit-body production has been shown to be dependent on the current
photoassimilates provided by the host plant (Lamhamedi et al. 1994).
This exchange of nutrients for C is the very core of the association. Unraveling the
C and nutrient fluxes between symbionts is therefore essential for its understanding.
17.2 Nitrogen
The ability of ECM fungi to take up inorganic nitrogen and transport nitrogen-
containing solutes to their host plant is well established (Chalot and Brun 1998;
Chalot et al. 2002). M roots and external hyphae have been found to have more
transporters and higher N uptake rates than nonmycorrhizal (NM) roots (Javelle
et al. 1999; Selle et al. 2005).
More specifically, ECM fungi can help increase ammonium (NH4þ) uptake. Most
of the tree species present in ECM-dominated ecosystems, as well as most ECM
fungi, prefer NH4þ to nitrate (NO3) as N source, presenting higher NH4þ uptake
rates and growth than the ones measured for NO3 (Eltrop and Finlay et al. 1992;
Marschner 1996; Anderson et al. 1999; Plassard et al. 2000; Fig. 17.1). The ECM
fungus Laccaria bicolor was found to have a widely expanded NH4þ transporter
family (AMT) when compared with other basidiomycetes, indicating a higher poten-
tial for its uptake (Lucic et al. 2008). Correspondingly, the uptake of NH4þ is
generally improved in ECM plants, whereas the same has less frequently been
observed for NO3 (Eltrop and Marschner 1996; Plassard et al. 2000). The impor-
tance of the external hyphae as NH4þ-absorbing structures in ECM roots has been
demonstrated. Hyphal NH4þ acquisition was observed to contribute with 45–73% of
total plant N uptake under N deficiency (Brandes et al. 1998; Jentschke et al. 2001),
and the 15N ammonium uptake by birch seedlings was greatly decreased when the
external mycelium was disrupted (Javelle et al. 1999).
Organic N may also play an important part in N nutrition of both ECM fungi and
their host plants. In pure culture, some ECM fungi have been shown to readily
assimilate amino acids (a.a.) and amides that appear to occur in significant amounts
in soil solution such as glutamine, glutamate, and alanine (Anderson et al. 1999;
Tibbett et al. 2000; Fig. 17.1). Recent evidence has been reported that this is also the
case under natural conditions (Treseder et al. 2008). Genes encoding a.a. transpor-
ters have been isolated in A. muscaria (Nehls et al. 1999) and Hebeloma cylin-
drosporum (Wipf et al. 2002), and evidence has been obtained of the existence of a
proton symport mechanism for a.a. uptake in Paxillus involutus (Chalot et al. 1996),
17 C:N Interactions and the Cost:Benefit Balance in Ectomycorrhizae 389
ERM IRM Interfacial Apoplast Plant Cell
Urea Urea
f
Protein +
NH4
Peptides Peptides b
Amino acids Amino acids Amino acids
Amino
Amino acids
acids
NH3
Gln Amino acids
NH3
a
d
Arg Arg NH3 NH3 NH3
c
+ +
NH4 NH4 + + +
Urea NH4 NH4 NH4 Gln
e +
NH4
NO3
–
NO3
– k
NADPH, ATP,
NADPH, ATP, C skeletons l
C skeletons Glucose
Glycogen
j Glucose6P
h
Sucrose Sucrose
Trehalose/ Trehalose/ g
mannitol mannitol i Fructose
Fig. 17.1 Possible N and C transfer mechanisms, and their interactions. (a) Most ECM fungi
prefer NH4þ as N source, which is taken up through plasma membrane fungal Amts. They can also
take and use NO3 and several forms of organic N. Evidence has been obtained of a proton
symport mechanism for a.a. uptake (Chalot et al. 1996), general a.a. permeases (Nehls et al. 1999;
Chalot et al. 2002, 2006), and transporters of a.a. (Nehls et al. 1999; Wipf et al. 2002), peptides
(Bendjia et al. 2006) and urea (Morel et al. 2008). (b) a.a. are thought to be the main sink for the
assimilated N, and the N form that is transported inside the mycelium and to the mantle, where the
absorption by the root system takes place. No transport mechanisms have been identified so far, but
a.a. transfer from the fungus to the plant has been confirmed (e.g., Blaudez et al. 2001). (c) The
direct transfer of arginine-derived NH4þ has also been suggested (Selle et al. 2005; Chalot et al.
2006). (d) Transfer of NH4þ-derived NH3 has also been hypothesized (Chalot et al. 2006). NH3
transfer to the apoplast may happen through passive diffusion, driven by a favorable concentration
gradient, or through ammonia-loaded vesicles which would fuse with the plasma membrane and
release ammonia into the interfacial apoplast. (e) Transfer of NH4þ from the apoplast to the plant
host cytoplasm might involve plant Amts or nonspecific channels such as aquaammoniaporins. (f)
The number of fungal a.a. and NH4þ transporters may be decreased at the plasma membrane in
symbiotic hyphae compared with those in extraradical hyphae. However, the fungus may also
regulate the supply of N to the plant by increasing or decreasing the number of transporters, and the
competition with the plant by N reuptake. (g) Sucrose is supposed to be carbohydrate released by
the plant into the interfacial apoplast. The mechanism is still unknown. Sucrose is then hydrolysed
into monosaccharides, either by plant-derived cell wall invertases, or nonenzymatic inversion due
to the acidity of the apoplast. (h) From the resulting glucose and fructose, the fungus preferentially
uses glucose. (i) Hexose phosphates are utilized for generation of energy/carbon skeletons or
storage and transport compounds (trehalose/mannitol or glycogen) (Deveau et al. 2008). (j)
Trehalose/mannitol are presumably used for long-distance carbohydrate transport towards ERM
hyphae. In basidiomycetes, transport is supposed to be enabled by dolipore-guarded cell–cell
connections. (k) The energy/carbon skeletons obtained from trehalose/mannitol in the ERM are,
among other uses, used in N uptake and assimilation, which will partly be channeled towards the
and general a.a. permeases in P. involutus and A. muscaria (Chalot et al. 2002,
2006, Nehls et al. 1999).
Some fungal species have also been shown to be able to use peptides or protein
as N source, namely through production of proteinases (Zhu et al. 1994; Anderson
et al. 1999; Bendjia et al. 2006; Fig. 17.1). Recently, an urea transporter was
isolated in P. involutus (Morel et al. 2008), and peptide transporters have been
identified in H. cylindrosporum and shown to mediate dipeptide uptake (Bendjia
et al. 2006).
Mycorrhization was observed to enhance the use of a.a., peptides or protein by
the host plant (Blaudez et al. 2001; Finlay et al. 1992; Tibbett et al. 2000) or to
enable its use in plants that are unable to do so when nonmycorrhizal (Abuzinadah
and Read 1989). The uptake of 14C glutamate by ECM plants has been observed to
increase by up to eight times (Blaudez et al. 2001). In addition, expression and
activity of the enzyme L-amino acid oxidase, with a potential role in N mineraliza-
tion from a.a. at the ecosystem level, has been found in ECM fungi (Nuutinen and
Timonen 2008).
17.2.1 N Assimilation, Transport, and Transference to the Plant
Following its uptake by the fungus, NH4þ is not accumulated as a free ion in the
mycelial tissue but quickly assimilated at considerable distances of the fungal
mantle. Several studies suggest a.a. as the main sink for the assimilated N, and
the N form that is transported inside the mycelium and to the mantle, where the
absorption by the root system takes place (Finlay et al. 1988; Ek et al. 1994;
Fig. 17.1).
N assimilation in ECM fungi, similarly to plants, can be made through the same
two main and alternative pathways: the NADP-glutamate-dehydrogenase (GDH, E.C.
1.4.1.4) and the glutamine synthetase (GS, E.C. 6.3.1.2)/glutamate synthase
(GOGAT, E.C. 1.4.1.13) pathways. Both can be present in ECM fungi, the relative
importance of each differing according to fungal species, the physiological state of
the mycelium, and the mycorrhizal partner (Martin and Botton 1993; Rudawska
et al. 1994). The uptake of both NH4þ and a.a. has been reported to involve
GS/GOGAT, and this seems to be the main assimilation pathway similarly to
what happens in plants (Wright et al. 2005; Morel et al. 2006).
Fig. 17.1 (continued) plant partner. (l) Rhizodermal cells can compete with fungal hyphae for
glucose and/or can restrict fructose content in the common apoplast by hexose uptake. This
competition for hexoses has been proposed to increase if the fungus fails to supply the plant
with adequate amounts of nutrients, and therefore serve as a regulatory mechanism linking C
transfer to the fungus with the fungal N supply (Nehls 2008). Modified from Chalot et al. (2006)
and Nehls et al. (2010). ERM extraradical mycelium; IRM intraradical mycelium; Gln glutamine;
Arg arginine
The main form of N transferred to the host plant is glutamine, followed by

glutamate, alanine, and aspartate-asparagine (Finlay et al. 1988, 1992; Blaudez
et al. 1998, 2001). The extension of the transference of other N compounds through
the mantle is not clear, but the direct transfer of NH4þ, arginine derived, as
hypothesized for AM symbiosis has also been suggested for ECM (Chalot et al.
2006; Fig. 17.1). In support of this hypothesis, a NH4þ importer of poplar and the
L. bicolor NH4þ transporter LbAMT2.2 were found to be highly upregulated in
ECM roots and in symbiotic tissues, respectively (Couturier et al. 2007; Selle et al.
2005). The transfer of NH3 has also been suggested (Chalot et al. 2006).
Both organic and inorganic N transfer may coexist. It has been suggested that the
transfer of organic N may be larger in conditions of C sufficiency, when larger
incorporation of NH4+ into C skeletons may be expected, and that transfer of N may
be increased under C deficiency (Chalot et al. 2006).
The physiological processes involved in the transfer of N from fungus to plant
within the symbiotic tissue are still poorly understood. N transporters from plant
and fungal partners have been isolated (Javelle et al. 2003a, b; Lucic et al. 2008),
but their role and regulation is still under investigation, as is their localization in
mycorrhizal tissues (M€ uller et al. 2007; Fig. 17.1). On the other hand, little is
known about the metabolic zonation of N assimilation in ECM. The GS-GOGAT
cycle may take place entirely in the fungus, and the newly formed glutamine be
exported into the plant where it is converted into the remaining amino acids. It may,
however, also be separated, and GS could be active in the fungus and GOGAT in
the root, in which case there could be a glutamine–glutamate shuttle across the
interface (Martin and Botton 1993).
17.3 Carbon
ECM associations have a deep effect in plant C metabolism, generally increasing

the C allocation to the roots (e.g., Jones et al. 1991; Rygiewicz and Andersen 1994;
Wu et al. 2002), although this is not always the case (Jones et al. 1998). An
important percentage of this C is allocated to mycorrhizae and fungal mycelia,
indicating that the fungal partner is an important sink for the hosts’ carbohydrates
(Wu et al. 2002). Furthermore, mycorrhizae may impose a higher C cost on their
hosts under field conditions than under laboratory conditions (Rygiewicz and
Andersen 1994). Correspondingly, the expression of hexose importers from ECM
fungi was found to be enhanced in symbiotic tissues, indicating enhanced fungal
hexose uptake in symbiosis, and to be sugar-regulated, being activated by increased
in vivo concentration of monosacharides at the plant–fungus interface (reviewed by
Nehls 2008), while the expression of hexose transporters in spruce (Nehls et al.
2000) and birch (Wright et al. 2000) root cells was decreased in symbiosis.
As a result, mycorrhization can upregulate the host plant’s rate of net photo-
synthesis and sucrose synthesis (Dosskey et al. 1990; Colpaert et al. 1996;
Loewe et al. 2000). Mycorrhizal effects on photosynthesis are, however, very
varied, and in some cases an explanation for them has not been found (Dosskey
et al. 1990).
17.3.1 C Transfer and Use by the Fungus
Sucrose is the main carbohydrate transported by the host plant, but ECM fungi are
unable to use it as C source. Its conversion into glucose and fructose seems to be a
precondition for its use by the fungus. Hexoses are the main carbon compounds
taken and used by ECM fungi, while the significance of other C sources is unclear
(Buscot et al. 2000; Nehls 2008; Fig. 17.1). ECM fungi are able to take up and use
both glucose and fructose, although they show a preference for glucose (Hampp
et al. 1995; Wiese et al. 2000). The hexose transporters from ECM fungi investi-
gated so far clearly favor glucose over fructose uptake (Nehls et al. 1998; Polidori
et al. 2007), and in A. muscaria evidence was found that the same hexose trans-
porters were responsible for carbohydrate uptake in symbiosis (Nehls et al. 1998).
Since so far ECM fungi have been found to have no sucrolytic capacity (Nehls
2008), it has been proposed that sucrolytic enzymes of the host plant are responsible
for sucrose hydrolysis. Plants possess two major types of sucrolytic enzymes:
invertases and sucrose synthase. Because in ECM the plasma membranes of the
plant and fungus do not come into direct contact, and exchanges take place across
the apoplast, the activity of cell wall-bound acid invertase has been hypothesized
to have a potentially major role in this (Nehls 2008), and therefore expected to
increase in M plants. In AM interactions, this has been observed repeatedly (e.g.,
Schaarschmidt et al. 2006), but not in ECM (Schaeffer et al. 1995; Wright et al.
2000; Corrêa et al. 2010). An alternative to a role of plant invertases could be that
nonenzymatic inversion of sucrose takes place, since ECM formation leads to
acidification of the already acidic apoplast (Martin and Selosse 2008).
The hexoses taken by the fungus are quickly converted to fungus-specific
compounds, therefore maintaining an hexose concentration gradient between
plant and fungal tissues, which is thought to maintain the sugar flow toward the
fungus (Fajardo-López et al. 2007; Fig. 17.1). Increased carbohydrate export to the
root and decreased levels of carbohydrates in ECM plants were observed, while
simultaneously the synthesis and contents of fungus-specific compounds such
trehalose and manitol, the two main carbohydrates accumulated in fungi, were
increased (Hampp et al. 1995; Martin et al. 1998; Fajardo-López et al. 2007;
Deveau et al. 2008; Corrêa et al. 2010). Trehalose biosynthesis was found to
occur mainly in hyphae of the plant–fungus interface (Fajardo-López et al. 2007;
Deveau et al. 2008; Fig. 17.1).
17.4 C–N Interactions
The supply of nutrients by the fungus to the plant is deeply connected to the supply
of C to the fungus. The assimilation of inorganic N into a.a. is an important sink for
carbohydrates in the fungal mycelium, and therefore dependent and closely
regulated by the availability of C metabolites, which are ultimately supplied from

host carbohydrates (Martin et al. 1998; Wallenda and Kottke 1998; Fig. 17.1). On
the other hand, plant C metabolism is dependent on N uptake by the root, and the
fungal partner, and its allocation to the shoot. Just as the integration of the plant C
and N metabolisms involve extensive coregulation between the root and the shoot,
and any change in activity of one of them implicates an adjustment in the other
(Foyer and Noctor 2002), so must the integration of the exchanges of N and C
between mycorrhizal partners.
N availability and the plant N needs seem to determinate the amount of C
delivered to the fungus. Both the potential capacity of ECM fungi to colonize the
roots and the growth of extramatrical mycelium increase with decreasing N avail-
ability, indicating that the supply of C to the fungus is higher at low N supply and
plant productivity (Wallander and Nylund 1991, 1992; H€ogberg et al. 2003; Nilsson
and Wallander 2003; Treseder 2004; Hobbie 2006; Corrêa et al. 2008, 2010).
Because the degree of mycorrhizal colonization was observed to be strongly
negatively correlated with shoot N concentration (Wallander and Nylund 1991;
Corrêa et al. 2008), it has been proposed that the N status of the plant regulates the C
supply to the fungus (Nilsson and Wallander 2003).
Increased N supply causes a switch from gluconeogenesis, that is, sucrose and
starch formation, to glycolisis (Wallenda et al. 1996; Wingler et al. 1994), and the
decreased sucrose production has been observed to negatively affect C delivery to
the fungus, resulting in decreased fungal biomass (Wallenda et al. 1996). Further-
more, evidence has been found of a correlation between the transference of 14C
from the plant to the fungus and of 15N from the fungus to the plant at various N
availabilities (Kyt€oviita 2005).
A regulatory mechanism linking N supply to the plant with C transfer from the
plant to the fungus has been proposed which assumes that the transport of N into the
plant–fungus interface decreases the competition between the fungus and the plant
for apoplastic hexoses, and that if the fungus fails to supply the plant with adequate
amounts of nutrients, it will reduce the C supply to the fungus (Nehls 2008;
Fig. 17.1). This control of the carbohydrate efflux by the host plant in symbiosis
has been considered essential to avoid fungal parasitism (Nehls 2008). Several
reports, however, indicate that ECM plants allocate more C to the fungus as the
nutrient availability decreases, even if the amount of nutrients supplied by the
fungus also decreases (Ingestad et al. 1986; Treseder and Allen 2002; H€ogberg
et al. 2003; Hobbie 2006; Corrêa et al. 2008, 2010).
The effects of mycorrhization on C and N metabolisms are, however, not limited
to changes in demand and supply of N and C. A readjustment of metabolic path-
ways has been observed to occur in mycorrhizal roots, starting very early following
contact, as indicated by changes in free a.a. profile, and expression and activities of
N metabolism enzymes. Namely, molecular and biochemical evidences have been
obtained of a deactivation of the root metabolism, and an activation of the fungal
metabolism, in response to mycorrhizae formation (Schaeffer et al. 1996; Blaudez
et al. 1998; Johansson et al. 2004; Duplessis et al. 2005; Frettinger et al. 2007;
Herrmann and Buscot 2007; Corrêa et al. 2010). In some cases, these changes have
been considered a consequence of a metabolic shift, with some metabolic functions
of the root being taken over by the fungus (Vézina et al. 1989; Johansson et al.
2004), namely N assimilation (Wingler et al. 1996). Those results have led to the
idea that ECM symbioses allow plants to slow-down regulative pathways and that
this may compensate the cost of photoassimilate transfer to the fungal partner.
However, recent evidence has been found that decreased metabolic activities in
mycorrhizal roots are not associated with decreased belowground C allocation
(Corrêa et al. 2010). In addition, these metabolic readjustments may not be perma-
nent but alternate between periods of plant metabolic deactivation/fungal activation
and plant activation/fungal deactivation in a cyclic pattern (Corrêa et al. 2010).
17.5 Cost/Benefit and the Symbiotic Continuum
Mycorrhizae are traditionally accepted as being mutualistic symbioses, therefore

resulting in a net beneficial outcome for both plant and fungal partners. However, in
an increasing number of reported cases mycorrhization was found to decrease plant
productivity (e.g., Dosskey et al. 1990; Colpaert et al. 1992, 1996; Eltrop and
Marschner 1996; Plassard et al. 2000). This has led some authors to question the
nature of the interaction, and to the growing belief that its effects on the host plant
can vary from the traditionally accepted mutualistic through to antagonistic in a
continuum of responses (Jonhson et al. 1997; Jones and Smith 2004). It has been
proposed that in any particular host–plant/fungus combination the response can
move along this continuum (Jonhson et al. 1997; Gange and Ayres 1999).
Central to this discussion is the evaluation of what are costs and benefits in
mycorrhizae, and the degree to which the benefits exchanged are by-products or
costly to the symbionts (Herre et al. 1999; Hoeksema and Schwartz 2002; Hoek-
sema and Kummel 2003; de Mazancourt et al. 2005). Mycorrhizae can have
multiple nonnutritional effects on the host plant that may determine increased
survival and fitness such as protection against root and shoot pathogens (e.g.,
Akema and Futai 2005), protection from toxic minerals (e.g., Hildebrandt et al.
2007; see Chaps. 14 and 18 in this volume), or resistance to drought (e.g., Bogeat-
Triboulot et al. 2004). However, it is generally considered that the main benefit to
the plant from mycorrhizae is an improved nutrition, while the cost is the C
expended in growth and maintenance of the fungus. The outcome of the relation-
ship is presumed to depend on the balance between the two, and negative effects of
mycorrhizal colonization are expected to occur when the net C costs for fungal
maintenance and growth exceed the net benefits obtained from improved nutrient
uptake (Jonhson et al. 1997; Tuomi et al. 2001; Jones and Smith 2004). The C and N
exchanges and balances in the association are, therefore, of central importance in
the evaluation of costs and benefits in ECM.
The balance between C cost and nutrient benefit is expected to be reflected in
plant productivity/growth, which can therefore be used as a benefit indicator. The
meaningfulness of growth as benefit indicator has been questioned, and the advan-
tages and disadvantages of this and other possible parameters have been previously
reviewed (Jones and Smith 2004). Nonetheless, growth remains the parameter most
often used to evaluate the plant response to mycorrhization, and in models that
attempt to explain the variation in that response (Schwartz and Hoeksema 1998;
Gange and Ayres 1999; Tuomi et al. 2001; Neuhauser and Fargione 2004; Morgan
et al. 2005; Janos 2007).
The mycorrhizal response seems to vary according to the nutrient availability
(Schwartz and Hoeksema 1998; Tuomi et al. 2001; Neuhauser and Fargione 2004;
Janos 2007). Mycorrhizae have been found to improve N nutrition of the host plant
when N was in limited supply, but not when it was abundant (Dickson et al. 1999;
B€ucking and Heyser 2000). In contrast, evidence was also found of negative effects
of mycorrhization on N uptake and growth, which were more pronounced as N
became more limited (Corrêa et al. 2008, 2010). Another factor that may determine
the mycorrhizal response is the degree of mycorrhizal colonization (Gange and
Ayres 1999; Tuomi et al. 2001). Negative correlations have been found between
plant biomass and the extension of fungal development, the mass of the fungal
mycelium produced or the relative growth rate of the mycelium (Dosskey et al.
1990; Colpaert et al. 1992). These two factors are, however, not independent, since
decreased N availability leads to increased mycorrhization and extraradical fungal
growth (Ingestad et al. 1986; Treseder and Allen 2002; H€ogberg et al. 2003;
Nilsson and Wallander 2003; Corrêa et al. 2008). Some models have been proposed
to explain the variation of mycorrhizal effects on plant productivity that considered
it to depend on the nutrient availability (Morgan et al. 2005; Janos 2007), the
balance between C cost and nutrient gain (Schwartz and Hoeksema 1998; Tuomi
et al. 2001; Neuhauser and Fargione 2004), and the degree of mycorrhization,
either alone or together with nutrient availability (Gange and Ayres 1999; Tuomi
et al. 2001).
Two main possibilities have been advanced as the cause of negative effects of
mycorrhizae on plant growth: (1) excessive fungal C drain and (2) nutrient retention
by the fungus. ECM fungi have a high potential to take up, accumulate, and
immobilize N (Colpaert et al. 1992, 1996; Wallenda and Kottke 1998; Plassard
et al. 2000). However, negative effects on plant growth have generally been
considered to be due to excessive C drain (Colpaert et al. 1992, 1996; Jonhson
et al. 1997; Plassard et al. 2000). Namely, increased rates of photosynthesis that
were not associated to increased growth relative to NM plants have been considered
evidence of this (Colpaert et al. 1996; Conjeaud et al. 1996).
On the other hand, differences in plant performance upon mycorrhization have
been proposed to be due to differences in cost efficiency, that is, nutrients acquired
per C expended. Because mycorrhizae are considered to be most beneficial in
conditions of nutrient deficiency, M plants have been proposed to be more cost
efficient, and grow better, under nutrient poor conditions, and NM plants at high
nutrient levels (Schwartz and Hoeksema 1998; Tuomi et al. 2001). However,
whether M roots or fungal hyphae can be more cost efficient than NM roots is
still a question. Cost efficiency has been calculated to be higher (Jones et al. 1991,
1998), equal (Tuomi et al. 2001) or lower (Jones et al. 1991) in M compared to NM
roots. Some authors have found the construction and maintenance costs lower for
fungal hyphae and M roots than NM roots, and this has been considered the reason
for the greater cost efficiency of M plants (Jones and Smith 2004; Jones et al. 1998,
1991). However, others have found them higher (Colpaert et al. 1996). In addition,
some results suggest that M roots may be less cost efficient than NM roots as P
becomes more limited (Jones et al. 1991, 1998) and in conditions of N limitation
(Colpaert et al. 1996).
Furthermore, although a lower cost efficiency at high levels of N could explain
negative effects of mycorrhization in those conditions, it does not explain situations
of decreased growth when N is limiting, which have also been observed (Ingestad
et al. 1986; Colpaert et al. 1999; Hobbie 2006; Corrêa et al. 2008).
Recent results indicate that in N-limited conditions, the C cost of mycorrhizae,
and by extension their cost efficiency, may have no influence on the growth of M
plants, and that differences in growth are exclusively due to differences in N
uptake (Corrêa et al. 2008; Hobbie et al. 2008). This is in accordance with the
model of Tuomi et al. (2001), which predicts that under nutrient limitation,
mycorrhizae may be beneficial and have a selective advantage, even when they
are less cost efficient.
Under N-limiting conditions and testing a wide range of combinations between
N availability, N concentration in plant tissues, and degree of mycorrhizal coloni-
zation, which resulted in a gradient of mycorrhizal effects from negative to
positive plant growth responses, the mycorrhizal effects on plant growth and N
uptake were very strongly and positively correlated, and no evidence was found of
a C limitation to growth (Fig. 17.2a). This indicated that the negative effects of
mycorrhization over host plant productivity were due to N retention by the fungal
partner and not to excessive C drainage (Corrêa et al. 2008). This supports the
idea that, under low nutrient supply, because growth is more limited by nutrients
than by C, carbohydrates accumulate in plant organs, becoming more advanta-
geous for plants to allocate more photosynthetic C belowground, and to the
mutualistic partner, if by doing so they can acquire more of the nutrients they
need (Kiers and Van der Heijden 2006). Furthermore, under nutrient-limited
conditions, the accumulation of carbohydrates can lead to a downregulation of
genes and enzymes responsible for photosynthesis, and the investment in the
mycorrhizal symbiosis could even enhance the hosts’ photosynthetic capacity
(Kiers and Van der Heijden 2006). Under these conditions, the C supply to the
fungus will not result in decreased growth, and will therefore not constitute a cost.
This is in agreement with the suggestion that mycorrhizal associations are based on
the exchange of excess resources or by-product benefits (Brundrett 2002; Kiers and
Van der Heijden 2006).
Evidence was also found that the response of growth to mycorrhization varies
according to the balance between the nutritional needs of the plant and the nutrient
supply by the fungus (Corrêa et al. 2008). This balance was accessed by combining
the mycorrhizal colonization (Myc: mg P. tinctorius DW/mg root DW) and the N
availability (N relative adition rate; Ingestad et al. 1986) in a single parameter, the
Mycorrhizal N Demand-Supply Balance (MDSN; Fig. 17.2b). The variation in
Fig. 17.2 (a) Correlations a 2.0

between the mycorrhizal
profits on N uptake (RNm/
RNNM) and on growth
(RGRm/RGRNM). The 1.5
RGRm / RGRNM
mycorrhizal profit, or the net
increase in total value
generated by investing in 1.0
mycorrhizae instead of in
nonmycorrhizal roots, was
calculated as the ratio
0.5
between the parameter for an
individual mycorrhizal plant
and the mean value for non
mycorrhizal plants grown in 0.0
the same experimental 0.0 0.5 1.0 1.5 2.0 2.5 3.0
conditions (r ¼ 0.58, n ¼ 67, RNm / RNNM
p < 0.001). (b) Correlations b 2
between RGRm/RGRNM and 1 .8
the mycorrhizal demand
supply balance (MDSN). 1 .6
RGRm / RGRNM
MDSN was calculated as the 1 .4

ratio between the average 1 .2
level of mycorrhizal 1
colonization (Myc;
P. tinctorius mg DW root mg 0 .8
DW1) and the nitrogen 0 .6
relative addition rate (RARN; 0 .4
Ingestad and Lund, 1979) for 0 .2
the considered period. The
0
plants were fed either 1.9 1 10 100
(open circles) or 3.8 mM
MDSN
NH4þ (closed circles) as N
source. The line dividing the
graphic area makes the
division between negative
(below 1) and positive
(above 1) mycorrhizal growth
profit. (Source: From Corrêa
et al. (2008), with permission
from the authors)
growth response had a curvilinear behavior, being negative at high and low values
of MDSN. Curvilinear correlations between degree of mycorrhizal colonization
and host plant growth had been previously observed in AM mycorrhizal plants
(Clapperton and Reid 1992) and predicted in several models, although the shape of
the predicted curve has varied (Gange and Ayres 1999; Janos 2007). This is
particularly important since it reveals that an evaluation of the mycorrhizal benefit
or detriment cannot be based on only one, or a limited amount, of data points,
but that M and NM plants should be compared over gradients of the conditions that
are being tested.
17.6 Conclusions
Over the last years, new knowledge has been gained on C and N metabolisms and
exchanges in ECM, mostly with the use of molecular tools. However, they remain
mostly unknown to us. Pieces of the puzzle are slowly being discovered, but we are
still far from fitting them into an integrative view of the system and its regulation.
Away from the molecular approach, knowledge has also been gathering on how the
C and N balances reflect on mycorrhizal cost:benefit, which will have important
implications on our understanding of the regulation of exchanges. The time has
come in which we need to start integrating the different approaches and results if we
are to understand the function, nature, and evolution of the symbiosis.
References
Abuzinadah RA, Read DJ (1989) The role of proteins in the nitrogen nutrition of ectomycorrhizal
plants IV. The utilization of peptides by birch (Betula pendula L.) infected with different
mycorrhizal fungi. New Phytol 112:55–60
Akema T, Futai K (2005) Ectomycorrhizal development in a Pinus thunbergii stand in relation to
location on a slope and effect on tree mortality from pine wilt disease. J For Res 10:93–99
Anderson IC, Chambers SM, Cairney JWG (1999) Intra- and interspecific variation in patterns of
organic and inorganic nitrogen utilization by three Australian Pisolithus species. Mycol Res
103:1579–1587
Bendjia M, Rikirsch E, M€ uller T, Morel M, Corratgé C, Zimmermann S, Chalot M, Frommer WB,
Wipf D (2006) Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum:
characterization of two di- and tri-peptide transporters (HcPTR2A and B). New Phytol 170:
401–410
Blaudez D, Chalot M, Dizengremel P, Botton B (1998) Structure and function of the ectomycor-
rhizal association between Paxillus involutus and Betula pendula. II. Metabolic changes during
mycorrhizal formation. New Phytol 138:543–552
Blaudez D, Botton B, Dizengremel P, Chalot M (2001) The fate of [14C] glutamate and [14C]
malate in birch roots is strongly modified under inoculation with Paxillus involutus. Plant Cell
Environ 24:449–457
Bogeat-Triboulot M, Bartoli F, Garbaye J, Marmeisse R, Tagu D (2004) Fungal ectomycorrhizal
community and drought affect root hydraulic properties and soil adherence to roots of Pinus
pinaster seedlings. Plant Soil 267:213–223
Brandes B, Godbold DM, Kuhn AJ, Jentschke G (1998) Nitrogen and phosphorus acquisition by
the mycelium of the ectomycorrhizal fungus Paxillus involutus and its effect on host nutrition.
Brundrett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytol
154:275–304
B€
ucking H, Heyser W (2000) Subcellular compartmentation of elements in nonmycorrhizal and
mycorrhizal roots of Pinus silvestris: an X-ray microanalytical study. I. The distribution of
phosphate. New Phytol 145:311–320
Buscot F, Munch JC, Charcosset JY, Gardes M, Nehls U, Hampp R (2000) Recent advances in
exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these
symbioses in ecosystems. FEMS Microbiol Rev 24:601–614
Chalot M, Brun A (1998) Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and
ectomycorrhizas. FEMS Microbiol Rev 22:21–44
Chalot M, Brun A, Botton B, S€ oderstr€

omm B (1996) Kinetics, energetics and specificity of the
general amino-acid transporter from the ectomycorrhizal fungus Paxillus involutus. Microbi-
ology 142:1749–1756
Chalot M, Javelle A, Blaudez D, Lambilliote R, Cooke R, Sentenac H, Wipf D, Botton B (2002)
An update on nutrient transport processes in ectomycorrhizas. Plant Soil 244:165–175
Chalot M, Blaudez D, Brun A (2006) Ammonia: a candidate for nitrogen transfer at the mycorrhi-
zal interface. Trends Plant Sci 11:263–266
Clapperton MJ, Reid DM (1992) A relationship between plant growth and increasing VA mycor-
rhizal inoculum density. New Phytol 120:227–234
Colpaert JV, Assche JÁ, Luitjens K (1992) The growth of the extramatrical mycelium of
ectomycorrhizal fungi and the growth response of Pinus sylvestris L. New Phytol 120:127–135
Colpaert JV, Van Laere A, Van Assche JA (1996) Carbon and nitrogen allocation in ectomycor-
rhizal and non-mycorrhizal Pinus sylvestris L. seedlings. Tree Physiol 16:787–793
Colpaert JV, Van Tichelen KK, Van Assche JÁ, Van Laere A (1999) Short-term phosphorus
uptake rates in mycorrhizal and non-mycorrhizal roots of intact Pinus sylvestris seedlings. New
Phytol 143:589–597
Conjeaud C, Scheromm P, Moussain D (1996) Effects of phosphorus and ectomycorrhiza on
maritime pine seedlings (Pinus pinaster). New Phytol 133:345–351
Corrêa A, Strasser RJ, Martins-Loução MA (2008) Response of plants to ectomycorrhizae in
N-limited conditions: which factors determine its variation? Mycorrhiza 18:413–427
Corrêa A, Magel E, Hampp R, Martins-Loução MA (2010) Carbon allocation in ectomycorrhizal
plants at limited and optimal N supply: an attempt at unraveling conflicting theories. Mycor-
rhiza. doi:10.1007/s00572-010-0309-3
Couturier J, Montanini B, Martin F, Brun A, Blaudez D, Chalot M (2007) The expanded family of
ammonium transporters in the perennial poplar plant. New Phytol 174:137–150
de Mazancourt C, Loreau M, Dieckman U (2005) Understanding mutualism when there is
adaptation to the partner. J Ecol 93:305–314
Deveau A, Kohler A, Frey-Klett P, Martin F (2008) The major pathways of carbohydrate
metabolism in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. New Phytol 180:
379–390
Dickson S, Smith SE, Smith FA (1999) Characterization of two arbuscular mycorrhizal fungi in
symbiosis with Allium porrum: colonization, plant growth and phosphate uptake. New Phytol
144:163–172
Dosskey MG, Linderman RG, Boersma L (1990) Carbon-sink stimulation of photosynthesis in
Douglas fir seedlings by some ectomycorrhizas. New Phytol 115:269–274
Duplessis S, Courty P, Tagu D, Martin F (2005) Transcript patterns associated with ectomycor-
rhiza development in Eucalyptus globulus and Pisolithus microcarpus. New Phytol 165:
599–611
Ek H, Andersson S, Arnebrant K, S€ om B (1994) Growth and assimilation of NH4þ and
oderstr€

NO3 by Paxillus involutus in association with Betula pendula and Picea abies as affected
by substrate pH. New Phytol 128:629–637
Eltrop L, Marschner H (1996) Growth and mineral nutrition of non-mycorrhizal and mycorrhizal
Norway spruce (Picea abies) seedlings grown in semi-hydroponic sand culture. I. Growth and
mineral nutrient uptake in plants supplied with different forms of nitrogen. New Phytol 133:
469–478
Fajardo-López MF, M€anner P, Willman A, Hampp R, Nehls U (2007) Increased trehalose
biosynthesis in Hartig net hyphae of ectomycorrhizas. New Phytol 174:389–398
Finlay RD, Ek H, Odham G, S€ oderstr€
om B (1988) Mycelial uptake, translocation and assimilation
of nitrogen from 15N-labelled ammonium by Pinus sylvestris plants infected with four different
ectomycorrhizal fungi. New Phytol 110:59–66
Finlay RD, Frostegård Å, Sonnerfeldt AM (1992) Utilization of organic and inorganic nitrogen
sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl.
ex Loud. New Phytol 120:105–115
Foyer CH, Noctor G (2002) Photosynthetic nitrogen assimilation: inter-pathway control and
signalling. In: Foyer CH, Noctor G (eds) Photosynthetic nitrogen assimilation and associated
carbon and respiratory metabolism. Advances in photosynthesis and respiration series. Kluwer,
Dordrecht, pp 1–22
Frettinger P, Derory J, Herrmann S, Plomion C, Lapeyrie F, Oelm€uller R, Martin F, Buscot F
(2007) Transcriptional changes in two types of pre-mycorrhizal roots and in ectomycorrhizas
of oak microcuttings inoculated with Piloderma croceum. Planta 225:331–340
Gange AC, Ayres RL (1999) On the relation between arbuscular mycorrhizal colonization and
plant “benefit”. Oikos 87:615–621
Hampp R, Schaeffer C, Wallenda T, St€ ulten C, Johann P, Einig W (1995) Changes in carbon
partitioning or allocation due to ectomycorrhiza formation: biochemical evidence. Can J Bot
73(suppl 1):S548–S556
Heinemeyer A, Ineson P, Ostle N, Fitter AH (2006) Respiration of the external mycelium in the
arbuscular mycorrhizal symbiosis shows strong dependence on recent photosynthates and
acclimation to temperature. New Phytol 171:159–170
Herre EA, Knowlton N, Mueller UG, Rehner SA (1999) The evolution of mutualisms: exploring
the paths between conflict and cooperation. Trends Ecol Evol 14:49–53
Herrmann S, Buscot F (2007) Cross talks at the morphogenetic, physiological and gene regulation
levels between the mycobiont Piloderma croceum and oak microcuttings (Quercus robur)
during formation of ectomycorrhizas. Phytochemistry 68:52–67
Hildebrandt U, Regvar M, Bothe H (2007) Arbuscular mycorrhiza and heavy metal tolerance.
Phytochemistry 68:139–146
Hobbie EA (2006) Carbon allocation to ectomycorrhizal fungi correlates with belowground
allocation in culture studies. Ecology 87:563–569
Hobbie EA, Colpaert JV, White MW, Ouimette AP, Macko SA (2008) Nitrogen form, availability,
and mycorrhizal colonization affect biomass and nitrogen isotope patterns in Pinus sylvestris.
Hoeksema JD, Kummel M (2003) Ecological persistence of the plant-mycorrhizal mutualism: a
hypothesis from species coexistence theory. Am Nat 162:S40–S50
Hoeksema JD, Schwartz MW (2002) Expanding comparative-advantage biological market mod-
els: contingency of mutualism on partners’ resource requirements and acquisition trade-offs.
Proc R Soc Lond B 270:913–919
H€ogberg MN, Bååth E, Nordgren A, Arnebrant K, H€ ogberg P (2003) Contrasting effects of
nitrogen availability on plant carbon supply to mycorrhizal fungi and saprotrophs- a hypothesis
based on field observations in boreal forest. New Phytol 160:225–238
Ingestad T, Lund A (1979) Nitrogen stress in birch seedlings. I. Growth technique and growth.
Ingestad T, Arveby AS, K€ahr M (1986) The influence of ectomycorrhiza on nitrogen nutrition and
growth of Pinus sylvestris seedlings. Physiol Plant 68:575–582
Janos DP (2007) Plant responsiveness to mycorrhizas differs from dependence upon mycorrhizas.
Javelle A, Chalot M, S€ oderstr€
om B, Botton B (1999) Ammonium and methylamine transport by
the ectomycorrhizal fungus Paxillus involutus and ectomycorrhizas. FEMS Microbiol Ecol
30:355–366
Javelle A, André B, Marini AM, Chalot M (2003a) High-affinity ammonium transporters and
nitrogen sensing in mycorrhiza. Trends Microbiol 11:53–55
Javelle A, Morel M, Rodrı́guez-Pastrana BR, Botton B, André B, Marini AM, Brun A, Chalot M
(2003b) Molecular characterization, function and regulation of ammonium transporters (Amt)
and ammonium metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus
Hebeloma cylindrosporum. Mol Microbiol 47:411–430
Jentschke G, Godbold DL, Brandes B (2001) Nitrogen limitation in mycorrhizal Norway spruce
(Picea abies) seedlings induced mycelial foraging for ammonium: implications for Ca and Mg
uptake. Plant Soil 234:109–117

ectomycorrhizal root tissue. MPMI 17:202–215
Jones MD, Smith SE (2004) Exploring functional definitions of mycorrhizas: are mycorrhizas
always mutualisms? Can J Bot 82:1089–1109
Jones MD, Durall DM, Tinker PB (1991) Fluxes of carbon and phosphorus between symbionts in
willow ectomycorrhizas and their changes with time. New Phytol 119:99–106
Jones MD, Durall DM, Tinker PB (1998) A comparison of arbuscular and ectomycorrhizal
Eucalyptus coccifera: growth response, phosphorus uptake efficiency and external hyphal
production. New Phytol 140:125–134
Jonhson NC, Graham JH, Smith FA (1997) Functioning of mycorrhizal associations along the
mutualism-parasitism continuum. New Phytol 135:575–585
Kiers ET, Van der Heijden MGA (2006) Mutualistic stability in the arbuscular mycorrhizal
symbiosis: exploring hypotheses of evolutionary cooperation. Ecology 87:1627–1636
Kyt€oviita M (2005) Role of nutrient level and defoliation on symbiotic function: experimental
evidence by tracing 14C/15N exchange in mycorrhizal birch seedlings. Mycorrhiza 15:65–70
Lamhamedi MS, Godbout C, Fortin JA (1994) Dependence of Laccaria bicolor basidiome
development on current photosynthesis of Pinus strobus seedlings. Can J For Res 24:
1797–1804
Leake JR, Read DJ (1990) Proteinase activity in mycorrhizal fungi. II. The effects of mineral and
organic nitrogen sources on induction of extracellular proteinase in Hymenoscyphus ericae
(Read) Korf, Kerman. New Phytol 116:123–128
Loewe A, Einig W, Shi L, Dizengremel P, Hampp R (2000) Mycorrhiza formation and elevated
CO2 both increase the capacity for sucrose synthesis in source leaves of spruce and aspen. New
Phytol 145:565–574
Lucic E, Fourrey C, Kohler A, Martin F, Chalot M, Brun-Jacob A (2008) A gene repertoire for
nitrogen transporters in Laccaria bicolor. New Phytol 180:343–364
Martin F, Botton B (1993) Nitrogen metabolism of ectomycorrhizal fungi and ectomycorrhiza.
Adv Plant Pathol 9:85–102
Martin F, Selosse MA (2008) The Laccaria genome: a symbiont blueprint decoded. New Phytol
180:296–310
Martin F, Boiffin V, Pfeffer PE (1998) Carbohydrate and amino acid metabolism in the Eucalyptus
globulus-Pisolithus tinctorius ectomycorrhiza during glucose utilization. Plant Physiol 118:
627–635
Morel M, Buée M, Chalot M, Brun A (2006) NADP-dependent glutamate dehydrogenase: a
dispensable function in ectomycorrhizal fungi. New Phytol 169:179–190
Morel M, Jacob C, Fitz M, Wipf D, Chalot M, Brun A (2008) Characterization and regulation of
PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus
involutus. F Gen Biol 45:912–921
Morgan JAW, Bending GD, White PJ (2005) Biological costs and benefits to plant-microbe
interactions in the rhizosphere. J Exp Bot 56:1729–1739
M€uller T, Avolio M, Olivi M, Bendjia M, Rikirsch E, Kasaras A, Fitz M, Chalot M, Wipf D (2007)
Nitrogen transport in the ectomycorrhiza association: the Hebeloma cylindrosporum-Pinus
pinaster model. Phytochemistry 68:41–51
Nehls U (2008) Mastering ectomycorrhizal symbiosis: the impact of carbohydrates. J Exp Bot 59:
1097–1108
Nehls U, Wiese J, Guttenberg M, Hampp R (1998) Carbon allocation in ectomycorrhizas:
identification and expression analysis of an Amanita muscaria monosaccharide transporter.
MPMI 11:167–176
Nehls U, Ecke M, Hampp R (1999) Sugar- and nitrogen-dependent regulation of an Amanita
muscaria phenylalanine ammonium lyase gene. J Bacteriol 181:1931–1933
Nehls U, Wiese J, Hampp R (2000) Cloning of a Picea abies monosaccharide transporter gene and
expression- analysis in plant tissues and ectomycorrhizas. Trees 14:334–338
Nehls U, G€ohringer F, Wittulsky S, Dietz S (2010) Fungal carbohydrate support in the ectomycor-
rhizal symbiosis: a review. Plant Biol 12:292–301
Neuhauser C, Fargione JE (2004) A mutualism-parasitism continuum model and its application to
plant mycorrhizae interactions. Ecol Modell 177:337–352
Nilsson LO, Wallander H (2003) Production of external mycelium by ectomycorrhizal fungi in a
norway spruce forest was reduced in response to nitrogen fertilization. New Phytol 158:
409–416
Nuutinen JT, Timonen S (2008) Identification of nitrogen mineralization enzymes, L-amino acid
oxidases, from the ectomycorrhizal fungi Hebeloma spp. and Laccaria bicolor. Mycol Res
112:1453–1464
Plassard C, Bonafos B, Touraine B (2000) Differential effects of mineral and organic N sources,
and of ectomycorrhizal infection by Hebeloma cylindrosporum, on growth and N utilization in
Pinus pinaster. Plant Cell Environ 23:1195–1205
Polidori E, Ceccaroli P, Saltarelli R, Guescini M, Menotta M, Agostini D, Palma F, Stocchi V
(2007) Hexose uptake in the plant symbiotic ascomycete Tuber brochii Vittadini: biochemical
features and expression pattern of the transporter TBHXT1. Fungal Genet Biol 44:187–198
Read DJ, Pérez-Moreno J (2003) Mycorrhizas and nutrient cycling in ecosystems–a journey
Rudawska M, Kieliszewska-Rokicka B, Debaud JC, Lewandowski A, Gay G (1994) Enzymes of
ammonium metabolism in ectendomycorrhizal and ectomycorrhizal symbionts of pine. Physiol
Plant 92:279–285
Rygiewicz PT, Andersen CP (1994) Mycorrhizae alter quality and quantity of carbon allocated
below ground. Nature 369:58–60
Schaarschmidt S, Roitsch T, Hause B (2006) Arbuscular mycorrhiza induces gene expression of
the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots. J Exp Bot 57:
4015–4023
Schaeffer C, Wallenda T, Guttenberger M, Hampp R (1995) Acid invertase in mycorrhizal and
non-mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.) seedlings. New Phytol
129:417–424
Schaeffer C, Johann P, Nehls U, Hampp R (1996) Evidence for an up-regulation of the host and a
down-regulation of the fungal phosphofructokinase activity in ectomycorrhizas of Norway
spruce and fly agaric. New Phytol 134:697–702
Schwartz MW, Hoeksema JD (1998) Specialization and resource trade: biological markets as a
model of mutualisms. Ecology 79:1029–1038
Selle A, Willmann M, Grunze N, Geßler A, Weiß M, Nehls U (2005) The high-affinity poplar
ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis. New Phytol 168:
697–706
Tibbett M, Hartley M, Hartley S (2000) Comparative growth of ectomycorrhizal basidiomicetes
(Hebeloma spp.) on organic and inorganic nitrogen. J Basic Microbiol 40:393–395
Treseder KK (2004) A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and
atmospheric CO2 in field studies. New Phytol 164:347–355
Treseder KK, Allen MF (2002) Direct nitrogen and phosphorus limitation of arbuscular mycor-
rhizal fungi: a model and field test. New Phytol 155:507–515
Treseder KK, Czimczik CI, Trumbore SE, Allison SD (2008) Uptake of an amino acid by
ectomycorrhizal fungi in a boreal forest. Soil Biol Biochem 40:1964–1966
Tuomi J, Kyt€oviita M, H€ardling R (2001) Cost efficiency of nutrient acquisition and the advantage
of mycorrhizal symbiosis for the host plant. Oikos 92:62–70
Vézina LP, Margolis HA, McAfee BJ, Delaney S (1989) Changes in the activity of enzymes
involved with primary metabolism due to ectomycorrhizal symbiosis on jack pine seedlings.
Wallander H, Nylund JE (1991) Effects of excess nitrogen on carbohydrate concentration and
mycorrhizal development of Pinus sylvestris L. seedlings. New Phytol 119:405–411
Wallander H, Nylund JE (1992) Effects of excess nitrogen and phosphorus starvation on the
extramatrical mycelium of ectomycorrhizas of Pinus sylvestris L. New Phytol 119:405–411
Wallenda T, Kottke I (1998) Nitrogen deposition and ectomycorrhizas. New Phytol 139:169–187
Wallenda T, Schaeffer C, Einig W, Wingler A, Hampp R, Seith B, George E, Marschner H (1996)
Effects of varied soil nitrogen supply on Norway spruce (Picea abies (L.) Karst.). II. Carbon
metabolism in needles and mycorrhizal roots. Plant Soil 186:361–369
Wiese J, Kleber R, Hampp R, Nehls U (2000) Functional characterization of the Amanita muscaria
monosaccharide transporter, AmMst1. Plant Biol 2:278–282
Wingler A, Einig W, Schaeffer C, Wallenda T, Hampp R, Wallander H, Nylund J (1994) Influence
of different nutrient regimes on the regulation of carbon metabolism in Norway spruce [Picea
abies (L.) Karst.] seedlings. New Phytol 128:323–330
Wingler A, Wallenda T, Hampp R (1996) Mycorrhiza formation on Norway spruce (Picea abies)
roots affects the pathway of anaplerotic CO2 fixation. Physiol Plant 96:699–705
Wipf D, Ludewig U, Tegeder M, Rentsch D, Koch W, Frommer WB (2002) Conservation of
amino acid transporters in fungi, plants and animals. Trends Biochem Sci 27:139–147
Wright DP, Scholes JD, Read DJ, Rolfe SA (2000) Changes in carbon allocation, expression of
carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus
Paxillus involutus (Batsch) Fr. Plant Cell Environ 23:39–49
Wright DP, Johansson T, Le Quéré A, S€ oderstr€
om B, Tunlid A (2005) Spatial patterns of gene
expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycor-
rhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil
microcosms. New Phytol 167:579–596
Wu B, Kazuhide N, Hogetsu T (2002) Spatiotemporal transfer of carbon-14-labelled photosyn-
thate from ectomycorrhizal Pinus densiflora seedlings to extraradical mycelia. Mycorrhiza
12:83–88
Zhu H, Dancik BP, Higginbotham KO (1994) Regulation of extracellular proteinase production in
an ectomycorrhizal fungus Hebeloma crustuliniforme. Mycologia 86:227–234
.
Chapter 18
Ectomycorrhizal Interaction Between
Cantharellus and Dendrocalamus
Rohit Sharma and Ram C. Rajak
18.1 Introduction
Studies on ectomycorrhizal mushroom diversity help in conservation of fungal and

plant species, indigenous forests, and sustainable use of fungi as nontimber forest
product. Considerable attention has been given to arbuscular mycorrhiza (AM)
fungal research but knowledge on ectomycorrhizal mushroom is rare. Moreover, it
has been studied scarcely in India. Sporadically, however researchers in India have
conducted forays to study ectomycorrhizal fungi, accumulated knowledge and
published.
Apart from two recognized hot spots viz., north eastern Himalayans and Western
Ghats, India is bestowed with several regions rich in fungal biodiversity. From year
2004 to 2008, mycological studies undertaken to collect mushrooms from Central
Indian dense forests of Madhya Pradesh and Chhattisgarh (under research project
sponsored by Department of Biotechnology, New Delhi, Government of India) has
been rewarding for authors, which yielded a large number of fleshy fungi found
to be ectomycorrhizal with various tree species (Sharma 2008; Sharma et al. 2008a,
2009a, b, c, 2010a, b, c). Several investigators have also conducted studies on
diversity of ectomycorrhizae (Lakhanpal 1996; Natarajan and Ravindran 2003a, b;
Pande et al. 2004; Valentine et al. 2004; Kranabetter et al. 2005; Natarajan et al.
2005a, b; Riviere et al. 2007). About 61 ectomycorrhizal mushrooms were collected
R. Sharma (*)
Microbial Culture Collection, Affiliated to National Centre for Cell Science, University of Pune,
Ganeshkhind, Pune 411 007, Maharashtra, India
e-mail: rsmushrooms@yahoo.co.uk
R.C. Rajak
SGH Center for Biotechnology, B-96, Priyadarshini Housing Society, Dumna Road, Jabalpur
482001, Madhya Pradesh, India
e-mail: rcmykes@yahoo.com

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_18,
406 R. Sharma and R.C. Rajak
Fig. 18.1 Cantharellus tropicalis basidiomata in natural forest

Source: Adapted from Sharma et al. 2010b
(Sharma et al. 2009c) including Russula, Lactarius, Scleroderma, Pisolithus,

Geaster, Boletus, Cantharellus and Astreaus. Cantharellus tropicalis (Fig. 18.1)
is a popular edible and medicinal mushroom sold in the local market of Balaghat
district, Madhya Pradesh, India. It is a high temperature species, which appears in
late rainy season during August and September. It has been recommended as energy
booster for pregnant women. C. tropicalis forms ectomycorrhiza (ECM) with
Dendrocalamus strictus (bamboo) and consistently found associated with bamboo
forest of Madhya Pradesh (Sharma 2008; Sharma et al. 2009c). The fruiting bodies
are harvested in large amount from Balaghat, Lamta, Nainpur, and Baihar of
Madhya Pradesh, India and nearby regions and often been taken to adjoining
districts in large quantities. It is leathery in texture, with good taste and greater
shelf-life. The seasonal collection may go up to 50 tons per year (based on personal
observation). The number of described Cantharellus species worldwide exceeds 70
(Danell 1999). Other taxonomically related species viz. Cantharellus minor, C.
cibarius, C. cinereus, C. friesii, C. lutescens, C. melanoxeros, C. tubaeformis, and C.
xanthopus are commonly called chanterelle mushrooms known world over for
edibility.
This chapter highlights various aspects of ectomycorrhizal association between
Cantharellus (isolated from fresh mushroom body using stipe tissue which is
aseptically transferred to ectomycorrhizal media) and bamboo species. It focusses
on simple technique for synthesis of ECM, in vitro ectomycorrhization with
Dendrocalamus seedlings, mass production of inoculum with low-cost substrate,
enzymatic studies on the acid phosphatase which forms an important criteria for
good ectomycorrhizal fungus and interaction studies with other microfungi isolated
from rhizosphere of bamboo and its potential to increase growth of seedlings
growing under green house conditions.
18 Ectomycorrhizal Interaction Between Cantharellus and Dendrocalamus 407
18.2 Ectomycorrhiza Formation and Growth Response

on Host Plant
18.2.1 Growth in Culture
Cantharellus is relatively easy to isolate as compared to other mushrooms viz.,

C. cibarius wherein Pseudomonas lives in association (Danell 1994). To isolate,
fruit body of a freshly collected mushroom is taken up for isolation. It is free from
any insect or disease infestation. Usually young basidiomata is used for the same. Stipe
of a clean basidiomata is broken vertically and a small tissue piece is aseptically
transferred into Petri dish containing Melin–Norkrans Agar Medium (MNM) with the
help of a sterile forcep (Sharma et al. 2009b). Wide mouth vials of 50ml capacity filled
with moistened used tea-leaves (pre-sterilized) were also used. It prevents bacterial
contamination as mushroom mycelia colonizes the cellulose, lignin rich substrate
faster (Sharma 2008). We also observed that acidic pH is more suitable to
C. tropicalis. When grown in liquid media C. tropicalis tends to bring final pH to
acidic (Sharma unpublished). It grew faster when the medium (agar and liquid media)
was supplemented with 0.2-0.5% malt extract. The mycelia of C. tropicalis is white
and grow sticking to medium surface. However, at later stage some portion of it turns
brown which may be due to aging (and should not be confused with contamination)
(Fig. 18.2). The mycelia also forms clamp connections in artificial medium
(Fig. 18.3). A detail study on physical and physiological factors affecting growth
of C. tropicalis has already been conducted (Sharma et al. unpublished).
18.2.2 Simplified Technique of Ectomycorrhizal Synthesis
There is a need to enhance our knowledge on interactions within fungal community

and explore potential of ectomycorrhizospheric environment for biotechnological
Fig. 18.2 Culture of C. tropicalis on modified FDA medium after 15 days

Fig. 18.3 Clamp connection of C. tropicalis mycelia in culture (100x)
purpose (Cairney and Meharg 2002; Whipps 2004; Cairney 2005). For this we need to
study the process of mycorrhization in controlled environment under laboratory
conditions. Before carrying out small- scale field experiments it is important to select
a technique which can suit Cantharellus mycelia for symbiotic association with host
plant seedlings. It is essential for development of an effective mycorrhizal symbiosis
towards exploitation of this symbiosis. Several devices have been developed for
in vitro ECM synthesis for performing physiological, biochemical, and structural
experiments using various ectomycorrhizal mushrooms (Danell 1994; Cairney and
Chambers 1999; Vaario et al. 1999, 2000; Yamada et al. 2001a; Danell 2002;
Theodorou and Reddell 2006). But most of the equipments were found bulky and
did not allow investigation of large number of experimental units at the same time.
Many aseptic methods involved large tubes and Petri dishes (Duddridge 1986) to
maintain aseptic conditions for whole seedling (Chilvers et al. 1986) or for only
roots (Duddridge 1986). Although there are numerous work on synthesis of pine
ectomycorrhiza (Marx et al. 1982), less work has been done with angiosperm and
none with monocots. Also techniques of synthesizing pine mycorrhiza are difficult
to apply to angiosperms such as eucalypts, bamboo, sal, dipterocarps, etc. A filter
paper-flask technique was developed by us, which was not bulky and requires
limited space, forms rapid ECM, and yet permits observation of ectomycorrhiza
formation (Sharma et al. 2009b). This technique enables bamboo shoots to grow
well and roots to make contact with Cantharellus mycelium in a short time. Root
and shoot parts extended within flask and formed typical ectomycorrhiza
(Fig. 18.4). It keeps root and shoots system under aseptic conditions. Externally,
ECM lacks root hairs and forms light-brown mantle with woven hyphae.
The filter paper-flask method permits observation of developing external myce-
lium before and after ectomycorrhizal formation. Fortin et al. (1980) suggested that
exudation of substances stimulating the growth of Pisolithus tinctorius and
18
Ectomycorrhizal Interaction Between Cantharellus and Dendrocalamus
Fig. 18.4 Filter paper-flask technique used for in vitro ectomycorrhiza formation
Source: Adapted from Sharma et al. 2009b
409
Cenococcum graniforme which should also be investigated for this association in

future studies. However, this technique has some features common with others
including culture technique that used a Petri dish lined with paper (Chilvers et al.
1986); a plastic pouch technique (Fortin et al. 1980) uses paper to support and nourish
roots; and a cellulose thimble technique (Littke et al. 1980) uses paper to help fungal
growth and transmission. Moreover, Vaario et al. (2000) described a simple in vitro
system for synthesis of Abies firma–Cenococcum geophilum ectomycorrhiza. This
technique developed by us (Sharma et al. 2009b) is not only useful for the production
of sufficient mycorrhizal seedlings for biochemical and physiological studies but can
produce mycorrhizal material for practical forestry applications. Moreover, this
technique permitted accurate evaluation of the colonization process of C. tropicalis.
The host plant–fungus association will help in studies on development of its fruit
bodies, physiological studies and other in vitro studies viz., effects of different organic
sources of carbon and nitrogen on ectomycorrhizal formation capability of Canthar-
ellus.
18.2.3 Ectomycorrhizal Interaction
In most instances, a fungus is considered ECM forming based on field observation.

Consistent association of basidiomata with one or more tree species forms the basis
of ECM forming fungi as an indirect method of ECM assessment. However, not all
ectomycorrhizal fungi found associated with adult trees in field form mycorrhizae
with young seedlings (Last et al. 1992). Pure culture synthesis technique has been
modified by several workers for different mushroom species. Few edible ectomy-
corrhizal mushrooms have been successfully cultivated under controlled condi-
tions. Numerous studies on ECM formation between host plant seedlings and
mushroom mycelia has been conducted and widely reviewed (Nezzar-Hocine
et al. 1998; Vaario et al. 1999, 2000; Yamada et al. 1999, 2001b; Dahlstrom et al.
2000; Guerin-Laguette et al. 2000a, b). On the other hand, Lyophillum shimeji
(Kawam) Hongo., C. cibarius Fr., and Tuber melanosporum Vitt. sporocarps have
been produced under laboratory, green house, and field conditions, respectively
(Danell and Camacho 1997). Tricholoma and Lactarius have been also successfully
cultivated in laboratory conditions. Thus, establishment of artificial cultivation
system for C. tropicalis and understanding its ectomycorrhizal interaction may
form the basis for reforestation program mushroom cultivation.
The mushroom mycelia used for the study was isolated by excising tissue blocks
from basidiomata stipe on MNM medium (Straatsma and van Griensven 1986). The
test tube or flask system used in this experiment contained a sterilized mixture of
sand þ used tea leaves (50% v:v) moistened with sterile distilled water. It was
cooled to room temperature and inoculated with C. tropicalis culture (Sharma et al.
unpublished). The seedlings (produced by germinating seeds on Petri dishes) were
inserted in growth system with C. tropicalis mycelia plug. Fungal colonization
of bamboo roots was observed to confirm presence of intercellular Hartig Net
confirming the symbiotic association (Harley and Smith 1983). After few weeks of
incubation, seedlings of D. strictus showed yellow-white mycelium in substrate.
A mycorrhiza-like relation was observed from root morphology between roots
of D. strictus and C. tropicalis and is a maiden example illustrating association
between ectomycorrhizal fungus and a monocot plant. Mycorrhizal synthesis with
other mushroom species and various host plants has also been achieved and
reported (Molina and Trappe 1982; Yamada and Katsuya 1995; Dahlstrom
et al. 2000). In a study, Theodorou and Reddell (2006) studied 11 species of
mycorrhizal fungi from stands of Eucalyptus spp., Allocasuarina spp., or Pinus
radiata D. Don and tested for their abilities to initiate ECM with Allocasuarina
littoralis (Salisb.) L. Johnson, Casuarina equisetifolia L., and C. cunninghamiana
Miq. in aseptic system. Moreover, Vaario et al. (1999) first reported in vitro
mycorrhizal formation of Abies firma Sieb, et Zucc. with P. tinctorius (Pers.)
Coker and Couch and improved the technique of ectomycorrhization of A. firma a
slow-growing species and P. tinctorius using a novel culture medium with both
sterilized and rerooted seedlings. Although in vitro mycorrhizal synthesis is
difficult in the genus Cantharellus. In this experiment we tried to form in vitro
ECM using low-cost substrate. Pinus densiflora formed ECM with 21 fungal
species, including two species of Russula, which were also found difficult to
manipulate in vitro (Yamada and Katsuya 1995). A mycorrhizal study indicated
that Morchella formed mycorrhiza-like interaction with four tree species of
Pinaceae (Dahlstrom et al. 2000).
Although different media are known to stimulate mycorrhization process, they
have also detected to affect hyphal branching and several plant- microbe inter-
actions. Extensive data are available concerning the effect of media or substrate
on the process. In our study, the formation of ECM was also influenced by solid/
liquid inoculum and environmental factors. Receptiveness was greatly influenced
by the physical, chemical, and biological characteristics of substrate as confirmed
by used tea leaves þ sand as substrate. During the experiment, certain seedlings
showed mortality (personal observation), which might be due to unsuitable
conditions. Such results have been reported for C. cibarius in vitro synthesis,
where CO2 content, exogenous glucose content, water drainage, and host
specificity have been discussed for such observations (Danell 1994). Most seed-
lings (including control) grew well throughout incubation and ectomycorrhizal
seedlings showed similar or slightly better growth as compared to control plants
(Sharma et al. unpublished).
Some fungi that form mycorrhizae in pure culture fail to form it in greenhouse or
natural soil conditions (Molina and Trappe 1982; Duddridge 1986; Molina et al.
1997) Mycorrhizal succession (contamination) to another symbiont is problematic
in cultivation of mycorrhizal mushrooms. Protection of inoculated fungus from
competitive air borne mycorrhizal fungi in green house conditions is significant for
their acclimatization. In present study, hairless or bifurcated root tips frequently
exhibited mantle or hyphal penetration between the cortical cells. Soon after inocu-
lation with pure cultures of Indian chanterelle, fungal, and/or bacterial contamination,
or both appeared within some synthesis units. Insufficient surface sterilization
period for seeds might be the cause. During our study, some contaminants appeared
on seed cotyledon which affected mycorrhizae formation between chanterelle
and seedlings and made system ineligible for further studies (personal observation).
18.2.4 Mass Multiplication of Inoculum
During plantation in forest areas/disturbed sites, it is observed that mycorrhizal

fungi should accompany them for better survival in new microcosm in which they
are introduced. Seedlings inoculated with effective mycorrhizal fungi in nursery
can establish a healthy ECM system before outplanting into forests or mine sites.
Inoculation of P. tinctorius significantly increased growth and survival of five
southern pine species planted at different sites (Marx et al. 1982). Moreover,
these can be stored at low temperature without damage (Lapeyrie and Bruchet
2006). During the study for large scale production the inoculum of C. tropicalis
produced was kept at room temperature and when inoculated formed ECM with
nursery seedlings of bamboo (Sharma et al. 2010a). The tea leaves based substrate
conserved moisture which helped the fungus to grow actively (personal observa-
tion). Previously several types of natural or laboratory-produced inocula (seedlings
with ectomycorrhiza or excised ectomycorrhiza, spores, crushed basidiomata) and
various methods of application have proved successful through the years (Marx
1980). T. melanosporum has been established in nursery beds with ECM formed
under laboratory conditions. Lamb and Richards (1974) demonstrated the use of
basidiospores of Rhizopogon, Scleroderma and Pisolithus as inoculum. Basidio-
spore inoculum of P. tinctorius and Rhizopogon successfully forms ECM in pine
seedlings (Molina and Trappe 1982; Bruns et al. 2009). However, Trappe (1977)
had recommended pure mycelial or vegetative inoculum of ECM fungi for forest
inoculation.
Pure culture inoculum of ectomycorrhizal fungi poses many difficulties for
wide-scale application thus restricting it to laboratory or green house experiments.
However, nursery beds of Pinus cembra have been successfully inoculated with
pure cultures of Suillus plorans. Theodorou (1971) and Theodorou and Bowen
(1973) inoculated P. radiata with isolates of Rhizopogon luteolus, Suillus granu-
latus, S. luteolus, and Cenococcum geophilum. Danell (1994) used peat/quartz sand
mixture (25%/75% v/v) and 20 ml ingested solution for outplanting in vitro-formed
Cantharellus mycorrhizae. Moreover, with four decades of mycorrhizal research,
peat moss, and vermiculite remain major substrate for inoculum preparation
(Garbaye et al. 1988; Nezzar-Hocine et al. 1998; Yamada et al. 2001a). Lapeyrie
and Bruchet (2006) have used liquid fermentation in airlift bioreactor to produce
inoculum of Pisolithus microcarpus.
Synthesis of Cantharellus ECM with Dendrocalamus has been achieved with
in vitro germinated seedlings in laboratory (Sharma et al. 2009b; Sharma et al.
unpublished) and green house (Sharma et al. 2008b). Moreover, mycelia of
Fig. 18.5 Cantharellus inoculum produced on sterilized waste tealeaves þ sand

Source: Adapted from Sharma et al. 2010a
Cantharellus forms mycorrhiza with laboratory grown seedlings and exhibited

positive effect on growth of Dendrocalamus seedlings (Sharma et al. 2008b;
Sharma et al. 2009b; Sharma et al. unpublished). Mass production of C. tropicalis
inoculum is essential for field application of D. strictus. A ratio of 1:1 used tea
leaves and sand moistened with a volume of sterile distilled water equal to approxi-
mately half the volume of dry substrate proved best (Sharma et al. 2010a). The
mycelia grew very fast through the substrate producing good fungal inoculums
(Fig. 18.5). Used tea leaves have previously been successfully used by Sharma et al.
(2003) for pure culturing of Pleurotus species in tubes, which reduced bacterial
contamination and other noncellulolytic fungus.
According to Kumar and Satyanarayana (2002), selection of mycorrhizal fungi
and performance of seedlings is dependent upon plantation site and plant species.
Ectomycorrhizal fungi are suitable inoculants for various trees (Table 18.1). A good
substrate allows penetration of mycelia, retains moisture, and low in production
cost also. Moreover, it allows smooth ECM formation by the fungus in host
seedlings. Moser (1958) produced ectomycorrhizal inoculum for inoculating P.
cembra with S. plorans (Rolland) Sing and also used pure culture of Suillus
placidus (Bon.) Sing., S. grevillei (Klotzsch) Sing., S. aeruginascens (Secr.)
Snell., Paxillus involutus, Phlegmacium glaucopus, Amanita muscaria, and Lactar-
ius porninsis Rolland. Takacs (1961) inoculated sterilized germinated grains of
cereals, cereal chaff, or peat moss to produce inoculum of A. verna (Bull. ex Fr.)
Lamarck, S. granulatus, S. lutea, Hebeloma crustuliniforme, Russula sp., Sclero-
derma verrucosum (Bull.) Pers., and S. vulgare. Park (1984) also grew mycelial
cultures of S. granulatus and C. geophilum in cereal grains. Several attempts to
produce effective inoculum of these fungi in wheat grains resulted in failure (Marx
1980). Vermiculite and peat moss moistened with modified MNM medium was
found to be an excellent substrate by Marx and Bryan (1975). A successful
commercial formulation of P. tinctorius mycelia has been developed by USDA,
Table 18.1 Commercial availability of vegetative mycelial inoculum of ectomycorrhizal fungi

Process Inoculum form Species (strain) Commercial source
Solid substrate Vermiculite Pisolithus tinctorius Abbott Laboratories, USA
fermentation (MycoRhizR)
Vermiculite Hebeloma Somycel and INRA,
crustuliniforme France
Laccaria laccata
Paxillus involutus
Vermiculite Laccaria laccata Sylvanspawn, Worthigton,
Paxillus involutus PA
Vermiculite Pisolithus tinctorius Mycorr Tech Inc.,
Pittsberg, PA
Submerged Alginate Hebeloma Biotal Ltd., Cordiff, UK
fermentation crustuliniforme
Laccaria laccata
Paxillus involutus
Thelephora terrestris
Alginate Hebeloma Rhone-Poulec and INRA,
crustuliniforme France
Alginate 11 ECM strains Biosynthetica Pvt. Ltd.,
(MycobeadR) Australia
Source: Kumar and Satyanarayana (2002)
Forest Service, and Abbott Laboratories (Marx et al. 1982). This inoculum trade-
marked as MycoRhiz®, is also grown in vermiculite–peat moss nutrient medium.
Pure cultures of fungi viz., S. granulatus, R. luteolus, Thelephora terrestris (Ehrh.)
Fr., and P. tinctorius improved survival and growth of seedlings (Marx 1980; Marx
et al. 1982).
During course of our studies, it was observed that mycelia of C. tropicalis
completely covered the substrate rich in cellulose and lignin within short incuba-
tion period. Sand helps in breaking the inoculum into pieces during application.
Good ECM formation can be attributed to better mixing of inoculum to soil with
sand allowing good aeration. However, Molina and Trappe (1982) have stressed on
the difficulty of transporting, spreading, and mixing of vermiculite inoculum as it
becomes heavy due to water saturation. Sand þ used tea leaves inoculum was found
promising as Cantharellus colonized entire root system and substrate. Moreover,
high quality tree seedlings cannot be produced unless conventional nursery is
substituted by modern forest nursery production techniques (Lamhamedi et al.
2009). This is possible when there is increase in technology transfer from research-
ers to nursery personnel.
18.2.5 Growth Response of Host Seedlings
Ectomycorrhizal mushrooms help in survival and growth of host seedlings. This

association increases plant ability for nutrient and water uptake (Whipps 2001).
It has been successfully demonstrated through various experiments that Pisolithus

and Rhizopogon increased growth of host seedlings (Cairney and Chambers 1999).
Apart from these, several other ectomycorrhizal mushrooms viz., Tuber, Lactarius,
Laccaria, Scleroderma, Canharellus, Paxillus., Hebeloma, and Tricholoma have
been reported experimentally to enhance growth of host seedlings. During our
studies in green house condition it was observed that although C. tropicalis formed
ECM with Dendrocalamus strictus, D. asper and Bambusa nutans seedlings they
were not stable, whereas fungus failed to form ECM with B. vulgaris. Further,
Sharma et al. (2008b) reported that uninoculated seedlings conditioned to cyclic
drought were smaller than mycelia inoculated seedlings. Natarajan et al. (1995)
while studying ECM with Acacia nilotica found that Laccaria fraternal and
P. tinctorius improved its growth. Chen et al. (2006) reported that in South China
inoculation of Scleroderma spores increased the growth of eucalypt plants. Similar
results have been reported by Khosla and Reddy (2008) with Pisolithus albus and
Eucalyptus tereticornis. Pande et al. (2007) reported that oak and pine seedlings
when inoculated with ectomycorrhizal fungi showed more growth. Since Canthar-
ellus is comparatively easily isolated in pure culture and have moderate growth rate,
they are relatively good for inoculation purpose. Current interest by researchers in
production of ectomycorrhizal inoculum in forestry programs focusses on selection
of potential strain for this purpose and also test inoculum effectiveness before field
inoculations. The mushroom mycelia forms numerous rhizomorphs in soil which
are essential for nutrient uptake and also help in stress tolerance during drought and
hence increase seedling growth.
18.3 Ecological Studies
18.3.1 General Ecology
C. tropicalis has been reported from older natural forests and plantations but has not
been reported from nurseries. However, during past several years their has been
overharvesting of Cantharellus from the bamboo forests of Madhya Pradesh
(Sharma unpublished). The observations are based on the sites visited by the author
during 2004–2008. This may lead to decrease in productivity of Cantharellus in the
region. The need for restricted harvesting and technique for harvesting has been
emphasized as the tribals and villagers lack the knowledge of sustainable harvest-
ing. It disturbs the below ground mycelia of mushroom and affects successive
sporocarp formation. Danell (1999) has highlighted that whatever estimates we
make is based on the above ground basidiomata and decrease in below ground
mycelia and ECM should also be emphasized.
The basidiomata are observed in the late rainy season when their is high temp-
erature and humidity. During laboratory experiments also it required high humi-
dity i.e. above 80% for sporocarp formation in controlled environment chamber
(Sharma et al. unpublished). In another study optimum growth pH of C. tropicalis

was acidic which can be correlated to low soil pH value which may be due to
secretion of metabolites due to litter degradation. Although their is lack of informa-
tion on ecology of C. tropicalis in Central India, increased interests among
researchers will trigger more work on it.
18.3.2 Antagonistic Interactions with Rhizosphere Fungi
The mycobiota of forest soils consist of AM fungi, ECM and saprotrophic decom-
poser fungi which supply nutrients to trees and decomposite woody plant litter.
Saprotrophic basidiomycetes are also abundant in bamboo forests. Ectomycorrhizal
fungal mycelia (found in forest soils associated with host trees) with its special
physiology can use either inorganic nutrients or utilize organic sources. ECM fungi
provide an increased surface area for absorption of nutrients and interactions with
other microorganisms (Smith and Read 1997). These interactions may be inhibi-
tory, stimulatory, competitive, mutualistic and is important in biogeochemical
cycling in ecosystems of forests. According to Leake and Johnson (2004), sapro-
trophs obtain most of their C from decaying organic matter while ectomycorrhizal
fungi obtain it from their host plants. Antagonistic interactions between rhizosphere
microorganisms and mycorrhizal fungi have significant function on mycorrhizal
systems (Stark and Kyt€ oviita 2005). Moreover, exudation and reabsorption of fluid
droplets at ECM hyphal tips influence vicinity environment (Sun et al. 1999).
The interactions of plants with soil microorganisms, both pathogens (nematodes
and fungi) and mutualists (nitrogen-fixing bacteria) are also influenced by mycorrhi-
zal fungi. Pathogenic fungi invade roots and mycorrhizal fungi and can alter host
response to these pathogens. Fitter and Garbaye (1994) observed that Laccaria
bicolor prevented spread of Fusarium oxysporum in Douglas–fir roots as a result of
flavonoids wall infusion. Wu et al. (2003) explored interactions between sapro-
trophic microbes and ECM fungi using a protein–tannin complex as N source by
red pine (Pinus resinosa). In a similar study, Olsson (1999) showed the role of fatty
acids for determination of distribution and interactions of mycorrhizal fungi in soil.
Protection of root system from endemic pathogens (such as Fusarium spp.) causing
root infection has been due to reduced phosphorus uptake. There is direct evidence
that mycorrhizal fungi may reduce the incidence and severity of root diseases
(Whipps 2004). In past role of ectomycorrhizal fungi in controlling plant diseases
has been realized. Moreover, tree seedlings has been infected by several pathogens
and hence these interactions can be exploited to restrict them (Finlay 2004).
Soil mycoflora was recovered from rhizosphere soil samples of D. strictus
collected from three sites of bamboo forests in Balaghat, Mandla, and Shahdol,
Madhya Pradesh, India. Pairwise combinations were made by plating mycelial
plugs of ECM and soil microfungi on opposite corners of Petri dish. Radial growth
toward other mycelium was determined by measuring colony radius. The cross
inoculation method (Figs. 18.6 and 18.7) showed that C. tropicalis was active
Fig. 18.6 Dual culture interaction between C. tropicalis and Aspergillus niger
Source: Sharma et al. 2010c
Fig. 18.7 Dual culture interaction between C. tropicalis and Alternaria sp.
Source: Sharma et al. 2010c
against rhizosphere soil microorganism (Sharma et al. 2010c). This resulted in

different types of interactions between fungi, but also in differences in the degree
of interactions. Overgrowth was the most common (45%) interaction, followed by
inhibition at a distance (29%), intermingling (17%), and contact inhibition (13%)
(Sharma et al. 2010c).
The inhibition of soil microfungi, mostly at a distance by C. tropicalis suggested
that it prevented invasion by potential competitors (Sharma et al. 2010c). The
inhibition of soil microfungi by C. tropicalis might be caused by production of
secondary metabolites. In some instances, antibiotics produced by ectomycorrhizal
fungi Amanita, Boletus, and Cenococcum spp. have been reported (Santoro and
Casida 1962). There are reports of in vitro inhibition of pathogenic fungi by several
ectomycorrhizal mushroom mycelia. Shaw et al. (1995) reported growth suppres-

sion of R. roseolus by several saprotrophic basidiomycetes. Furthermore growth of
S. granulatus (L.:Fr.) Rouss, has been shown to be inhibited by rhizoplane fungi of
Pinus halepensis (Girlanda et al. 1995). Baar and Stanton (2000) attributed low
investment of N in mycelial biomass for reduced competition of some ECM fungi.
Hardly any sporocarps of saprotrophic basidiomycetes found to occur in bamboo
forest but species of Ramaria, Clavaria, and Clitocybe have been collected from
bamboo forests and can be studied for their competitiveness with chanterelle. In a
study, Clitocybe marginella Harmaja inhibited the growth of C. geophilum and
L. bicolor (Baar and Stanton 2000). Leake et al. (2001) observed limited effect of
mycorrhiza on growth of saprotroph. In our studies, ectomycorrhizal fungi sup-
pressed soil microfungi indicating that Cantharellus mycelia has higher competi-
tiveness than soil microfungi, which may be due to alkaloids, terpenes,
polysaccharides produced by Cantharellus mycelia. Low competitiveness of
some microfungi viz., Curvularia, Alternaria, Mucor, Fusarium, and Penicillium,
etc. suggest that they were unable to compete with Cantharellus in the acidified
soil.
Ectomycorrhizal fungi show inhibitory effects on root pathogenic fungi but as
Johansson et al. (2004) puts in, their interactions with saprophytic fungi have
received little attention. In another study, Tricholoma sp., P. involutus, Hebeloma
cylindrosporum, and L. bicolor demonstrated inhibition of a range of pathogens
in vitro (Morin et al. 1999). When grown in coculture Werner and Zadworny (2003)
observed suppression of Mucor hiemalis by L. laccata and inhibition of growth of
Trichoderma virens in coculture (Werner et al. 2002). Antifungal and antibacterial
action of ECM fungi Pisolithus and Scleroderma was tested in vitro against eight
fungi and six bacteria. Both showed higher activity against all fungi except some
Aspergillus spp. (Vaidya et al. 2005). Significant progress has been made toward
understanding of interaction (Zak 1971; Stark and Kytöviita 2005; Sampangira-
maiah and Perrin 1990; Natarajan and Govindasamy 1990) but more extensive
research is required to enhance our knowledge on interactions within fungal
community and exploring potential for manipulating ectomycorrhizosphere envi-
ronment for biotechnological purposes (Bruns and Bidartondo 2002; Cairney and
Meharg 2002). The intensity of interactions between different soil fungi and ECM
fungus C. tropicalis highlights the potential importance of interactions on function-
ing of these organisms in forest ecosystems.
18.3.3 Acid Phosphatase Production
Growth enhancements of host plant associated with ectomycorrhizal fungi has been
correlated with increased nutrient uptake by ectomycorrhiza. They solubilize insol-
uble forms of nutrients and have a significant role in carbon, nitrogen, and phos-
phorus cycling in forested ecosystems (Cullings et al. 2008). Activities of
phosphatase, laccase, glucuronidase, cellobiohydrolase, N-acetyl-glucosamine,
leucine aminopeptidase, xylosidase, and b- glucosidase were found responsible for

increased nutrient uptake (Courty et al. 2007; Mosca et al. 2007), an advantage for
enhancing nutrient acquisition (Cameron et al. 2006). There are several reports of
acid phosphatase activity of Amanita, Hebeloma, Tricholoma, etc. (Alvarez et al.
2005; Buée et al. 2005, 2007; Courty et al. 2005) which help in selecting effective
mycorrhizal symbiont for inoculation in reforestation. According to Antibus et al.
(1986) external factors affect production and activity of acid and alkaline phospha-
tase and hence efficiency of ectomycorrhizal fungi but inadequate data is known on
enzyme activities (Courty et al. 2005).
C. tropicalis was found to use a broad range of phosphorus sources during
in vitro studies conducted by Sharma (2008). When it was grown in defined
media, it releases phosphatase (probably acid phosphatase) as media has acidic
pH. Activity of acid phosphatase in ectomycorrhizal fungus C. tropicalis under
controlled conditions has been studied by Baghel et al. (2009). The pH had strong
effect on production of wall-bound acid phosphatase and maximum was observed at
pH 5 followed by 7 (Sharma et al. 2010d). Culture pH strongly influences extracel-
lular acid phosphatase production. Generally, ectomycorrhizal phosphatase has a
pH optimum approaching that of native soil (Antibus et al. 1986). Acid phosphatase
production was not affected by various temperatures tested except at 40 2 C
which showed marked reduction in enzyme production. Of nine carbon sources
used, citric acid supported highest acid phosphatase production (Sharma et al.
2010d). All tested nitrogen sources supported enzyme production and highest
with yeast extract. Among heavy metals and trace elements studied, ferric chloride
did not inhibit acid phosphatase production. Potassium di-hydrogen phosphate and
di-ammonium hydrogen phosphate produced significantly high enzyme when sup-
plemented as sole phosphorus source.
Different temperature environments have considerable effect on the physiologi-
cal and ecological consequences of ectomycorrhizal associations (Tibbett and
Cairney 2007). Like other secondary metabolites, acid phosphatase production is
directly related with mycelial growth of fungus and regulated by several abiotic and
biotic factors. Moreover, trace elements reduce enzyme activity by interacting with
the enzyme–substrate complex, by denaturing the enzyme protein, or interacting
with the protein active group (Nannipieri 1995). Trace elements (metal ions) are
assumed to inactivate enzymes by reacting with sulfhydryl groups, a reaction
analogous to formation of a metal sulfide. It has been generally recognized that
copper and cadmium are more toxic than other metals (Hattori 1992). However,
Gibson and Mitchell (2005) showed that copper has no effect on the wall-bound
phosphatase activity up to a level. Baxter and Dighton (2005) found that phospha-
tase enzymes are differentially expressed under contrasting phosphorus condition
by different ectomycorrhizal fungi. Piloderma has also shown species-specific
substrate preferences in response to organic and inorganic sources of phosphorus
(Rosling and Rosenstock 2008). However, ECM fungi differ greatly in their
capacity to produce acid phosphatase due to differential potentiality to utilize phos-
phorus, which can also be affected by season and succession (Meyselle et al. 1991).
Moreover, Courty et al. (2006) found less seasonal differences in ectomycorrhizal
acid phosphatase activity while working with Lactarius quietus, Cortinarius anom-
alus, and Xerocomus chrysenteron.
18.3.4 Activity of Acid Phosphatase
Ectomycorrhizal fungi use organic forms of soil nutrients through production of

extracellular enzymes (Aučina et al. 2007) as an adaptation for plants to colonize
soils (Read and Perez-Moreno 2003). In forest ecosystems, P is one of the most
important growth-limiting nutrients for plants. In soil, there may be many sources
of P viz., in solution as orthophosphate, ionically bound or bound in organic
compounds. The major part of soil P (sometimes as much as 90%) is sequestered
in the organic compounds phosphomonoesters and phosphodiesters (Nygren 2008).
The P uptake by forest trees has been shown to be greatly enhanced in plants
colonized by ectomycorrhizal fungi (Conn and Dighton 2000; Courty et al. 2005).
The phosphatase enzyme capabilities of ectomycorrhizal fungi are continuously
distributed between species rather than restricted to a particular taxonomic group
(Nygren 2008). Sheathing mycorrhizal fungi have been shown to possess phospha-
tase enzymes which can hydrolyze inositol hexaphosphate. Phosphatase production
by basidiomycete fungi in liquid culture is independent of P in the medium.
Saprophytic basidiomycetes tend to incorporate hydrolyzed phosphate into their
biomass. In contrast, mycorrhizal fungi release more hydrolyzed phosphate into
solution than they absorb (Dighton 1983).
Ectomycorrhizal fungi are able to secrete hydrolytic enzymes involved in the
degradation of organic matter (Burns and Dick 2002; Lindahl et al. 2005). Acid
phosphatases solubilize insoluble forms of P not readily available to uninfected
plant roots (Tibbett et al. 1998). These enzymes are generally bound to the outer
cell walls (Rast et al. 2003). Phosphatase activities of ectomycorrhizal fungi can
vary between species, resulting in different efficiency of P utilization of host plant
(Dighton 1983). These enzymes are in direct contact with soil environment but are
able to adapt to various soil conditions and maintain activity. However, it is known
that soil components, pH, and trace elements modify the conformation of enzymes
and affect their activities. Activities of acid phosphatase are found to differ signifi-
cantly amongst ECM synthesized with different fungi and among different species
of the same fungi (Buée et al. 2005, 2007; Courty et al. 2006).
C. tropicalis showed maximum growth in acidic culture medium (Sharma
2008; Baghel et al. 2009). The p-nitrophenol phosphatase (p-NPPase) activity of
C. tropicalis isolate showed a stable activity at a pH range of 3–4 with optimum
being 4 even though the mycelial biomass production was less as compared with
pH 3 (Table 18.2). The activity of p-NPPase for C. tropicalis isolate dropped
significantly above pH 4.0. Experiments conducted at higher pH up to 12 to detect
any alkaline phosphatase activity yielded negative results (Baghel et al. 2009).
Baghel et al. (2008) have also studied the acid phosphatase activity of soil of
these bamboo forests. Surface-bound phosphoesterases activities of P involutus,
Table 18.2 Effects of pH and temperature on the acid phosphatase activity of C. tropicalis
pH study Temperature study
Enzyme Enzyme activity Incubation temperature Enzyme activity
incubation pH (mg g1 mdw) (2 C) (mg g1 mdw)
1 0.30 0.01 a 5 3.73 0.05 a
2 0.97 0.03 b 10 3.57 0.05 b
3 3.90 0.28 cd 15 4.50 0.14 c
4 4.16 0.54 d 20 5.67 0.33 d
5 3.71 0.15 cf 25 6.66 0.08 e
6 3.81 0.26 cf 30 11.13 0.81 f
7 3.55 0.18 f 35 8.80 0.33 g
812 40 18.48 0.55 h
Initial pH was 5.5; culture was 15 days old; results are average of mycelial dry weights (mdw) with
standard deviations; values within a column followed by the same alphabet (viz., a, b, c, etc.) do
not differ significantly at p < 0.05
Source: Adapted from Baghel et al. (2009)
Austropaxillus boletinoides, Descolea antartica, C. geophilum, and P. tinctorius

have been reported at pH 3–7 (Alvarez et al. 2005). Investigations by Antibus et al.
(1986) indicated that Hebeloma, Paxillus, Entoloma, and C. geophilum were
typified by sharp decrease in phosphatase activity above pH 5.
18.4 Conclusion
Several devices have been developed for in vitro ectomycorrhizal synthesis but
most of them have been found to be bulky and does not allow handling of several
experimental units simultaneously. Although considerable attention has been given
to synthesis of pine ECM, the techniques used were found difficult to apply to
angiosperms. A filter paper-flask technique developed by the authors was quite
handy, requires limited space, forms rapid ECM, and permits observation of ECM
formation. The technique was found highly successful in a maiden attempt of
ectomycorrhization in a monocot plant, D. strictus by C. tropicalis (Sharma et al.
2009b). The technique not only appears to be useful for the production of sufficient
mycorrhizal seedlings for biochemical and physiological studies but can also
produce desired quantity of mycorrhizal material for practical forestry applications.
In depth, understanding of the structure of ECM and its functioning in the perfor-
mance of tailored seedlings in nursery and field conditions are the ultimate objec-
tives of research on in vitro ectomycorrhization. However, further development of
the techniques involved in artificial synthesis of ECM is desirable.
Ectomycorrhization was influenced by solid or liquid inoculum, physical, chem-
ical and biological characteristics of the substrate as confirmed by used tea leaves
and sand mixture. The substrate stored sufficient amount of moisture which helped
the fungus to grow actively. Several types of natural or laboratory produced inocula
and various methods of application have been reported to be successful through the
years. Ectomycorrhization helps in survival and growth of host seedlings and also
increases host ability for nutrient and water uptake. The mycobiota of forest soils
consists of AM fungi, ECM fungi, and saprotrophic decomposers and pathogens.
ECM fungi interact with other microorganisms, the outcome of which may be
inhibitory or stimulatory, some competitive, while others mutualistic, which are
important in biogeochemical cycling in forest ecosystems. Antagonistic interac-
tions with mycorrhizal fungi have significant effect on mycorrhizal systems. In the
present studies, C. tropicalis was found highly antagonistic to some rhizosphere soil
microorganisms. Growth enhancement of host plant associated with ECM fungi has
been often correlated with increased nutrient uptake by ectomycorrhizal which is
due to secretion of surface-bound extracellular enzymes including phosphatases,
laccases, xylosidase, cellulohydrolase, etc. There are several reports of acid phos-
phatase activity of ECM fungi, which help in selecting effective mycorrhizal
symbionts for field inoculation of seedlings in reforestation program. However,
there are inadequate data on diversity and distribution of enzymes activities in
native ectomycorrhizal communities.
Work in future will focus on several aspects which are essential for complete
study of the mushroom. Bacteria affect functioning of ectomycorrhizal symbioses
including establishment, mobilization of minerals, nitrogen fixation, and antago-
nism of pathogens (Frey-Klett et al. 2007). Few attempts have been made to
characterize bacterial communities associated with ECM (Mogge et al. 2000;
Bertaux et al. 2005; Burke et al. 2008). In future studies, the bacterial population
associated with this mushroom has to be studied along with its role which will
highlight their beneficial role in the life cycle of Cantherellus. Moreover, Danell
et al. (1993) has already demonstrated the role of Pseudomonas on culturing of
C. cibarius. ECM fungi are able to alleviate the stress for plants caused by heavy
metal contamination of soil. Analyzes of molecular response of ECM fungi to these
pollutants will lead to new insights in the study of C. tropicalis. There is a need to
investigate the kinetics, energetics, and specificity of amino acid transporter from
the ECM fungus C. tropicalis.
The amplification of internal transcribed spacer (ITS) of ribosomal RNA genes
using PCR and subsequent RFLP or sequence analysis offers promise for improved
species or strain level identification of ectomycorrhizal fungi. Intrageneric homo-
geneity and intraspecific heterogeneity of these characters are however, frequent.
Therefore, there is a need to study intraspecific variation among various strains of
Cantharellus which holds the future of our research. The size and spatial distribu-
tion of mycelial individuals of C. tropicalis could be studied using RAPD and
microsatellite data and genetically distinct individuals can be recognized which will
help in knowing its genetic makeup in the region. In fact, the present trend is to use
the advance molecular tools and built-up capability to manipulate the symbionts to
our advantage (Krishna 2005). There is a need to study field plantation and study its
interaction with other fungi and organisms. All these studies will help to gather
information which will be suitable to study the biology of C. tropicalis.
Finally, sustainable forest management and the establishment of plantation of

fast growing tree species are key issues in present day tropical forestry. Bamboos
are the most important feature of tropical forests along with dipterocarps and other
broad leaf trees. Thus, management of the existing natural forests for sustained
timber supply, research in to the role of ectomycorrhizas in seedling establishment,
and growth of plant is need of hour.
References
Alvarez M, Godoy R, Heyser W, Hartel S (2005) Anatomically physiological determination of

surface bound phosphatase activity of ectomycorrhizae of Nothofagus oblique. Soil Biol
Biochem 37:125–132
Antibus RK, Kroehler CJ, Linkins AE (1986) The effects of external pH temperature and substrate
concentration on acid phosphates activity of ectomycorrhizal fungi. Can J Bot 64:2383–2387
Aučina A, Rudawska M, Leski T, Skridaila A, Riepsas E, Iwanski M (2007) Growth and
mycorrhizal community structure of Pinus sylvestris seedlings following the addition of forest
litter. Appl Environ Microbiol 73:4876–4883
Baar J, Stanton NL (2000) Ectomycorrhizal fungi challenged by saprotrophic basidiomycetes and
soil micro fungi under different ammonium regimes in vitro. Mycol Res 104:691–697
Baghel RK, Sharma R, Pandey AK (2008) Acid phosphatase activity in rhizospheric soils of
tropical bamboo forests. J Mycol Plant Pathol 38(3):596–598
Baghel RK, Sharma R, Pandey AK (2009) Activity of acid phosphatase in the ectomycorrhizal
fungus Cantharellus tropicalis under controlled conditions. J Trop For Sci 21(3):222–226
Baxter JW, Dighton J (2005) Phosphorus source alters host plant response to ectomycorrhizal
diversity. Mycorrhiza 15:513–523
Bertaux J, Schmid M, Hutzler P, Hartmann A, Garbaye J, Frey-Klett P (2005) Occurrence and
distribution of endobacteria in the plant associated mycelium of the ectomycorrhizal fungus
Laccaria bicolor S238N. Environ Microbiol 7:1786–1795
Bruns TD, Bidartondo MI (2002) Molecular windows into the below-ground interactions of
ectomycorrhizal fungi. Mycologist 16:47–50
Bruns TD, Peay KG, Boynton PJ, Grubisha LC, Hynson NA, Nguyen NH, Rosenstock NP (2009)
Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr spore
burial experiment. New Phytol 181(2):463–470
Buée M, Vairelles D, Garbaye J (2005) Year-round monitoring of diversity and potential meta-
bolic activity of the ectomycorrhizal community in a beech forest subjected to two thinning
regimes. Mycorrhiza 15:235–245
Buée M, Courty PE, Mignot D, Garbaye J (2007) Soil niche effect on species diversity and
catabolic activities in an ectomycorrhizal fungal community. Soil Biol Biochem 39:1947–1955
Burke DJ, Dunham SM, Kretzer AM (2008) Molecular analysis of bacterial communities asso-
ciated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycor-
rhizal fungi. FEMS Microbiol Ecol 65:299–309
Burns GB, Dick RP (2002) Enzymes in the environment; activity ecology and applications. Marcel
Dekker, New York
Cairney JWG (2005) Basidiomycete mycelia in forest soils: dimensions, dynamics and roles in
nutrient distribution. Mycol Res 109(1):7–20
Cairney JWG, Chambers SM (1999) Ectomycorrhizal fungi-key genera in profile. Springer-
Verlag, Berlin
Cairney JWG, Meharg AA (2002) Interactions between ectomycorrhizal fungi and soil sapro-
trophs: implications for decomposition of organic matter in soils and degradation of organic
pollutants in the rhizosphere. Can J Bot 80(8):803–809
Cameron W, Margaret SP, Johnston E, Ramage CM, Edwards DG, Cawthray GR, Lambers H
(2006) Functional significance of dauciform roots: exudation of carboylates and acid phospha-
tase under phosphorus deficiency in Caustis blakei (Cyperaceae). New Phytol 170:491–500
Chen YL, Kang LH, Dell B (2006) Inoculation of Eucalyptus urophylla with spores of Sclero-
derma in a nursery in South China: comparison of field soil and potting mix. For Ecol Manage
222:439–449
Chilvers GA, Douglas PA, Lapeyrie FF (1986) A paper sandwich technique for rapid synthesis of
Conn C, Dighton J (2000) Litter quality influences on decomposition, ectomycorrhizal community
structure and mycorrhizal root surface acid phosphatase activity. Soil Biol Biochem
32:489–496
Courty PE, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005) Activity profiling of ectomycor-
rhiza communities in two forest soils using multiple enzymatic tests. New Phytol 167
(1):309–319
Courty PE, Pouysegur R, Bùee M, Garbaye J (2006) Laccase and phosphatase activities of the
dominant ectomycorrhizal types in a lowland oak forest. Soil Biol Biochem 38(6):1219–1222
Courty P-E, Bréda N, Garbaye J (2007) Relation between oak tree phenology and the secretion of
organic matter degrading enzymes by Lactarius quietus ectomycorrhizas before and during
bud break. Soil Biol Biochem 39(7):1655–1663
Cullings K, Ishkhanova G, Hensen J (2008) Defoliation effects on enzyme activites of the
ectomycorrhizal fungus Suillus granulatus in a Pinus contorta (lodgepole pine) stand in
Yellostone National Park. Oecologia 158(1):77–83
Dahlstrom JL, Smith JE, Weber NS (2000) Mycorrhiza like interaction by Morchella with species
of the Pinaceae in pure culture synthesis. Mycorrhiza 9:279–285
Danell E (1994) Formation and growth of the ectomycorrhiza of Cantharellus cibarius. Mycor-
rhiza 5:89–97
Danell E (1999) Cantharellus. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal fungi-key
genera in profile. Springer-Verlag, Berlin, pp 253–267
Danell E (2002) Current research on chantharelle cultivation in Sweden. In: Hall I, Wang Y,
Danell E, Zambonelii A (eds) Edible mycorrhizal mushrooms and their cultivation. Crop &
Food Research, Christchurch, pp 1–4
Danell E, Camacho FJ (1997) Successful cultivation of the golden chanterelle. Nature 385:303
Danell E, Sadhna A, Ternstrom A (1993) Pseudomonas fluorescens in association with fruit bodies
of the ectomycorrhizal mushroom Cantharellus cibarius. Mycol Res 97(9):1148–1152
Dighton J (1983) Phosphatase production by mycorrhizal fungi. Plant Soil 71:455–462
Duddridge JA (1986) The development and ultrastructure of ectomycorrhizae III. Compatible and
incompatible interactions between Suillus grievllei (Klotzsch) Sing, and species of ectomycor-
rhizal hosts in vitro in the absence of exogenous carbohydrate. New Phytol 103:457–464
Finlay RD (2004) Mycorrhizal fungi and their multifunctional role. Mycologist 18(2):91–96
Fitter AH, Garbaye J (1994) Interactions between mycorrhizal fungi and other soil organisms.
Plants Soil 159:123–132
Fortin JA, Piche Y, Lalonde M (1980) Technique for the observation of early morphological
changes during ectomycorrhiza formation. Can J Bot 58:361–365
Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhiza helper bacteria revisited. New Phytol
176:22–36
Garbaye J, Delwaulle JC, Diangana D (1988) Growth response of eucalypts in the Congo to
ectomycorrhizal inoculation. For Ecol Manage 24(2):151–157
Gibson BR, Mitchell DT (2005) Phosphatses of ericoid mycorrhizal fungi: kinetic properties and
the effect of copper on activity. Mycol Res 109(4):478–486
Girlanda M, Varese GC, Luppi-Mosca AM (1995) In vitro interactions between saprotrophic
microfungi and ectomycorrhizal symbionts. Allionia 33:81–86
Guerin-Laguette A, Plassard C, Mousain D (2000a) Effects of experimental conditions on mycor-
rhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-
body formation of the Saffron milk cap under controlled soil less conditions. Can J Microbiol
46:790–799
Guerin-Laguette A, Vaario L, Gill WM, Lapeyrie F, Matsushita N, Suzuki K (2000b) Rapid

in vitro ectomycorrhizal infection on Pinus densiflora roots by Tricholoma matsutake.
Mycoscience 41:389–393
Harley JL, Smith SE (1983) Mycorrhizal symbiosis. Academic Press Inc., London, p 483
Hattori H (1992) Influence of heavy metals on soil microbial activities. Soil Sci Plant Nutr
38:93–100
Johansson J, Paul L, Finlay RD (2004) Microbial interactions in the mycorrhizosphere and their
significance for sustainable agriculture. FEMS Microbiol Ecol 18:1–13
Khosla B, Reddy S (2008) Response of ectomycorrhizal fungi on the growth and mineral nutrition
of Eucalyptus seedlings in bauxite mined soil. Am Eurasian J Agric Environ Sci 3(1):123–126
Kranabetter JM, Friesen J, Gamiet S, Kroeger P (2005) Ectomycorrhizal mushroom distribution by
stand age in western hemlock–lodgepole pine forests of North-Western British Columbia. Can
J For Res 35:1527–1539
Krishna KR (2005) Mycorrhizas: a molecular analysis. Oxford and IBH Publishing Co. Pvt. Ltd.,
New Delhi
Kumar S, Satyanarayana T (2002) Production of inoculum of ectomycorrhizal fungi. In: Mukerji
KG, Manoharachary C, Chamola BP (eds) Techniques in mycorrhizal studies. Kluwer Aca-
demic Publishers, Netherlands, pp 143–166
Lakhanpal TN (1996) Mushrooms of India Boletaceae. In: Mukerji KG (ed) Studies in crypto-
gamic botany, vol I. APH Publishing Corporation, Delhi
Lamb RJ, Richards BN (1974) Survival of potential of sexual and asexual spores of ectomycor-
rhizal fungi. Trans Br Mycol Soc 62:181–191
Lamhamedi MS, Abourouh M, Fortin JA (2009) Technological transfer: the use of ectomycor-
rhizal fungi in conventional and modern forest tree nurseries in northern Africa. In: Khasa D,
Piche Y, Coughlan AP (eds) Advances in mycorrhizal science and technology. NRC Research
Press, Ottawa, p 197
Lapeyrie FF, Bruchet G (2006) Some factors influencing viability of ectomycorrhizal fungal
inoculum. New Phytol 100(4):585–593
Last FT, Natarajan K, Mohan V, Mason PA (1992) Sequences of sheathing (ecto)mycorrhizal
fungi associated with man-made forests, temperate and tropical. In: Read DJ, Lewis DH,
Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. CAB International, Wallingford,
pp 214–219
Leake JR, Johnson D (2004) Networks of power and influence: the role of mycorrhizal mycelium
in controlling plant communities and agro ecosystem functioning. Can J Bot 82(8):1016–1045
Leake JR, Donnelly DP, Saunders EM, Boddy L, Read DJ (2001) Rates and quantities of carbon
flux to ectomycorrhizal mycelium following 14C pulse labeling of Pinus sylvestris L. seedlings:
effects of litter patches and interaction with a wood-decomposer fungus. Tree Physiol
21:71–82
Lindahl BD, Finlay RD, Cairney JWG (2005) Enzymatic activities of mycelia in mycorrhizal
fungal communities. In: Dighton J (ed) The fungal community, its organization and role in the
ecosystem. Marcel Dekker, New York, pp 331–348
Littke WR, Bledsoe CS, Nadkarni NM, Edmonds RL (1980) Technique for rapid mycorrhizal
colonization of container-grown Douglas-fir by Hebeloma crustuliniforme. Soil Biol Biochem
12:575–578
Marx DH (1980) Ectomycorrhiza fungus inoculations, a tool for improving forestation practices.
In: Mikola P (ed) Tropical mycorrhiza research. Oxford University Press, Oxford, pp 13–71
Marx DH, Bryan WC (1975) Growth and ectomycorrhizal development of loblolly pine seedlings
Marx DH, Ruehle JL, Kennedy DS, Cordell CE, Riffle JW, Ruehle JW, Molina RJ, Pawac WH,
Nauratil S, Tinus RW, Goodwin OC (1982) Commercial vegetative inoculum of Pisolithus
tinctorius and inoculation techniques for development of ectomycorrhizae on container grown
seedlings. For Sci 28:373–400
Meyselle JP, Gay G, Debaud JC (1991) Intraspecific genetic variation of acid phosphatase activity
in monokaryotic and dikaryotic populations of ectomycorrhizal fungus Hebeloma cylindros-
porum. Can J Bot 69:8089–8113
Mogge B, Loferer C, Agerer R, Hutzler P, Hartmann A (2000) Bacterial community structure and
colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluores-
cent in situ hybridization and confocal laser scanning microscopy. Mycorrhiza 9:271–278
Molina R, Trappe JM (1982) Applied aspects of ectomycorrhiza. In: Subbarao NS (ed) Advances
in agricultural microbiology. Oxford and IBH Publishing Co., New Delhi, pp 305–324
Molina R, Smith JE, McKay D, Melville LH (1997) Biology of the ectomycorrhizal genus
Rhizopogon. New Phytol 137:519–528
Morin C, Samson J, Dessureault M (1999) Protection of black spruce seedlings against Cylindro-
cladium root rot with ectomycorrhizal fungi. Can J Bot 77(1):169–174
Mosca E, Montecchio L, Scattolin L, Garbaye J (2007) Enzymatic activities of three ectomycor-
rhizal types of Quercus robur L. in relation to tree decline and thinning. Soil Biol Biochem 39
(11):2897–2904
Moser M (1958) Die Kiinstliche Mycorrhizaimp fung an Forstpflanzen. I. Erfahrungen bei der
Reinkulture von Mycorrhizapilzen. Forest wiss Centralbl 77:32–40
Nannipieri P (1995) The potential use of soil enzymes as indicators of productivity, sustainability
and pollution. In: Pankhurst CE, Doube BM, Gupta VVSR, Gracel PR (eds) Soil biota:
management in sustainable farming systems. CSIRO Publishing, East Melbourne, pp 238–244
Natarajan K, Govindasamy V (1990) Antagonism of ectomycorrhizal fungi to some common root
pathogens. In: Jalali BL, Chand H (eds) Current trends in mycorrhizal research. Sankat
Mochan Art Press, Hisar, India, pp 98–99
Natarajan K, Ravindran C (2003a) Two new species of the genus Entoloma from South India.
Mycotaxon 85:143–146
Natarajan K, Ravindran C (2003b) Two new species of the genus Pholiota from South India.
Mycotaxon 85:271–275
Natarajan K, Nagarajan G, Sudhakara Reddy M (1995) In vitro mycorrhization and growth
response of Acacia nilotica seedlings by inoculation with ectomycorrhizal fungi. Ind J Micro-
biol 35(1):35–38
Natarajan K, Senthilarasu G, Kumaresan V, Riviere T (2005a) Diversity in ectomycorrhizal fungi
of a dipterocarp forest in Western Ghats. Curr Sci 88(12):1893–1895
Natarajan K, Narayan K, Ravindran C, Kumaresan V (2005b) Biodiversity of agarics from Nilgiri
Biosphere Reserve, Western Ghats, India. Curr Sci 88(12):1890–1893
Nezzar-Hocine H, Pen-In R, Halli-Hargas R, Chevalier G (1998) Ectomycorrhizal associations
with Cedrus atlantica (Endl) Manetti ex Carriere-I, mycorrhizal synthesis with Tricholoma
tridentinum Singer var. cedretorum Bon. Mycorrhiza 8:47–51
Nygren C (2008) Functional diversity in nutrient acquisition by ectomycorrhizal fungi. PhD thesis,
Swedish University of Agricultural Sciences, Uppsala
Olsson PA (1999) Signature fatty acids provide tools for determination of the distribution and
interactions of mycorrhizal fungi in soil. FEMS Microbiol Ecol 29(4):303–310
Pande V, Palni UT, Singh SP (2004) Species diversity of ectomycorrhizal fungi associated with
temperate forest of Western Himalaya: a preliminary assessment. Curr Sci 86(12):1619–1623
Pande V, Palni UT, Singh SP (2007) Effect of ectomycorrhizal fungal species on the competitive
outcome of two major forest species. Curr Sci 92(1):80–84
Park JE (1984) Inoculum potential of ectomycorrhizal fungi in forest soil from South-West Oregon
and Northern California. For Sci 30:300–304
Rast DM, Baumgartner D, Mayer C, Hollenstein GO (2003) Cell wall-associated enzymes in
fungi. Phytochemistry 64:339–366
Riviere T, Diedhiou AG, Diabate M, Senthilarasu G, Natarajan K, Verbeken A, Buyck B, Dreyfus B,
Bena G, Ba Amadou M (2007) Genetic diversity of ectomycorrhizal basidiomycetes from
African and Indian tropical rain forests. Mycorrhiza 17(5):415–428
Rosling A, Rosenstock N (2008) Ectomycorrhizal fungi in mineral soil. Minerol Mag 72
(1):127–130
Sampangiramaiah K, Perrin R (1990) Interactions between isolates of ectomycorrhizal Laccaria

spp. and root rot fungi of conifers. In: The proceedings of national conference on mycorrhiza,
Harayana Agricultural University, Hissar, India, pp 124–125
Santoro T Jr, Casida LE (1962) Elaboration of antibiotics by Boletus luteus and certain other
mycorrhizal fungi. Can J Microbiol 8:43–48
Sharma R (2008) Studies on ectomycorrhizal mushrooms of M.P. and Chhattisgarh. PhD thesis,
R.D. University, Jabalpur, India
Sharma ND, Nema S, Rai BK (2003) A simple technique for obtaining pure culture of Pleurotus
spp. J Mycopathol Res 41(1):119–120
Sharma R, Rajak RC, Pandey AK (2008a) Some ectomycorrhizal mushrooms of Central India – I.
Russula. J Mycopathol Res 46(2):201–212
Sharma R, Rajak RC, Pandey AK (2008b) Growth response of Dendrocalamus seedlings by
inoculation with ectomycorrhizal fungi. Middle East J Sci Res 3(4):200–206
Sharma R, Rajak RC, Pandey AK (2009a) Some ectomycorrhizal mushrooms of Central India – II.
Lactarius. J Mycopathol Res 47(1):43–47
Sharma R, Rajak RC, Pandey AK (2009b) Simple technique for ectomycorrhizal formation
between Cantharellus and Dendrocalamus strictus. Taiwan J For Sci 24(2):141–148
Sharma R, Rajak RC, Pandey AK (2009c) Ectomycorrhizal mushrooms in Indian tropical forests.
Biodiversity 10(1):25–30
Sharma R, Rajak RC, Pandey AK (2010a) Mass multiplication of ectomycorrhizal Cantharellus
inoculum for large scale tailoring nursery inoculations of bamboo seedlings. Asian J Sci Res,
published online
Sharma R, Rajak RC, Pandey AK (2010b) Some ectomycorrhizal mushrooms of Central India-V.
Pisolithus, Scleroderma, Geastrum, Cantharellus. J Mycopathol Res, in press
Sharma R, Rajak RC, Pandey AK (2010c) Evidence of antagonistic interactions between rhizo-
sphere and mycorrhizal fungi associated with Dendrocalamus strictus (Bamboo). J Yeast
Fungal Res 1(7):112–117
Sharma R, Baghel RK, Pandey AK (2010d) Dynamics of acid phosphatase production of the
ectomycorrhizal mushroom Cantharellus tropicalis. Afr J Microbiol Res 4(18), in press
Shaw TM, Dighton J, Sanders FE (1995) Interactions between ectomycorrhizal and saprotrophic
fungi on agar and in association with seedlings of lodgepole pine (Pinus contorta). Mycol Res
99:159–165
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn. Academic Press, London
Stark S, Kyt€oviita M-M (2005) Evidence of antagonistic interactions between rhizosphere micro-
organisms and mycorrhizal fungi associated with birch (Betula pubescens). Acta Oecologica
28(2):149–155
Straatsma G, van Griensven LJLD (1986) Growth requirements of mycelial cultures of the
mycorrhizal mushroom, Cantharellus cibarius Fr. Trans Br Mycol Soc 87:135–141
Sun YP, Unestam T, Lucas SD, Johanson KJ, Kenne L, Finlay R (1999) Exudation-reabsorption in
a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms.
Takacs EA (1961) Inoculation de especies de pinos con hongos formadores de micorizas.
Silvicultura (Uruguay) 15:5–17
Theodorou C (1971) Introduction of mycorrhizal fungi into soil by spore inoculation of seed. Aust
For 35:17–22
Theodorou C, Bowen GD (1973) Introduction of seeds and soil with basidiospores of mycorrhizal
fungi. Soil Biol Biochem 5:765–771
Theodorou C, Reddell P (2006) In vitro synthesis of ectomycorrhizas on Casuarinaceae with a
range of mycorrhizal fungi. New Phytol 118(2):279–288
Tibbett M, Chambers SM, Cairney JWG (1998) Method for determining extracellular and surface-
bound phosphatase activities in ectomycorrhizal fungi. In: Varma A (ed) Mycorhhiza manual.
Springer, New York, pp 217–226
Tibbett M, Cairney WG (2007) The cooler side of mycorrhizas: their occurrence and functioning
at low temperatures. Can J Bot 85(1):51–62
Trappe JM (1977) Selection of fungi for ectomycorrhizal inoculation in nurseries. Annu Rev
Phytopathol 15:203–222
Vaario L, Tanaka M, Ide Y, Gill WM, Suzuki K (1999) In vitro ectomycorrhiza formation between
Abies firma and Pisolithus tinctorius. Mycorrhiza 9:177–183
Vaario L, Gill WM, Tanaka M, Ide Y, Suzuki K (2000) Aseptic ectomycorrhizal synthesis between
Abies firma and Cenococcum geophilum in artificial culture. Mycoscience 41:395–399
Vaidya GS, Shrestha K, Wallander H (2005) Antagonistic study of ectomycorrhizal fungi
isolated from Baluwa forest (Central Nepal) against with pathogenic fungi and bacteria.
Sci World 3(3):49–52
Valentine LL, Fiedler TL, Hart AN, Petersen CA, Berninghausen HK, Southworth D (2004)
Diversity of ectomycorrhizas associated with Quercus garryana in southern Oregon. Can J
Bot 82:123–135
Werner A, Zadworny M (2003) In vitro evidence of mycoparasitism of the ectomycorrhizal
fungus Laccaria laccata against Mucor hiemalis in the rhizosphere of Pinus sylvestris.
Mycorrhiza 13(1):41–47
Werner A, Zadworny M, Idzikowska K (2002) Interaction between Laccaria laccata and Tricho-
derma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro. Mycor-
rhiza 12(3):139–145
Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52:487–511
Whipps JM (2004) Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Can
J Bot 82:1198–1227
Wu T, Sharda JN, Koide RT (2003) Exploring interactions between saprotrophic microbes and
ectomycorrhizal fungi using a protein-tannin complex as an N source by red pine (Pinus
resinosa). Special issue: functional genomics of plant–pathogen interactions. New Phytol
159(1):131–139
Yamada A, Katsuya K (1995) Mycorrhizal association of isolates from sporocarps and ectomycor-
rhizas with Pinus densiflora seedlings. Mycoscience 40:455–463
Yamada A, Maeda K, Ohmasa M (1999) Ectomycorrhiza formation of Tricholoma matsutake
isolates on seedlings of Pinus densiflora in vitro. Mycoscience 40:455–463
Yamada A, Ogura T, Ohmasa M (2001a) Cultivation of mushrooms of edible ectomycorrhizal
fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis. I. Primordium and
basidiocarp formation in open-pot culture. Mycorrhiza 11:59–66
Yamada A, Ogura T, Ohmasa M (2001b) Cultivation of mushrooms of edible ectomycorrhizal
fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis. II. Morphology of
mycorrhizas in open-pot soil. Mycorrhiza 11:67–81
Zak B (1971) Characterization and classification of mycorrhizae of Douglas-fir-II, Pseudostuga
menziesii þ Rhizopogon vinicolor. Can J Bot 49:1079–1084
Chapter 19
Edible Ectomycorrhizal Fungi: Cultivation,
Conservation and Challenges
Alka Karwa, Ajit Varma and Mahendra Rai
19.1 Introduction
A mycorrhiza (Gk: fungus roots) is a symbiotic association between a fungus and

the roots of a vascular plant (Frank 1885; Brundrett 2004). In a mycorrhizal
association, the fungus colonizes roots of the host plants, either intracellularly as
in endomycorrhizal fungi, also known as arbuscular mycorrhiza (AM), or extracel-
lularly as in ectomycorrhizal fungi (ECM). Most of the plants are equally dependent
on mycorrhizal fungi, and without them, the plants become stunted and yellow,
often due to a lack of phosphorus (Tarafdar and Marschner 1994; Schweiger et al.
1995; Kahiluoto and Vestberg 1998; Redecker et al. 2000). “Plants without mycor-
rhizal fungi are competitively inferior. Graham stated that mycorrhizal fungi
function as an auxiliary root system to provide additional nutrients” (Harvey
et al. 1987; Graham et al. 1994, 1999). The mutualistic association provides the
fungus with relatively constant and direct access to carbohydrates, such as glucose
and sucrose supplied by the plant (Cook 1977; Harrison 2005). The carbohydrates
are translocated from their source, usually leaves, to root tissue and on to the fungal
partners. In return, the plant gains the benefits of the higher absorptive capacity of
mycelium for water and mineral nutrients due to comparatively large surface area
of mycelium:root ratio (Tarafdar and Marschner 1994; Schweiger et al. 1995;
Kahiluoto and Vestberg 1998), thus improving the plant’s mineral absorption
capabilities (Selosse et al. 2006). Plant roots alone may be incapable of taking up
phosphate ions that are demineralized, for example, in soils with a basic pH (Abbott
and Robson 1991). The mycelium of the mycorrhizal fungus can, however, access
these phosphorus sources and make them available to the plants they colonize
(Li et al. 2006). Thus, the mechanisms of increased absorption are both physical
A. Karwa (*) and M. Rai

Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India
e-mail: alka0404@gmail.com
A. Varma
Amity Institute of Microbial Technology, Amity University, Noida, Uttar Pradesh, India
e-mail: ajitvarma@aihmr.amity.edu

Soil Biology 25, DOI 10.1007/978-3-642-15196-5_19,
430 A. Karwa et al.
and biochemical. Mycorrhizal mycelia are much smaller in diameter than the
smallest root hairs, and thus can explore a greater volume of soil, providing a larger
surface area for absorption (Tuomi et al. 2001). Also, the cell membrane chemistry
of fungi is different from that of plants (Ogawa 1985; Agerer 1995; Unestam and
Sun 1995) that include organic acid excretion which aids in ion displacement
(Griffiths and Caldwell 1992; http://cropsoil.psu.edu/sylvia/mycorrhiza.htm).
Mycorrhizae are thus especially beneficial for the plant partner in nutrient-poor
soils (Malajczuk et al. 1982).
19.2 Occurrence
The concept that plants have varying degrees of dependence on mycorrhizal

associations has gained acceptance long back (Janos 1980; St John 1980; Brundrett
1991; Marschner 1995). Mycorrhizae are present in 95% of plant families (Trappe
1987; Wang and Qiu 2006), with endomycorrhizae being the ancestral and predom-
inant form (Wang and Qiu 2006) and indeed the most prevalent symbiotic associa-
tion found in the entire plant kingdom (Harrison 2005). The structure of
endomycorrhizae has been highly conserved since their first appearance in the
fossil record (Pirozynski and Dalpé 1989; Remy et al. 1994; Redecker et al.
2000; Dotzler et al. 2006; Strullu-Derrien and Strullu 2007) with both the develop-
ment of ectomycorrhizae and the loss of mycorrhizae, evolving convergently on
multiple occasions (Wang and Qiu 2006). Both endomycorrhiza and ectomycor-
rhiza have the ability to be mycorrhizal fungi. In endomycorrhiza, the hyphae of the
fungus penetrate the outer cells of the plant root and extend into the surrounding soil
(Schultze et al. 1997; Addy et al. 2005; Schultz and Boyle 2005). Whereas in
ectomycorrhiza, the hyphae surround, but do not penetrate the roots. Endomycor-
rhiza are much more common than ectomycorrhiza. The fungal component of
endomycorrhiza is a zygomycete. While only about 30 species of zygomycetes
are known to be involved in endomycorrhizal relationships, the zygomycetes are
associated with more than 200,000 species of plants (85%) (Wang and Qiu 2006).
Basidiomycetes are the most common fungal components of ectomycorrhiza,
although some ascomycetes also form ectomycorrhizal relationships (Hosaka et al.
2007; Wilson et al. 2007). More species of fungi are involved in ectomycorrhiza (at
least 5,000), but most are only associated with a single species of plant. Further-
more, the total number of plants involved in ectomycorrhiza is limited to a few
thousand (only 10%). The most common plants associated with ectomycorrhiza are
trees and shrubs growing in temperate regions. These trees include teak, shorea,
bamboo, acacia, pines, firs, oaks, beeches, and willows (Rose 1980; Malloch and
Malloch 1981; Harley and Harley 1987; Brundett et al. 1990; Redhead 1997;
Tedersoo et al. 2007). These plants tend to be more resistant to extreme tempera-
tures, drought, and other harsh environmental conditions. Some ectomycorrhizal
fungi may provide protection from acidic precipitation. Mycorrhizal plants are
often more resistant to diseases, such as those caused by microbial soil-borne
19 Edible Ectomycorrhizal Fungi: Cultivation, Conservation and Challenges 431
pathogens, and to the effects of drought (Brundrett and Kendrick 1988). These
effects are perhaps due to the improved water and mineral uptake in mycorrhizal
plants.
19.3 Role of Ectomycorrhizas
Ectomycorrhizas consist of a hyphal sheath, or mantle, covering the root tip and a
hartig net of hyphae surrounding the plant cells within the root cortex. In some
cases, the hyphae may also penetrate the plant cells, in which case the mycorrhiza is
called an ectendomycorrhiza. Outside the root, the fungal mycelium forms an
extensive network within the soil and leaf litter (Meyer 1973; Harvey et al. 1976;
Garbaye 1994). Nutrients can be shown to move between different plants through
the fungal network (sometimes called the wood wide web). Carbon has been shown
to move from birch trees into fir trees, thereby promoting succession in ecosystems
(Simard et al. 1997). The ectomycorrhizal symbiosis represents one of the most
prominent and ecologically crucial mutualistic associations in terrestrial habitats
(Marks et al. 1968; Vogt et al. 1981; Hunt and Fogel 1983). ECMs occur in most of
the temperate and boreal ecosystems and in large forested areas of tropical and
subtropical regions (Smith and Read 1997; Cairney and Chambers 1999; Verbeken
and Buyck 2001; Comandini et al. 2006; Wang and Qiu 2006; Rinaldi et al. 2008;
Sharma 2008; Baghel et al. 2009). The hyphae of most ECM fungal species
proliferate in the duff layer of the forest floor, but some also inhabit mineral soil,
and still others prefer decaying wood as a substrate (Goodman and Trofymow
1998). Some ECM fungi require large amounts of carbohydrate, which they acquire
from their plant hosts, and so are dependent on mature trees that can meet their
carbohydrate demands (Deacon and Fleming 1992).
19.4 Mycorrhizal Mushrooms
There is a wide taxonomic and structural diversity of higher fungi. The reproductive
structures of larger fungi include epigeous mushrooms, puffballs, coral fungi, crust
fungi, etc., and subterranean (hypogeal) fungi called truffles, or truffle-like fungi.
Most of these categories contain ectomycorrhizal mushrooms. The fact to be noted
is that each species of host plant is not restricted to just one mycorrhizal fungus. For
example, Douglas fir can form mycorrhizas with hundreds of different mycorrhizal
fungi. Similarly, some oaks can form ectomycorrhizas with a wide range of fungi,
such as Amanita caesarea, Amanita phalloides, Boletus edulis, as well as the
Périgord black truffle Tuber melanosporum, and it is not unusual to find half a
dozen different ECM fungi competing for space on the roots of a suitable host (Hall
and Wang 1998). A list of putative 955 mycorrhizal edible and/or medicinal
mushrooms has been provided by Hall et al. (2003). This suggests that most
432 A. Karwa et al.
mushrooms are ectomycorrhizal. Examples of these mushrooms are A. caesarea,

Astraeus hygrometricus, B. edulis, Cantharellus cibarius, Lactarius deliciosus,
Russula virescens, and Tricholoma matsutake. The mushrooms mentioned in
Table 19.1 belong to the clades as classified by Moncalvo et al. (2002) and families
Table 19.1 Epigeous and hypogeous mushrooms with confirmed ectomycorrhizal relations
Sr Order/family Epigeous mushrooms (gilled mushrooms, Hypogeous and semihypogeous
no puffballs, etc.) mushrooms (truffles, sclerodermas, etc.)
1 Amanitaceae Amanita, Limacella Ammarrendia, Torrendia
2 Boletales Boletellus, Boletochaete, Boletus, Austrogaster, Austrogautieria, Astaeus,
Austroboletus, Aureoboletus, Gastroboletus, Alpova,
Psiloboletinus, Rubinoboletinus, Gymnopaxillus, Gymnogaster,
Fuscoboletinus, Paxillus, Horakiella, Melanogaster,
Astropaxillus, Gomphidus, Rhizopogon, Sclerogaster,
Gyroporus, Chroogomphus, Scleroderma, Chamonixia,
Calostoma, Leccinum, Heimielia, Hysterogaster, Mycoamaranthus,
Paragyrodon, Phylloporus, Octaviania, Velligaster,
Phlebopus, Pisolithus, Poryphyrellus, Truncocolumella
Scleroderma, Strobilomyces,
Tylopilus, Suillus, Xanthoconium,
Xerocomus
3 Cantharellaes Cantharellus, Cantharellula, Cratrellus
4 Clavariaceae Clavaria, Clavicorna, Clavulina,
Clavulinopsis, Clavaridelphus,
Ramariopsis, Aphelaria
5 Cortinariaceae Cortinarius, Naucoria, Rozites, Cortinarius, Hymenogaster,
dermocybe Quadrispora, Stephanopus,
Protoglossum, Destuntzia,
Thaxeterogaster, Setchelliogaster
6 Elaphomycetaceae Elaphomyces, Pseudotulostoma
7 Endogonaceae Endogone, Densospora, Yougimyces,
Peridiospora
8 Entolomataceae Some species of Entoloma Rhodogaster, Richoniella
9 Gomphales, Gomphus, Boletopsis, Bankera, Aroramyces, Hysterangium,
Hysterangiales Clavaridelphus, Hydnum, Hydnellum, Chondrogaster, Gautieria,
Sarcodon, Ramaria, Phellodon Gummiglobus, Mesophellia,
Malajczukia, Austrogautiera,
Gallacea, Protrubera, Trappea,
Phallogater, Catoreum
10 Hydnangiaceae Laccaria Hydnangium, Podohydnangium,
Gigaspera
11 Hygrophoraceae Hygrophorus, Gliophorus, Humidicutis
12 Hymenogastraceae Hebeloma, Phaeocollybia Hymenogaster
13 Inocybaceae Inocybe, Auritella Auritella
14 Lyophyllum Lyophyllum
15 Pezizales Geopora, Helvella, Hydnotyra, Tuber, Kalaharituber , Terfezia,
Pulvinulla, Geopora, Sarcosphaera, Turmania, Dingleya,
Tirmania, Tricharina, Sphaerozone, Labyrinthomyces, Pachyphloeus,
Genea, Wilcoxina Redellomyces, Eremiomyces
16 Russulales Russula, Lactarius Stephanospora, Cystangium,
Arcangeliella, Leucogaster,
Gymnomyces, Zelleromyces
17 Thelephorales Thelephora, Sarcodon, Phellodon,
Bankera
18 Tricholomataceae Tricholoma, Leucopaxillus
as described by Matheny et al. (2006) and Hibbett et al. (2007). A survey of

literature provides evidence that many researchers have contributed in the field of
identity and taxonomy of ectomycorrhizal fungi (Arora 1986, 1991; LoBuglio et al.
1996; Erland and Taylor 1999; Kõljalg et al. 2000; Moncalvo et al. 2000; Bougher
and Lebel 2001; Humpert et al. 2001; Peinter et al. 2001; Binder and Bresinsky
2002; Selosse et al. 2002; Tehler et al. 2003; Urban et al. 2003; Binder et al. 2005;
Douhan and Rizzo 2005; Ferdman et al. 2005; Henkel et al. 2006; Matheny and
Bougher 2006; Matheny et al. 2006; Tedersoo et al. 2006; Trocha et al. 2006;
Watling 2006; Barroetaveña et al. 2007; Hibbett et al. 2007; Hosaka et al. 2007;
Wilson et al. 2007). Many of them are the confirmed ECM associations by fungal
isolation and resynthesis under controlled conditions (Warcup 1980, 1985; Kropp
and Trappe 1982; Malajczuk et al. 1982; Molina and Trappe 1982; Bougher and
Malajczuk, 1985; Godbout and Fortin 1985; Reddell and Milnes 1992; Massicotte
et al. 1994; Thomson et al. 1994; Cripps and Miller 1995; McGee 1996; Kawai
1997; Lu et al. 1998; Reddell et al. 1999; Baxter and Dighton 2001; Yamada
et al. 2001a, b; Yun and Hall 2004; Brundret et al. 2005; Henkel et al. 2006).
However, the medicinal properties of mycorrhizal mushrooms have not been well-
investigated.
Mycorrhizal mushrooms, some of which are among the world’s most expensive
foods, are of considerable interest to the chef and the gourmet (Table 19.2). Most of
these ectomycorrhizal mushrooms are found only in the North of the equator and
fruit for short periods during the year. There has been a marked decline in the
harvests of some edible mycorrhizal mushrooms over the past century (Hall et al.
2003). Possible reasons for this decline include deforestation, the loss of host plants
within forests due to pests or disease, changed forest management practices such as
planting more densely than occurs in natural forests, the replacement of natural
forests with plantations of species that are poor hosts for edible mycorrhizal mush-
rooms, global warming since the last ice age, soil compaction by hordes of pickers,
acid rain, and, for truffles, the loss of expertise during two World Wars as to
Table 19.2 Market value of commercially important ectomycorrhizal edible mushrooms

Mushroom species Common Estimated world Approx. price US$/kg
name production (t)
B. edulis Porcini 20,000–100,000 20–200 (Hall et al. 1998a;
http://en.wikipedia.org/wiki/
Boletus_edulis, 2010)
C. cibarius Chanterelle 220,000 8–19 (Danell 2000; Warner 2010,
http://basiceating.blogspot.com/
2010/04/Chantharelle-
cantharellus.cibarius.html)
T. matsutake Matsutake 1,000 90–2,000 (Ashkenazi and Jacob
2003, http://en.wikipedia.org/
wiki.matsutake, 2010)
Tuber melanogaster Black Truffle 150 1,000–3,490 (Maison de la 2010)
T. magnatum White Truffle 10,200 10,200 (Maison de la 2010)
434 A. Karwa et al.
where and how to find them. There has been a spectacular increase of interest
and commercial activity concerned with dietary supplements, functional foods, and
other products that are “more than just food.” On the contrary, there has been a
marked decline in the harvests of some edible mycorrhizal mushrooms over the past
century, which is illustrated by the official figures of Eurasian countries. This makes
the cultivation of edible mycorrhizal mushrooms all the more attractive.
19.5 Production and Forest Management
Wild edible mushroom harvest generates millions of dollars each year and consists
largely of ectomycorrhizal fungi, such as pine mushrooms (Tricholoma magnivelare),
chanterelles (Cantharellus formosus and Cantharellus subalbidus), and boletes
(B. edulis) (Danell and Camacho 1997). Pine mushrooms are the most commercially
important wild forest mushroom and are exported exclusively to Japan (de Geus
1995), while chanterelles, boletes, and others are primarily exported to parts of North
America and Europe (de Geus 1995). Known commercial mycorrhizal mushroom
(particularly tubers) sites are located across all the regions of France (de Geus 1995;
Freeman 1997; Trowbridge and Macadam 1999; Ehlers and Frederickson 2000;
Berch and Wiensczyk 2001; Kranabetter et al. 2002), in forests from 20 to more
than 200 years old (Hosford and Ohara 1995; Norvell 1995; Redhead 1997; Pilz et al.
1998). Forest practices, such as logging, site preparation, tree selection, fire, fertiliza-
tion, pesticide use, brushing and spacing, and grazing, influences mushroom presence,
reproduction, and productivity. Ectomycorrhizal fungi require living roots, and there-
fore living trees, to survive. As a result, timber harvesting, particularly clearcutting,
profoundly reduces mushroom production (Smith et al. 2002) until the mature forest
becomes reestablished. In some areas, gap area size significantly affects the produc-
tion of fruiting bodies in forests (Durall et al. 1999, 2006). Sporocarp diversity
declines significantly in forests as soil compaction from machinery and trampling
can damage the mycelium and reduce mushroom productivity (Colgan et al. 1999).
Forest management techniques that promote mushroom production have been
studied in many countries. To encourage matsutake mushroom production in
Japanese forests, for example, various silviculture treatments have been applied.
Overstorey trees are thinned, tree species composition is altered, nonhost under-
storey shrubs and herbs are cut, and organic litter is removed from the forest floor
(Hosford et al. 1997). In North America, such intense management of forests for
pine mushroom production does not occur. Studies in Europe show that nitrogen
deposits from air pollution (Arnolds 1991) and applications of nitrogen fertilizers
(Termorshuizen 1993) reduce the productivity of edible ectomycorrhizal fungi.
Information on the effects of pesticide application or grazing on edible mushrooms
is currently not available for any country. More research is required to determine
how silviculture techniques could be used to promote the fruiting of economically
important fungi in forests across the globe.
19.5.1 Ectomycorrhizal Mushrooms and Wildlife Association
A highly evolved beneficial relationship exists between ectomycorrhizal fruiting

bodies, host trees, and wildlife. Fruiting bodies of ectomycorrhizal fungi are an
important food source for many temperate forest mammals and invertebrates (Fogel
1975; Fogel and Trappe 1978; Bruns 1984; Lawrence 1989; Cazares and Trappe
1994; Johnson 1994; Janos and Sahley 1995; North and Trappe 1996). In the
process of consuming the fruiting body, fungal inoculum is dispersed throughout
the animal’s range, thereby exposing the ectomycorrhizal host trees to a higher
diversity of inocula. Aboveground sporocarps generally disperse their spores
through the forest by means of air and water currents. Below-ground fruiting
bodies, by contrast, depend on animals for spore dispersal. Animals are attracted
to the aromatic compounds produced by truffles and false-truffles (i.e., true truffles
belong to the class Ascomycete; false-truffles belong to the class Basidiomycete),
which lead them to excavate and consume these mushrooms. The spores in the
fruiting bodies ingested by the animal pass through the gut and are deposited with
fecal pellets (Cazares and Trappe 1994; Johnson 1994; Currah et al. 2000). Some
small mammals, such as the northern flying squirrel and the California red-backed
vole, use truffles as their primary food source (Amaranthus et al. 1994). These
mammals, in turn, are important prey for other species of animals (Forsman et al.
1984). Cavities in downed and standing dead wood are commonly used by small
mammals to store food when foraging (Bunnell et al. 1999) and are therefore
important not only for wildlife survival but also as sources of ectomycorrhizal
fungal inocula for the surrounding forest. Retaining standing and downed coarse
woody debris is thus important for the dispersal of spores from both above- and
belowground ectomycorrhizal fruiting bodies (Klironomos and Hart 2001).
19.6 Cultivation of Edible Mycorrhizal Mushrooms
As is evident, about half of the world’s species of edible mushrooms are mycorrhi-
zal, and among them are some of the world’s most expensive foods. A few of these
mushrooms have well-established worldwide markets measured in billions of
dollars. All of the mycorrhizal mushrooms are seasonal, best-eaten fresh, and do
not preserve well (Hall et al. 2007). Of more than 300 ectomycorrhizal mushrooms
eaten around the world, those held in the highest regard are the Périgord black
truffle (Hall et al. 1994), Italian white truffle (Hall et al. 1998a), porcini (Hall et al.
1998b), chanterelle (C. cibarius; Danell 2001), and matsutake (T. matsutake; Wang
et al. 1997). However, many of the other edible mycorrhizal mushrooms like
Caesar’s mushroom (Amanita caesaria), honshimeji (Lyophilum shimeji), Bur-
gundy truffle (Tuber uncinatum), Oregon white truffle (Tuber gibbosum), and
saffron milk cap (L. deliciosus) also have significant local markets. Figure 19.1
showed the different edible ectomycorrhizal mushrooms of high culinary demand.
436 A. Karwa et al.
a b
c d
Fig. 19.1 Edible ectomycorrhizal mushrooms of high culinary demand: (a) Perigord truffle Tuber
melanosporum, (b) Russula brevipesm, (c) Cantharellus cibarius, (d) Boletus edulis, (e) Tuber
magnatum, (f) Russula emetica, (g) Cantharellus cibarius, (h) Boletus edulis
19.6.1 Truffles
A truffle is a subterranean fungal fruiting body that develops underground and relies
on mycophagy for spore dispersal (FEMAT 1993). There are 70 known varieties of
truffles, 32 of which are found in Europe, but the fruiting bodies of some are highly
prized as a food. They have a pungent, intense, earthy fragrance and lend a unique
flavor to food, sometimes referred to as “Black gold” or “black diamonds” because
of their scarcity and worth. Edible truffles are held in high esteem in international
haute cuisine. They prefer argillaceous or calcareous soils, which are well drained
and neutral or alkaline (Martin et al. 2010). Truffles fruit throughout the year,
depending on the species, and can be found buried between the leaf litter and the
soil. One of the best-known is the French truffles or the Périgord black truffle (T.
melanosporum). This mushroom is found in the forests of southern France, northern
Italy, and northeastern Spain on the roots of, for example, oaks and hazels. It is used
very widely in gourmet cooking where it either imparts a flavor of its own to a dish
or enhances the flavor of other foods it is cooked with. These truffles are considered
as one of the great foods of the world, and when fresh in season, prices can be as
high as $2,500 per kg.
At the beginning of the twentieth century, production of the Périgord black
truffle has been estimated to have been between 1,000 and 2,000 tons, but in the last
decade, it is reported to be less than 150 tons (Olivier 2000, Lefevre and Hall 2001).
The earliest cultivation of truffles was achieved by Joseph Talon (father of truffle
raising) in the early 1808s (Hall et al. 1994; Hall and Wang 1998). He found that
transplanted oak seedlings from the rooting zones of trees produced Périgord black
truffles. Despite its lack of sophistication, Talon’s technique was widely used for
150 years. In some last decades, new attempts for mass production of truffles have
been started. There are truffle-growing areas in the United States, Spain, Sweden,
New Zealand, Australia, Chile, and the UK. Truffles have long eluded the modern
techniques of domestication known as trufficulture. Although the field of trufficul-
ture has greatly expanded since its inception in 1808, several species still remain
uncultivated. Cultivation of Terfezia (Gutierrez, unpublished) (Morte et al. 2000)
and various species of Tuber, including T. borchii (bianchetto) (Zambonelli et al.
2002), T. melanosporum (Lefevre and Hall 2001; Hall et al. 2003), and T. uncina-
tum (Chevalier and Frochot 1997) through sporal suspensions has been reported at
different times.
Lately, the genome sequence of the Périgord black truffle has been published in
March 2010 (Martin et al. 2010).
19.6.2 Russula
The genus Russula (Pers. Ex. Fr.) S.F. Gray accounts in a large measure for
monsoon mushroom flora in forests. It is cosmopolitan and largely ectomycorrhizal
genus with a wide range of Gymnosperms and Angiosperms (Richardson 1970;
Agerer 2002). A number of Russula species form ectomycorrhizas with different
tree species (Arora 1986). Russula brevipes and its morphotypes/ectomycorrhiza
have been described and illustrated (Niazi et al. 2006) from moist temperate forests
of Pakistan, associated with Pinus wallichiana. Russula species is widely
distributed and mainly associated with species of Abies, Picea, Tsuga, and Pseu-
dosuga (Stanis 1979; Pillukat and Agerer 1992; Kraigher et al. 1995; Kernaghan
et al. 1997). It is more commonly found in Himalayan forests under conifers in late
fall and can easily be identified by its large fruit size. Yamada, since 1997, has
studied the cultivation of edible EMF. Among many species, Russula nigiricans
and Russula mariae were successfully induced to synthesize mycorrhiza in vitro
(Yamada 2003).
The mushroom contains very useful phytochemicals such as phenolics, flavo-
noids, ergosterol, and b-carotene. The sesquiterpene lactone named russulactaror-
ufin along with lactarorufin-A and 24-ethyl-cholesta-7,22E-diene-3b,5a,6b-triol
have been isolated and characterized (Suri et al. 1997). The wild species proved
438 A. Karwa et al.
to have antioxidant potential and free radical scavenging activity. The combination
of bioactive substances and rich nutritional composition (high contents in protein
and carbohydrates, low content in fat) in the mushroom should be useful to
consumers in encouraging them to utilize the nutritive potential of this edible
wild mushroom (Chen et al. 2010).
19.6.3 Cantharellus
C. cibarius, commonly known as the golden chanterelle, is probably the best known
species of the genus Cantharellus. It is orange or yellow, meaty and funnel-shaped
with smooth cap, and gill-like ridges that run almost all the way down its stipe,
which tapers down seamlessly from the cap. It has a fruity smell, reminiscent of
apricots, and a mildly peppery taste and is considered an excellent food mushroom.
Chanterelles are common in northern parts of Europe and North America, Asia, and
in Africa (Boa 2004). Chanterelles are associated with conifers and oaks (Arora
1979; Metzler 1992). In Scotland, chanterelles grow in mixed forest (silver birch
and scots pine), especially when the forest has plenty of moist, mossy undergrowth.
Chanterelles are versatile and can be added as an ingredient to most dishes. They
can also be pickled in brine and can last from 6 to 12 months. In most places, they
are dry stored. Fresh chanterelles can generally be stored up to 10 days in a
refrigerator.
Nils (1979) first proved that spores of C. cibarius can be germinated in vitro.
Danell attempted to cultivate the species (Danell and Camacho 1997). Up to
6.7% of its tissues contain vitamin C; as for carotene, they can be as much as
23.1%. Research has suggested that the golden chanterelle may have potent
insecticidal properties that are harmless against humans and yet protect the
mushroom body against insects and other potentially harmful organisms
(Philpot 1965).
19.6.4 Boletus
Boletus has a cosmopolitan distribution, concentrated in cool-temperate to subtropical

regions (Marais and Kotzé 1977; Eicker 1990; Masuka 1996; Hall et al. 1998b; Hall
et al. 2003; Orlovich et al. 2004; Oria-de-Rueda et al. 2008). It has been reported from
Europe – from northern Scandinavia, south to the extremities of Greece and Italy –
and North America, where its southern range extends as far south as Mexico (Tylukti
1987), China (Tylukti 1987), Nepal (Giri and Rana 2007), and India (Adhikary et al.
1999). The mushroom’s habitat consists of areas dominated by Pinus spp.(Vozzo and
Hackskaylo 1961; Froidevaux and Amiet 1975), spruce (Picea spp.) (Ceruti et al.
1988), hemlock (Tsuga spp.) fir (Abies spp.) trees (Gobl 1977), chestnut, chinquapin,
beech, Keteleeria spp., Lithocarpus spp., and oak (Wang et al. 1995; Gross et al. 1998;
Quan and Lei 2000; Agueda et al. 2006, 2008; Fu et al. 2009). The mushroom has
been noted to commonly cooccur with Amanita muscaria or Amanita rubescens,
although it is unclear whether this is due to a biological association between the
species or because of similarities in growing season, habitat, and ecological require-
ments (Hall et al. 1998b; Hall et al. 2003; Peinter et al. 2007).
Estimates suggest the total annual worldwide consumption of B. edulis and
closely related species (B. aereus, B. pinophilus, and B. reticulatus) has increased
from 20,000 to 100,000 tons in the current decade (Hall et al. 1998b, 2003; Agueda
et al. 2008). They are widely exported and sold in dried form, reaching countries
where they do not occur naturally, such as Australia and New Zealand. In autumn,
the price of porcini typically ranges between $20 and $80 per kilogram, although
the scarcity of fruit bodies sometimes elevates the wholesale price to over $200 per
kilogram (Sitta 2000; Hall et al. 2003; Boa 2004; Sitta and Floriani 2008; Drumeva
and Gyosheva 2009).
As with other strictly mycorrhizal fungi, B. edulis has eluded attempts to
cultivate it (Arora 1986; Chang and Miles 2004). The results of some studies
suggest that unknown components of the soil microflora might be required for
B. edulis to successfully establish a mycorrhizal relationship with the host plant
(Veselkov 1975; Ceruti et al. 1988; Fitter and Garbaye 1994).
B. edulis fruit bodies contain ergosterol and ergosterol peroxide (Mattila et al.
2002; Ey et al. 2007; Ribeiroa et al. 2008), with a wide spectrum antimicrobial and
anti-inflammatory activity, and cytotoxicity to various tumor cell lines grown in
laboratory culture (Lucas et al. 1957; Krzyczkowskia et al. 2008). However, some
investigations in the United States do not support this (Lamaison and Polese 2005).
The mushroom also contains mitogenic lectin with antiviral properties against the
human immunodeficiency virus enzyme reverse transcriptase (Zheng et al. 2007),
Vaccinia virus (Kandefer-Szersen et al. 1980) and tobacco mosaic virus grown in
culture (Piraino 2006; Li et al. 2009).
19.6.5 Cultivation of Other Species
While some plants can live in the absence of the fungal partner, at least under
amended (fertilized) environments, mycorrhizal fungi cannot live in the absence of
the host (Harley and Smith 1983). In poor, dry tropical soils, however, mycorrhizal
associations are vital for plant growth and survival (Munyanziza 1994). The normal
reproductive cycle of mushroom-producing mycorrhizal fungi involves the follow-
ing stages: (1) spore germination, (2) mycelium growth, (3) infection of the specific
host, and (4) fruiting-body (mushroom) production and sporulation. Without the
right host, the fungus does not reach stages 3 and 4.
Plants infected following inoculation with spore suspensions (Hall and Wang
1998; Hall et al. 2002) or pure cultures prepared either from fruiting bodies (Sisti
et al 1998) or mycorrhizal root tips (Kagan-Zur 2002) have led to the formation of
edible mycorrhizal mushroom fruiting bodies in the field, including L. deliciosus
440 A. Karwa et al.
(Poitou et al. 1989; Wang et al. 2002a, b), Lyophyllum shimeji (“Kyoto scientists
grow hon-shimeji mushrooms,” http://www.kippo.or.jp/KansaiWindowhtml/
News/1996e/19961119_NEWS.HTML), Rhizopogon rubescens (Wang et al.
2002a, b), Suillus granulatus (Poitou et al. 1989), Terfezia (Morte et al. 2000),
and various species of Tuber (Chevalier and Frochot 1997; Lefevre and Hall 2001;
Zambonelli et al. 2002; Hall et al. 2003); Danell and Camacho (1997) also produced
C. cibarius in pots in the greenhouse. Despite these successes, fewer than a dozen of
the many hundreds of edible mycorrhizal mushrooms have ever been cultivated
with any degree of success (Wang et al. 2002a, b), and this includes important
commercial species such as B. edulis (Hall et al. 1998a, b), T. matsutake (Yun and
Hall 2004), and the Italian white truffle (Tuber magnatum) (Hall et al. 1998a;
Gregori 2002). T. matsutake was first found to form a typical Hartig net showing
ectomycorrhiza, which subsequently synthesized mycorrhiza. Other tested EMF
also produced mycorrhizal seedlings. Among them, Lactarius akahatsu, R. rubes-
cens, Tricholoma portentosum, and T. saponaceum were successfully induced to
fruit with juvenile pine seedlings of around of 1-year old (Yamada 2003).
Consequently, supplies of most commercially important edible mycorrhizal
mushrooms are still restricted to those that can be harvested from the wild during
autumn or winter. For these, we are left to speculate on the reasons why we have not
been able to establish viable infections on a suitable host plant or why fruiting
bodies simply fail to form.
19.7 Conservation
In spite of their importance, discussions of ectomycorrhizal mushroom conserva-

tion are not widespread. Many factors may be influencing the more prominent
decline in these mushrooms compared to saprophytic and lignicolous fungi (Leake
et al. 2002). Mycorrhiza formation and especially fruit body formation is sensitive
to soil disturbance. The amount of organic matter available in forest stands influ-
ences the types of mycorrhizae present on tree roots (Harvey et al. 1976). Similarly,
chemical weeding, which is often done in forests or agroforestry (Amakiri 1977),
may have consequences on mycorrhiza fungi and hence on mushroom production
(Iloba 1980). A broader appreciation for the conservation of ectomycorrhizal
mushrooms is needed as these fungi are crucial to many ecosystem functions and
have great ecological and economic value. For the in vitro culture, usually isolation
of the mycelia is difficult. Mycelial growth on synthetic medium is poor, and fruit
bodies do not form easily in pure cultures. Many attempts to cultivate mycorrhizal
mushrooms have failed. Fungal propagules including spores and hyphae do not
remain viable for long periods. In nature, these fungi are more or less obligate root
symbionts.
Much concern about the conservation of EMF diversity has come from Europe
where populations have declined over the last three decades (Arnolds 1988;
Arnolds 1991). Ectomycorrhizal fungal spores are lost to predation, germination

under unsuitable conditions, and weathering (Miller et al. 1994). The most dramatic
fungal declines have been associated with increases in various types of pollution
(Arnolds 1988). Though mushrooms like Tricholoma and Lactarius are not culti-
vable, Cantharellus and Truffles can be cultivated using symbiotic relationship as a
technique forming mycorrhiza in the field or by applying the same methods used for
cultivating saprophytic mushrooms to use substrates and nutrients in bottles or bags
as culture medium. Environmental conditioning of the wooded area by thinning the
undergrowth and by plowing the litter layer can also increase the number of
mycelia. Controlled burning of trees to induce formation of new fungal colonies
is yet another way practiced to grow these mushrooms.
In fact, in the tropics, about 95% tree species from endomycorrhizas do not form
mushrooms (Lapeyrie and H€ ogberg 1994; Mason and Wilson 1994). Clearly, in the
domestication of mycorrhizal fungi for mushroom production on trees in agrofor-
estry systems, the tree component has to be made of the species forming ectomy-
corrhizas. For increased mushroom diversity and sustained production, the
domestication of mycorrhizal mushrooms will mean the creation of an agroforestry
system having trees of different species and age groups supplied with the right
spectrum of fungal partners (Mason et al. 1987; Termoshuizen 1991).
19.7.1 Challenges Ahead
One possible reason behind the failure in cultivating ectomycorrhizal mushrooms is

simply the assumption that they are mycorrhizal when their relationship with their
hosts is more complicated. Out of about 2,500 recorded species of edible mush-
rooms, the most expensive and sought after mushrooms belong to the mycorrhizal
group and include T. melanosporum, T. magnatum, T. matsutake, B. edulis,
C. cibarius, Russula emetica, and A. caesarea. Over the past century, harvests of
many mycorrhizal mushrooms have declined dramatically, which has prompted
interest in the development of methods for their cultivation. So far, only a few
species of truffles have been produced in commercial quantities, although methods
have been developed that may see the cultivation of species such as C. cibarius,
L. shimeji, and L. deliciosus. Despite this, many of the most expensive mycorrhizal
mushrooms, including T. magnatum Pico & Vitt. and T. matsutake, have defied
cultivation (Yun and Hall 2004).
The links between wild edible fungi and tree hosts are well known for economi-
cally important species such as B. edulis and Tuber spp. Cantharellus spp. form
mycorrhizae with many tree species in tropical countries. There is an expanding
body of information about many other edible fungus–tree associations, but this has
not been assembled in the form of a database that would, for example, allow for
predictive searches. The search for matsutake in Asia was assisted by a knowledge
of its tree hosts, and this approach would assist in prospecting for other wild edible
442 A. Karwa et al.
fungi. Knowledge about the mycorrhizal partners of edible species of Amanita,

Lactarius, and Russula is steadily increasing. However, our understanding of the
ecological conditions favored by edible ectomycorrhizal mushroom is restricted to
a handful of fungi and a paucity of publications (Chevalier and Frochot 1997; Wang
et al. 1997; Hall et al. 1998a; Morte et al. 2000; Sbrana et al. 2000; Lefevre and Hall
2001; Hall et al. 2003). Clearly, there is a need for considerable study in this area to
determine whether an edible mycorrhizal mushroom is found on young or old trees –
the so-called early or late-stage mushrooms (Visser 1995; Redecker et al. 2001);
define the optimum edaphic and climatic conditions tolerated by the fungus;
monitor the effect of changing environmental conditions on mycorrhizal commu-
nity structures (Lilleskov et al. 2002); their relationship with other belowground
organisms (Duplessis et al. 2002); and identify the factors required to trigger
fruiting (Hall et al. 2002). There is a considerable body of literature on truffles
(Federation-Française-des-Trufficulteurs 2001). Researchers have focused much
attention to the complex relationships between biological, social, and economic
issues, a welcome move towards establishing a sound basis for sustainable produc-
tion of wild edible fungi.
19.8 Conclusions
Ectomycorrhizal mushrooms constitute an important resource being exploited on a

commercial scale. In future, ectomycorrhizal mushrooms may play important role
as medicines and as bioindicators of sustainable ecosystems practices. Traditionally
gathered for household consumption, they have won international attention. There
has been a marked decline in the harvests of some edible mycorrhizal mushrooms
over the past century. Possible reasons for this decline include deforestation, the
loss of host plants within forests due to pests or disease, deforestation as well as
changed forest management practices such as planting more densely than occurs in
natural forests, the replacement of natural forests with plantations of species that are
poor hosts for edible mycorrhizal mushrooms, global warming since the last ice
age, soil compaction by hordes of pickers, acid rain, etc. There has been a spectac-
ular increase of interest and commercial activity concerned with dietary supple-
ments and functional foods. This makes the cultivation of edible mycorrhizal
mushrooms all the more attractive. Biochemical and molecular studies also might
prove crucial to our understanding of these issues. The domestication of edible
ectomycorrhizal mushrooms is therefore gathering some momentum in a few parts
of the world. This needs to build on the understanding of the relation between
the partners and the environment under which the cultivation can be optimized.
Cultivating ectomycorrhizal mushrooms requires special management of forestry
systems. This will depend on incorporating appropriate tree species, practicing
limited soil disturbance, understanding the mycorrhizal partnership, and forest
dynamics.
References
Abbott LK, Robson AD (1991) Factors influencing the occurrence of vesicular-arbuscular mycor-
rhizas. Agric Ecosyst Environ 35:121–150
Addy HD, Piercey MM, Currah RS (2005) Microfungal endophytes in roots. Can J Bot 83:1–13
Adhikary RK, Baruah P, Kalita P, Bordoloi D (1999) Edible mushrooms growing in forests of
Arunachal Pradesh. Adv Hortic For 6:119–123
Agerer R (1995) Anatomical characteristics of identified ectomycorrhizas: an attempt towards
a natural classification. In: Varma A, Hock B (eds) Mycorrhiza. Springer Verlag, Berlin,
pp 685–734
Agerer R (2002) Color atlas of ectomycorrhiza, 12th edn. Einhorn Verlag Edward, Dientenurger,
Germany
Agueda B, Parlade J, Fernandez-Toiran LM, Cisneros O, de Miguel AM, Modrego MP, Martinez-
Pena F, Pera J (2008) Mycorrhizal synthesis between Boletus edulis species complex and
rockroses (Cistus sp.). Mycorrhiza 18(8):443–449
Agueda B, Parladé J, de Miguel AM, Martı́nez-Peña F (2006) Characterization and identification
of field ectomycorrhizae of Boletus edulis and Cistus ladanifer. Mycologia 98(1):23–30
Amakiri MA (1977) Effect of herbicides on microbial populations and activities in soils under teak
(Tectona grandis L.) plantation. East Afr Agric For J 42(4):420–426
Amaranthus MP, Trappe JM, Bednar L, Arthur D (1994) Hypogeous fungal production in mature
Douglas-fir forest fragments and surrounding plantations and its relation to coarse woody
debris and animal mycophagy. Can J For Res 24:2157–2165
Arnolds E (1988) The changing mycromycete flora in the Netherlands. Trans Br Mycol Soc 90
(3):391–406
Arora D (1979) Mushroom demystified. Ten Speed Press, Berkeley. ISBN 0-89815-009-4
Arora D (1986) Mushrooms demystified: a comprehensive guide to the fleshy fungi, 2nd edn. Ten
Speed Press, Berkeley
Arora D (1991) All That the Rain Promises, and More: A Hip Pocket Guide to Western Mushrooms.
Berkeley: Ten Speed Press, 1991. ISBN 0898153883
Ashkenazi M, Jacob J (2003) Food culture in Japan. Greenwood Publishing Group, p 49. ISBN
0313324387
Baghel RK, Sharma R, Pandey AK (2009) Activity of acid phosphatase in the ectomycorrhizal
fungus Cantharellus tropicalis under controlled conditions. J Trop For Sci 21(3):218–222,
ISSN 0128-1283
Barroetaveña C, Cázares E, Rajchenberg M (2007) Ectomycorrhizal fungi associated with pon-
derosa pine and Douglas-fir: a comparison of species richness in native western North
American forests and Patagonian plantations from Argentina. Mycorrhiza 17:355–373
Baxter JW, Dighton J (2001) Ectomycorrhizal diversity alters growth and nutrient acquisition of
grey birch (Betula populifolia) seedlings in host-symbiont culture conditions. New Phytol
152:139–149
Berch SM, Wiensczyk AM (2001) Ecological description and classification of some pine mush-
room (Tricholoma magnivelare) habitat in British Columbia. B.C. Ministry of Forests and
Southern Interior Forest Extension and Research Partnership. Victoria, B.C. Research Report
No. 19
Binder M, Bresinsky A (2002) Derivation of a polymorphic lineage of gasteromycetes from
boletoid ancestors. Mycologia 94:85–98
Binder M, Hibbett DS, Larsson KH, Larsson E, Langer E, Langer G (2005) The phylogenetic
distribution of resupinate forms across the major clades of mushroom-forming fungi (Homo-
basidiomycetes). Syst Biodivers 3:113–157
Boa E (2004) Wild edible fungi: a global overview of their use and importance to people (non-
wood forest products). Food & Agriculture Organization of the UN, Rome, Italy, pp 96–97
444 A. Karwa et al.
Bougher NL, Lebel T (2001) Sequestrate (Truffle-like) fungi of Australia and New Zealand. Aust
Syst Bot 14:439–484
Bougher NL, Malajczuk N (1985) A new species of Descolea (Agaricales) from Western Australia
and aspects of its ectomycorrhizal status. Aust J Bot 33:619–627
Brundret M, Malajczuk N, Mingqin G, Daping X, Snelling S, Dell B (2005) Nursery inoculation of
Eucalyptus seedlings in Western Australia and Southern China using spores and mycelial
inoculum of diverse ectomycorrhizal fungi from different climatic regions. For Ecol Manage
209:193–205
Brundrett MC (1991) Mycorrhizas in natural ecosystems. In: Macfayden A, Begon M, Fitter AH
(eds) Advances in ecological research, vol 21. Academic Press, London, pp 171–313
Brundrett MC (2004) Diversity and classification of mycorrhizal associations. Biol Rev
79:473–495
Brundrett MC, Kendrick WB (1988) The mycorrhizal status, root anatomy, and phenology of
plants in a sugar maple forest. Can J Bot 66:1153–1173
Brundrett MC, Murase G, Kendrick B (1990) Comparative anatomy of roots and mycorrhizae of
common Ontario trees. Can J Bot 68:551–578
Bruns TD (1984) Insect mycophagy in the boletales: fungivore diversity and the mushroom
habitat. In: Wheeler Q, Blackwell M (eds) Fungus–insect relationships: perspectives in ecology
and evolution. Columbia University, New York, pp 91–129
Bunnell FL, Kremsater LL, Wind E (1999) Managing to sustain vertebrate richness in forests of
the Pacific Northwest: relationships within stands. Environ Rev 7:97–146
Cairney JWJ, Chambers SM (1999) Ectomycorhizal fungi: key genera in profile. Springer,
Germany
Cazares E, Trappe JM (1994) Spore dispersal of ectomycorrhizal fungi on a glacier forefront by
mammal mycophagy. Mycologia 86(4):507–510
Ceruti A, Tozzi M, Reitano G (1988) Micorize di sintesi tra Boletus edulis, Pinus sylvestris e Picea
excelsa (in Italian). Allionia (Turin) 28:117–124
Chang ST, Miles PG (2004) Mushrooms: cultivation, nutritional value, medicinal effect, and
environmental impact, 2nd edn. CRC Press, Boca Raton, FL, p 131
Xin-Hua Chen, Le-Xian Xia, Hong-Bo Zhou, Guan-Zhou Qiu (2010) Chemical composition and
antioxidant activities of Russula griseocarnosa sp. nov. J Agric Food Chem 58(11):6966–6971
Chevalier G, Frochot H (1997) La Truffe de Bourgogne. Pétrarque, Levallois-Perret
Colgan W III, Carey AB, Trappe JM, Molina R, Thysell D (1999) Diversity and productivity of
hypogeous fungal sporocarps in a variably thinned Douglas-fir forest. Can J For Res
29:1259–1268
Comandini O, Contu M, Rinaldi AC (2006) An overview of Cistus ectomycorrhizal fungi.
Cook R (1977) The biology of symbiotic fungi. Wiley, London
Cripps CL, Miller OK (1995) Ectomycorrhizae formed in vitro by quaking aspen: including
Inocybe lacera and Amanita pantherina. Mycorrhiza 5:1432–1890
Currah RS, Smreciu EA, Lehesvirta T, Niemi M, Larsen KW (2000) Fungi in the winter diets of
northern flying squirrels and red squirrels in the boreal mixedwood forest of northeastern
Alberta. Can J Bot 78:1514–1520
Danell E (2000) Cantharellus. In: Cairney JWG, Chambers SM (eds) Ectomycorrhizal fungi: key
genera in profile. Springer, Berlin, pp 253–267
Danell E (2001) Current research on chanterelle cultivation in Sweden. Second international
workshop on edible mycorrhizal mushrooms. http:/www.crop.cri.nz/whats_on/mushroom_
conf/Index.htm
Danell E, Camacho FJ (1997) Successful cultivation of the golden chanterelle. Nature 385:303
de Geus N (1995) Botanical forest products in British Columbia: an overview. B.C. Ministry of
Forests, Victoria
Deacon JW, Fleming LV (1992) Interactions of ectomycorrhizal fungi. In: Allen MF (ed)
Mycorrhizal functioning: an integrative plant-fungal process. Chapman and Hall, New York,
pp 249–300
Dotzler D, Krings M, Taylor TN, Agerer R (2006) Germination shields in Scutellospora (Glomer-
omycota: Diversisporales, Gigasporaceae) in the 400 million-year-old Rhynie chert. Mycol
Prog 5:178–184
Douhan GW, Rizzo DM (2005) Phylogenetic divergence in a local population of the ectomycor-
rhizal fungus Cenococcum geophilum. New Phytol 166:263–271
Drumeva M, Gyosheva M (2009) The macromycetes fungi of Bulgaria. World Wildlife Fund.
http://www.worldwildlife.org/bsp/publications/europe/bulgaria/bulgaria1.html
Duplessis S et al (2002) Living together underground, a molecular glimpse of the ectomycorrhizal
symbiosis. In: Osiewacz HD (ed) Molecular biology of fungal development. Marcel Dekker,
Durall DM, Gamiet S, Simard SW, Kudrna L, Sakakibara SM (2006) Effects of clearcut logging
and tree species composition on the diversity and community composition of epigeous fruit
bodies formed by ectomycorrhizal fungi Canadian Journal of Botany, 84:966–980
Durall DM, Jones MD, Wright EF, Kroeger P, Coates KD (1999) Species richness of ectomycor-
rhizal fungi in cutblocks of different sizes in the interior Cedar-Hemlock forests of northwest-
ern British Columbia: sporocarps and ectomycorrhizae. Can J For Res 29:1322–1332
Ehlers T, Frederickson S (2000) An inventory and characterization of pine mushroom habitats in
Galena and Fosthall small business enterprises. B.C. Ministry of Forests, Arrow Forest District,
Small Business Forest Enterprise Program. Unpublished report
Eicker A (1990) Commercial mushroom production in South Africa. Bulletin 418. Department of
Agricultural Development, Pretoria
Erland S, Taylor AFS (1999) Resupinate ectomycorrhizal fungl genera. In: Cairney JWG,
Chambers SM (eds) Ectomycorrhizal fungi key genera in profile. Springer Verlag, Berlin,
pp 347–363
Ey J, Sch€omig E, Taubert D (2007) Dietary sources and antioxidant effects of ergothioneine.
J Agric Food Chem 55(16):6466–6474
Ferdman Y, Aviram S, Roth-Bejerano N, Trappe JM, Kagan-Zur V (2005) Phylogenetic Studies of
Terfezia pfeilii and Choiromyces echinulatus (Pezizales) support new genera for southern
African truffles: Kalaharituber and Eremiomyces. Mycol Res 109:237–245
Fitter AH, Garbaye J (1994) Interaction between mycorrhizal fungi and other organisms. In:
Robson AD, Abbott LK, Malajczuk N (eds) Management of mycorrhizas in agriculture,
horticulture and forestry. Kluwer Academic Publishers, Dordrecht, Netherlands
Fogel R (1975) Insect mycophagy: a preliminary bibliography. U.S. Department of Agriculture
Forest Service, p 16
Fogel R, Trappe JM (1978) Fungus consumption (mycophagy) by small animals. Northwest Sci 52
(1):1–31
Forest Ecosystem Management Assessment Team [FEMAT] (1993) Forest ecosystem manage-
ment: an ecological, economic, and social assessment. U.S. Department of Agriculture Forest
Service, Pacific Northwest Region, Portland, Oregon
Forsman ED, Meslow EC, Wight HM (1984) Distribution and biology of the potted owl in Oregon.
Wildlife Monogr 87:1–64
Frank AB (1885) Uber € die auf W€ urzelsymbiose beruhende Ehrn€ahrung gewisser B€aum durch
unterirdische Pilze. Berichte der Deutschen Botanischen Gesellschaft 3:128–145
Freeman S (1997) An estimate of pine mushroom production in the Nahatlatch watershed. Forest
Renewal BC. Unpublished Report
Froidevaux L, Amiet R (1975) Ecto mycorrhizae endo mycorrhizae of Pinus mugo plus Boletus
edulis ssp edulis and Pinus cembra plus Suillus variegatus formed in pure culture. Eur J For
Pathol 5(1):57–61
Fu SC, Tan Q, Chen MJ, Shang XD, Cai LY, Zhang MY (2009) Mycorrhizal synthesis involving
Boletus edulis and Pinus massoniana. Acta Edulis Fungi 16(1):31–41
Garbaye J (1994) Helper bacteria: a new dimension to the mycorrhizal symbiosis. Tansley review
no. 76. New Phytol 128:197–210
Giri A, Rana P (2007) Some higher fungi from Sagarmatha National Park (SNP) and its adjoining
areas, Nepal. Sci World 5(5):67–74
446 A. Karwa et al.
Gobl F (1977) Mycorrhiza in Austrian Douglas fir stands (in German). Centralblatt fur das
Gesamte Forstwesen 94(4):185–194
Godbout C, Fortin JA (1985) Synthesized ectomycorrhizae of aspen: fungal genus level of
structural characterization. Can J Bot 63:252–262
Goodman DM, Trofymow JA (1998) Comparison of communities of ectomycorrhizal fungi in old-
growth and mature stands of Douglas-fir at two sites on southern Vancouver Island. Can J For
Res 28:574–581
Graham RT, Harvey AE, Jurgensen MF, Jain TB, Tonn JR, Page-Dumroese DS (1994) Managing
coarse woody debris in forests of the rocky mountains. U.S. Department of Agriculture Forest
Service, Intermountain Research Station, Ogden, Utah. Research Paper INT-RP-477
Graham RT, Jain TB, Harvey AE (1999) Fuel: logs, sticks, needles, duff and much more. In:
Proceedings, joint fire science conference and workshop. Crossing the millennium: integrating
spatial technologies and ecological principles for a new age in fire management. L.F. Neunsch
Gregori G (2002) Problems and expectations with the cultivation of Tuber magnatum. In: Hall IR
et al (eds) Edible mycorrhizal mushrooms and their cultivation, Proceedings of the second
international conference on edible mycorrhizal mushrooms, cd-rom, New Zealand Institute for
Crop & Food Research Limited
Griffiths RP, Caldwell BA (1992) Mycorrhizal mat communities in forest soils. In: Read DJ,
Lewis DH, Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. CAB International,
Wallingford, CT, pp 98–105
Gross E, Thomazini-Casagrande LI, Caetano FH (1998) A scanning electron microscopy study of
ectomycorrhizae on Pinus patula Schiede and Deppe. Naturalia (Rio Claro) 23(1):93–101
Gutiérrez A, Morte A, Honrubia M (2003) Morphological characterization of the mycorrhiza
formed by Helianthemum almeriense Pau with Terfezia claveryi Chatin and Picoa lefebvrei
(Pat.) Maire. Mycorrhiza. 2003, 13(6):299–307
Hall IR, Wang Y (1998) Methods for cultivating edible ectomycorrhizal mushrooms. In: Varma A
(ed) Mycorrhiza manual, Springer laboratory manual. Springer Verlag, Heidelberg, pp 99–114
Hall IR, Brown G, Byars J (1994) The black truffle: its history, uses and cultivation, 2nd edn. New
Zealand Institute for Crop & Food Research Limited, Christchurch, New Zealand
Hall IR et al (1998a) Ectomycorrhizal fungi with edible fruiting bodies 3 Tuber magnatum. Econ
Bot 52:192–200
Hall IR et al (1998b) Ectomycorrhizal fungi with edible fruiting bodies. 2. Boletus edulis. Econ
Bot 52:44–56
Hall IR et al (2002) Edible Mycorrhizal mushrooms and their cultivation. Proceedings of the
second international conference on edible Mycorrhizal mushrooms, CD-ROM, New Zealand
Institute for Crop & Food Research Limited
Hall IR, Wang Y, Amicucci A (2003) Cultivation of edible ectomycorrhizal mushrooms. Trends
Biotechnol 21(10):433–438
Hall IR, Brown G, Zambonelli A (2007) Taming the Truffle: the history, lore, and science of the
ultimate mushroom. Timber Press, Portland, p 304
Harley JL, Harley EL (1987) A check-list of mycorrhiza in the British flora. New Phytol (Suppl)
105:1–102
Harley JL, Smith SE (1983) Mycorrhizal symbiosis. Academic Press, London, p 483
Harrison MJ (2005) Signaling in the arbuscular mycorrhizal symbiosis. Annu Rev Microbiol
59:19–42
Harvey AE, Larsen MJ, Jurgensen MF (1976) Distribution of ectomycorrhizae in a mature
douglas-fir/larch soil in western Montana. For Sci 22:393–633
Harvey AE, Jurgensen MF, Larsen MJ, Graham RT (1987) Relationships among soil microsite,
ectomycorrhizae, and natural conifer regeneration of old-growth forests in western Montana.
Can J For Res 17:58–62
Henkel TW, James TY, Miller SL, Aime ML, Miller OK Jr (2006) The mycorrhizal status
of Pseudotulostoma volvata (Elaphomycetaceae, Eurotiales, Ascomycota). Mycorrhiza
16:241–244
Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, Eriksson OE, Huhndorf S, James T,
Kirk PM, L€ucking R, Lumbsch HT, Lutzoni F, Matheny PB, McLaughlin DJ, Powell MJ,
Redhead S, Schoch CL, Spatafora JW, Stalpers JA, Vilgalys R, Aime MC, Aptroot A, Bauer R,
Begerow D, Benny GL, Castlebury LA, Crous PW, Dai YC, Gams W, Geiser DM, Griffith
GW, Gueidan C, Hawksworth DL, Hestmark G, Hosaka K, Humber RA, Hyde KD, Ironside
JE, Kõljalg U, Jyrtznabm CP, Larsson KH, Lichtwardt R, Longcore J, Miadlikowska J,
Miller A, Moncalvo JM, Mozley-Standridge S, Oberwinkler F, Parmasto E, Reeb V,
Rogers JD, Roux C, Ryvarden L, Sampaio JP, Sch€ ußler A, Sugiyama J, Thorn RG, Tibell L,
Untereiner WA, Walker C, Wang Z, Weir A, Weiß M, White MM, Winka K, Yao YJ, Zhang N
(2007) A higher-level phylogenetic classification of the fungi. Mycol Res 111:509–547
Hosaka K, Castellano MA, Spatafora JW (2008) Biogeography of hysterangiales (phallomyceti-
dae, basidomycota). Mycol Res 112:488–462
Hosford D, Ohara H (1995) Ecological study of Tricholoma magnivelare shiros in central
Washington. In: Schnepf C (ed) Dancing with an elephant. Proceedings: the business and
science of special forest products, Western Forestry and Conservation Association, Portland,
Oregon, pp 111–116
Hosford D, Pilz D, Molina R, Amaranthus M (1997) Ecology and management of the commer-
cially harvested American matsutake mushroom. U.S. Department of Agriculture Forest
Service, Pacific Northwest Research Station, Portland, Oregon. General Technical Report
PNW GTR-412
Humpert AJ, Muench EL, Giachini AJ, Castellano MA, Spatafora JW (2001) Molecular phylo-
genetics of Ramaria and related genera: evidence from nuclear large subunit and mitochondrial
small subunit rDNA sequences. Mycologia 93:465–477
Hunt GA, Fogel R (1983) Fungal hyphal dynamics in a western Oregon douglas-fir stand. Soil Biol
Biochem 15:614–649
Iloba C (1980) The use of chemicals for weed control in forest tree nurseries. In: Mikola P (ed)
Tropical mycorrhiza research. Clarendon Press, Oxford, pp 121–123
Janos DP (1980) Vesicular arbuscular mycorrhizae affect lowland tropical rain forest plant growth.
Ecology 61:151–162
Janos DP, Sahley CT (1995) Rodent dispersal of vesicular-arbuscular mycorrhizal fungi in
Amazonian Peru. Ecology 76(6):1852–1858
Johnson CN (1994) Mycophagy and spore dispersal by a rat-kangaroo: consumption of ectomy-
corrhizal taxa in relation to their abundance. Funct Ecol 8:464–468
Kagan-Zur V (2002) Tuber melanosporum research in Israel. In: Hall IR et al (eds) Edible
mycorrhizal mushrooms and their cultivation. Proceedings of the second international confer-
ence on edible mycorrhizal mushrooms, CD-ROM, New Zealand Institute for Crop & Food
Research Limited
Kahiluoto H, Vestberg M (1998) The effect of arbuscular mycorrhiza on biomass production and
phosphorus uptake from sparingly soluble sources by leek (Allium porrum L.) in Finnish field
soils. Biol Agric Hortic 16:65–85
Kandefer-Szersen M, Kawecki Z, Salata B, Witek M (1980) Mushrooms as a source of substances
with antiviral activity (in Polish). Acta Mycologica 16(2):215–220
Kawai M (1997) Artificial ectomycorrhiza formation on roots of air-layered Pinus densiflora
saplings by inoculation with Lyophyllum shimeji. Mycologia 89:228–232
Kernaghan G, Currah RS, Bayer RJ (1997) Russulaceous ectomycorrhizas of Abies lasicarpa and
Picea engelmanii. Can J Bot 75:1843–1850
Klironomos JN, Hart MM (2001) Animal nitrogen swap for plant carbon. Nature 410:651–652
Kõljalg U, Dahlberg A, Taylor AFS, Larsson E, Hallenberg N, Stenlid J, Larsson KH, Fransson
PM, Kårén O, Jonsson L (2000) Diversity and abundance of resupinate thelephoroid fungi as
ectomycorrhizal symbionts in Swedish boreal forests. Mol Ecol 9:1985–1996
Kraigher H, Agerer A, Javornik B (1995) Ectomycorrhizae of Lactarius lygniotus on Norway
spruce, characterized by anatomical and molecular tools. Mycorrhiza 5:175–180
448 A. Karwa et al.
Kranabetter JM, Trowbridge R, Macadam A, McLennan D, Friesen J (2002) Ecological descrip-

tions of pine mushroom (Tricholoma magnivelare) habitat and estimates of its extent in
northwestern British Columbia. For Ecol Manage 158:249–261
Kropp BR, Trappe JM (1982) Ectomycorrhizal fungi of Tsuga heterophylla. Mycologia
74:479–488
Krzyczkowskia W, Malinowskaa E, Suchockib P, Klepsa J, Olejnikd M, Herold F (2008) Isolation
and quantitative determination of ergosterol peroxide in various edible mushroom species.
Food Chem 113(1):351–355
Lamaison JL, Polese JM (2005) The great encyclopedia of mushrooms. K€onemann, Koln,
Germany, p 28
Lapeyrie F, H€ogberg P (1994) Harnessing symbiotic associations: ectomycorrhizas. In: Leakey
RRB, Newton AC (eds) Tropical trees: the potential for domestication and the rebuilding of
forest resources. HMSO, London
Lawrence JF (1989) Mycophagy in the Coleoptera: feeding strategies and morphological adapta-
tions. In Wilding, N., Collins, M., Hammond, P.M. and Webber, J.F., eds, Insect - Fungus
Interactions. Royal Entomological Society Symposium Series. 14:1–23
Leake JR et al (2002) Interactions between ecto-mycorrhizal and saprotrophic fungi. In: Van der
Heijden MGA, Sanders IR (eds) Mycorrhizal ecology, ecological studies analysis and synthe-
sis. Springer, Berlin, pp 345–372
Lefevre C, Hall IR (2001) In: Mehlenbacher SA (ed) The global status of truffle cultivation.
Proceedings of the fifth international congress on hazelnut (556), Acta Hortic, pp 513–520
Li H, Smith SE, Holloway RE, Zhu Y, Smith FA (2006) Arbuscular mycorrhizal fungi contribute
to phosphorus uptake by wheat grown in a phosphorus-fixing soil even in the absence of
positive growth responses. New Phytol 172(3):536–543
Li D, Zhao WH, Kong BH, Ye M, Chen HR (2009) Inhibition effects of the extract and
polysaccharide in macrofungus on TMV. J Yunnan Agri Univ 24(2):175–180
Lilleskov EA et al (2002) Belowground ectomycorrhizal fungal community change over a
nitrogen deposition gradient in Alaska. Ecology 83:104–115
LoBuglio KF, Berbee ML, Taylor JW (1996) Phylogenetic origins of the asexual mycorrhizal
symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among Ascomycetes. Mol
Phylogenet Evol 6:287–294
Lu X-H, Malajczuk N, Dell B, Dell B (1998) Mycorrhiza formation and growth of Eucalyptus
globulus seedlings inoculated with spores of various ectomycorrhizal fungi. Mycorrhiza
8:81–86
Lucas EH, Montesono R, Pepper MS, Hafner M, Sablon E (1957) Tumor inhibitors in Boletus
edulis and other holobasidiomycetes. Antibiot Chemother 7:1–4
Maison de la (2010) Truffle-Truffe fraiche Noire (in French). http://en.wikipedia.org/wiki/Truffle-
(fungus)
Malajczuk N, Molina R, Trappe J (1982) Ectomycorrhiza formation in Eucalyptus I. Pure culture
synthesis, host specificity and mycorrhizal compatibility with Pinus radiata. New Phytol
91:467–482
Malloch D, Malloch B (1981) The mycorrhizal status of boreal plants: species from northeastern
Ontario. Can J Bot 59:2167–2172
Marais LJ, Kotzé JM (1977) Notes on ectotrophic mycorrhizae of Pinus patula in South Africa.
South Afr For J 100:61–71
Marks GC, Ditchborne N, Foster RC (1968) Quantitative estimates of mycorrhizal populations in
radiata pine forests. Aust For 32:26–38
Marschner H (1995) Mineral nutrition of higher plants. Academic Press, London
Martin F, Kohler A, Murat C, Balestrini R, Coutinho P M, Jaillon O, Montanini B, Morin E, Noel
B, Percudani R, Porcel B, Rubini A, Amicucci A, Amselem J, Anthouard V, Arcioni S,
Artiguenave F, Aury J-M, Ballario P, Bolchi A, Brenna A, Brun A, Buee M, et al. (2010)
The Black Truffle Genome Uncovers Evolutionary Origins and Mechanisms of Symbiosis.
Nature 464:1033–1038
Mason P, Last FT, Wilson J, Deacon JW, Fleming LV, Fox FM (1987) Fruiting and succession of
ecto-mycorrhizal fungi. In: Fungal infection of plants. (Ed GF Pegg and PG Ayres) 253–268.
Cambridge University Press
Mason PA, Wilson J (1994) Harnessing symbiotic associations: vesicular arbuscular mycorrhizas.
In: Leakey RRB, Newton AC (eds) Tropical trees: the potential for domestication and the
rebuilding of forest resources. HMSO, London, pp 165–175
Massicotte HB, Molina R, Luoma DL, Smith JE (1994) Biology of the ectomycorrhizal genus.
Rhizopogon II. Patterns of host-fungus specificity following spore inoculation of diverse hosts
grown in monoculture and dual culture. New Phytol 126:677–690
Masuka AJ (1996) Dynamics of mushroom (Boletus edulis) production in pine plantations in
Zimbabwe. J Appl Sci Southern Afr 2(2):69–76
Matheny PB, Bougher NL (2006) The new genus Auritella from Africa and Australia (Inocyba-
ceae, Agaricales): molecular systematics, taxonomy and historical biogeography. Mycol Progr
5:2–17
Matheny PB, Curtis JM, Hofstetter V, Aime MC, Moncalvo JM, ZaiWei G, ZhuLiang Y, Slot JC,
Ammirati JF, Baroni TJ, Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW, Seidl MT,
Aanen DK, Denitis M, Daniele GM, Desjardin DE, Kropp BR, Norvell LL, Parker A, Vellinga EC,
Vilgalys R, Hibbett DS (2006) Major clades of Agaricales: a multilocus phylogenetic
overview. Mycologia 98:982–995
Mattila P, Lampi AM, Ronkainen R, Toivo J, Piironen V (2002) Sterol and vitamin D2 contents in
some wild and cultivated mushrooms. Food Chem 76(3):293–298
McGee PA (1996) The Australian zygomycetous mycorrhizal fungi: the genus Densospora gen.
nov. Aust Syst Bot 9:329–336
Meyer FH (1973) Distribution of ectomycorrhizae in native and man-made forests. In: Marks GC,
Kozlowski TT (eds) Ectomycorrhizae, their ecology and physiology. Academic Press,
Miller SL, Torres P, McClean TM (1994) Persistence of basidiospores and sclerotia of ectomy-
corrhizal fungi and Morchella in soil. Mycologia 86:89–95
Molina R, Trappe JM (1982) Patterns of ectomycorrhizal host specificity and potential amoung
pacific northwest conifers and fungi. Forest Sci 28:423–458
Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R (2000) Phylogenetic relationships
of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Syst Biol
49:278–305
Moncalvo JM, Vilgalys R et al (2002) One hundred and seventeen clades of euagarics. Mol
Phylogenet Evol 23:357–400
Morte A et al (2000) Effect of drought stress on growth and water relations of the mycorrhizal
association Helianthemum almeriense – Terfezia claveryi. Mycorrhiza 10:115–119
Munyanziza E (1994) Miombo trees and mycorrhizae: ecological strategies, a basis for afforesta-
tion. Ph.D. thesis, Wageningen Agricultural University, The Netherlands, p 193
Niazi R, Iqbal SH, Khalid AN (2006) Biodiversity of Mushrooms and ectomycorrhizas. 1. Russula
brevipes Peck, and its ectomycorrhiza – a new record from Himalayan moist temperate forest
of Pakistan. Pakistan J Bot 38(4):1271–1277
Nils F (1979) Germination of spores of Cantharellus cibarius. Mycologia 71(1):216–219
North M, Trappe JM (1996) Relative abundance and animal consumption of fungal sporocarps in
Pacific Northwest young- mature- and old-growth forests. In: Szaro TM, Bruns TD (eds)
Proceedings, 1st international conference on mycorrhizae, Berkeley, California, p 93
Norvell LL (1995) Loving the chanterelle to death? The ten-year Oregon chanterelles project.
McIlvainea 12(1):6–25
Ogawa M (1985) Ecological characters of ectomycorrhizal fungi and their mycorrhizae – an
introduction to the ecology of higher fungi. JARQ 18:305–314
Oria-de-Rueda J, Martin-Pinto P, Olaizola J (2008) Bolete productivity of cistaceous scrublands in
northwestern Spain. Econ Bot 62(3):323–330
450 A. Karwa et al.
Orlovich D, Stringer A, Yun W, Hall I, Prime G, Danell E, Weden C, Bulman S (2004) Boletus
edulis Bull. Ex Fries in New Zealand. Aust Mycol Soc Newslett 1(1):6
Peinter U, Bougher NL, Castellano MA, Moncalvo J-M, Moser MM, Trappe JM, Vilgalys R
(2001) Multiple origins of sequestrate fungi related to Cortinarius (Cortinariaceae). Am J Bot
88:2168–2179
Peinter U, Iotti M, Klotz P, Bonuso E, Zambonelli A (2007) Soil fungal communities in a
Castanea sativa (chestnut) forest producing large quantities of Boletus edulis sensu lato
(porcini): where is the mycelium of porcini? Environ Microbiol 9(4):880–889
Philpot R (1965) Viennese cookery. Hodder & Staughton, London, pp 139–140
Pillukat A, Agerer R (1992) Studies an Ektomycorrhizen XL. Vergleichende Untersuchungen zur
bambezogenen variabilitat der Ektomycorrhizen von Russula ochroleuca. Z Mykol
58:211–242
Pilz D, Molina R, Liegel L (1998) Biological productivity of chanterelle mushrooms in and near
the Olympic Peninsula Biosphere Reserve. Ambio 9:8–13
Piraino FF (2006) Emerging antiviral drugs from medicinal mushrooms. Int J Med Mushrooms 8
(2):101–114
Pirozynski KA, Dalpé Y (1989) Geological history of the Glomaceae with particular reference to
mycorrhizal symbioses. Symbiosis 7:1–36
Poitou N et al (1989) Mycorrhization controlee et culture experimentale du champ de Boletus (¼
Suillus) granulatus et Lactarius deliciosus. In: Grabbe K, Hilber O (eds) Proceedings of the
twelfth international congress on the science and cultivation of edible fungi, Braunschweig, pp
551–563
Quan L, Lei ZP (2000) A study on effect of ectomycorrhizae on promoting Castanea mollissima
resistance to drought and its mechanism (in Chinese). For Res 13(3):249–256
Reddell P, Milnes AR (1992) Mycorrhizas and other specialised nutrient-acquisition strategies:
their occurrence in woodland plants from Kakadu and their role in rehabilitation of waste rock
dumps at a local uranium mine. Aust J Bot 40:223–242
Reddell P, Gordon V, Hopkins MS (1999) Ectomycorrhizas in Eucalyptus tetrodonta and
E. miniata forest communities in tropical northern Australia and their role in the rehabilitation
of these forests following mining. Aust J Bot 34:435–444
Redecker D, Kodner R, Graham LE (2000) Glomalean fungi from the Ordovician. Science
289:1920–1921
Redecker D et al (2001) Small genets of Lactarius xanthogalactus, Russula cremoricolor and
Amanita francheti in late-stage ectomycorrhizal successions. Mol Ecol 10:1025–1034
Redhead SA (1997) The pine mushroom industry in Canada and the United States: why it exists
and where it is going. In: Palm ME, Chapela IH (eds) Mycology in sustainable development:
expanding concepts, vanishing borders. Parkway Publishers, Boone, NC, pp 15–54
Remy W, Taylor TN, Hass H, Kerp H (1994) 4 hundred million year old vesicular-arbuscular
mycorrhizae. Proc Natl Acad Sci 91(25):11841–11843
Ribeiro Bárbara, Andrade PB, Silva BM, Baptista P, Seabra RM, Valentão P (2008) Comparative
study on free amino acid composition of wild edible mushroom species. Journal of agricultural
and food chemistry. 56(22):10973–9.
Richardson MJ (1970) Studies on Russula emetica and other agarics in a Scot pine plantation.
Trans Br Mycol Soc 55:217–229
Rinaldi AC, Comadini O, Kuyper TW (2008) Ectomycorrhizal fungi diversity: separating the
wheat from the chaff. Fungal Divers 33:1–45
Rose SL (1980) Mycorrhizal associations of some actinomycete nodulated nitrogen fixing plants.
Can J Bot 58:1449–1454
Sbrana C et al (2000) Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains
isolated from Tuber borchii ascocarps. Can J Microbiol 46:259–268
Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686
Schulze W, Schulze ED, Pate JS, Gillison AN (1997) The nitrogen supply from soil and insects
during growth of the pitcher plants Nepenthes mirabilis, Cephalotus follicularis and Darling-
tonia californica. Oecologia 112:464–471
Schweiger PF, Robson AD, Barrow NJ (1995) Root hair length determines beneficial effect of a
Glomus species on shoot growth of some pasture species. New Phytol 131:247–254
Selosse MA, Bauer R, Moyersoen B (2002) Basal hymenomycetes belonging to the Sebacinaceae
are ectomycorrhizal on temperate deciduous trees. New Phytol 155:183–195
Selosse MA, Richard F, He X, Simard SW (2006) Mycorrhizal networks: des liaisons danger-
euses? Trends Ecol Evol 21(11):621–628
Sharma R (2008) Studies on ectomycorrhizal mushrooms of M.P. and Chhattisgarh. PhD thesis,
R.D. University, Jabalpur
Simard SW, Perry DA, Jones MD, Myrold DD, Durall DM, Molina R (1997) Net transfer of
carbon between ectomycorrhizal tree species in the field. Nature 388:579–582
Sisti D et al (1998) In vitro mycorrhizal synthesis of micropropagated Tilia platyphyllos Scop.
plantlets with Tuber borchii Vittad. Mycelium in pure culture. Acta Hortic 457:379–387
Sitta N (2000) Presence of Tylopilus into dried “Porcini” mushrooms from China (in Italian).
Micol Ital 29(1):96–99
Sitta N, Floriani M (2008) Nationalization and globalization trends in the wild mushroom
commerce of Italy with emphasis on porcini (Boletus edulis and allied species). Econ Bot 62
(3):307–322
Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic Press, London
Smith JE, Molina R, Huso MMP, Luoma DL, McKay D, Castellano MA, Lebel T, Valachovic V
(2002) Species richness, abundance, and composition of hypogeous and epigeous ectomy-
corrhizal fungal sporocarps in young, rotation-age, and old-growth stands of Douglas-fir
(Pseudotsuga menziesii) in the Cascade Range of Oregon, USA. Can J Bot 80(2):186–204
St John TV (1980) A survey of mycorrhizal infection in an Amazonian rain forest. Acta Amazon
10:527–533
Stanis VF (1979) Russula species occurring n the boreal region of Ontario and Quebec Canada.
M.Sc. thesis, Department of Botany, University of Toronto, Toronto, Ontario
Strullu-Derrien C, Strullu DG (2007) Mycorrhization of fossil and living plants. CR Palevol
6:483–494
Suri OP, Shah R, Satti NK, Suri KA (1997) Russulactarorufin, a lactarane skeleton sesquiterpene
from Russula brevipes. Phytochemistry 45(7):1453–1455
Susan M (1992) Texas mushrooms: a field guide, 1st edn. University of Texas Press, Austin
Olivier JM (2000) Progress in the cultivation of truffles. In: Van Griensven LJLD (ed) Mushroom
science XV: science and cultivation of edible fungi, vol 2. Balkema, Rotterdam, pp 937–942
Tarafdar JC, Marschner H (1994) Efficiency of VAM hyphae in utilisation of organic phosphorus
by wheat plants. Soil Sci Plant Nutr 40:593–600
Tedersoo L, Hansen K, Perry BA, Kjller R (2006) Molecular and morphological diversity of
Pezizalean ectomycorrhiza. New Phytol 170:581–596
Tedersoo L, Suvi T, Beaver K, Kõljag U (2007) Ectomycorrhizal fungi of the Seychelles: diversity
patterns and host shifts from the native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia
bijuga (Caesalpiniaceae) to the introduced Eucalyptus robusta (Myrtaceae), but not Pinus
caribea (Pinaceae). New Phytol 175:321–333
Tehler A, Little DP, Farris JS (2003) The full-length phylogenetic tree from 1551 ribosomal
sequences of chitinous fungi, Fungi. Mycol Res 107:901–916
Termorshuizen AJ (1993) The influence of nitrogen fertilisers on ectomycorrhizas and their fungal
carpophores in young stands of Pinus sylvestris. For Ecol Manage 57:179–189
Termoshuizen AJ (1991) Succession of mycorrhizal fungi in stands of Pinus sylvestris in the
Netherlands. J Veg Sci 2:255–264
Thomson BD, Grpe TS, Malajczuk N, Hardy GES (1994) The effectiveness of ectomycorrhizal
fungi in increasing the growth of Eucalyptus globulus Labill in relation to root colonization and
hyphal development in soil. New Phytol 126:517–524
452 A. Karwa et al.
Trappe JM (1987) Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an
evolutionary standpoint. In: Safir GR (ed) Ecophysiology of VA mycorrhizal plants. CRC
Press, Boca Raton, FL, pp 5–25
Trocha LK, Rudawska M, Leski T, Dabert M (2006) Genetic diversity of naturally established
ectomycorrhizal fungi of norway spruce seedlings under nursery conditions. Microb Ecol
52:418–425
Trowbridge R, Macadam A (1999) Ecological description and classification of highly productive
pine mushroom sites in Northwestern British Columbia. In: Moss P (ed) Studies of the pine
mushroom (Tricholoma magnivelare) in the Skeena-Bulkley Region. Northwest Institute of
Bioregional Studies, Smithers, BC
Tuomi J, Kytoviita M, Hardling R (2001) Cost efficiency of nutrient acquisition of mycorrhizal
symbiosis for the host plant. Oikos 92:62–70
Tylukti EE (1987) Mushrooms of Idaho and the Pacific Northwest. Non-gilled Hymenomycetes,
vol 2. The University of Idaho Press, Moscow, Idaho, pp 9–10
Unestam T, Sun YP (1995) Extramatrical structures of hydrophobic and hydrophilic ectomycor-
rhizal fungi. Mycorrhiza 5:301–311
Urban A, Weiss M, Bauer R (2003) Ectomycorrhizas involving sebacinoid mycobionts. Mycol
Res 107:3–14
Verbeken A, Buyck B (2002) Diversity and ecology of tropical ectomycorrhizal fungi in Africa.
In: Watling R, Frankland JC, Ainsworth AM, Isaac S, Robinson CH, eds. Tropical mycology,
Vol. 1. Macromycetes. Wallingford, UK: CABI Publishing, 11–24
Veselkov IM (1975) Artificial propagation of Boletus edulis in forests. PacтитeльHьie Pecуpcы
Poccии 11:574–578
Visser S (1995) Ectomycorrhizal fungal succession in Jack pine stands following wildfire. New
Phytol 129:389–401
Vogt KA, Edmonds RL, Grier CC (1981) Biomass and nutrient concentrations of sporocarps
produced by mycorrhizal and decomposer fungi in Abies amabilis stands. Oecologia
50:170–175
Vozzo JA, Hackskaylo E (1961) Mycorrhizal fungi on Pinus virginiana. Mycologia 53
(5):538–539
Wang B, Qiu YL (2006) Phylogenetic distribution and evolution of mycorrhizas in land plants.
Wang Y, Sinclair L, Hall IR, Cole ALJ (1995) Boletus edulis sensu lato: a new record for New
Zealand. New Zeal J Crop Hort Sci 23:227–231
Wang Y et al (1997) Ectomycorrhizal fungi with edible fruiting bodies 1. Tricholoma matsutake
and related fungi. Econ Bot 51:311–327
Wang Y et al (2002a) Some ectomycorrhizal mushrooms. In: Hall IR et al (eds) Edible mycorrhi-
zal mushrooms and their cultivation. Proceedings of the second international conference on
edible mycorrhizal mushrooms, CD-ROM, New Zealand Institute for Crop & Food Research
Limited
Wang Y et al (2002b) The cultivation of Lactarius deliciosus (saffron milk cap) and Rhizopogon
rubescens (shoro) in New Zealand. In: Hall IR et al (eds) Edible mycorrhizal mushrooms and
their cultivation. Proceedings of the second international conference on edible mycorrhizal
mushrooms, CD-ROM, New Zealand Institute for Crop & Food Research Limited
Warcup JH (1980) Ectomycorrhizal associations of Australian indigenous plants. New Phytol
85:531–535
Warcup JH (1985) Ectomycorrhizal formation by Glomus tubiforme. New Phytol 99:267–272
Warner Jeremy (2010) http://basiceating.blogspot.com/2010/04/Chantharelle-cantharellus.cibarius.
html retrieved on 02/06/10
Watling R (2006) The sclerodermoid fungi. Mycoscience 47:18–24
Wilson AW, Hobbie EA, Hibbet DS (2007) The ectomycorrhizal status of Calostoma cinnabarinum
determined using isotopic, molecular and morphological methods. Can J Bot 85:385–393
Yamada A (2003) Studies in ecology and mycorrhizal taxonomy of ectomycorrhizal fungi in Pinus
densiflora forests and cultivation of ectomycorrhizal mushrooms. Nippon Kin Gakkal Kaiho 44
(1):9–18
Yamada A, Ogura T, Ohmasa M (2001a) Cultivation of mushrooms of edible ectomycorrhizal
fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis I. Primordium and
basidiocarp formation in open pot culture. Mycorrhiza 11:59–66
Yamada A, Ogura T, Ohmasa M (2001b) Cultivation of mushrooms of edible ectomycorrhizal
fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis II. Morphology of
mycorrhizas in open pot soil. Mycorrhiza 11:67–81
Yun W, Hall IR (2004) Edible ectomycorrhizal mushrooms: challenges and achievements.
Can J Bot 82(8):1063–1073
Zambonelli A et al (2002) Tuber borchii cultivation: an interesting perspective. In: Hall IR et al
(eds) Edible mycorrhizal mushrooms and their cultivation. Proceedings of the second inter-
national conference on edible mycorrhizal mushrooms, cd-rom, New Zealand Institute for
Crop & Food Research Limited
Zheng S, Li C, Ng TB, Wang HX (2007) A lectin with mitogenic activity from the edible wild
mushroom Boletus edulis. Process Biochem 42(12):1620–1624
Index
A Bioremediation, 213–217
Accumulative in situ passive monitor, 312–313 Biotechnological potential, 347–363
Acid phosphatase, 406, 418–420 Boletaceae, 4
Active monitors, 309, 314–316 Boletus, 6, 7, 9
Adhesion proteins, 165 Boletus edulis, 431, 433, 436
Agar cultures, 44, 45 Brazil, 6
Agricultural and nursery practices, 341 BSA, 8
Agrobacterium, 123–136 By-product benefits, 396
Agrobacterium-mediated transformation
(AMT), 125–128, 130–136 C
Alginate-bead inoculum, 46, 48, 52–53, 56 Calcium, 5
Alginated bead mycelial inoculum, 7 Calvatia, 7, 9
Amanita, 7 Cantharellus, 405–423
Amanitaceae, 4 Cantharellus cibarius, 432, 433, 436, 438
Amino acids, 388–391 Catecholate, 349, 350, 352, 353, 356
Ammonium transporters (Amt), 388, 389 Cenococcum geophilum, 242, 247
AMT. See Agrobacterium-mediated Chlorinated aromatic hydrocarbons (CAHs),
transformation 209, 220–221
Antagonism, 416–418, 422 Citrate, 238
Antioxidative, 438 Commercial inoculum, 57
Apoptosis, 2, 112–116 Community, 255–258, 261, 262, 265, 270, 271,
Application of spores, 54 273–276, 278–288, 290, 291
Application of vegetative inoculum, 55–56 homomorphic model, 279, 280
Arbuscular mycorrhizas, 3 Conservation, 429–442
Artificial synthesis, 410 Controlled ectomycorrhization, 145, 150
Avoidance mechanism, 237 Controlled mycorrhization, 324–325, 334, 335
Cost/benefit, 391, 394–397
B Cost efficiency, 395, 396
Basidiospores, 50–51, 54 Costs, 391, 394–397
Beads of alginate gel, 149 Crude oil, 217
Benefits, 394–397 Culinary, 435, 436
Bioavailability of toxic metals, 233 Cultivation, 429–442
Biocides, 326, 341
Biodegradation, 210, 213, 214, 221 D
Biodiversity, 405 Defence response, 160
Biogeochemical cycling of metals, 231, Degree of mycorrhization, 395
232, 248 Dendroremediation, 211
Biological invasions, 10 Developmental system, 259–260

Soil Biology 25, DOI 10.1007/978-3-642-15196-5,
456 Index
Developmental system (cont.) Food webs, 232, 240, 245, 249

epistemic status, 260–261, 264 Forest ecosystem, 301–317
homomorphic model, 264, 269, 279 Forests, 19, 20, 25, 26, 35
2,4-Dichlorophenol, 212 Fruiting body, 435, 436, 439
Dipterocarpaceae, 3 Functional dynamic module (FDM), 262–270,
Diversity indexes, 309 272, 276, 278–286, 289, 291
DNA, 109–113 Functional genomics, 131, 185, 192
Functional niche, 258, 262–264, 266, 270,
E 275, 278
ECM associated bacterium, 240 Fungal pure cultures, 44–45
Ecological hierarchy, 256 Fungal translocation of metal elements, 235
epistemic status, 262–268 Fungicides, 324, 326–330, 332, 334–341
Ecological indicator, 307, 309–314
Ecotoxicological concerns, 231, 243 G
Ectomycoremediation, 209–224 Gene knock-down, 195–197, 199, 200
Ectomycorrhiza, 3–12, 123–136, 177–200, Genes associated to ectomycorrhizal
371–382, 405–423 symbiosis, 158–170
Ectomycorrhizae (ECM) Glutamate-dehydrogenase (GDH), 390
anatomotypes, 25, 31, 32, 34, 35 Glutamine synthetase (GS), 390, 391
colonization, 19–35 Glutathione, 237
fungi, 19, 20, 24, 34, 35 GOGAT, 390, 391
inoculations, 24, 25 Growth pouches, pots, 45, 48–49
Ectomycorrhizal community, 211, 215, 217,
223, 224 H
Ectomycorrhizal establishment, 160, 168 Heat shock proteins, 116
Ectomycorrhizal fungi (EMF), 43–45, 47, 48, Heavy metals, 209, 211, 217, 221–223
50, 52–55, 57, 58, 123–136, 144–146, Hebeloma crustulinifome, 7
152, 177–200, 255–291, 323–341, Herbicides, 324, 326–329, 331, 333, 335–337,
347–363 339–341
dimension, 270–272 Heterotrophic leaching, 233
disturbance and succession, 256, 284–288 Hexose transporters, 391, 392
homomorphic model, 278–280, 282, 285, Hopea
289, 291 H. helferi, 6
horizontal location, 273 H. nervosa, 5, 9
individual, 270, 271, 273, 280 H. odorata, 6, 7, 9
life time, 271 Hydrophobins, 109–111, 117
location in time, 275–276 Hydroxamate, 349–354, 356, 361, 362
mathematical modeling, 288
research directions, 288–291 I
resources for, 276–277 Indonesia, 6, 7
species composition, 67–81 Inoculation, 43–58
use by other organisms, 276–278 beds, 8
vertical location, 273 techniques, 43–58
Ectomycorrhizal genes, 87–91 Inoculum formulation, 145, 147
Ectomycorrhizal symbiosis, 324 Inoculum types, 44, 45, 49–53, 56, 58
Ectomycorrhizosphere, 213–216, 221 Insecticides, 324, 326–329, 331, 333, 336,
Eucalyptus citriodora, 6 337, 341
Evolutionary hotspots of metal tolerance, 247 Inter-simple sequence repeats (ISSR)
markers, 75
F Ionizing radiation, 223
Field inoculation, 56–57
Flavonoids, 372, 374–375 K
Floating culture technique, 7, 11 Kalimantan, 9
Index 457
L N
Laccaria, 124, 125, 134–136, 196–200 Natural capital, 259, 266, 267, 289, 291
Laccase, 216, 217 role of ectomycorrhizal fungi, 286–288
Lactarius, 7 Natural inoculum, 50
Lactarius, 337–340 N availability, 393, 395, 396
Lignin degradation, 216, 220 Neotropics, 35
Lignin peroxidase, 216, 217 New technique, 409–410
Liquid cultures, 45, 51, 52 Nitrogen, 4
Litter decomposition, 233–235 Non-sterile techniques, 45, 48–49
Low molecular weight organic acids Non-timber forest products, 3
(LMWOA), 348, 355–359 Nutrient media, 44–45
M O
Magnesium, 5 Oddoux medium, 8
Malaysia, 4, 6, 9 Oxalic acid, 232, 233, 238
Management, 256, 259, 260, 264, 266–268,
278, 280, 283, 285–287, 289 P
Mancozeb, 5 Pachlewski’s medium, 8
Manganese peroxidase, 216, 217 PAHs. See Polycyclic aromatic hydrocarbons
Mass inoculums, 406 Parashorea tomentella, 5, 9
MDSN. See Mycorrhizal N demand-supply Passive monitor, 309–314
balance Pathogens, 12
Metabolic process, 92 Paxillus spp., 219, 221, 222
Metabolic readjustments, 394 PCBs. See Polychlorinated biphenyls
Metal binding capacity of ECMF, 236 PCR-RFLP, 10
Metal-chelating agents, 347–363 Peat swamp forest, 9, 12
Metal coordination, 237 Pest control, 323–325
Metallothionein (MT), 237, 238, 348, 359–363 Pesticide toxicity, 331
Metal tolerance, 232, 233, 236–243, 247–249 Petri dishes, 44–47
Metal toxicity, 232, 236–242, 246–249 Petroleum hydrocarbons (PHC), 213, 215,
Microbial community, 211, 213–215, 218, 217–218
223, 224 Phosphorus, 4
Microorganisms, 213, 214, 217 Photodamage, 5
Mine tailings, 9, 11 Phylogenetic analyses, 68
Mitochondrial large sub-unit rRNA, 69 Phytochelatins (PCs), 348, 359, 360, 362, 363
Modified Melin–Norkrans (MMN), 8 Phytoextraction, 210, 221, 222
Molecules that control the interactions between Phytohormones, 376–380
symbionts, 373 Phytoremediation, 210–224
Morphotypes, 34 Pinus, 8, 411, 412, 416
Mother tree inoculation, 8 Pisolithus
MT. See Metallothionein P. albus, 8
Mutualism, 394, 396 P. aurantioscabrosus, 6
Mycelium-based fungal inoculums, 146 P. tinctorius, 6, 7
Mycobioindication, 301–317 Pisolithus spp., 214, 215
Mycorrhizae, 125, 136, 197–200, 410–412 Plant-microbe interaction, 157, 158, 163, 165,
Mycorrhizal inoculum potential, 314–317 168, 169
Mycorrhizal mushrooms, 431–436, 439–442 Plant productivity, 393–396
Mycorrhizal N demand-supply balance Polychlorinated biphenyls (PCBs), 209, 212,
(MDSN), 396 214, 216, 220, 221
Mycorrhizal response, 395 Polycyclic aromatic hydrocarbons (PAHs),
Mycorrhizal tablets, 8, 9, 11 209, 212, 214, 216, 218–219
Mycorrhized forest plant, 340 Population genetics, 241–244, 249
Mycorrhizosphere, 213–216, 218, 223, 224 Populus spp., 211, 217, 220, 222
458 Index
Potassium, 4 Signalling molecules, 158, 160, 161, 168, 170

Precipitation of metals, 232, 236 South America, 19–35
Proteinases, 390 South-east Asia, 3, 7, 11
Proteins associated to ectomycorrhizal Spatial distribution of genets, 75, 78
symbiosis, 158, 166, 170 Spore-based fungal inoculums, 145
Protein synthesis, 88, 100, 101, 117 Spore tablets, 7, 10
Pseudoreplication, 5 Sporocarps and ectomycorrhizas, 67–70,
Pure culture, 11 73–75, 78–80
SPU. See Service production unit
R SRAPs. See Symbiosis regulated acidic
Radionuclides, 223 polypeptides
Reduced glutathione, 348, 359, 360, 362 Stable isotope, 5
Reforestation programmes, 3, 4 Sterile techniques, 45, 47–48
Reforestation schemes, 7, 9 Sterols, 380–381
Remediation, 209–224 Stress factor, 301–306, 313, 316
Responsive in situ mycoindicator, 309–312 Strobilomyces, 9
Reverse genetics, 134–136, 188, 196 Substrate inoculum, 49, 52
Rhizoremediation, 210 Suillus luteus, 241–243
Rhizosphere, 210, 213–216, 224 Suillus spp., 215, 217, 219, 221
Rhizospheric signals, 159–163 Sumatra, 10
RNA, 88, 105, 109–113 Superoxide dismutase, 237
RNA interference (RNAi), 177 Symbiosis, 87–117
RNA silencing, 177–200 Symbiosis regulated acidic polypeptides
Rubber agroforests, 10 (SRAPs), 109
Russula, 6, 7, 68, 69, 73–80 Synthesis of ectomycorrhizae, 45, 48
Russula brevipes, 436, 437
Russulaceae, 4
T
S Terpenes, 371, 373, 375–376
Salix spp., 211, 222, 223 Test tubes, 46–48
Scale, 255–291 Thailand, 6
homomorphic model, 264, 269, 276, Thelephoraceae, 4, 7, 9
279–280, 282 Thiamin, 5
and management, 267 Thiol-peptides, 359–361, 363
and productivity, 261–262 Timber production, 3
Scleroderma, 6–10 Transcription regulation, 88
Scleroderma columnare, 7, 10 Transformation, 123–136
Secondary metabolites, 371–382 Trehalose, 388, 389, 392
Service production unit (SPU), 263, 264, 267, 2,4,6-Trinitrotoluene (TNT), 212, 216, 220
278, 286–288, 291 Trophic-dynamic module (TDM), 258,
Shorea 259, 284
S. academica, 8 Tropical ectomycorrhizal fungi, 67–81
S. balangeran, 7, 9 Tropical forests, 3
S. glauca, 9 Tropical rainforests, 68, 69, 74–76, 81
S. lamellata, 10 Tuber melanosporum, 431, 436
S. leprosula, 8, 9
S. macroptera, 6 U
S. parvifolia, 6 Ubiquinone, 237
S. pinanga, 7, 8 Up-scaling, 255–291
S. roxburghii, 9 application to ectomycorrhizae, 269–284
S. selanica, 8, 10 production, 268
S. seminis, 5 research directions, 288–291
Siderophore, 348–356, 358–363 Urea, 389, 390
Index 459
V W
Vegetative inoculum, 43, 50–52, 55–56 Weathering, 232–233, 235, 236, 238
Vermiculite-peat inoculums, 148–149 White rot fungi (WRF), 216, 217, 220, 221
Vietnam, 12

Biodiversity and Biotechnology of Ectomycorrhizae PDF

Uploaded by

Copyright:

Available Formats

Biodiversity and Biotechnology of Ectomycorrhizae PDF

Uploaded by

Document Information

Original Title

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

Biodiversity and Biotechnology of Ectomycorrhizae PDF

Uploaded by

Copyright:

Available Formats

Soil Biology

Diversity and Biotechnology

Cover design: SPi Publisher Services

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Amravati, Maharashtra, India Mahendra Rai

Part I Diversity, Morphology and Applications

1 The Importance of Ectomycorrhizas for the Growth

2 The Ectomycorrhizal Symbiosis in South America: Morphology,

3 Ectomycorrhizal Inoculum and Inoculation Techniques . . . . . . . . . . . . . . 43

Part II Biotechnological Aspect of ECM

4 Systematics and Ecology of Tropical Ectomycorrhizal Fungi

5 The Molecular Ectomycorrhizal Fungus Essence in Association:

6 Agrobacterium tumefaciens-Mediated Transformation

7 Biotechnological Processes Used in Controlled Ectomycorrhizal

8 Signaling in Ectomycorrhizal Symbiosis Establishment . . . . . . . . . . . . . . 157

9 RNA Silencing in Ectomycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Part III Functions and Interactions

10 Ectomycoremediation: An Eco-Friendly Technique

11 Metal Elements and the Diversity and Function

12 A Conceptual Framework for Up-Scaling Ecological Processes

13 Mycobioindication of Stress in Forest Ecosystems . . . . . . . . . . . . . . . . . . . 301

14 Effects of Pesticides on the Growth of Ectomycorrhizal Fungi

15 Metal-Chelating Agents from Ectomycorrhizal Fungi

16 Ectomycorrhiza and Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . 371

17 C:N Interactions and the Cost:Benefit Balance

18 Ectomycorrhizal Interaction Between Cantharellus

19 Edible Ectomycorrhizal Fungi: Cultivation, Conservation,

Acioli-Santos, Bartolomeu Departamento de Micologia, Universidade Federal

Al Sayegh Petkovšek, Samar ERICo Velenje, Ecological Research & Industrial

Alvarez Crespo, Maria C. Laboratorio de Micologı́a Molecular, Departamento

Amadou, Bâ IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Laboratoire

Antoine, Galiana IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Labor-

Baptista, Paula CIMO / Escola Superior Agrária, Instituto Politécnico de

Becerra, Alejandra G. Instituto Multidisciplinario de Biologı́a Vegetal

Bernard, Dreyfus IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2,

Brearley, Francis Q. Department of Environmental and Geographical Sciences,

Bücking, Heike Biology and Microbiology Department, South Dakota State

Corrêa, Ana Departamento de Microbiologı́a del Suelo y Sistemas Simbióticos,

Dahm, Hanna Department of Microbiology, Institute of General and Molecular

Daniel, Mousain INRA. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2,

Ezékiel, Baudoin IRD. UMR 113 CIRAD/INRA/IRD/SUP-AGRO/UM2, Labor-

Gheghel, Felicia UMR 1136 Interactions Arbres/Microorganismes, INRA

Golińska, Patrycja Department of Microbiology, Institute of General and

Iordache, Virgil Department of Systems Ecology, University of Bucharest,

Karwa, Alka Department of Biotechnology, SGB Amravati University,

Kemppainen, Minna J. Laboratorio de Micologı́a Molecular, Departamento de

Kothe, Erika Microbial Phytopathology, Institute of Microbiology, Friedrich

Kraigher, Hojka Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana,

Lima, Cláudia E.P. Departamento de Micologia, Universidade Federal de

Lino-Neto, Teresa BioFIG, Departamento de Biologia, Universidade do Minho,

Machuca, Ángela Department of Plant Science and Technology, Universidad

Maia, Leonor C. Departamento de Micologia, Universidade Federal de Pernam-

Marin, Miguel Department of Physiology, Genetics and Microbiology, University

Martins-Loução, Maria-Amélia Faculdade de Ciências, Centro de Biologia

Neagoe, Aurora Research Center for Ecological Services (CESEC), University

Pardo, Alejandro G. Laboratorio de Micologı́a Molecular, Departamento de

Rai, Mahendra Department of Biotechnology, SGB Amravati University,

Rajak, Ram C. SGHG Center for Biotechnology, Priydarshini Society, Dumna