References

Soil Biology 

Series Editor: Ajit Varma 9

Volumes published in the series 

Volume 1 

A. Singh, O.P. Ward (Eds.) 

Applied Bioremediation and Phytoremediation 

2004 

Volume 2 

A. Singh, O.P. Ward (Eds.) 

Biodegradation and Bioremediation 

2004 

Volume 3 

F. Buscot, A. Varma (Eds.) 

Microorganisms in Soils: Roles in Genesis and Functions 

2005 

Volume 4 

S. Declerck, D.-G. Strullu, J.A. Fortin (Eds.) 

In Vitro Culture of Mycorrhizas 

2005 

Volume 5 

R. Margesin, F. Schinner (Eds.) 

Manual for Soil Analysis – 

Monitoring and Assessing Soil Bioremediation 

2005 

Volume 6 

H. König, A. Varma (Eds.) 

Intestinal Microorganisms of Termites 

and Other Invertebrates 

2006 

Volume 7 

K.G. Mukerji, C. Manoharachary, J. Singh (Eds.) 

Microbial Activity in the Rhizosphere 

2006 

Volume 8 

P. Nannipieri, K. Smalla (Eds.) 

Nucleic Acids and Proteins in Soil 

2006

Barbara J.E. Schulz 

Christine J.C. Boyle 

Thomas N. Sieber (Eds.) 

Microbial Root 

Endophytes 

With 29 Figures, 4 in Color 

123

PD Dr. Barbara J. E. Schulz 

Technical University of Braunschweig 

Institute of Microbiology 

Spielmannstraße 7 

38106 Braunschweig 

Germany 

e-mail: b.schulz@tu-bs.de 

Dr. Thomas N. Sieber 

Swiss Federal Institute of Technology 

Department of Environmental Sciences 

Institute of Integrative Biology 

Forest Pathology and Dendrology 

8092 Zürich 

Switzerland 

e-mail: thomas.sieber@env.ethz.ch 

Library of Congress Control Number: 2005938057 

Dr. Christine J. C. Boyle 

Augustastraße 32 

02826 Görlitz 

Germany 

e-mail: c.boyle@tu-bs.de 

ISSN 1613-3382 

ISBN-10 3-540-33525-0 Springer Berlin Heidelberg New York 

ISBN-13 978-3-540-33525-2 Springer Berlin Heidelberg New York 

This work is subject to copyright. All rights reserved, whether the whole or part of the 

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Preface 

Healthy plant roots are not only colonized by mycorrhizal fungi and rhizobial 

bacteria, but also by a myriad of other microorganisms, including 

endophytic bacteria and fungi. Comparatively little is known about these 

endophytic microorganisms, which do not cause apparent disease, but 

colonize root tissues inter- and/or intracellulary. Although there had been 

previous research studying both bacterial and fungal endophytes, it was 

in the mid-1980s that numerous investigators began studying these groups 

of microorganisms more intensively. Initially, most work on endophytes 

centered on the diversity of isolates and correlations with ecological factors.Recentlyithasbecomeclearthatsomeoftheseinteractionswith 

endophytic bacteria and fungi can be latently pathogenic and/or mutualistic. 

In mutualistic interactions, the endophyte may improve growth of the 

host, convey stress tolerance, induce systemic resistance, or supply the host 

with nutrients. On the other hand, most endophytes are also able to grow 

saprotrophically, e.g., from surface-sterilized tissues on media containing 

dead organic substrates. Thus, it has become obvious that endophytes have 

multiple life history strategies and that these can be extremely plastic, as 

will become clear to the readers of the subsequent 19 chapters. 

This book is the first to deal with bacterial and fungal root endophytes, 

their diversity, life history strategies, interactions, applications in agriculture 

and forestry, and also with methods for isolation, cultivation, and both 

conventional and molecular methods for identification and detection. The 

first chapter deals with the question: What are endophytes? However, it 

also introduces the reader to the subjects treated in the subsequent chapters. 

We hope that readers will not only find this book informative, but 

will also be provoked to further study these fascinating interactions, and 

in particular to better understand the mechanisms regulating them. It will 

become apparent that we are still far from understanding the factors that 

determine whether a plant-microbial interaction remains asymptomatic, 

leads to disease, or is mutualistic.

VI Preface 

We would like to thank our colleagues for their contributions and their 

work to make this book a successful unity, to Jutta Lindenborn of Springer 

for her friendly help and advice, and to Ajit Varma for the invitation to edit 

abookinthisseries. 

Braunschweig, Barbara Schulz 

Görlitz and Zürich, Christine Boyle 

June 2006 and Thomas Sieber

Contents 

1 WhatareEndophytes? 1 

Barbara Schulz, Christine Boyle 

1.1 Introduction and Definitions ............................................ 1 

1.2 Colonisation .................................................................. 2 

1.3 Assemblages and Adaptation ............................................ 4 

1.4 Life History Strategies ..................................................... 6 

1.5 Balanced Antagonism...................................................... 7 

1.6 Conclusions ................................................................... 9 

References ............................................................................. 10 

Part I Endophytic Bacteria 

2 Spectrum and Population Dynamics 

of Bacterial Root Endophytes 15 

Johannes Hallmann, Gabriele Berg 

2.1 Introduction .................................................................. 15 

2.2 Population Density ......................................................... 15 

2.3 Bacterial Spectrum ......................................................... 16 

2.4 Bacterial Diversity .......................................................... 21 

2.5 Factors Influencing Colonisation....................................... 21 

2.5.1 Methodology....................................................... 21 

2.5.2 Geography .......................................................... 22 

2.5.3 Plant Species ....................................................... 22 

2.5.4 Plant Genotype .................................................... 23 

2.6 Interactions ................................................................... 24 

2.6.1 Plant Pathogens ................................................... 24 

2.6.2 Plant Symbionts................................................... 25 

2.6.3 Plant Defence Mechanisms .................................... 25 

2.6.4 Agricultural Practices ........................................... 25 

2.7 Potential Human Pathogens Among Root Endophytes.......... 26 

2.8 Conclusions ................................................................... 27 

References ............................................................................. 28

VIII Contents 

3 Bacterial Endophytes as Elicitors of Induced Systemic Resistance 33 

Joseph W. Kloepper, Choong-Min Ryu 

3.1 Introduction and Terminology ......................................... 33 

3.2 Scope of Endophytes that Elicit Induced Resistance 

and Pathosystems Affected............................................... 34 

3.3 Internal Colonization of Endophytes 

that Elicit Induced Resistance ........................................... 39 

3.4 Plant Responses to Endophytic Elicitors............................. 41 

3.5 Implementation in Production Agriculture: 

Two Case Studies ............................................................ 44 

3.6 Conclusions ................................................................... 49 


4 Control of Plant Pathogenic Fungi with Bacterial Endophytes 53 

Gabriele Berg, Johannes Hallmann 

4.1 Introduction .................................................................. 53 

4.2 Spectrum of Indigenous Endophytic Bacteria 

with Antagonistic Potential Towards Fungal Plant Pathogens 54 

4.3 Mode of Action of Antagonistic Bacteria ............................ 58 

4.3.1 Antibiosis ........................................................... 58 

4.3.2 Competition........................................................ 59 

4.3.3 Lysis................................................................... 59 

4.3.4 Induction of Plant Defence Mechanisms .................. 60 

4.3.5 Plant Growth ....................................................... 60 

4.4 Control Potential of Endophytic Bacteria............................ 60 

4.5 Enhancing Biocontrol Efficiency ....................................... 61 

4.6 Conclusions ................................................................... 65 


5 Role of Proteins Secreted by Rhizobia in Symbiotic 

Interactions with Leguminous Roots 71 

Maged M. Saad, William J. Broughton, William J. Deakin 

5.1 Introduction .................................................................. 71 

5.2 Bacterial Protein Secretion Systems................................... 73 

5.2.1 Type I Secretion Systems ....................................... 73 

5.2.2 Type II Secretion Systems ...................................... 77 

5.2.3 Type III Secretion Systems..................................... 77 

5.2.4 Type IV Secretion Systems..................................... 82 

5.3 Conclusions ................................................................... 83 


Contents IX 

6 Research on Endophytic Bacteria: Recent Advances 

with Forest Trees 89 

Richa Anand, Leslie Paul, Chris Chanway 

6.1 Introduction .................................................................. 89 

6.2 Bacterial Endophytes of Forest Trees.................................. 91 

6.3 Endophytic Bacteria of Conifers........................................ 92 

6.4 Modes and Sites of Entry.................................................. 95 

6.5 Mechanisms of Plant Growth Promotion............................ 97 

6.6 Future Work................................................................... 102 


Part II Endophytic Fungi 

7 Biodiversity of Fungal Root-Endophyte Communities 

and Populations, in Particular of the Dark Septate Endophyte 

Phialocephala fortinii s. l. 107 

Thomas N. Sieber, Christoph R. Grünig 

7.1 Introduction .................................................................. 107 

7.2 Species Diversity of Root Endophyte Communities.............. 108 

7.2.1 Geography and Climate......................................... 109 

7.2.2 Soil .................................................................... 114 

7.2.3 Multitrophic Interactions ...................................... 115 

7.2.4 Natural and Anthropogenic Disturbances ................ 117 

7.3 Dark Septate Endophytes................................................. 119 

7.3.1 History ............................................................... 119 

7.3.2 Biodiversity......................................................... 119 

7.3.3 Diversity of Phialocephala fortinii........................... 121 

7.4 Conclusions ................................................................... 125 


8 Endophytic Root Colonization by Fusarium Species: 

Histology, Plant Interactions, and Toxicity 133 

CharlesW.Bacon,IdaE.Yates 

8.1 Introduction .................................................................. 133 

8.2 Plant and Fungus Interactions .......................................... 134 

8.2.1 Hemibiotrophic Characteristics.............................. 138 

8.2.2 Histology ............................................................ 139 

8.2.3 Mycotoxins.......................................................... 143 

8.2.4 Mycotoxins and Host Relationships......................... 144 

8.2.5 Physiological Interactions and Defense Metabolites... 145 

8.3 Summary....................................................................... 146 


X Contents 

9 Microbial Endophytes of Orchid Roots 153 

Paul Bayman, J. Tupac Otero 

9.1 Introduction .................................................................. 153 

9.2 Habits and Types of Orchid Roots ..................................... 153 

9.3 Bacteria as Epiphytes and Endophytes of Orchid Roots ........ 154 

9.4 Orchid Endophytes or Orchid Mycorrhizal Fungi? ............... 155 

9.5 Problems with the Taxonomy of Orchid Endophytic Fungi.... 157 

9.6 Host Specificity of Orchid Endophytes ............................... 158 

9.7 Endophytic Fungi in Roots of Terrestrial, 

Photosynthetic Orchids ................................................... 158 

9.8 Endophytic Fungi in Roots of Myco-Heterotrophic Orchids .. 167 

9.9 Endophytic Fungi in Roots of Epiphytic 

and Lithophytic Orchids .................................................. 169 

9.10 Endophytic Fungi in Epiphytic Orchid Roots: 

Importance to Plant Hosts................................................ 171 

9.11 Conclusions ................................................................... 172 


10 Fungal Endophytes in Submerged Roots 179 

Felix Bärlocher 

10.1 Introduction .................................................................. 179 

10.2 Aquatic Hyphomycetes .................................................... 180 

10.3 Fungi in Submerged Roots ............................................... 181 

10.4 Conclusions and Outlook................................................. 186 


11 Nematophagous Fungi as Root Endophytes 191 

Luis V. Lopez-Llorca, Hans-Börje Jansson, José Gaspar Maciá Vicente, 

Jesús Salinas 

11.1 Introduction .................................................................. 191 

11.2 Nematophagous Fungi..................................................... 191 

11.2.1 Nematode Parasites .............................................. 192 

11.2.2 Mycoparasites...................................................... 195 

11.2.3 Root Endophytes.................................................. 195 

11.3 Concluding Remarks....................................................... 202 


12 Molecular Diversity and Ecological Roles of Mycorrhiza-Associated 

Sterile Fungal Endophytes in Mediterranean Ecosystems 207 

Mariangela Girlanda, Silvia Perotto, Anna Maria Luppi 

12.1 Introduction .................................................................. 207

Contents XI 

12.2 Diversity of DSE Associates of Ecto- and Endo-Mycorrhizal 

Plants in Mediterranean Ecosystems in Northern Italy ......... 209 

12.3 Ecological Relationships with Conventional Mycorrhizal 

and Pathogenic Symbionts ............................................... 214 

12.4 Conclusions ................................................................... 219 


13 Oidiodendron maius: Saprobe in Sphagnum Peat, 

Mutualist in Ericaceous Roots? 227 

Adrianne V. Rice, Randolph S. Currah 

13.1 Introduction .................................................................. 227 

13.2 Oidiodendron maius as a Saprobe...................................... 230 

13.3 Ericoid Mycorrhizas........................................................ 234 

13.4 Oidiodendron maius as an Ericoid Mycorrhizal Fungus ........ 237 

13.5 Significance and Relevance............................................... 239 

13.6 Conclusions ................................................................... 241 


14 Mycorrhizal and Endophytic Fungi of Epacrids (Ericaceae) 247 

John W.G. Cairney 

14.1 Introduction .................................................................. 247 

14.2 Endophytes of Epacrid Roots............................................ 248 

14.3 Diversity and Spatial Distribution 

of Endophyte Taxa in Epacrid Root Systems........................ 251 

14.4 Mycorrhizal Status of Endophytes from Epacrids................. 253 

14.5 Saprotrophic Potential of Mycorrhizal Fungi....................... 254 

14.6 Symbiotic Functioning of Mycorrhizal Fungi ...................... 255 

14.7 Conclusions ................................................................... 257 


15 Mutualistic Interactions with Fungal Root Endophytes 261 

Barbara Schulz 

15.1 Introduction .................................................................. 261 

15.2 Colonisation and Histology.............................................. 262 

15.3 Secondary Metabolites..................................................... 264 

15.4 Growth Enhancement...................................................... 265 

15.5 Disease Suppression........................................................ 269 

15.6 Stress Tolerance.............................................................. 271 

15.7 Factors Determining the Status of the Interaction................ 272 

15.8 Conclusions ................................................................... 273 


XII Contents 

16 Understanding the Roles of Multifunctional Mycorrhizal 

and Endophytic Fungi 281 

Mark C. Brundrett 

16.1 How Mycorrhizal Fungi Differ from Endophytes ................. 281 

16.1.1 Definitions .......................................................... 281 

16.1.2 Roles of Endophytes and Mycorrhizal Fungi............. 282 

16.2 Endophytic Activity by Mycorrhizal Fungi.......................... 283 

16.2.1 Glomeromycotan (Vesicular-Arbuscular 

Mycorrhizal) Fungi............................................... 283 

16.2.2 Ectomycorrhizal Fungi.......................................... 286 

16.2.3 Fungi in Orchids .................................................. 287 

16.2.4 Ericoid Mycorrhizal Fungi ..................................... 290 

16.2.5 Endophytic Fungi in Mycorrhizal Roots................... 290 

16.3 Issues with the Identification and Categorisation 

of Fungi in Roots ............................................................ 291 

16.4 Evolution of Root-Fungus Associations.............................. 292 

16.5 Conclusions ................................................................... 293 


Part III Methods 

17 Isolation Procedures for Endophytic Microorganisms 299 

Johannes Hallmann, Gabriele Berg, Barbara Schulz 

17.1 Introduction .................................................................. 299 

17.2 Surface Sterilisation ........................................................ 300 

17.2.1 Pre-treatment ...................................................... 300 

17.2.2 Sterilising Agents ................................................. 301 

17.2.3 Surfactants.......................................................... 305 

17.2.4 Rinsing............................................................... 305 

17.2.5 Sterility Check and Optimisation............................ 305 

17.3 Culture of Tissue and Plant Fluid of Sterilised Roots 

on Nutrient Medium ....................................................... 306 

17.3.1 Segments ............................................................ 306 

17.3.2 Maceration of Root Tissue ..................................... 306 

17.3.3 Centrifugation of Root Tissue ................................ 307 

17.4 Vacuum and Pressure Extraction ...................................... 308 

17.5 Media............................................................................ 309 

17.5.1 Media for Isolating Bacteria................................... 310 

17.5.2 Media for Isolating Fungi ...................................... 310 

17.5.3 Supplements........................................................ 310 

17.5.4 Selective Media .................................................... 311

Contents XIII 

17.6 Cultivation-Independent Methods..................................... 311 

17.7 Quantification of Colonisation.......................................... 313 

17.8 Conclusions ................................................................... 313 


18 Microbial Interactions with Plants: a Hidden World? 321 

Guido V. Bloemberg, Margarita M. Camacho Carvajal 

18.1 Introduction .................................................................. 321 

18.2 Microscopic Techniques for Studying 

Plant-Microbe Interactions .............................................. 322 

18.2.1 Light Microscopy and Enzymatic Reporters ............. 322 

18.2.2 Scanning Electron Microscopy ............................... 324 

18.2.3 Epifluorescence Microscopy 

and the Application of Auto-Fluorescent Proteins...... 325 

18.3 Visualisation of Bacterium-Plant Interactions ..................... 327 

18.4 Most Recent Developments in Visualising 

Plant-Microorganism Interactions..................................... 330 

18.5 Visualisation of Plant-Fungus Interactions ......................... 331 

18.6 Future Perspectives ......................................................... 333 


19 Application of Molecular Fingerprinting Techniques 

to Explore the Diversity of Bacterial Endophytic Communities 337 

LeoS.vanOverbeek,JimvanVuurde,JanD.vanElsas 

19.1 Introduction .................................................................. 337 

19.2 Colonisation by Bacterial Endophytes................................ 338 

19.3 Shifts of Bacterial Endophyte Communities........................ 338 

19.4 Molecular methods to Study Bacterial Endophytes .............. 340 

19.5 Molecular Fingerprinting of Endophyte Communities.......... 341 

19.5.1 Basic Concept of Molecular Fingerprinting .............. 341 

19.5.2 Sample Preparation .............................................. 342 

19.5.3 Nucleic Acid Extraction......................................... 343 

19.5.4 PCR and Molecular Community Fingerprinting........ 344 

19.5.5 Group-Specific Molecular 

Community Fingerprinting ................................... 345 

19.5.6 Molecular Identification of Species and Genes .......... 348 

19.6 Integration of Detection Techniques .................................. 348 

19.6.1 Polyphasic Approach ............................................ 348 

19.7 Conclusions ................................................................... 349 


Subject Index 355

Contributors 

Anand, Richa 

Faculty of Land and Food Sciences, Faculty of Forestry, University of British 

Columbia, Vancouver, British Columbia, Canada V6T 1Z4; Current address: 

Department of Forest Mycology and Pathology, Swedish University of Agricultural 

Sciences (SLU), Box 7026, 750 07 Uppsala, Sweden 

Bacon, Charles W. 

Richard B. Russell Research Center, ARS, United States Department of 

Agriculture, Toxicology and Mycotoxin Research Unit, SAA, P.O. Box 5677, 

Athens, GA 30604, USA 

Bärlocher, Felix 

63BYorkStreet,DepartmentofBiology,MountAllisonUniversity,Sackville, 

New Brunswick, E4L 1G7, Canada 

Bayman, Paul 

Departamento de Biologia, Universidad de Puerto Rico – Rio Piedras, PO 

Box 23360, San Juan, PR 00931, USA 

Berg, Gabriele 

Graz University of Technology, Department of Environmental Biotechnology, 

Petersgasse 12, 8010 Graz, Austria 

Bloemberg, Guido V. 

Leiden University, Institute of Biology, Wassenaarseweg 64, 2333AL Leiden, 

The Netherlands 

Boyle, Christine 

Augustastraße 32, 02826 Görlitz, Germany 

Broughton, William J. 

Université de Genève, Laboratoire de Biologie Moléculaire des Plantes 

Supérieures, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland

XVI Contributors 

Brundrett, Mark C. 

School of Plant Biology, Faculty of Natural and Agricultural Sciences, The 

University of Western Australia, Crawley, WA 6009, Australia 

Cairney, John W.G. 

Centre for Plant and Food Sciences, Parramatta Campus, University of 

Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia 

Carvajal, Margarita M. Camacho 

Leiden University, Institute of Biology, Wassenaarseweg 64, 2333AL Leiden, 


Chanway, Chris 

Faculty of Land and Food Systems, Faculty of Forestry, University of British 

Columbia, Vancouver, British Columbia, Canada V6T 1Z4 

Currah, Randolph S. 

Department of Biological Sciences, University of Alberta, Edmonton, AB, 

T6G 2E9, Canada 

Deakin, William J. 


Supérieures, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland 

Elsas, Jan D. van 

Groningen University, Department of Microbial Ecology, Biological Center, 

Kerklaan 30, 9751 NN, Haren, The Netherlands 

Girlanda, Mariangela 

Dipartimento di Biologia Vegetale and IPP - Torino, Viale PA Mattioli 25, 

10125 Torino, Italy 

Grünig, Christoph R. 

Swiss Federal Institute of Technology, Department of Environmental Sciences, 

Institute of Integrative Biology, Forest Pathology and Dendrology, 

8092 Zürich, Switzerland 

Hallmann, Johannes 

Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Nematologie 

und Wirbeltierkunde, Toppheideweg 88, 48161 Münster, Germany

Contributors XVII 

Jansson, Hans-Börje 

Departamento de Ciencias del Mar y Biología Aplicada, Universidad de 

Alicante, Apdo. 99, 03080 Alicante, Spain 

Kloepper, Joseph W. 

Department of Entomology and Plant Pathology, Auburn University, Auburn, 

AL 36849, USA 

Lopez-Llorca, Luis V. 



Luppi, Anna Maria 

Dipartimento di Biologia Vegetale and IPP, Viale PA Mattioli 25, 10125 

Torino, Italy 

Otero, J. Tupac 

Universidad Nacional de Colombia-Palmira, Departamento de Ciencias 

Agrícolas, AA 237, Palmira, Valle del Cauca, Colombia 

Overbeek, Leo S. van 

Plant Research International B.V, Droevendaalsesteeg 1, 6708 PB, Wageningen, 


Paul, Leslie 

Faculty of Land and Food Sciences, Systems, University of British Columbia, 

Vancouver, British Columbia, Canada V6T 1Z4 

Perotto, Silvia 

Dipartimento di Biologia Vegetale and IPP - Torino, Viale PA Mattioli 25, 


Rice, Adrianne V. 

Northern Forestry Centre, Canadian Forest Service, Natural Resources 

Canada, 5320-122 St., Edmonton, AB, T6H 3S5 Canada 

Ryu, Choong-Min 

Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam 

Noble Parkway, Ardmore, OK 73401, USA

XVIII Contributors 

Saad, Maged M. 



Salinas, Jesús 



Schulz, Barbara 

Institute of Microbiology, Technical University of Braunschweig, Spielmannstraße 

7, 38106 Braunschweig, Germany 

Sieber, Thomas N. 

Swiss Federal Institute of Technology, Department of Environmental Sciences, 

Institute of Integrative Biology, Forest Pathology and Dendrology, 

8092 Zürich, Switzerland 

Vicente, José Gaspar Maciá 



Vuurde, Jim van 

Plant Research International B.V, Droevendaalsesteeg 1, 6708 PB, Wageningen, 


Yates, Ida E. 

Richard B. Russell Research Center, ARS, United States Department of 

Agriculture, Toxicology and Mycotoxin Research Unit, Athens, GA 30604, 

USA

Abbreviations 

ACC 1-aminocyclopropane-1-carboxylate 

AHL acyl homoserine lactones 

AM arbuscular mycorrhiza 

AMF arbuscular mycorrhizal fungi 

ARA acetylene reduction activity 

AUDPC area under the disease progress curve 

BAC bacterial artificial chromosome 

BCA biocontrol agents 

BRD root border cells 

BrdU bromide oxyuridine 

Cfu colony forming units 

CLSM confocal laser scanning microscopy 

CMA corn meal agar 

CMV Cucumber mosaic virus 

CPS capsular polysaccharides 

CTAB cetyl trimethyl ammonium bromide 

DGGE denaturing gradient gel electrophoresis 

DIC differential interference microscopy 

DON deoxynivalenol 

DSE dark septate endophytes 

DSF dark septate fungi 

DSM dark sterile mycelia 

ECFP enhanced cyan fluorescent protein 

ECM ectomycorrhizae 

ECM extracellular material 

EGFP Enhanced GFP 

ELISA enzyme-linked immunosorbent assay 

EPS extra-cellular polysaccharides 

EYFP Enhanced Yellow Fluorescent Protein 

FISH fluorescence in situ hybridization 

GFP Green fluorescent protein 

GSP general secretory pathway 

GUS β-glucuronidase

XX Abbreviations 

IAA indole acetic acid 

ICR induced systemic resistance 

ISSR inter-simple sequence repeat 

ITS internal transcribed spacer 

ITS-RFLP internal transcribed spacer-restriction fragment length 

polymorphism 

LPS lipo-polysaccharides 

MLH multilocus haplotypes 

MRA Mycelium radicis atrovirens 

NDFA nitrogen derived from the atmosphere 

PAH polyaromatic hydrocarbon 

PAR photosynthetically active radiation 

PBM peribacteroid membrane 

PCR polymerase chain reaction 

PCR-RFLP polymerase chain reaction -restriction fragment length 

polymorphism 

PDA potato dextrose agar 

PGPR plant growth promoting rhizobacteria 

PR pathogenesis-related 

PVP polyvinylpyrrolidone 

RAPD random amplified polymorphic DNA 

RDNA 16S rRNA gene 

RFLP restriction fragment length polymorphism 

RFP red fluorescent protein 

RGR relative growth rate 

SAR systemic acquired resistance 

SEM Scanning electron microscopy 

SPB sterile phosphate buffer 

SPS Surface polysaccharides 

SSCP single strand conformational polymorphism 

TGGE temperature gradient gel electrophoresis 

TNV tobacco necrosis virus 

ToMoV Tomato mottle virus 

TRFLP terminal restriction fragment length polymorphism 

TSA tryptic soya agar 

VAM vesicular arbuscular mycorrhiza 

VOC volatile organic compound 

VWT variable white taxon 

WA Western Australia

1 

What are Endophytes? 

Barbara Schulz, Christine Boyle 

1.1 

Introduction and Definitions 

Taken literally, the word endophyte means “in the plant” (endon Gr. = 

within, phyton = plant). The usage of this term is as broad as its literal 

definition and spectrum of potential hosts and inhabitants, e.g. bacteria 

(Kobayashi and Palumbo 2000), fungi (Stone et al. 2000), plants (Marler 

et al. 1999) and insects in plants (Feller 1995), but also for algae within 

algae (Peters 1991). Any organ of the host can be colonised. Equally variable 

is the usage of the term “endophyte” for variable life history strategies 

of the symbiosis, ranging from facultatively saprobic to parasitic to 

exploitive to mutualistic. The term endophyte is, for example, used for 

pathogenic endophytic algae (Bouarab et al. 1999), parasitic endophytic 

plants (Marler et al. 1999), mutualistic endophytic bacteria (Chanway 1996; 

Adhikari et al. 2001; Bai et al. 2002) and fungi (Carroll 1988; Jumpponen 

2001; Sieber 2002; Schulz and Boyle 2005), and pathogenic bacteria and 

fungi in latent developmental phases (Sinclair and Cerkauskas 1996), but 

also for microorganisms in commensalistic symbioses (Sturz and Nowak 

2000). 

Some authors also designate the interactions of mycorrhizal fungi with 

the roots of their hosts as being endophytic (reviewed by Sieber 2002). 

However, we concur with Brundrett (2004; see Chap. 16 by Brundrett), 

who distinguishes mycorrhizal from endophytic interactions; the former 

having synchronised plant-fungus development and nutrient transfer at 

specialised interfaces. Nevertheless, as we will see in this book, distinctions 

between mycorrhizal and non-mycorrhizal fungi are not always clear-cut 

[see Chaps. 9 (Bayman and Otero), 12 (Girlanda et al.), 13 (Rice and Currah), 

14 (Cairney), and 15 (Schulz)]. Not only can mycorrhizal fungi become 

pathogenic, but, for example, dark septate endophytes (DSE) can assume 

mycorrhizal functions [Jumpponen and Trappe 1998; see Chaps. 7 (Sieber 

Barbara Schulz: Technical University of Braunschweig, Institute of Microbiology, Spielmannstraße 

7, 38106 Braunschweig, Germany, E-mail: b.schulz@tu-bs.de 

Christine Boyle: Augustastraße 32, 02826 Görlitz, Germany 

Soil Biology, Volume 9 

Microbial Root Endophytes 

B.Schulz,C.Boyle,T.N.Sieber(Eds.) 

© Springer-Verlag Berlin Heidelberg 2006

2 B. Schulz, C. Boyle 

and Grünig), and 15 (Schulz)]. In addition, there are also cases in which 

fungal root endophytes seem to be saprobes, e.g. Oidiodendron maius (see 

Chap.13byRiceandCurrah)andPhialocephala fortinii (Jumpponen and 

Trappe 1998; Jumpponen et al. 1998; see Chap. 15 by Schulz). 

Although there are diverse uses for the word endophyte, “endophytes” 

are most commonly defined as those organisms whose “...infections are 

inconspicuous, the infected host tissues are at least transiently symptomless, 

and the microbial colonisation can be demonstrated to be internal...” 

(Stone et al. 2000). Although these authors used this definition to describe 

fungal endophytes, it is equally applicable to bacterial endophytes. 

It is important to remember that the definition describes a momentary 

status. Thus it includes an assemblage of microorganisms with different 

life history strategies: those that grow saprophytically on dead or senescing 

tissues following an endophytic growth phase (Stone 1987; see Chap. 8 by 

Bacon and Yates), avirulent microorganisms as well as latent pathogens and 

virulent pathogens in the early stages of infection (Sinclair and Cerkauskas 

1996; Kobayashi and Palumbo 2000). Unfortunately, taken literally, it can 

include all pathogens at some stage of their development. Since the plant 

host responds to at least some infections with mechanical defence reactions 

(Narisawa et al. 2004; see Chap. 15 by Schulz), there is merit to Petrini’s 

additional characterisation of endophytic interactions as not “causing apparent 

harm” (Petrini 1991), which presumably refers to an absence of 

macroscopically visible symptoms. Aware of the determinative discrepancies, 

we will nevertheless use the term “endophyte” to describe those 

bacteria and fungi that can be detected at a particular moment within the 

tissues of apparently healthy plant hosts (Schulz and Boyle 2005). 

1.2 

Colonisation 

In spite of the fact that bacteria are prokaryotes and fungi are eukaryotes, 

they share many attributes of their associations with plant hosts, e.g. both 

colonise root tissues inter- and intra-cellularly, and often systemically (Table 

1.1). They do, however, differ somewhat in their modes of colonisation. 

Bacteria primarily colonise intercellularly (Hinton and Bacon 1995; Hallmann 

et al. 1997), though they have also been found intracellularly, e.g. 

Azoarcus spp. (Hurek et al. 1994). They are frequently found in the vascular 

tissues of host plants (Kobayashi and Palumbo 2000), which is advantageous 

for distribution, whereas asymptomatic colonisation by fungi may 

be inter- and intra-cellular throughout the root. Although DSE sometimes 

colonise the vascular cylinder in asymptomatic interactions (Barrow 2003), 

such colonisation is frequently associated with pathogenicity (Bacon and 

Hinton 1996; Schulz and Boyle 2005).

1WhatareEndophytes? 3 

Table 1.1. Characteristics of the interactions of bacterial vs. fungal endophytes with plant roots (see also all other book chapters) 

Criteria Bacteria Fungi 

Host spectrum Broad, depends on habitat, host, season Broad, depends on habitat, host, season 

Mode and site of infection Passive through wounds and other tissue openings Active: through stomata, cell wall or wounds 

or active with enzymes or vectors, e.g. insects 

Nutritional source 

Host exudates, dead cortex cells, plant debris Storage material in the spores, 

during first stage infection 

dead cortex cells, plant debris, host exudates 

Nutritional source 

Components of the symplast and apoplast Components of the symplast and apoplast 

during colonisation, i.e. 

during a “steady state” status 

Growth in root Inter- and/or intra-cellular, slow, low colonisation densities Inter- and/or intracellular, often extensive 

Growth from roots into the shoot Yes Sometimes 

Systemic growth in roots Possible Possible 

Tissue colonised Primarily intercellular, also vascular tissue Usually not within vascular tissue 

Specialised structures 

Nodules, glands Sometimes 

for nutrient access 

Physiological status Only little data available Balanced antagonisms, active interaction 

Outcome of the interaction Commensalism, mutualism or latent pathogenicity Commensalism, mutualism or latent pathogenicity 

Benefits 

A reliable supply of nutrients and protection from A reliable supply of nutrients and protection from 

for the microbial symbiont environmental stresses, passive transfer and spread environmental stresses, advantages for reproduction 

between hosts via vectors, e.g. insects 

and colonisation at host senescence 

Potential benefits 

Induced resistance, improved growth (N-fixation, Induced resistance, improved growth 

for the plant symbiont 

phytohormones), synthesis of metabolites antagonistic to (phytohormones, improved access to minerals and 

plant pathogens and parasites 

nutrients), synthesis of metabolites antagonistic to 

predators and antagonists 

Reproduction Usually passive transfer and spread between hosts via Active and passive following host senescence, 

vectors, e.g. insects, but also active, e.g. Pseudomonads sometimes with vectors


The assemblages of fungi that colonise plant roots are diverse (Vandenkoornhuyse 

et al. 2002). In contrast to endophytic growth in the aboveground 

plant organs, endophytic growth of fungi within the roots has 

frequently been found to be extensive (Stone et al. 2000; Schulz and Boyle 

2005; see Chap. 11 by Lopez-Llorca et al.). Root colonisation can be both 

inter- and intra-cellular, the hyphae often forming intracellular coils, e.g. 

DSE (Jumpponen and Trappe 1998; Stone et al. 2000; Sieber 2002), the basidiomycete 

Piriformospora indica (Varma et al. 2000), or Oidiodendron 

maius (see Chap. 13 by Rice and Currah) and Heteroconium chaetospira 

(Usuki and Narisawa 2005), which can even form characteristic ericoid 

mycorrhizal infection units (see Chap. 14 by Cairney). DSE may also form 

ectendomycorrhiza (Lubuglio and Wilcox 1988) and ectomycorrhizal-like 

structures (Wilcox and Wang 1987; Fernando and Currah 1996; Kaldorf et 

al. 2004; see Chap. 15 by Schulz). 

Many orchid roots are systemically and mycoheterotrophically colonised 

by fungi of the genus Rhizoctonia (Ma et al. 2003; see Chap. 16 by Brundrett) 

and Leptodontidium (Bidartondo et al. 2004). In some cases, e.g. Fusarium 

verticillioides (= F. moniliforme), colonisation by an avirulent strain was 

found to be systemic and intercellular, whereas pathogenic strains also 

colonised intracellularly (Bacon and Hinton 1996). Latent pathogens, e.g. 

Cryptosporiopsis sp. (Kehr 1992; Verkley 1999) may occasionally penetrate 

the vascular bundles (Schulz and Boyle 2005). 

Bacteria usually invade the roots passively, e.g. at open sites on roots such 

as lateral root emergence or wounds (Kobayashi and Palumbo 2000), even 

achieving systemic colonisation from a single site of entry (Hallmann et al. 

1997). Although colonisation densities of nonpathogenic endophytic bacteria 

are rarely as high as those of pathogenic bacteria, they are highest in the 

root tissue; perhaps because this is the primary site of infection (Kobayashi 

and Palumbo 2000; Hallmann et al. 1997; see Chap. 2 by Hallmann and 

Berg). 

1.3 

Assemblages and Adaptation 

Both fungal and bacterial endophytes have been isolated from the roots of 

almost all hosts studied to date [Petrini 1991; Stone et al. 2000; Kobayashi 

and Palumbo 2000; Sieber 2002; see Chaps. 2 (Hallmann and Berg), 

3 (Kloepper and Ryu), and 7 (Sieber and Grünig)]. The assemblages of 

endophytes that colonise a particular host vary both with habitat and host, 

some even being adapted to very specialised habitats, e.g. the aquatic fungi 

that colonise submerged roots (see Chap. 10 by Bärlocher). Recent molecular 

methods enable better analyses of the geographical distribution of given


groups of microorganisms, for example that of the DSE [Jumpponen 1999, 

see Chaps. 7 (Sieber and Grünig), 12 (Girlanda et al.), and 15 (Cairney)]. 

Both diversity and colonisation density frequently increase during the 

course of the vegetation period (Smalla et al. 2001), since horizontal transmission 

predominates (Carroll 1988, 1995; Petrini 1991; Guske et al. 1996; 

Hallmann et al. 1997; Arnold and Herre 2003, see Chap. 2 by Hallmann and 

Berg). Particularly asexual sporulation increases in autumn at the end of 

the vegetation period. 

Communities of endophytes inhabiting a particular host may be ubiquitous, 

or have what is frequently referred to as host specificity (e.g. Carroll 

1988; Petrini 1996; Stone et al. 2000; Berg et al. 2002; Cohen 2004). We concur 

with Carroll (1999) and Zhou and Hyde (2001) that the term “specificity” 

should be reserved for organisms that will only grow in one host (Schulz 

and Boyle 2005). If this is not the case, this phenomenon could be termed 

host preference (Carroll 1999) or host-exclusivity (Zhou and Hyde 2001). 

Whether the interaction represents specificity, preference or exclusivity, 

an adaptation of host and endophyte to one another has occurred. The 

adaptation may not only be to a particular host, but to endophytic growth 

in one plant organ, e.g. in the roots in contrast to the shoots [Petrini 1991; 

Hallmann et al. 1997; Sieber 2002; Schulz and Boyle 2005; see Chaps. 2 (Hallmann 

and Berg), and 7 (Sieber and Grünig)]. 

It is often extremely difficult to know whether or not a particular fungus 

or bacterium that has been detected in healthy plant tissue has actually 

been growing within the host tissue or has been incidentally isolated, i.e. 

is normally found on other substrates. As reviewed by Schulz and Boyle 

(2005) and in Chap. 17 by Hallmann et al., there are four methods presently 

in use for detecting and identifying fungi and bacteria in plant tissue: 

(1) histological observation (see Chap. 6 by Anand et al.), most recently 

in combination with molecular methods (see Chap. 18 by Bloemberg and 

Carvajal), (2) surface sterilisation of the host tissue and isolation of the 

emerging fungi on appropriate growth media, (3) detection by specific 

chemistry, e.g. immunological methods (see Chap. 18 by Bloemberg and 

Carvajal), or (4) by direct amplification of fungal DNA from colonised 

plant tissues [Vandenkoornhuyse et al. 2002; see Chaps. 17 (Hallmann et 

al.), and 19 (van Overbeek et al.)], having first ascertained that there are 

no fungal residues on the plant surface (Arnold et al. 2006). Methods for 

quantification are reviewed by Sieber (2002), Schulz and Boyle (2005) and 

in Chap. 17 (Hallmann et al.).


1.4 

Life History Strategies 

Organismsdetectedatanyonemomentinasymptomaticplanttissueand 

arbitrarily named “endophytes” include microorganisms with different 

life history strategies. Endophytes represent, both as individuals and collectively, 

a continuum of mostly variable associations: mutualism, commensalism, 

latent pathogenicity, and exploitation. The phenotypes of the 

interactions are often plastic, depending on the genetic dispositions of the 

two partners, their developmental stage and nutritional status, but also on 

environmental factors (see Chap. 12 by Girlanda et al.). The role of genetic 

disposition was demonstrated by Freeman and Rodriguez (1993): a single 

mutation resulted in loss of a virulence factor, transforming a pathogenic 

fungus, Colletotrichum magna, intoanendophyte.Similarly,avirulence 

genes and the machinery of pathogenicity may be lacking or suppressed in 

bacterial endophytes (Kobayashi and Palumbo 2000). 

Just as fungi have been found to develop ectomycorrhiza in one host 

and what appear to be ericoid mycorrhiza in another host (Villarreal- 

Ruiz et al. 2004), a mycorrhizal fungus can grow endophytically in the 

roots of a non-host (see Chap. 12 by Girlanda et al.). The importance 

of a particular combination of host and microorganism as well as their 

reciprocal influences also becomes apparent when a fungal or bacterial 

pathogen is inoculated into a non-host and is no longer virulent, colonising 

as an asymptomatic endophyte (Carroll 1999; Kobayashi and Palumbo 2000; 

Schulz and Boyle 2005) The influence of the host plant in determining the 

mycorrhizal, endophytic or even pathogenic character of a DSE association 

is likely to be a prime factor. In plant communities, the multiple mutualistic 

potential of these fungi, establishing hyphal links or inoculum reservoirs, 

may favour inter-plant interactions (see Chap. 12 by Girlanda et al.). 

Interactions are frequently complex, involving more than two partners. 

Endophytic bacteria and fungi may interact not only with the plant host, 

but also with other organisms, including mycorrhizal fungi (see Chap. 9 

by Bayman and Otero) and metazoa. For example, nematophagous fungi, 

which are ubiquitous organisms in soils, not only can switch from a saprophytic 

to a parasitic stage to kill and digest living nematodes, but can also 

grow endophytically in plant roots (see Chap. 11 by Lopez-Llorca et al.). 

Mutualistic interactions involving fungi and bacteria that endophytically 

colonise plant roots benefit the microbial partner with a reliable supply of 

nutrients as well as protection from environmental stresses. As reported 

in this book, benefits for the host plant may include improved growth [see 

Chaps. 6 (Anand et al.), 13 (Rice and Currah), 15 (Schulz), and 19 (van 

Overbeek et al.)], induced resistance [see Chaps. 3 (Kloepper and Ryu), 

4 (Berg and Hallmann), 6 (Anand et al.), and 15 (Schulz)], biocontrol of


plant parasitic nematodes (see Chap. 11 by Lopez-Llorca et al.) and of fungi 

in agriculture [see Chaps. 3 (Kloepper and Ryu), 4 (Berg and Hallmann), 

15 (Schulz)] and forestry (see Chap. 6 by Anand et al.), as well as microbial 

synthesis of metabolites antagonistic to predators [Schulz et al. 2002; Schulz 

and Boyle 2005; see Chaps. 6 (Anand et al.), 8 (Bacon and Yates), 15 (Schulz), 

and 19 (van Overbeek et al.)] When synthesized in agricultural crops in 

situ, mycotoxins synthesised by endophytes, e.g. Fusarium verticillioides in 

maize, are potentially problematic for human consumption of these crops 

(see Chap. 8 by Bacon and Yates). 

Factors responsible for improving plant growth are the microbial synthesis 

of phytohormones [Tudzynski 1997; Tudzynski and Sharon 2002; 

Kobayashi and Palumbo 2000; see Chaps. 6 (Anand et al.) and 15 (Schulz)], 

access to minerals and/or other nutrients from the soil (Caldwell et al. 2000; 

Barrow 2003; see Chap. 13 by Rice and Currah), bacterial fixation of atmospheric 

nitrogen, which has been demonstrated not only for the noduleforming 

members of the Rhizobiaceae, but also for non-nodule-forming 

bacteria, e.g. Acetobactor and Azoarcus (Reinhold-Hurek and Hurek 1998; 

see Chap. 5 by Saad et al.). In the associations of nitrogen-fixing rhizobia 

with legumes, some of the same signalling molecules are involved as in 

the interactions of mycorrhizal fungi with their hosts, e.g. flavonoids and 

nod-factors (Lapopin and Franken 2000; Martin et al. 2001; Mirabella et 

al. 2002; Imaizumi-Anraku et al. 2005; see Chap. 5 by Saad et al.). And as 

has recently been shown, plastid membrane proteins involved in the first 

signalling interactions are crucial for the entry of both symbionts into the 

host roots (Imaizumi-Anraku et al. 2005). 

1.5 

Balanced Antagonism 

According to Heath (1997), only a few fungi are actually capable of causing 

disease in any one plant, since they must first cross several barriers and 

overcome other plant defences. This must also be true for bacteria. Thus, 

one question has motivated many investigations: how does the endophyte 

manage to exist, and often to grow, within its host without causing visible 

disease symptoms? We have proposed a working hypothesis based on observations 

from the interactions studied thus far (Schulz et al. 1999; Schulz 

and Boyle 2005). Asymptomatic colonisation is a balance of antagonisms 

between host and endophyte (Fig. 1.1). Endophytes and pathogens both 

possess many of the same virulence factors: the endophytes studied thus far 

produced the exoenzymes necessary to infect and colonise the host (Sieber 

et al. 1991; Petrini et al. 1992; Ahlich-Schlegel 1997; Boyle et al. 2001; Lumyong 

et al. 2002), even though only some of these endophytes are presumably


Fig.1.1. Hypothesis: a balance of antagonisms between endophytic virulence and plant 

defence response results in asymptomatic colonisation (reproduced with permission from 

Schulz and Boyle 2005) 

latent pathogens. The majority can produce phytotoxic metabolites (Schulz 

et al. 2002; Schulz and Boyle 2005). The host can respond with the same defence 

reactions as to a pathogen, i.e. with preformed and induced defence 

metabolites [Yates et al. 1997; Schulz et al. 1999; Mucciarelli et al. 2003; see 

Chaps. 3 (Kloepper and Ryu), and 11 (Lopez-Llorca et al.)], and general 

defence responses (Narisawa et al. 2004; Schulz and Boyle 2005). As long as 

fungal virulence and plant defence are balanced, the interaction remains 

asymptomatic. In all of these interactions we are referring to a momentary 

status, an often fragile balance of antagonisms. 

If the host-pathogen interaction becomes imbalanced, either disease results 

or the fungus is killed. In some cases, the virulence of weak pathogens 

such as Pezicula spp. (Kehr 1992) is sufficient for disease development only 

when the host is stressed or senescent. Whether the interaction is balanced 

or imbalanced depends on the general status of the partners, the virulence 

of the fungus, and the defences of the host – both virulence and defence 

being variable and influenced by environmental factors, nutritional status 

and developmental stages of the partners. Although this hypothesis has 

been developed to explain the interactions of fungal endophytes with their


hosts, further studies may well provide evidence that it is also applicable to 

endophytic bacteria. 

Balanced antagonistic interactions are plastic in expression, depending 

on the momentary status of host and endophyte, but also on biotic and abiotic 

environmental factors and on the tolerance of each of the partners to 

these factors. In particular, many endophytes seem to be masters of phenotypic 

plasticity: infecting as a pathogen, colonising cryptically, and finally 

sporulating as a pathogen or saprophyte. This necessitates a balance with 

the potential for variability, which means that these endophytic interactions 

are creative, having the potential for evolutionary development – the 

symbioses can evolve both in the direction of more highly specialised mutualisms 

and in the direction of more highly specialised parasitisms and 

exploitation. Indeed, there is evidence that mycorrhizal fungi may have 

evolved from the endophytic activity of saprophytic fungi (see Chap. 16 

by Brundrett), but also that plastids that have evolved from endosymbiotic 

bacteria facilitate further symbioses with other bacterial and fungal 

symbionts (Imaizumi-Anraku et al. 2005). 

1.6 

Conclusions 

The usage of the term “endophyte” is as broad as its literal definition 

and spectrum of potential hosts and inhabitants. The most common usage 

of the term “endophyte” for organisms whose infections are internal and 

inconspicuous, and in which the infected host tissues are at least transiently 

symptomless, is equally applicable to bacterial prokaryotes and fungal 

eukaryotes. 

Endophytes include an assemblage of microorganisms with different life 

history strategies: those that, following an endophytic growth phase, grow 

saprophytically on dead or senescing tissue, avirulent microorganisms, 

incidentals, but also latent pathogens and virulent pathogens at early stages 

of infection. These parasitic interactions may vary from mutualistic to 

commensalistic to latently pathogenic and exploitive. Phenotypes of the 

interactions are often plastic, depending on the genetic dispositions of the 

two partners, their developmental stage and nutritional status, but also on 

environmental factors. 

We have proposed a working hypothesis based on observations from the 

interactions studied thus far to explain asymptomatic microbial colonisation 

as a balance of antagonisms between host and endophyte (Fig. 1.1; 

Schulz and Boyle 2005). This often fragile balance of antagonism is a momentary 

status and depends on the general status of the partners, the 

virulence of the fungus and defences of the host, environmental fac-


tors, nutritional status, as well as the developmental stages of the partners. 

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2 

Spectrum and Population Dynamics 

of Bacterial Root Endophytes 

Johannes Hallmann, Gabriele Berg 

2.1 

Introduction 

Since the first reports regarding the existence of bacteria residing in plant 

roots (Trevet and Hollis 1948; Philipson and Blair 1957), numerous publicationshavedescribedthespectrumandpopulationdynamicsofindigenous 

endophytic root endophytes for various plant species (Bell et al. 1995; 

Gardner et al. 1982; Hallmann et al. 1997a, 1999; Hallmann 2003; Mahaffee 

and Kloepper 1997a, 1997b; McInroy and Kloepper 1995; Misaghi and 

Donndelinger 1990; Sturz et al. 1997). But what do we really know about 

the spectrum and population dynamics of those bacteria residing in the 

endorhiza? What are the potential risks associated with these bacteria? 

Answers to these questions will not only improve our understanding of 

plant/endophytic bacterial interactions, but are a prerequisite for any future 

commercialisation. This chapter reviews our current knowledge of 

(1) population density, bacterial spectrum and bacterial diversity of endophytic 

root bacteria, (2) factors influencing the population dynamics 

of indigenous and introduced bacterial endophytes, (3) bacterial interactions 

with biotic and abiotic factors, and (4) potential risks associated with 

endophytic bacteria. 

2.2 

Population Density 

Population densities of indigenous endophytic bacteria in roots are found 

to be about 10 5 cfu g −1 fresh root weight (Hallmann et al. 1997a). This is 

higher than in any other plant organ, as the population density usually 

decreases acropetally, with average densities of 10 4 cfu g −1 fresh weight in 

Johannes Hallmann: Federal Biological Research Centre for Agriculture and Forestry, Institute 

for Nematology and Vertebrate Research, Toppheideweg 88, 48161 Münster, Germany, 

E-mail: j.hallman@bba.de 

Gabriele Berg: Graz University of Biotechnology, Department of Environmental Technology, 






16 J. Hallmann, G. Berg 

the stem and 10 3 cfu g −1 fresh weight in leaves. Generative organs such 

asflowers,fruitsandseedsarecolonisedbyevenlowernumbersand,in 

many cases, are below the detection limit. However, depending on plant 

species, methodology and other factors, reported population densities can 

vary significantly. Common densities reported for indigenous endophytic 

bacteria in roots range from 10 4 to 10 6 cfu g −1 for cotton and sweetcorn 

(McInroy and Kloepper 1994), 10 3 to 10 6 cfu g −1 for sugar beet (Jacobs et al. 

1985), 4.0×10 2 to 1.3×10 4 cfu g −1 for cotton (Hallmann et al. 1997a, Misaghi 

and Donndelinger 1990), 10 5 cfu g −1 for potato (Krechel et al. 2002) and 

10 5 cfu g −1 for pine seedlings (Shishido et al. 1995). Some authors have even 

reported population densities up to 10 10 cfu g −1 without negative effects on 

plant growth (Dimock et al. 1988; McInroy and Kloepper 1994). However, 

these high densities are considered exceptional; population densities above 

10 7 cfu g −1 are known frombacterial plant pathogens to cause pathogenicity 

(Tsiantos and Stevens 1986; Grimault and Prior 1994). Within the first 

3 weeks after seeding the population density generally increases to an 

optimum carrying capacity of about 10 5 cfu g −1 and then remains at this 

level for the rest of the growth period (Mahaffee and Kloepper 1997a, 1997b; 

McInroy and Kloepper 1995; Krechel et al. 2002). Even artificial inoculation 

of a highly concentrated bacterial suspension into the root tissue does not 

increase population density in the long run (Frommel et al. 1991). However, 

the above figures account only for culturable bacteria, which are only part 

of the total endophytic bacterial community. Their relative population size 

is still unknown as reliable data are lacking; but from other environments 

it is known that culturable bacteria represent between 0.001% and 15% of 

the total bacterial population. 

2.3 

Bacterial Spectrum 

Studying the bacterial spectrum requires bacterial identification. Before 

1990 this was laborious, using a combination of morphological and physiological 

characters often supported by ready-made tests such as API identification 

strips (Arrow Scientific, Lane Cove, Australia), or Biolog (Biolog, 

Hayward, CA) tests. With the fatty acid method described by Sasser (1990), 

bacterial identification and thus analysis of bacterial spectra became considerably 

more efficient, allowing time-course studies in which hundreds 

of bacteria could be identified (McInroy and Kloepper 1995; Mahaffee and 

Kloepper 1997a, 1997b). Nowadays, sequencing of 16S rDNA genes and 

alignment with public databases allows rapid and accurate species identification 

(Krechel et al. 2002; Reiter et al. 2002; Sessitsch et al. 2004). While 

this approach still relies on bacterial isolation (see Chap. 17 by Hallmann et

2 Spectrum and Population Dynamics of Bacterial Root Endophytes 17 

al.), newly developed molecular methods use universal or specific primers 

to characterise either the endophytic community, certain bacterial groups 

or particular species, therefore enabling complete analysis of culturable as 

well as non-culturable bacteria (see Chap. 19 by van Overbeek et al.). 

As mentioned for bacterial density, the spectrum of indigenous endophytic 

bacteria is affected by similar biotic and abiotic factors. However, it 

also seems to be influenced by niche specialisation, differences in colonisation 

pathway (seed-borne, rhizosphere, above-ground plant organs), or 

some form of mutual exclusion operating between different bacterial populations 

(Hallmann 2001; Sturz et al. 1997). Table 2.1 gives an overview 

of endophytic bacteria commonly isolated from the roots of some cultivated 

plants. Although far from complete, it shows the broad spectrum of 

bacterial endophytes that occur in plant roots. Table 2.2 provides a more 

comprehensive list of bacterial species isolated from roots, comprising 219 

species representing 71 genera, with Bacillus and Pseudomonas being the 

most common genera. 

Table 2.1. Spectrum of endophytic bacteria most commonly isolated from roots of various 

cultivated plant species 

Plant Bacterial taxa Reference 

Alfalfa Pseudomonas, Erwinia-like bacteria Gagné et al. 1987 

Carrot Agrobacterium, Pseudomonas, Staphylococcus Surette et al. 2003 

Clover Agrobacterium, Bacillus, Methylobacterium, 

Pseudomonas, Rhizobium 

Sturz et al. 1997 

Cotton Bacillus, Burkholderia, Clavibacter, Erwinia, Chen et al. 1995; 

Phyllobacterium, Pseudomonas 

Hallmann et al. 1999; 

Misaghi and 

Donndelinger 1990 

Cucumber Agrobacterium, Bacillus, Burkholderia, 

Mahaffee and Kloepper 

Chryseobacterium, Clavibacter, Curtobacterium, 1997a, 1997b; McInroy 

Enterobacter, Micrococcus, Paenibacillus, 

Phyllobacterium, Pseudomonas, Serratia, 

Stenotrophomonas 

and Kloepper 1995 

Grapevine Enterobacter, Pseudomonas, Rahnella, Rhodococcus, 

Staphylococcus 

Bell et al. 1995 

Maize Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Lalande et al. 1989; 

Corynebacterium, Curtobacterium, Enterobacter, McInroy and Kloepper 

Micrococcus, Phyllobacterium, Pseudomonas, 

Serratia 

1995 

Potato Agrobacterium, Arthrobacter, Bacillus, 

Krechel et al. 2002; 

Chryseobacterium, Enterobacter, Micrococcus, 

Pantoea, Pseudomonas, Stenotrophomonas, 

Streptomyces 

Sturz 1995


Table 2.1. (continued) 

Canola Acidovorax, Agrobacterium, Aureobacterium, 

Bacillus, Cytophaga, Chryseobacterium, 

Flavobacterium, Micrococcus, Rathayibacter, 

Pseudomonas 

Red clover Agrobacterium, Bacillus, Methylobacterium, Pantoea, 

Pseudomonas, Rhizobium, Xanthomonas 

Germida et al. 1998; 

Graner et al. 2003; 

Misko and Germida 

2002 

Sturz et al. 1997 

Rice Serratia, Azoarcus Gyaneshwar et al. 2001; 

Hurek et al. 1994 

Rough 

lemon 

Bacillus, Corynebacterium, Enterobacter, 

Pseudomonas, Serratia 

Gardner et al. 1982 

Soybean Bacillus Bai et al. 2002 

Sugar beet Bacillus, Corynebacterium, Erwinia, Lactobacillus, 

Peudomonas, Xanthomonas 

Jacobs et al. 1985 

Sugar cane Acetobacter Cavalcante and 

Döbereiner 1988 

Tomato Bacillus, Burkholderia, Chryseobacterium, Kluyvera, 

Micrococcus, Pseudomonas, Serratia 

Munif 2001 

Wheat Bacillus, Flavobacterium, Microbispora, Micrococcus, 

Micromonospora, Nacardiodes, Rathayibacter, 

Streptomyces 

Diverse 

species 

Coombs and Franco 

2003; 

Germida et al. 1998 

Streptomyces Sardi et al. 1992 

Table 2.2. Endophytic bacteria isolated from roots of various plants 

Acetobacter diazotrophicus 13 , A. pasteurianus 9 

Acidovorax delafieldii 7,9,10 , A. facilis 10 

Acinetobacter baumannii 10 , A. calcoaceticus 10 , A. wolffi 5 , A. radioresistens 10 

Aeromonas salmonicida 9 

Agrobacterium radiobacter 7,8,9,10,12 , A. rhizogenes 12 , A. rubi 7,9 , A. tumefaciens 12,15 

Alcaligenes piechaudii 9,12 , A. xylosoxydans 9,12 

Arthrobacter atrocyaneus 10,11 , A. aurescens 11 , A. crystallopodietes 12 , A. ilicis 15 , 

A. mysorens 10,12 , A. nicotianae 12 , A. pascens 10,11 

Aureobacterium barkeri12 , A. esteroaromaticum10,11 , A. liquefaciens11 , A. saperdae12 , 

A. testaceum12 Azospirillum amazonense13 , A. brasiliense9,13 , A. lipoferum13 Bacillus alvei12 , B. amyloliquefaciens12 , B. azotoformans12,14 , B. brevis6,12,14,15 , B. cereus7,12 , 

B. circulans10,14 , B. citinusporus11 , B. coagulans12,15 , B. fastidiosus2 , B. gordonae6 , 

B. insolitus2,14 , B. laterosporus6,7,11,12 , B. lentus12 , B. licheniformis6 , B. longisporus6 , 

B. macerans12 , B. megaterium6,7,10,11,12,14,15 , B. mycoides11 , B. pumilus6,7,11,12 , 

B. sphaericus7,12 , B. subtilis7,12,14 , B. throphaeus6 , B. thuringiensis12



Bordetella avium 14 , B. bronchiseptica 8 

Brevibacterium acetylicum 11 

Brevundimonas diminuta 9 , B. vesicularis 7,9 

Burkholderia cepacia 7,8,9,12 , B. gladioli 8,10,12 , B. pickettii 7,8,9,12 , B. solanacearum 12 

Cellulomonas cellulans 12 , C. flavigena 10 , C. turbata 12,15 

Chryseobacterium balustinum 9,11 , C. indologenes 7,11 , C. meningosepticum 7,11 

Citrobacter freundii 5 , C. koseri 12 

Clavibacter michiganensis 2,7,11,12,15 

Comamonas acidovorans 7,8,9,10,11 , C. terrigena 2 , C. testosteroni 9,12,14 

Curtobacterium allidum15 , C. citreum14 , C. flaccumfaciens2,10,12,14,15 , C. luteum14,15 , 

C. pusillum2,12,15 Cytophaga aquatilis10 , C. johnsonae9,10,11 Enterobacter aerogenes 5 , E. asburiae 8,10,12 , E. cancerogenus 12 , E. cloacae 2,5,8,12 , 

E. intermedius 11 , E. taylorae 7,8,12 , E. sakazakii 5 

Erwinia carotovora 9,12 , E. chrysanthemi 9 

Escherichia coli 12,14 , E. vulneris 12 

Flavimonas oryzihabitans 12 

Flavobacterium aquatile 11 , F. indologenes 12 , F. meningosepticum 12 , F. resinovorum 9,10,11 

Gluconobacter oxidans 9 

Gordonia polyisoprenivorans 3 

Herbaspirillum spp. 13 

Hydrogenophaga flava 12 , H. pseudoflava 10,12 

Kingella kingae 15 

Klebsiella ozaenae 2 , K. planticola 12 , K. pneumoniae 2,12 , K. terrigena 12 

Kluyvera ascorbata 12 , K. cryocrescens 8,9,10,12 

Kocuria kristinae 11 

Lactobacillus kefir 10 

Leuconostoc pseudomesenteroides 10 

Methylobacterium extorquens14 , M. fuijsawaense12 , M. mesophilicum12 , M. radiotolerans12 , 

M. rhodinum14 Microbacterium imperiale12 , M. laevaniformans12 Micrococcus agilis 12 , M. halobius 9,10,11 , M. kristinae 12 , M. luteus 8,9,10,11,12 , M. lylae 10,12 , 

M. roseus 12 , M. varians 10,12,15 

Micromonospora endolithica 3 , M. peucetica 3 

Moraxella bovis 2 , M. phenylpyruvica 10 

Mycobacterium aichiense3 , M. bohemicum3 , M. cookii3 , M. flavescens3 , M. heidelbergense3 , 

M. palustre3 Neisseria flavescens, N. mucosa9



Nocardia pseudobrasiliensis 3 

Nocardiodes albus 4 

Ochrabactrum anthropi 12 

Paenibacillus alvei 10 , P. pabuli 12 , P. polymyxa 12 

Pantoea agglomerans 2,5,11,12,14,15 , P. ananas 12 , P. stewartii 9 

Paracoccus denitrificans 7 

Pedicoccus pentosaceus 9 

Photobacterium angustum 9 

Phyllobacterium myrsinacearum 7,8,9,12,14 , P. rubiacearum 7,8,9,10,12 

Plesiomonas shigelloides 10 

Providencia spp. 5 

Pseudomonas aeruginosa 5,9 , P. cepacia 5 , P. chlororaphis 6,7,10,11,12 , P. cichorii 2,6,12,15 , 

P. coronofaciens 12 , P. corrugata 2,6,10,14,15 , P. flectens 10 , P. fluorescens 5,6,7,9,11,12 , P. fragi 14 , 

P. fulva 14 , P. marginalis 2,6,9,12 , P. mendocina 6,9,12 , P. pseudoalcaligenes 6 , 

P. putida 2,5,6,7,9,11,12 , P. rubrisubalbicans 9,12 , P. saccharophila 8,10,12 , P. savastanoi 6,10,12 , 

P. stutzeri 9 , P. syringae 2,6,9,11,12 , P. tolaasii 14,15 , P. vesicularis 12,14 , P. viridiflava 6,11 

Rahnella aquatilis 2 

Rhizobium etli 10 , R. japonicum 12 , R. leguminosarum 14 , R. loti 14 , R. meliloti 15 

Rhodococcus coprophilus 3 , R. luteus 2 

Salmonella cholerasuis 7 

Serratia liquefaciens 5,12 , S. marcescens 12 , S. plymuthica 12 

Shigella spp. 5 

Shingobacterium heparinum 11 

Sphingomonas capsulata 9 , S. paucimobilis 9,12,15 , S. thalpophilum 15 

Staphylococcus capitis 12 , S. epidermidis 12 , S. haemolyticus 11 

Stenotrophomonas maltophilia 9,11,12 

Streptomces argenteolus 4 , S. bikiniensis 4 , S. caviscabies 4 , S. cyaneus 11 , S. galilaeus 4 , 

S. halstedii 11 , S. maritimus 4 , S. pseudovenezuelae 4 , S. scabies 4 , S. setonii 4 , S. tendae 4 , 

S. thermolineatus 3 , S. violaceusniger 11 

Variovorax campestris, V. paradoxus 7,9,12 

Vibrio cholerae 9 

Xanthobacter agilis 9,10 

Xanthomonas agilis 7 , X. campestris 2,8,9,12,14,15 , X. oryzae 15 

Yersinia frederiksenii 12 , Y. pseudotuberculosis 10 

a References: 

1 Adhikari et al. 2001; 2 Bell et al. 1995; 3 Conn and Franco 2004; 4 Coombs and Franco 2003; 

5 Gardner et al. 1982; 6 Germida and Siciliano 2001; 7,8,9 Hallmann et al. 1997b, 1998, 1999; 

10 Hallmann 2003; 11 Krechel et al. 2002; 12 McInroy and Kloepper 1995; 13 Reis et al. 2000; 

14,15 Sturz et al. 1997, 1999


2.4 

Bacterial Diversity 

Diversity indices allow the compression of bacterial spectrum and bacterial 

density into a single number to facilitate comparisons between plant 

species, habitats, etc., as well as elucidation of changes in community relationships 

(Mahaffee and Kloepper 1997a, 1997b). Of the many diversity 

indices used in ecology, only few can be applied to endophytic bacteria. 

Three of the more commonly used indices are genera richness, Hill’s modified 

Shannon’s index N1, and Hill’s modified Simpson’s index N2, with N2 

more than N1 representing very abundant species (Ludwig and Reynolds 

1988). Using these indices for the endorhiza of field-grown cucumber, Mahaffee 

and Kloepper (1997a, 1997b) observed that all three indices tended to 

increase over the growing season, reaching their highest values at the final 

sampling date 70 days after planting. However, density varied between two 

consecutive years, indicating the importance of climatic conditions for the 

community structure of bacterial root endophytes. For potatoes, Krechel et 

al. (2002) observed the highest bacterial diversity at flowering. During that 

period the plant undergoes massive physiological changes that probably 

increase nutrient availability and thus bacterial diversity. Other biotic and 

abiotic factors that influence bacterial diversity are discussed later in this 

chapter. There is little question, though, that a better understanding of the 

population dynamics of bacterial root endophytes will enhance our ability 

to take advantage of their beneficial potential to enhance plant growth and 

health. 

2.5 

Factors Influencing Colonisation 

The enormous spectrum and high diversity of endophytic bacteria found 

in different plant species begs the question: what biotic and abiotic factors 

influence bacterial colonisation? 

2.5.1 

Methodology 

Methodology is especially important in describing the spectrum of culturable 

bacteria, as different methods will give different results. Key factors 

affecting the bacterial spectrum recovered are (1) length of surface sterilisation, 

(2) concentration of the sterilising agent and (3) the method itself. 

Comparison of the trituration method with the pressure bomb technique


(see Chap. 17 by Hallmann et al.) revealed significantly larger populations 

of endophytic bacteria in cotton roots using the first method (Hallmann et 

al. 1997b). However, total number of bacterial genera and species recovered 

was greater using the pressure bomb technique, mainly because a higher 

number of less commonly occurring species was recovered. These results 

suggest that the two techniques sample two different microhabitats, i.e. 

the pressure bomb technique more effectively recovering mainly vascular 

colonists while the trituration method recovers both vascular colonisers 

and bacteria residing in the root cortex. An even higher bacterial spectrum 

can be identified using molecular methods that detect culturable as well as 

non-culturable bacteria (see Chap. 17 by Hallmann et al.). 

2.5.2 

Geography 

Geographical regions are believed to differ in their bacterial spectra. Munif 

(2001) compared the bacterial spectrum of tomato roots grown under temperate 

conditions in Bonn, Germany, with that of tomato roots grown under 

tropical conditions in Bogor, Indonesia. In Germany, 38 species comprising 

21 genera were isolated, whereas in Indonesia, 50 species comprising 

32 genera were isolated. Twenty-four bacterial species were exclusively isolated 

from tomato roots in Germany and 38 species exclusively from tomato 

roots in Indonesia. However, 14 species were isolated from both regions. 

Interestingly, the most abundant species in both geographical regions were 

Pseudomonas putida and Bacillus megaterium. These two bacterial species 

are also commonly reported in other regions of the world (Tables 2.1, 2.2). 

Howisitpossiblethatabacterialspeciescolonisessuchdifferentregions 

and still dominates the endophytic spectrum? And are populations from 

different regions distinguishable? Using molecular fingerprint techniques, 

fluorescent Pseudomonas strains collected worldwide could be regionally 

grouped suggesting that adaptation to their specific environment has occurred 

(Cho and Tiedje 2000). But adaptations also seem to occur within 

a given environment. For example, Pseudomonas fluorescens strains isolated 

from the endorhiza of potato are distinguishable from P. fluorescens 

strains isolated from the rhizosphere of the same plant (Berg et al. 2005). 

2.5.3 

Plant Species 

The effect of plant species on the bacterial spectrum being recovered has 

only been marginally studied. Plant specificity of the bacterial community 

was shown for the rhizosphere by Smalla et al. (2001) and Berg et al. (2002).


As endophytic bacteria represent a subset of the rhizosphere colonisers, 

one would expect that different plant species growing side by side would be 

colonised by a different spectrum of endophytic bacteria. This hypothesis 

was confirmed by McInroy and Kloepper (1995), who showed that the bacterial 

spectrum of sweet corn and cotton grown next to each other differed. 

Although the total number of genera was similar for both plant species, 

some genera, such as Alcaligenes, Aureobacterium, Cellulomonas, Comamonas, 

Erwinia, Ochrobactrum, andYersinia, were recovered only from 

cotton roots, while other genera, such as Arthrobacter, Citrobacter, Flavimonas, 

Microbacterium and Stenotrophomonas, were recovered exclusively 

from sweetcorn roots. Plant-species-specific factors such as root architecture, 

surface structure, and composition of the root exudates as well as 

non-plant factors such as mycorrhization or wounding probably influence 

the bacterial spectrum prior to colonisation. Following root colonisation, 

size of the intercellular space, nutrient composition within the apoplastic 

fluid, and the plant’s response to endophytic colonisation are probably 

the main factors determining bacterial selectivity and thus the bacterial 

spectrum found inside the roots. 

2.5.4 

Plant Genotype 

Does the plant genotype affect the spectrum of endophytic bacteria? What 

about cultivars resistant to bacterial plant pathogens? For the latter, no 

differences were seen in the population densities of endophytic bacteria of 

two grapevine cultivars resistant and susceptible to Agrobacterium tumefaciens, 

the causal agent of crown gall (Bell et al. 1995). However, resistance 

varied in some colonised cultivars (Conn et al. 1997), suggesting that not 

only the host genotype but also the associated bacterial endophytes may 

contribute to plant resistance, as suggested by Bird et al. (1980). Differences 

in the endophytic spectrum were also reported to occur in potato 

tubers of four potato cultivars (Sturz et al. 1999). Although the two bacterial 

species Curtobacterium flaccumfaciens and Pseudomonas cichorii occurred 

in all four cultivars, cultivar-specific preferences were observed. 

For example, C. flaccumfaciens dominated the endophytic spectrum of 

potato ‘Kennebec’ but was rarely found in ‘Butte’, whereas P. cichorii was 

dominant in ‘Butte’ and less frequently isolated from the other three cultivars. 

In addition, some bacterial species were exclusively isolated from 

one cultivar, but not from others. Similar to plant species, plant genotypes 

also vary in their biochemical composition, which may thus affect bacterial 

spectra. Similarly, Graner et al. (2003) found noticeable differences in 

endophytic bacterial populations of oil-seed rape cultivars that differ in


their susceptibility to the fungal wilt pathogen Verticillium longisporum. 

Genotype-specific differences were also shown for wheat cultivars: modern 

cultivars supported a more diverse endophytic community than ancient 

land races (Germida and Siciliano 2001). Furthermore, endophytic bacteria 

isolated from different canola cultivars had different carbon utilisation 

profiles (Misko and Germida 2002). 

2.6 

Interactions 

Ecological theory states that when a disruptive force such as pathogen 

infestation affects a community, the diversity of that community initially 

increases and its membership becomes highly variable before reaching 

a new equilibrium (Mahaffee and Kloepper 1997a; Petratis et al. 1989). But 

does this also apply to endophytic bacteria of roots? Plant roots are continuously 

challenged by soil-borne pathogens, mutualistic symbionts and 

various soil conditions. As a result, plant defence mechanisms might be induced. 

How do these parameters affect the endophytic bacterial spectrum? 

2.6.1 

Plant Pathogens 

While the beneficial effects of endophytic bacteria to control plant pathogens 

are well documented [see Chaps. 3 (Kloepper and Ryu) and 4 (Berg 

and Hallmann)], very little is known about how plant pathogens affect 

the spectrum and diversity of bacterial root endophytes. Regarding fungal 

pathogens, Mahaffee et al. (1997a, 1997b) reported that root infection 

by Rhizoctonia solani promoted colonisation of the two introduced bacterial 

endophytes Enterobacter asburiae JM22 and Pseudomonas fluorescens 

89B-27. Interactions between plant pathogenic bacteria and endophytic 

bacteria have so far been described only for above ground plant organs. 

Inoculation of potatoes with Erwinia carotovora subsp. atroseptica caused 

an increase in endophytic bacterial diversity of infected plants compared 

with healthy plants (Reiter et al. 2002); similarly, infestation of citrus by 

Xylella fastidiosa was positively correlated with the occurrence of Methylobacterium 

spp. (Araújo et al. 2002). A third group of plant pathogens, 

plant parasitic nematodes, significantly increased the total number of bacterial 

root endophytes (Hallmann et al. 1998; Hallmann 2003), and also 

affected the bacterial spectrum (Hallmann 2003). In cotton roots infested 

with the root-knot nematode Meloidogyne incognita, 11 species were exclusively 

isolated from roots of infested plants, while 15 species occurred 

only in non-infested plants.


2.6.2 

Plant Symbionts 

Interactions between endophytic bacteria and mutualistic plant symbionts 

have been best studied for nodule bacteria. Sturz et al. (1997) reported that 

bacterial density and diversity was lower in root nodules of red clover than 

in tap roots, but root nodules yielded a higher number of species exclusively 

colonising this specific niche (Sturz et al. 1997). Coinoculation of 

Rhizobium leguminosarum BV trifolii with endophytic bacteria promoted 

root nodulation. In other cases, endophytic bacteria reduced or inhibited 

root colonisation by nitrogen-fixing bacteria (Bacilio-Jiménez et al. 2001), 

indicating that those interactions seem to be strain-specific. Little is yet 

known about the interaction between endophytic bacteria and other symbionts 

such as fungal endophytes and mycorrhizal fungi. Mycorrhizal fungi 

are at least known to influence rhizosphere bacteria (Azacón-Aguilar and 

Barea 1992) and similar effects might apply to endophytic bacteria. 

2.6.3 

Plant Defence Mechanisms 

A question frequently asked is: why are endophytic bacteria not inhibited 

by a plant defence response? And if there is a plant defence response, 

how does this affect the bacterial spectrum? To answer these questions, 

potato plant resistance was experimentally induced by Rhizobium etli G12 

(Hallmann 2003). R. etli G12 was applied to one-half of a split potato root 

system and the endophytic bacterial spectrum was analysed for the other 

(“induced”) half of the split root system and compared with that of nontreated 

plants. Total bacterial density was significantly higher in treated 

(1.6×10 4 cfu g −1 ) than in non-treated (3.2×10 3 cfu g −1 ) roots. Seventeen 

bacterial species were isolated from treated roots compared with 14 species 

from non-treated roots. The results indicated that, under the conditions 

of bacteria-mediated induced plant defence responses, the density and 

spectrumofbacterialrootendophytesisincreased. 

2.6.4 

Agricultural Practices 

Besides the plant itself and plant-associated microorganisms, agricultural 

practices can also influence the spectrum and population dynamics of bacterial 

root endophytes. Seghers et al. (2004) showed that different fertiliser 

treatments influenced the endophytic community of maize roots, whereas


the tested herbicide treatments did not. High N-fertilisation inhibited the 

colonisation of sugarcane by Acetobacter diazotrophicus (Fuentes-Ramírez 

et al. 1999), while application of nitrogen-containing chitin as an organic 

amendment supported endophytic species in cotton roots that otherwise 

did not occur (Hallmann et al. 1999). For the latter, it was shown that 

the endophytic spectrum was completely different from that of the rhizosphere, 

indicating that the bacterial composition of the rhizosphere is not 

the only factor determining the endophytic spectrum. Differences in plant 

biochemistry due to the organic amendment, such as enhanced chitinase 

and peroxidase concentrations (Hallmann 2003), might have changed the 

plants’ preference for certain bacterial endophytes. 

2.7 

Potential Human Pathogens Among Root Endophytes 

By definition, endophytic bacteria reside within the plant without causing 

visible disease symptoms (see Chap. 1 by Schulz and Boyle). However, the 

distinction between harmless and harmful bacteria is not always clear. 

Potentially harmful bacteria might colonise the plant latently or reside as 

dormant stages, becoming harmful only at later growth stages or when 

a critical density, known as “quorum sensing”, is reached (Eberl 1999). 

Since the beneficial attributes of endophytic bacteria are covered elsewhere 

in this book [see Chaps. 3 (Kloepper and Ryu), 4 (Berg and Hallmann), 

and 6 (Anand et al.)], we will focus on potentially human pathogenic 

bacteria. In general, mechanisms of pathogenicity and antagonism are 

very similar, and sometimes only the expression of one metabolite or total 

bacterial numbers dictates whether the bacteria are harmful or harmless 

(Suckstorff and Berg 2003; Berg et al. 2006). For example, type III secretion 

systems, known for their role in bacterial pathogenicity, are present in 

many plant-associated Pseudomonas strains and may confer induced resistance, 

plant growth promotion or biological control (Preston et al. 2001). 

A few bacterial species isolated from inside plant roots are also known to be 

pathogenic to humans These include strains of Bacillus cereus, Burkholderia 

cepacia, Serratia marcescens and Stenotrophomonas maltophilia,whichnot 

only have excellent antagonistic properties against plant pathogens, but are 

also known to cause human diseases, especially of debilitated or immunosuppressed 

individuals (LiPuma 2003; Minkwitz and Berg 2001; Vandamme 

and Mahenthiralingam 2003; Wolf et al. 2002). Staphylococcus species, also 

known to be human pathogens, have been isolated from the potato endorhiza 

(Krechel et al. 2002; Reiter et al. 2002). In some cases, humanpathogenic 

isolates differ from the harmless isolates occurring in plants by 

the expression of certain toxins, in other cases no such distinction has yet


been found. Reiter et al. (2002) showed, based on 16S rDNA sequences, high 

homology of endophytic bacterial isolates from potato leaves with those of 

human pathogens such as Enterobacter amnigenus, Enterobacter cloacae, 

Stenotrophomonas maltophilia, Staphylococcus xylosus and Ochrobactrum 

anthropi. E. cloacae, S. maltophilia,andO. anthropi have also been reported 

to occur in plant roots (McInroy and Kloepper 1995). 

Nevertheless, the presence of human-pathogenic bacteria in plant roots 

can be a health concern and therefore should be carefully monitored. As 

a result of several Salmonella outbreaks attributed to alfalfa sprout contamination 

in Finland and the United States (van Beneden et al. 1999), alfalfa 

seeds were found to be contaminated and surface sterilisation did not 

eliminate the enteric pathogen. Gandhi et al. (2001) detected a Salmonella 

Stanley strain inside alfalfa sprouts to a depth of 18 µm without finding cells 

on the surface, thus confirming the endophytic properties of this pathogen. 

Strains isolated from patients infected with Salmonella enterica during an 

alfalfa-sprout-associated outbreak and labelled with the green fluorescent 

protein (GFP) successfully colonised alfalfa seedlings (Dong et al. 2003). An 

inoculation with very few cells was sufficient to colonise the plant interior; 

however, strains of S. enterica differed greatly in their ability to invade the 

plant interior and to colonise alfalfa roots. This demonstrates the need for 

further investigations to ensure food safety in the future. 

2.8 

Conclusions 

In conclusion, of the plants thus far studied, the spectrum and diversity of 

endophytic bacteria in the roots varies greatly. What about the endophytic 

bacterial spectrum of plants growing under extreme climatic conditions, 

such as halophytes and xerophytes? Survival mechanisms developed by 

those bacteria may have some interesting industrial or pharmaceutical applications. 

Newly developed cultivation-independent methods have made 

clear that there is much more diversity among endophytic bacteria than at 

first expected. The major factors influencing bacterial diversity and colonisation 

have been discussed and their potential to manage endophytic communities 

towards increased benefits for plants and human health have been 

outlined. However, the potential risks of endophytic bacteria, especially of 

those strains known also to be potential human pathogens, need further 

exploration.



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1944

3 

Bacterial Endophytes as Elicitors 

of Induced Systemic Resistance 

Joseph W. Kloepper, Choong-Min Ryu 

3.1 

Introduction and Terminology 

As indicated elsewhere in this book (e.g. Chap. 1 by Schulz and Boyle), 

the question of what are endophytes can be answered in different ways. 

For the purposes of this chapter, only those endophytes that could be 

isolated from surface-sterilized plant tissue or extracted from within the 

plant, as proposed by Hallmann et al. (1997), will be discussed. All of the 

rhizobacteria discussed here were isolated by grinding tissues of surfacesterilized 

plants, while maintaining sterility controls. It was subsequently 

discovered that some of these bacterial strains elicited systemic protection 

against pathogens when the bacteria were inoculated onto seeds or into the 

potting mix. 

Application to crops of many plant-associated bacteria, including some 

endophytic bacteria, results in a reduction in the incidence or severity of 

diseases. This phenomenon is referred to as biological control. The most 

commonly reported mechanism of biological control is antagonism, where 

the bacterium causes a reduction in the pathogen population or its diseaseproducing 

potential. Antagonism includes the more specific mechanisms 

of predation, competition, and antibiosis. Antagonism is discussed in detail 

in Chap. 4 by Berg and Hallmann. 

An alternative mechanism for biological control is that bacterial metabolites 

affect the plant in such a way as to increase the plant’s resistance to 

pathogens, a process termed induced systemic resistance (ISR). Resistance 

can also be elicited in plants by the application of chemicals or necrosisproducing 

pathogens, and this process is termed systemic acquired resistance 

(SAR). Pieterse et al. (1998) proposed that ISR and SAR can be differentiatednotonlybytheelicitorbutalsobythesignaltransductionpathways 

that are elicited within the plant. Accordingly, ISR is elicited by rhizobac- 

Joseph W. Kloepper: Department of Entomology and Plant Pathology, Auburn University, 

Auburn, AL 36849, USA, E-mail: kloepjw@auburn.edu 

Choong-Min Ryu: Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam 

Noble Parkway, Ardmore, OK 73401, USA 





34 J.W. Kloepper, C.-M. Ryu 

teria or other nonpathogenic microorganisms, while SAR is elicited by 

pathogens or chemical compounds. Further, the signal transduction pathway 

of ISR is independent of salicylic acid but dependent on jasmonate 

and ethylene, while the pathway of SAR is dependent on salicylic acid and 

shows variable dependency on jasmonate and ethylene. Recent discoveries 

show that some rhizobacteria, including some of the endophytic strains 

discussed in this chapter, elicit systemic protection that may be dependent 

on salicylic acid and independent of jasmonate or ethylene. Hence, ISR 

cannot be separated from SAR based on signal transduction pathways. In 

this chapter, ISR is used to describe the phenomenon whereby application 

of bacteria to one part of the plant results in a significant reduction in the 

severity or incidence of a disease following inoculation of a pathogen to 

another part of the plant. 

3.2 

Scope of Endophytes that Elicit Induced Resistance 

and Pathosystems Affected 

The first indication that endophytic bacteria could elicit ISR dates to 1991 

(Wei et al. 1991). Pseudomonas fluorescens strain G8-4, which was later 

designated 89B-61 and found to colonize plants internally, elicited systemic 

protection against cucumber anthracnose following application to 

cucumber seeds. 

In efforts to find other strains of endophytic bacteria that elicited ISR, 

the research group at Auburn University performed isolations from cucumber 

plants in the field or from cucumber seeds. Bacillus pumilus strain 

INR7 was isolated from a surface-sterilized stem of a surviving cucumber 

plant in a field heavily infested with cucurbit wilt disease, caused by 

Erwinia tracheiphila. In two field trials, treatment with INR7 resulted in 

significant growth promotion relative to the nontreated control (Wei et al. 

1996). In addition, the severity of angular leaf spot, following inoculation 

with Pseudomonas syringae pv. lachrymans, and the severity of naturally 

occurring anthracnose were significantly reduced by INR7. The cumulative 

yield of marketable cucumber fruit was also significantly enhanced by 

INR7 in both field trials. In the same study, strain 89B-61 also increased 

plant growth and yield and reduced the incidence of both angular leaf spot 

and anthracnose. In a subsequent field trial, INR7 reduced the severity of 

cucurbit wilt (Zehnder et al. 2001). 

Erwinia tracheiphila is completely dependent on the striped cucumber 

beetle and the spotted cucumber beetles for survival and transmission. The 

finding that cucumber treated with strain INR7 exhibited reduced severity 

of cucurbit wilt in the field led to investigations aimed at determining if


ISR changed beetle feeding activity. In a 2-year field study, the number 

of beetles feeding on cucumber was significantly reduced following treatment 

with strain INR7 (Zehnder et al. 1997b). In both years of the study, 

the season-long average number of cucumber beetles per plant was significantly 

lower on plants treated with INR7 than on nonbacterized plants. 

ReducedbeetlefeedingonplantstreatedwithstrainINR7wasconfirmedin 

subsequent greenhouse studies (Zehnder et al. 1997a). Beetle preference for 

nontreated plants was evident within the first 24 h of releasing the beetles 

into cages containing cucumber plants. After feeding for 17 days, beetle 

damage remained significantly lower on cotyledons and stems of plants 

treated with INR7 than on nontreated plants. 

Elicitation of altered beetle behavior and feeding preferences in the field 

and greenhouse following seed treatment with INR7 was unexpected. As 

summarized by Zehnder et al. (1997a), cucumber beetle feeding behavior is 

influenced by a group of secondary plant metabolites called cucurbitacins, 

which are bitter compounds toxic to most insects. Cucumber beetles consume 

cucurbitacins without toxicity, apparently as an evolutionary adaptation 

that protects the cucumber beetles from predation. The beetles seek 

out cucurbitacins, and concentrations of 1 ng cause cucumber beetles to 

demonstrate arrested feeding behavior, whereby the beetles feed intensely 

on a single plant without moving from plant to plant. Hence, as an explanation 

for reduced beetle feeding on plants treated with INR7, Zehnder et al. 

(1997a) reasoned that elicitation of ISR by INR7 might be accompanied by 

reduced production of cucurbitacins by cucumber plants. Support for this 

hypothesis was found in a study (Zehnder et al. 1997a) in which treatment 

of cucumber with strain INR7 resulted in significantly reduced production 

of cucurbitacin C. Collectively, the results from studies on cucumber beetles 

demonstrate that specific endophytic bacteria can elicit unexpected yet 

important physiological changes in plants. 

Serratia marcescens strain 90-166 colonizes roots internally (Press et al. 

2001) and has been shown to elicit ISR against various diseases of cucumber. 

Elicitation of ISR against Fusarium wilt was demonstrated using a splitroot 

system (Liu et al. 1995a). Root systems of seedlings were mechanically 

separated into two halves, with each half then being placed into a separate 

pot. Strain 90-166 was applied to one pot and the pathogen to the other 

pot. Numbers of dead plants and severity of the disease were significantly 

reduced by strain 90-166 over a 6-week experimental period. In another 

study (Liu et al. 1995b), strain 90-166 was found to elicit ISR against angular 

leaf spot. Treatment of seeds or cotyledons with strain 90-166 resulted in 

significant reductions in numbers and size of angular leaf spot lesions when 

the pathogen was inoculated 3 weeks after planting. ISR against angular 

leaf spot was also elicited when 90-166 was injected into cotyledons 1 week 

before pathogen inoculation. When 90-166 was injected into cotyledons,


there was a reduction of 1.8 log units in the population of the pathogen 

inside leaves. 

The capacity of strain 90-166 to elicit protection against cucumber anthracnose 

over a 5-week period was evaluated by Liu et al. (1995c). Strain 

90-166 was applied to seeds at the time of planting, and Colletotrichum 

orbiculare was inoculated onto the first, second, third, fourth, or fifth leaf. 

There was approximately 1 week between each leaf stage. Treatment with 

strain 90-166 resulted in a significant reduction in the mean total lesion 

diameter when the pathogen was inoculated onto the fifth leaf, indicating 

that ISR elicited by the strain persists for at least 5 weeks on cucumber. 

Elicitation of ISR by strain 90-166 in tobacco against wildfire, caused by 

P. syringae pv. tabaci, was also demonstrated by Press et al. (1997). Stem 

injection of tobacco with strain 90-166 resulted in a significant decrease 

in disease severity when the pathogen was sprayed onto leaves 10 days 

after bacterial treatment. Raupach et al. (1996) reported that strain 90-166 

also elicited ISR against Cucumber mosaic virus (CMV) on cucumber and 

tomato. On cucumber, seed treatment with 90-166 completely prevented 

development of CMV symptoms when the virus was inoculated onto cotyledons. 

On tomato, the effect of 90-166 was to delay symptom development 

over time. The area under the disease progress curve (AUDPC) was significantly 

reduced by strain 90-166. 

Elicitation of ISR against viruses has also been reported for other strains 

of endophytic bacteria. Zehnder et al. (2000) conducted a greenhouse 

screen of PGPR (plant growth promoting rhizobacteria) for the potential 

to elicit ISR against CMV on tomato. PGPR were applied as seed treatments 

and as drenches upon transplanting 2 weeks after seeding. CMV 

was rub-inoculated with carborundum onto leaves 1 week after transplanting. 

From among 26 tested strains, three strains of endophytes were 

selected (Bacillus subtilis strain IN937b, Bacillus pumilus strain SE34, and 

Bacillus amyloliquefaciens strain IN937a). All of the selected strains significantly 

reduced disease incidence in each of five experiments. In the 

same study, Zehnder et al. (2000) conducted two field trials to evaluate 

the effects of strains IN937b, SE34, and IN937a on CMV. Treatment with 

all three endophytic bacterial strains resulted in significant reductions in 

the AUDPC compared to the nonbacterized control in both years of testing. 

In another study with CMV in tomato, Murphy et al. (2003) used various 

two-strain combinations, where one strain was B. subtilis strain GB03, 

which is not reported to be an endophyte, and various endophytic bacteria, 

including strains IN937a, IN937b, SE34, and INR7. Spores of the bacteria 

were formulated on chitosan as a carrier and this preparation was mixed 

into potting mix. All of the bacterial treatments significantly reduced disease 

severity based on symptoms, decreased disease incidence based on


enzyme-linked immunosorbent assay (ELISA), and decreased virus accumulation, 

compared to controls. 

Three field trials were conducted with various formulations of the endophytic 

strains IN937a, IN937b, and SE34 (Murphy et al. 2000) to determine 

their capacity to elicit ISR against Tomato mottle virus (ToMoV), which is 

vectored by the silver whitefly (Bremisia argentifolii). The plots were inoculated 

with ToMoV by natural movement of viruliferous whitefly adults 

from adjacent plantings of ToMoV-resistant tomato germplasm that was 

inoculated prior to transplanting into the field. The incidence and severity 

of ToMoV were significantly reduced by one or more of the formulations 

of each endophyte. The number of whitefly nymphs detected on plants was 

significantly reduced by strains IN937a and IN937b. Hence, as in the case 

with cucurbit wilt of cucumber, some endophytic bacteria can elicit ISR 

against both a plant disease and its insect vectors. 

ISR elicited by the endophytes B. pumilus strain SE34, S. marcescens strain 

90-166, and Pseudomonas fluorescens strain 89B-61 has been shown to reduce 

the severity of blue mold of tobacco, caused by Peronospora tabacina 

(Zhang et al. 2002a, 2002b, 2004). In one study (Zhang et al. 2002b), strains 

SE34, 90-166, and 89B-61 elicited ISR in detached leaf and microtiter plate 

bioassays as well as in pot trials in the greenhouse. In the pot assay, application 

of all three strains as a soil drench to 4-week-old plants of three 

tobacco cultivars resulted in significant reductions in the mean percentage 

of leaf area with lesions caused by P. tabacina inoculated onto leaves 

1 week after bacterial treatment. Sporulation of the pathogen on lesions 

was significantly decreased by treatment with the three strains in pot trials. 

Strains SE34, 90-166, and 89B-61 also significantly reduced disease severity 

in the detached leaf (injection of a bacterial suspension into petioles) and 

microtiter plate bioassays (application of bacterial suspensions to roots). 

Sporulation of the pathogen was significantly reduced by both strains in 

the detached leaf bioassay. 

In another study (Zhang et al. 2004), strains SE34 and 90-166 were used 

to explore the relationship between elicitation of plant growth promotion 

and ISR. Application of the endophytes as a seed treatment alone elicited 

significantly enhanced tobacco plant growth but not disease protection. 

When the strains were applied as seed treatments followed by a soil drench, 

both plant growth promotion and ISR were elicited. Overall, the results from 

this study indicated that while there was a relationship between growth 

promotion and ISR, elicitation of growth promotion can occur without 

elicitation of ISR; however, when ISR was elicited, growth promotion was 

also elicited with the bacterial strains used in the study. 

Endophytic bacteria have also elicited ISR against tomato late blight, 

caused by Phytophthora infestans (Yan et al. 2002). Application of strains 

SE34 and 89B-61 by incorporation into the potting medium at the time of


planting elicited significant reductions in disease severity when P. infestans 

wasinoculatedontoleaves5weeksafterplanting. 

Greenhouse screening of endophytic Bacillus spp. that have elicited ISR 

on some crops was conducted in Thailand (Jetiyanon and Kloepper 2002) 

as a first step toward employment of endophyte-elicited ISR in tropical 

agriculture. This study used four different host/pathogen systems: tomato 

and Ralstonia solanacearum, long cayenne pepper (Capsicum annuum 

var. acuminatum) andColletotrichum gloeosporioides, greenkuangfutsoi 

(Brassica chinensis var. parachinensis)andRhizoctonia solani, and cucumber 

and CMV. The goal of the study was to find mixtures of endophytic 

spore-forming bacteria that elicited ISR in all four host/pathogen systems. 

Seven individual strains and 11 combinations of 2 strains were tested. One 

strain (B. amyloliquefaciens IN937a) and four mixtures (IN937a + B. subtilis 

IN937b; IN937b + B. pumilus SE34; IN937b + B. pumilus SE49; and IN937b 

+ B. pumilus INR7) significantly reduced incidence or severity of all four 

diseases. The results are noteworthy for two reasons. First, they indicate 

that ISR elicited by specific endophytic bacterial strains can protect hosts 

under tropical conditions. Second, the results show that mixtures of two 

bacterial strains are superior to individual strains for eliciting significant 

protection in multiple hosts against different pathogens. 

FurtherevidencefortheconceptofusingstrainmixturesofBacillus spp. 

to increase the repeatability of plant growth promotion or elicitation of ISR 

by bacteria was reported in a follow-up field investigation (Jetiyanon et al. 

2003). Field tests were conducted in Thailand to find mixtures of bacteria 

that could protect several different hosts against the multiple diseases 

that are typical under the multi- or inter-cropping agricultural conditions 

predominant in Thailand. In tests conducted during the rainy and dry 

seasons, some two-strain mixtures of endophytes more consistently protected 

against disease than did a single strain. In each season, the mixture 

of strains IN937a and IN937b significantly protected against all the tested 

diseases (southern blight of tomato, CMV on cucumber, and anthracnose 

of long cayenne pepper). The same mixture of endophytes also resulted in 

significant yield increases of all crops during the rainy season. 

Endophytic bacteria have also been shown to elicit ISR in conifers. 

Enebak and Carey (2000) tested the potential elicitation of ISR on loblolly 

pine (Pinus taeda) bystrainsB. sphaericus SE56 and B. pumilus strains 

INR7, SE34, SE49, and SE52 against Cronartium quercuum f. sp. fusiforme, 

which causes fusiform rust. Bacteria were applied at seeding, and suspensions 

of C. quercuum basidiospores from field-collected telia on water 

oak (Quercus nigra) were sprayed onto the pine seedlings at five different 

times. Six months after the final application of basidiospores, the incidence 

of fusiform rust was determined by noting the presence or absence of the 

typical symptoms of main-stem swellings or galls. The experiment was


conducted annually for 2 years. All strains except SE49 resulted in significant 

reductions in disease incidence in either one of the 2 years or in the 

pooled data from both years. 

Before concluding this discussion of case studies of endophytic bacteria 

that have been shown to elicit ISR, a note should be made about Azospirillum 

spp. Azospirillum brasilense is a well characterized endophyte, and nearly 

all strains of this species have been shown to promote growth of many 

crop species (Bashan and de-Bashan 2002). Because many of the strains of 

Bacillus spp. cited above that elicit ISR also elicit plant growth promotion, 

one might expect that A. brasilense would also elicit ISR. However, this 

is not the case. Bashan and de-Bashan (2002) investigated the potential 

of A. brasilense to elicit ISR in tomato against bacterial speck, caused by 

P. syringae pv. Tomato, and concluded that this endophyte does not elicit 

ISR. 

3.3 

Internal Colonization of Endophytes 

that Elicit Induced Resistance 

In most of the studies discussed in the previous section, extensive microbial 

ecology studies to determine the extent of internal colonization of plant 

tissues by the applied endophytes were not carried out. Typically, isolations 

are performed near the location where the pathogen was applied. Such 

isolation is done to test one of the suggested tenants of ISR: that there 

is physical separation of the pathogen and the inducing agent. According 

to this tenant, physical separation is required to differentiate ISR from 

antagonism as a mechanism for protection against pathogens. While testing 

for physical separation has validity, it also creates an inherent problem 

with endophytic bacteria that exhibit systemic colonization of plants. An 

endophyte that can move within the plant and colonize petioles could, 

theoretically, still elicit ISR at a level sufficient to reduce disease severity 

ofafoliarpathogen.However,basedonthetenantofphysicalseparation, 

one could not state that ISR was the operable mechanism by which disease 

severity was reduced. Hence, some endophytic bacteria might actually elicit 

plant defense although they are not spatially separated from the pathogen. 

An example illustrating the limited internal colonization of well characterized 

endophytes that elicit ISR is P. fluorescens strain 89B-61, which 

was initially designated as strain G8-4. Because this strain has reactions 

in biochemical tests that are intermediate between P. putida and P. fluorescens, 

some publications before 1997 refer to 89B-61 as P. putida.Later 

publications designate the strain as P. fluorescens basedonrepeatedfatty 

acid analyses. Chen et al. (1995) found that strain 89B-61 significantly


reduced the severity of Fusarium wilt of cotton. In this system, 89B-61 

was stab-inoculated into seedling stems 13 days prior to inoculation with 

Fusarium oxysporum f. sp. vasinfectum atapoint1.5cmabovethepoint 

of bacterial inoculation. Using a rifampicin-resistant mutant of 89B-61, no 

movement up the stem from the point of bacterial inoculation was detected. 

In a separate greenhouse study, Kloepper et al. (1992) reported that seed 

treatment of cucumber with strain G8-4 (89B-61) significantly reduced lesion 

numbers and size of anthracnose following challenge inoculation of 

leaves with C. orbiculare. This induced resistance was associated with colonization 

of internal root tissues at log 4.0 cfu/g at 14 days after emergence; 

however, the bacteria were not isolated from stems or leaves. Elicitation of 

induced resistance in cucumber by 89B-61 was confirmed in field studies 

(Wei et al. 1996), where application of the bacterium as a seed treatment 

resulted in significant reductions in severity of anthracnose and angular 

leaf spot. 

Quadt-Hallmann et al. (1997) used isolation, ELISA, and immunogold 

labeling with P. fluorescens 89B-61-specific polyclonal antibodies to investigate 

the pattern of internal colonization of cotton by the ISR-eliciting 

strain 89B-61. Results from isolation studies indicated that, following treatment 

of seeds, 89B-61 colonized roots internally at a mean population of 

1.1×10 3 cfu/g, while the bacterium was not recovered from stems, cotyledons, 

or leaves. With ELISA, strain 89B-61 was detected outside and inside 

roots but not inside stems, cotyledons, or leaves. Electron microscopy with 

immunogold labeling revealed that internal colonization of roots by 89B-61 

was restricted mainly to intercellular spaces of the epidermis. Interestingly, 

the colonization pattern was quite distinct from that of another bacterium 

(Enterobacter asburiae strain JM22) that does not elicit ISR. JM22 colonized 

throughout the root cortex, including inside the vascular stele, in intercellular 

spaces close to the conducting elements, as was previously found in 

other plant species (Quadt-Hallmann and Kloepper 1996). It was suggested 

that the internal colonization of cotton by 89B-61 and JM22 could be considered 

as representative of two fundamental options for how endophytic 

bacteria colonize plants after application to seeds or soils. The first pattern 

is that of 89B-61 and consists of limited internal colonization of roots. The 

second pattern, demonstrated by JM22, consists of extensive internal root 

colonization and ultimately in some vascular colonization. 

Internal colonization of roots by S. marcescens strain 90-166 was demonstrated 

by Press et al. (2001) in an investigation into the role of iron in 

elicitation of ISR. Cucumber root colonization by the wild-type strain was 

compared to that by a mutant deficient in siderophore production. While 

the total root population sizes (external and internal colonization) were statistically 

equivalent, the internal population size was significantly greater 

with the wild-type than with the siderophore-negative mutant. Because the


mutant failed to elicit ISR against anthracnose, while ISR was elicited by 

the wild-type strain, it was concluded that capacity to elicit ISR was related 

to the population size of the bacterium inside roots. 

3.4 

Plant Responses to Endophytic Elicitors 

Investigations aimed at determining how plants respond to inoculation 

with endophytes that elicit ISR is one approach to studying mechanisms 

of ISR by such bacteria. During the previously discussed study on cotton 

colonization by strain 89B-61 (Quadt-Hallmann et al. 1997), the epidermal 

cell walls of plant cells adjacent to cells of 89B-61 in the intercellular space 

developed electron-opaque appositions of an amorphous matrix. 

Two cytological studies were conducted by Benhamou et al. (1996, 1998) 

with B. pumilus strain SE34. In the first study (Benhamou et al. 1996), 

colonization of pea roots by Fusarium oxysporum f. sp. pisi was restricted 

totheepidermisandoutercortexofrootstreatedwithSE34,whilein 

nonbacterized roots, the pathogen colonized the cortex, endodermis, and 

the paratracheal parenchyma cells. This reduction in fungal colonization by 

SE34wasassociatedwithstrengtheningoftheepidermalandcorticalcell 

walls. In addition, roots treated with SE34 exhibited newly formed barriers 

beyondthesiteoffungalinfection.Thesebarrierswerecellwallappositions 

that contained large amounts of callose and were infiltrated with phenolic 

compounds. Phenolic compounds were detected in transmission electron 

microscopy using gold-complexed laccase and were found to accumulate 

in host cell walls, in intercellular spaces, and on the surface of and inside 

the invading pathogen hyphae. 

In another study (Benhamou et al. 1998), the effect of SE34 alone or in 

combination with chitin on structural and cytochemical changes of tomato 

infected with F. oxysporum f. sp. radicis-lycopersici was investigated. Treatment 

with SE34 reduced the severity of typical symptoms, including wilting 

of seedlings and numbers of brown lesions on lateral roots. This disease 

protection by strain SE34 was associated with more limited fungal colonization 

of roots and with marked changes in host physiology. Physiological 

changes elicited by strain SE34 included an increase in host cell wall density, 

the accumulation of polymorphic deposits at sites of potential pathogen 

penetration, and the occlusion of epidermal cells and intercellular spaces 

with an osmophilic, amorphous material that appeared to trap the invading 

fungal hyphae. The extent and magnitude of the physiological changes in 

the host elicited by SE34 were enhanced by the addition of chitosan. Interestingly, 

the overall chitin component of the pathogen was structurally 

preserved in roots treated with SE34 with or without chitosan at the time


when hyphal degradation was apparent. This suggests that synthesis of 

chitinase in bacteria-treated roots is not an early event in the cascade of 

physiological steps in signal transduction that lead to induced resistance. 

Benhamou et al. (1998) concluded: “According to our cytological observations, 

the induction of resistance triggered by B. pumilus strain SE34 

involves a sequence of events including first the elaboration of structural 

barriers and the production of toxic substances such as phenolics and phytoalexins, 

and second the synthesis and accumulation of other molecules 

including chitinases and other hydrolytic enzymes such asβ-1,3-glucanases 

which probably contribute to the release of oligosaccharides that, in turn, 

can stimulate other defense reactions.” 

Jeun et al. (2004) conducted a cytological comparison of cucumber plants 

in which systemic resistance had been elicited by bacteria or by chemicals. 

In this study, the endophytes 89B-61 and 90-166 were used to elicit ISR 

against C. orbiculare. Significantly fewer numbers of anthracnose lesions 

developed on plants treated with 89B-61 and 90-166 than on the control 

(chemical treatment). Cytological studies using fluorescent microscopy revealed 

a higher frequency of autofluorescent epidermal cells, which are 

related to accumulation of phenolic compounds, at the sites of fungal penetration 

in plants treated with either strain and inoculated with C. orbiculare. 

In addition, callose-like structures (β-1,3-glucan polymers) were frequently 

deposited at the site of fungal penetration of the leaves of plants treated 

with either strain. 

Investigations on plant responses to elicitation of ISR can also examine 

the signal transduction pathway of plants to determine general biochemical 

pathways in plants during ISR. As previously discussed, according to the 

model pathway for signal transduction (Pieterse et al. 1998), ISR pathways 

are independent of salicylic acid, but dependent on ethylene, jasmonic acid, 

and the regulatory gene npr-1. Further, according to the model, ISR elicited 

by bacteria does not result in the accumulation of pathogenesis-related (PR) 

proteins. PR proteins are accumulated during SAR elicited by pathogens 

and chemicals, and SAR is dependent upon salicylic acid. 

Afewstudiesonsignalpathwayshavebeenreportedwithendophytic 

bacteria that elicit ISR. In the tomato late blight system, ISR was elicited 

by B. pumilus strain SE34 on NahG lines, which breakdown endogenous 

salicylic acid, but not in the ethylene-insensitive NR/NR line or in the 

jasmonic acid-insensitive df1/df1 line (Yan et al. 2002). These results are 

consistent with the model of Pieterse et al. (1998). Similar results were 

reported by Zhang et al. (2002a). In the tobacco blue mold system, B. pumilus 

strain SE34, as well as two strains of Gram-negative bacteria, elicited ISR 

on both wild-type and NahG transgenic tobacco lines, as evidenced by 

significant reductions in the severity of blue mold on bacterized plants 

compared to nonbacterized plants.


Different results were found with strain 89B-61 (Park and Kloepper 

2000), which elicits ISR in tobacco against wildfire caused by P. syringae pv. 

tabaci. In this system, a transgenic line of tobacco with a β-glucuronidase 

(GUS) reporter gene fused to the PR-1a promoter had significantly reduced 

severity of wildfire compared to nonbacterized controls. Elicitation of ISR 

by strain 89B-61 was associated with a significant increase in GUS activity 

in microtiter plate and whole plant bioassays. Hence, with strain 89B-61, 

elicitation of ISR results in activation of the PR-1a gene, which is activated 

during SAR but not during bacterial-induced ISR according to the model 

of Pieterse et al. (1998). 

Signal pathways in ISR elicited by P. fluorescens strain CHA0 in Arabidopsis 

against Peronospora parasitica were investigated by Iavicoli et al. 

(2003) using various transgenic and mutant plant lines: NahG (for degradation 

of salicylic acid), sid2-1 (lacks production of salicylic acid), npr1-1 

(nonexpressor of PR genes), jar1-1 (insensitive to jasmonic acid), ein2-1 

(insensitive to ethylene), eir1-1 (insensitive to ethylene/auxin), and pad2-1 

(phytoalexin-deficient). ISR was elicited by strain CHA0 in all lines except 

jar1-1, eir1-1,andnpr1-1. 

In another study of signaling pathways, Ryu et al. (2003b) used endophytic 

bacterial strains in Arabidopsis to elicit ISR against two different 

pathovars of P. syringae (pvs. tomato and maculicola). Strains SE34, 90- 

166, and 89B-61 elicited ISR against both pathogens. Strain SE34 elicited 

a salicylic acid-independent pathway against one pathovar and salicylic 

acid-dependent pathway against a different pathovar. Additional tests of 

strains 89B-61 and SE34 on various mutant lines of Arabidopsis (Ryu et al. 

2003b) revealed that, in agreement with the model of Pieterse et al. (1998), 

ISRelicitedbybothstrainswasdependentonNPR1andISRelicitedby 

SE34 was dependent on jasmonic acid and ethylene. However, in contrast 

to the model, ISR elicited by strain 89B-61 was independent of ethylene and 

jasmonic acid, and ISR by strain 90-166 was dependent on jasmonic acid 

but independent of ethylene. 

Endophyte strains 90-166 and SE34 also elicited ISR against CMV in 

Arabidopsis (Ryu et al. 2004b). Strains 90-166 and SE34 reduced disease 

severity in NahG plants, indicating that ISR elicited by strains 90-166 and 

SE34 was independent of salicylic acid. Further investigation on the signal 

pathway of ISR against CMV elicited by strain 90-166 with lines NahG, 

npr1, andfad3-2 fad7-2 fad8 (insensitive to jasmonic acid) indicated that 

ISR against CMV by strain 90-166 is independent of salicylic acid and NPR1, 

but is dependent on jasmonic acid (Ryu et al. 2004b). 

Collectively, the results on signaling pathways of ISR elicited by endophytic 

bacteria indicate that different pathways are elicited by various 

strains. Further, the specific signal transduction pathway that is activated 

during ISR depends on the host plant and, at least in one case, on the 

pathogen used on a given host.


A new approach to investigations on plant responses to endophytic bacteriawasrecentlyopenedbythefindingthatvolatileorganiccompounds 

produced by the endophyte B. amyloliquefaciens IN937a elicit plant growth 

promotion (Ryu et al. 2003a) and ISR (Ryu et al. 2004a). Significant growth 

promotion of Arabidopsis by IN937a was observed in I-plates, which have 

a raised plastic divider separating agar on each half of the dish, thus preventing 

movement of soluble compounds. When IN937a was placed on 

onesideofanI-plate,Arabidopsis plants growing on the other side exhibited 

enhanced growth, presumably as a result of volatiles produced by 

the bacteria. Characterization of the volatile organic compounds (VOCs) 

produced by IN937a, coupled with bioassays of fractions of VOCs, revealed 

that 2,3-butanediol and acetoin elicited plant growth promotion. In a separate 

study (Ryu et al. 2004a), exposure of Arabidopsis to VOCs from strain 

IN937a resulted in significantly less disease caused by Erwinia carotovora 

subsp. carotovora. Tests with various mutant lines of Arabidopsis revealed 

that elicitation of ISR by VOCs of IN937a is independent of jasmonic acid, 

ethylene, salicylic acid, and npr1. Such a pattern of signal pathway has not 

been reported with ISR elicited by bacteria and, therefore, it is likely that 

VOCs of IN937a elicit a distinct, and as yet uncharacterized, pathway in 

Arabidopsis. 

3.5 

Implementation in Production Agriculture: 

Two Case Studies 

The principle that endophytic bacteria can elicit ISR or plant growth promotion 

has been extended to use in production agriculture and horticulture 

through the development of two products. These two products are discussed, 

not as endorsements of the products, but as case studies indicating 

that our growing scientific knowledge of endophytic bacteria can be put to 

practical use. 

In the first case study, an agricultural product has been developed using 

the capacity of a single endophytic strain of Bacillus spp. to elicit 

both ISR and plant growth promotion. The product is Yield Shield, which 

is produced by Gustafson, LLC. Yield Shield consists of a spore preparation 

of the B. pumilus strain listed in this review as INR7 (Table 3.1) 

and designated by Gustafson as GB34 (http://www.gustafson.com/Labels/ 

yield shield label.pdf). The product received registration from the United 

States Enivironmental Protection Agency (EPA) in 2003 for use on soybeans 

to protect against Rhizoctonia solani and Fusarium spp. Seed treatment of 

soybean with Yield Shield and strain INR7 results in significant seedling 

growth promotion and in ISR, which is apparent both by a significant de-


Table 3.1. Endophytic bacterial strains that have been reported to elicit induced systemic 

resistance (ISR) in at least two publications 

Strain no. and 

identification 

IN937a 

Bacillus amyloliquefaciens 

IN937b 

Bacillus 

subtilis 

90-166 

Serratia 

marcescens 

Effectsreportedandsystemsused a Reference 

Reduced incidence or severity of vegetable diseases 

(caused by Cucumber mosaic virus (CMV), Sclerotium 

rolfsii, Ralstonia solanacearum, Colletotrichum 

gloeosporioides,andRhizoctonia solani) in greenhouse 

and field trials in Thailand 

Jetiyanon et al. 

2002; 

Jetiyanon and 

Kloepper 2003 

Reduced incidence of CMV on tomato in the greenhouse Zehnder et al. 

2000 

When applied to tomato with the non-endophyte Bacillus 

subtilis GB03, reduced severity of CMV in the greenhouse 

Reduced incidence and severity of tomato mottle virus in 

the field. Also reduced numbers of the white fly vector 

feeding on plants 

Volatile organic compounds of the strain elicit growth 

promotion of Arabidopsis and ISR against Erwinia 

carotovora subsp. carotovora 

Component of the product, BioYield (Gustafson LLC, 

http://www.gustafson.com) 

Murphy et al. 

2003 

Murphy et al. 

2000 

Ryu et al. 

2003a, 2004 

Kloepper et al. 

2004 


2000 

When applied to tomato with the non-endophyte 

B. subtilis GB03, reduced severity of CMV in the 

greenhouse 


the field. Also reduced numbers of the white fly vector 

feeding on plants 

Reduced incidence and delayed development of 

symptoms of Fusarium wilt of cucumber, caused by 

Fusarium oxysporum f. sp. cucumerinum 

Reduced severity of bacterial angular leaf spot of 

cucumber, caused by Pseudomonas syringae pv. 

lachrymans 

Reduced severity of anthracnose, caused by 

Colletotrichum orbiculare, in two cucumber cultivars 

over a 5-week period after bacterial treatment 

Reduced incidence of CMV on cucumber and tomato 

and reduced the area under the disease progress curve 

(AUDPC) 

Decreased severity of tobacco wildfire, caused by 

Pseudomonas syringae pv. tabaci 

Murphy et al. 

2003 

Murphy et al. 

2000 

Liu et al. 1995a 

Liu et al. 1995b 

Liu et al. 1995c 

Raupach et al. 

1996 

Press et al. 

1997





SE34 Bacillus 

pumilus 


Wild-type strain reduced severity of cucumber 

anthracnose. A siderophore-negative mutant did not elicit 

ISR and colonized roots internally at lower populations 

than the wild-type 

Decreased severity of tobacco blue mold, caused by 

Peronospora tabacina, in NahG transgenic tobacco lines 

that degrade salicylic acid 

Decreased severity of tobacco blue mold in microtiter 

plate assays and detached leaf assays. Reduced pathogen 

sporulation 

Reduced symptoms of Pseudomonas syringae pvs. tomato 

and maculicola on Arabidopsis 


Peronospora tabacina 


(caused by cucumber mosaic virus, Sclerotium rolfsii, 

Ralstonia solanacearum, Colletotrichum gloeosporioides, 

and Rhizoctonia solani) in greenhouse and field trials in 

Thailand 






sporulation 

Decreased severity of tobacco blue mold when applied as 

both seed treatment and drench in the greenhouse. 

Elicitation of ISR was associated with growth promotion 

Decreased severity of tomato late blight, caused by 

Phytophthora infestans, and decreased germination of 

sporangia and zoospores of the pathogen 

Press et al. 

2001 

Zhang et al. 

2002a 

Zhang et al. 

2002b 

Ryu et al. 

2003b 

Zhang et al. 

2004 

Jetiyanon et. 

al. 2002; 

Jetiyanon and 

Kloepper 2003 

Zhang et al. 

2002a 

Zhang et al. 

2002b 

Zhang et al. 

2004 

Yan et al. 2002 


2000 



greenhouse 


the field 

Reduced incidence of Fusiform rust, caused by 

Cronartium quercuum f. sp. fusiforme,onloblollypine 

Murphy et al. 

2003 

Murphy et al. 

2000 

Enebak and 

Carey 2000





INR7 

Bacillus 

pumilus 

(available as 

the product 

Yield Shield; 

Gustafson 

LLC) 

89B-61 

Pseudomonas 

fluorescens 

(earlier 

referred to 

as G8-4) 


Restricted colonization of pea roots by F. oxysporum f. sp. 

pisi and induced formation of structural barriers in the 

plant 

Reduced damage of tomato roots to F. oxysporum f. sp. 

radicis-lycopersici and induced structural and 

cytochemical barriers in the plant 




(caused by CMV, Sclerotium rolfsii, Ralstonia 

solanacearum, Colletotrichum gloeosporioides,and 

Rhizoctonia solani) in greenhouse and field trials in 

Thailand 

Decreased severity of anthracnose (caused by 

Colletotrichum orbiculare) and angular leaf spot (caused 

by Pseudomonas syringae pv. lachrymans) on cucumber 

in field trials 

Decreased the incidence of cucurbit wilt disease, caused 

by Erwinia tracheiphila,infieldtrials 

Decreased numbers of cucumber beetles on plants in the 

field 

Decreased beetle feeding activity and transmission of 

E. tracheiphila on cucumber in cages where beetles had 

a choice between bacterial-treated and nontreated plants 



greenhouse 

Reduced incidence of Fusiform rust, caused by 

Cronartium quercuum f. sp. fusiforme,onloblollypine 




in field trials 


Peronospora tabacina 

Decreased severity of Fusarium wilt of cotton, caused by 

Fusarium oxysporum f. sp. vasinfectum 

Following seed treatment of cotton, 89B-61 reached an 

internal root population of 1.1×10 3 cfu/g. Bacteria were 

not detected inside cotyledons, stems, or leaves 

Benhamou et 

al. 1996 

Benhamou et 

al. 1998 

Ryu et al. 

2003b 

Jetiyanon et. 

al. 2002; 

Jetiyanon and 

Kloepper 2003 

Wei et al. 1996 

Zehnder et al. 

2001 


1997b 


1997a 

Murphy et al. 

2003 

Enebak and 

Carey 2000 


Zhang et al. 

2004 

Chen et al. 

1995 

Quadt- 

Hallmann et 

al. 1997





CHA0 

Pseudomonas 

fluorescens 





Decreased severity of tomato late blight, caused by 

Phytophthora infestans, and decreased germination of 

sporangia and zoospores of the pathogen when applied to 

seeds 






sporulation 

Decreased severity of tobacco wildfire, caused by 

Pseudomonas syringae pv. tabaci. Activated the promoter 

for PR1a [a pathogenesis-related (PR) protein] 



Reduced number of lesions of Colletotrichum orbiculare 

on cucumber and increased deposition of callose-like 

polymers on leaf cells at the site of pathogen penetration 

Reduced mean numbers and size of anthracnose lesions 

on cucumber 

Colonized roots internally at log 4.0 cfu/g at 2 weeks after 

emergence when applied as seed treatments. Bacteria 

were not detected in leaves or stems 

Reduced severity of Tobacco necrosis virus (TNV) on 

tobacco. 

Severity of TNV on tobacco was reduced equivalently by 

the wild-type strain and a transgenic strain carrying 

pchAB genes for synthesis of salicylic acid. 

Reduced sporulation of Peronospora parasitica on 

Arabidopsis 


Yan et al. 2002 

Zhang et al. 

2002a 

Zhang et al. 

2002b 

Park and 

Kloepper 2000 

Ryu et al. 

2003b 

Jeun et al. 2004 


Kloepper et al. 

1992 

Maurhofer et 

al. 1994 

Maurhofer et 

al. 1998 

Iavicoli et al. 

2003 

a In all cases, the stated reductions in disease incidence or severity and the effects on insects 

are statistically significant at P ≤ 0.05 

crease in incidence and severity of R. solani inoculated onto stems at a point 

where INR7 does not colonize, and by a systemic increase in lignification 

of plant cell walls (C.-M. Ryu and C.-H. Hu, unpublished). It should be 

emphasized that Yield Shield is a unique case for a rhizobacterium that


elicits ISR in that economically significant efficacy sufficient to warrant the 

costs of product development and EPA registration was shown for a single 

bacterial strain. 

In the second case study, the product consists of a two-strain mixture 

of Bacillus spp., where one strain (IN937a) is an endophyte that elicits ISR 

(Table 3.1) and the other strain (GB03) is a non-endophyte. The product is 

BioYield and is also produced by Gustafson. The development of BioYield 

was recently reviewed (Kloepper et al. 2004). The underlying concept was 

to develop a biological formulation consisting of components known to exert 

different mechanisms for control of diseases. The selected components 

and their mechanisms were chitosan (as a carrier) for nematode control 

via promotion of indigenous soil predators and antagonists to root-knot 

nematodes, B. subtilis strain GB03 for control of soil-borne pathogens via 

production of the antibiotic iturin, and one of several tested endophytic 

Bacillus spp. that elicit ISR. The most unexpected finding was that the 

three-component combination (chitosan plus two bacterial strains) exhibited 

more consistent, and a greater magnitude of, growth promotion 

and systemic protection against pathogens than did any of the individual 

components (Kloepper et al. 2004). Based on the results, the two-strain 

combination of B. amyloliquefaciens strain IN937a and B. subtilis strain 

GB03 was selected for product development. 

3.6 

Conclusions 

As discussed in this review, selected strains of nonpathogenic endophytic 

bacteria can elicit ISR in plants, leading to reductions in severity of various 

diseases. Research on such endophytes has concentrated both on delineating 

the pathosystems where protection results and in understanding 

plant responses that occur during the signal transduction pathways that 

culminate in disease protection. In many cases, elicitation of ISR by endophytic 

bacilli is associated with increased plant growth, and the relationship 

between ISR and growth promotion should be further investigated. Elucidation 

of specific bacterial determinants that account for elicitation of 

ISR is just beginning, and further work is needed to understand why one 

strain of a given bacterial species can elicit ISR while another strain of 

the same species cannot. It is encouraging that implementation of ISR by 

endophytic bacilli is beginning, even while some basic questions remain to 

be answered.



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4 

Control of Plant Pathogenic Fungi 

with Bacterial Endophytes 

Gabriele Berg, Johannes Hallmann 

4.1 

Introduction 

Interest in biological control has increased over the past years, driven by 

the need for alternatives to chemicals – which have often lost their activity 

due to the development of resistant pathogen populations – and 

to public pressure to develop production systems favourable to the environment 

(Whipps 2001). In this respect antagonistic bacteria provide an 

environmentally sound alternative to protect plants against attack by fungal 

pathogens (Whipps 1997; Bloemberg and Lugtenberg 2001). In the past, 

rhizosphere bacteria have been shown to be effective antagonists against 

a broad spectrum of fungal pathogens (Weller 1988; Emmert and Handelsman 

1999; Kurze et al. 2001). More recent studies have indicated that 

bacteria colonising the root interior can even improve plant growth and 

plant health (Frommel et al. 1991; Sturz et al. 1999; see Chap. 3 by Kloepper 

and Ryu), and seem to be excellent candidates for use as biological control 

agents (BCAs) (Chen et al. 1995; Sturz et al. 1997; Downing and Thomson 

2000; Sturz et al. 2000; Adhikari et al. 2001; Tjamos et al. 2004; see Chap. 3 

by Kloepper and Ryu). 

Besides induced resistance (see Chap. 3 by Kloepper and Ryu), little is 

known about other mechanisms used by antagonistic endophytic bacteria 

towards fungal pathogens, such as antibiosis, competition and lysis. Furthermore, 

endophytic bacteria are known to promote plant growth by the 

production of plant hormones, enhanced nutrient availability and nitrogen 

fixation (Whipps 2001; Hurek and Reinhold-Hurek 2003). For example, 

plant hormones produced by endophytic bacteria seem to be essential for 

bryophyte development (Hornschuh et al. 2002). 

So far, most information about the community structure of endophytic 

bacteria with antagonistic properties has been obtained using cultivationdependent 

approaches [Chen et al. 1995; Sturz et al. 1999; see Chaps. 2 

Gabriele Berg: Graz University of Biotechnology, Department of Environmental Technology, 

Petersgasse 12, 8010 Graz, Austria, E-mail: gabriele.berg@TUGraz.at 

Johannes Hallmann: Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für 

Nematologie und Wirbeltierkunde, Toppheideweg 88, 48161 Münster, Germany 





54 G. Berg, J. Hallmann 

(Hallmann and Berg) and 15 (Schulz)]. However, recently developed cultivation-independent 

methods (reviewed by Smalla 2004) analyse endophytic 

communities directly from root tissue. Techniques using bacterial 

DNAprovideinformationonthestructuraldiversityoftheentireendophytic 

community, while techniques using bacterial RNA identify metabolically 

active bacteria, e.g. those which are of interest after pathogen infection. 

Primers are available that specifically recognise bacterial taxa with 

high percentages of antagonistic strains, such as Pseudomonas, Serratia 

or Burkholderia (Widmer et al. 1998; Salles et al. 2001). Unfortunately, 

the methodology does not distinguish between antagonistic and nonantagonistic 

strains. However, genes involved in bacterial antagonism, 

such as those involved in the expression of antibiotics, siderophores or 

phytohormones, are now being cloned and in the near future may lead to 

the development of primers targeting antagonism and specific microarrays 

(Raaijmakers et al. 1997; De Souza and Raaijmakers 2003; Zhou 

2003). Overall, a polyphasic approach combining cultivation-dependent 

and cultivation-independent methods is recommended to best gain insights 

into the dynamics of antagonistic endophytic communities as well 

as plant/endophyte/pathogen interactions (Garbeva et al. 2001; Krechel et 

al. 2002; Reiter et al. 2002; Sessitsch et al. 2004). Understanding those interactions 

provides a platform from which to develop endophytic bacteria 

as biocontrol agents. 

However, formulation, application, risk assessment, fruit quality and 

potential side-effects are further questions that need to be properly answered 

before endophytic bacteria can be released as BCAs. This chapter 

discusses four key aspects of biological control of fungal pathogens using 

endophytic bacteria: (1) the spectrum of indigenous bacterial antagonists 

in plant roots, (2) modes of action, (3) use of BCAs, and (4) strategies to 

enhance biocontrol efficiency. 

4.2 

Spectrum of Indigenous Endophytic Bacteria with 

Antagonistic Potential Towards Fungal Plant Pathogens 

Antagonists are naturally occurring organisms with the potential to interfere 

with pathogen infection, growth, and survival (Chernin and Chet 

2002). A better understanding of the spectrum of indigenous antagonistic 

bacteria will (1) increase our knowledge of plant/endophyte interactions, 

(2) facilitate screening efforts for effective biocontrol organisms, (3) allow 

breeding of cultivars supporting a high level of antagonistic bacteria, and 

(4) may even lead to management strategies for increasing the antagonistic 

potential of endophytic bacteria. This chapter focuses on the spectrum of


antagonistic endophytic bacteria, and discusses the factors that influence 

and regulate them. 

Antagonistic communities can be studied from a quantitative or qualitative 

perspective. Commonly used methods for bacterial identification 

include morphological and physiological characterisation, fatty methylester 

analysis and molecular techniques based on specific primers and/or 

(partial) sequencing of 16S rDNA. While the first method is limited to 

culturable bacteria, the latter method can also identify non-culturable endophytic 

bacteria, e.g. in a clone library. Antagonistic activity of endophytic 

bacteriaisgenerallytestedbyinvitroinhibitionoffungalpathogensindual 

cultures and then confirmed in bioassays on host plants. Quantitative analysis 

to detect antagonistic bacteria is time-consuming as all the endophytic 

bacteria have to be screened for their antagonistic potential. Primers developed 

for gene sequences involved in antagonistic activity may, in the 

future, be able to recognise antagonists without cultivation. 

Using cultivation-dependent methods, the proportion of antagonistic 

endophytes can vary between 0%, as shown for the pathosystem Phytophthora 

cactorum–potato (Sessitsch et al. 2004) and 50%, for Verticillium 

longisporum–oilseed rape (Graner et al. 2003) (Table 4.1). Major factors 

determining the percentage of antagonists are most likely plant species, 

pathogen infestation, habitat and vegetation period (Sessitsch et al. 2004; 

Berg et al. 2005). In conclusion, these data confirm that a significant portion 

of the indigenous endophytic bacteria in plant roots have antagonistic 

potential towards fungal pathogens. 

Table 4.1. Proportion of antagonistic endophytic bacteria in different pathosystems 

Plant Plant pathogens Proportion of 

antagonists (%) 

Reference 

Rice Achlya klebsiana 25 Adhikari et al. (2001) 

Pythium spinosum 75 

Potato Verticillium dahliae 

Rhizoctonia solani 

9 Krechel et al. (2002) 

Potato Verticillium dahliae 13 Berg et al. (2004) 

Rhizoctonia solani 9.7 

Potato Verticillium dahliae 2 Sessitsch et al. (2004) 

Rhizoctonia solani 3 

Phytophthora cactorum 0 

Streptomyces scabies 43 

Xanthomonas campestris 29 

Oilseed rape Verticillium longisporum 50 Graner et al. (2003) 

Tomato Verticillium dahliae 12 Tjamos et al. (2004)


But what are the main bacterial species conferring antagonism towards 

fungal plant pathogens? Although a high diversity of antagonistic endophytic 

bacteria is found in general, some genera harbour more antagonistic 

strains than others, e.g. Bacillus, Curtobacterium, Methylobacterium, 

Paenibacillus, Pseudomonas, Serratia, Stenotrophomonas and Streptomyces 

(Sturz et al. 1999; Garbeva et al. 2001; Krechel et al. 2002; Reiter et al. 2002; 

Sessitsch et al. 2004). Antagonistic species have been isolated from a number 

of different plant species, but most studies have been done on potato. 

Table 4.2 lists antagonistic species found in potato in five studies. Surprisingly, 

a total of 51 different species comprising 27 genera were identified! 

The proportion and composition of indigenous endophytic bacteria with 

antagonistic capacity is influenced by a variety of biotic and abiotic factors, 

with the plant itself being a major factor (Germida et al. 1998). As shown by 

Table 4.2. Endophytic bacterial species of potato with antagonistic properties towards plant 

pathogenic fungi a 

Species with antagonistic properties 

Agrobacterium tumefaciens 

Amycolatopsis meditarranei 

Arthrobacter ilicis 

Bacillus aquamarinus 

Bacillus cereus 

Bacillus megaterium 

Clavibacter michigenensis 

Curtobacterium albidum 

Curtobacterium flaccumfaciens 

Curtobacterium luteum 

Erwinia persicinus 

Flavobacterium sp. 

Frateuria aurantia 

Frigoribacterium sp. 

Kingella kingae 

Kitasatosporia cystargenia 

Methylobacterium sp. 

Micrococcus varians 

Paenibacillus sp. 

Pantoaea agglomerans 

Pantoaea anantis 

Pseudomonas cichorii 

Pseudomonas corrugata 

Pseudomonas fluorescens 

Pseudomonas graminis 

Pseudomonas migulae 

Pseudomonas orientalis 

Pseudomonas putida 

Pseudomonas rhodesiae 

Pseudomonas reactans 

Pseudomonas straminea 

Pseudomonas synthaxa 

Pseudomonas syringae 

Pseudomonas tolaasii 

Pseudomonas veronii 

Psychrobacter immobilis 

Ralstonia paucula 

Rhizobium meliloti 

Rhizomonas suberifaciens 

Sphigobacterium thalophilum 

Sphingomonas adhaesiva 

Stenotrophomonas maltophilia 

Streptomyces turgidiscabies 

Streptomyces bottropensis 

Streptomyces diastatochromogenes 

Streptomyces galilaeus 

Streptomyces griseus 

Streptomyces lavendulae 

Streptomyces setonii 


Xanthomonas campestris 

Xanthomonas oryzae 

a According to Sturz et al. (1999), Krechel 

et al. (2002), Reiter et al. (2002), Sessitsch 

et al. (2004), and A. Krechel et al., unpublished 

data


Zinniel et al. (2002) for endophytic bacteria in above-ground plant organs, 

the bacterial spectrum of 4 agronomic crop species and 27 prairie plant 

species varied greatly. A similar effect of the plant species on the bacterial 

spectrum can also be expected for antagonistic root endophytes. There is 

even an effect of the cultivar or plant genotype on the bacterial spectrum 

(see Chap. 2 by Hallmann and Berg). For example, the oilseed rape cultivar 

‘Express’, which is tolerant to V. longisporum,containedahigherproportion 

of bacteria with proteolytic, cellulolytic and phosphatase activity than 

the susceptible cultivar ‘Libraska’ (Graner et al. 2003). Modern wheat cultivars 

have a more diverse endophytic community than ancient land races, 

and are more aggressively colonised by endophytic pseudomonads that 

produce antifungal metabolites (Germida and Siciliano 2001). Breeder selection 

for higher productivity might have selected plants that support an 

endophytic microflora antagonistic to fungal pathogens. Finally, the plant 

tissue itself can also vary in its antagonistic bacteria. Sturz et al. (1999) 

made the observation that the outer tissue of a potato tuber yielded higher 

relative densities of endophytic bacteria antagonistic to soilborne fungal 

pathogens than deeper layers of the potato. Furthermore, the antifungal 

potential of bacterial endophytes was highest for isolates recovered from 

the outermost layer of the tuber. The authors assumed that, in certain 

communities of endophytic bacteria, antagonism against fungal pathogens 

may be related to bacterial adaptation to location within a host plant, and 

may be tissue-type and tissue-site specific, indicating that in plant tissue 

exposed to the soil, antagonistic endophytes become more prominent. Is 

this a result of coevolution or a process controlled by the plant itself? Such 

questions still await proper answers. 

Besides the plant itself, several biotic factors affect endophytic communities 

and the proportion of antagonists (Reiter et al. 2002; Sessitsch et al. 

2002, 2004). In this context, it was shown that pathogen stress had a greater 

impact than plant genotype on bacterial diversity. Furthermore, Sessitsch 

et al. (2004) found a different spectrum of antagonistic bacteria in good 

and poor growing potatoes in the field. 

Fluctuations in antagonistic bacterial communities of plant roots may 

also be caused by abiotic factors such as temperature, rainfall, cropping 

practice or soil amendments (Mocali et al. 2003). Plants are able to select 

specific bacterial genotypes in response to soil conditions (Siciliano et 

al. 2001). Therefore, the bacterial community in the soil is an important 

factor affecting the composition of indigenous endophytic bacteria. Soil 

amendments can modify the bacterial spectrum in the soil as well as in the 

roots (Hallmann et al. 1999) and enhance the efficacy of biocontrol agents 

(Ahmed et al. 2003). 

In conclusion, the spectrum and diversity of endophytic bacterial communities 

in plant roots is never a stable scenario, rather it has its own


dynamics in response to biotic as well as abiotic factors. This offers new 

opportunities for managing the indigenous spectrum of antagonistic endophytic 

bacteria to increase the benefits to the plant by means of plant 

breeding, soil amendments or application of endophytic BCAs. 

4.3 

Mode of Action of Antagonistic Bacteria 

Modes of action of antagonistic towards fungal pathogens have been intensively 

studied for plant growth-promoting rhizobacteria, as reviewed 

by Fravel (1988), Whipps (2001), Lugtenberg et al. (2001), and Bloemberg 

et al. (2001). Presumably, endophytic bacteria use similar mechanisms for 

the control of fungal plant pathogens. However, their hidden life within 

the plant tissue makes it much more difficult to study such mechanisms 

(see Chap. 18 by Bloemberg and Camacho Carvajal). Furthermore, it is 

often difficult to distinguish between direct antagonism (such as antibiosis), 

competition and lysis, and indirect mechanisms such as induced resistance 

and improved plant growth (see also Chap. 3 by Kloepper and 

Ryu). 

4.3.1 

Antibiosis 

Antibiosis describes the ability of an endophytic bacterium to inhibit 

pathogen growth by the production of antibiotics or toxins. Although 

the vast majority of endophytic bacteria show antibiosis against fungal 

pathogens in vitro (Krechel et al. 2002; Sturz et al. 1999), very little is known 

about the significance of antibiosis controlling fungal pathogens within the 

root tissue. Examples for antifungal substances released by endophytic 

bacteria include iturin A (produced by Bacillus subtilis) and pyrrolnitrin 

(produced by Serratia plymuthica) (Cho et al. 2002). Further support for 

the hypothesis that these antifungal metabolites represent the underlying 

mechanisms in situ could be achieved by antibiotic-deficient mutants that 

fail to express biocontrol activity. Unfortunately, no such studies have yet 

been carried out. 

However, just as microbial antagonists utilise a diverse arsenal of mechanisms 

to dominate interactions with fungal pathogens, pathogens have 

surprisingly diverse responses to counteract these antagonisms (Duffy and 

Défago 1997). These responses include detoxification, antibiotic resistance, 

active efflux of antibiotics, and repression of biosynthetic genes expressing 

proteins involved in biocontrol (Duffy et al. 2003). Again, most work in


this area has been carried out on rhizosphere bacteria, and almost nothing 

is known about the regulation of antifungal metabolites expressed by 

endophytic bacteria. However, since antibiosis seems to be a mechanism 

of fungal control used by endophytic bacteria, metabolites released by the 

bacteria into plant tissue must be carefully monitored to ensure that they 

pose no risk regarding fruit quality and consumer health. 

4.3.2 

Competition 

Competition is considered an important factor in the control of fungal 

pathogens by endophytic bacteria, since both organisms colonise similar 

niches and utilise the same nutrients. Conclusive data demonstrating 

competition as a major control mechanism of endophytic bacteria are still 

lacking. Work on rhizosphere bacteria has shown that, under iron-limiting 

conditions, bacteria produce siderophores with high affinity for ferric iron. 

By binding available iron these bacteria deprive fungal pathogens of iron, 

thus restricting their growth (O’Sullivan and O’Gara 1992). Siderophores 

are also commonly produced by endophytic bacteria (Krechel et al. 2002), 

indicating that similar mechanisms may occur in the endorhiza. 

4.3.3 

Lysis 

Cell wall lysis is another potential mechanism whereby endophytic bacteria 

can control fungal pathogens. This mechanism is well established in the 

biocontrol of fungal pathogens by rhizosphere bacteria. Endophytic bacteria 

isolated from potato roots express high levels of hydrolytic enzymes 

such as cellulase, chitinase and glucanase (Krechel et al. 2002). Pleban et al. 

(1997) analysed the importance of lytic enzymes in antagonism of Bacillus 

cereus strain 65 towards the soilborne fungal pathogen Rhizoctonia 

solani. B. cereus strain 65, originally isolated from surface-sterilised seeds 

of Sinapis arvensis, was shown to excrete a chitinase of 36 kDa, responsible 

for the observed protection of cotton seedlings from root rot disease caused 

by R. solani. Additionally, chitinolytic Bacillus subtilis strains were able to 

reduce symptoms of Verticillium dahliae in several host plants (Tjamos et 

al. 2004). An endophytic chitinase-producing isolate of Actinoplanes missouriensis 

and its culture filtrates were shown to suppress Plectosporium 

tabacinum on lupins (El-Tarabily 2003). The importance of hydrolytic enzymes 

other than chitinases as biocontrol mechanisms is still unknown.


4.3.4 

Induction of Plant Defence Mechanisms 

Induction of plant defence mechanisms by endophytic bacteria plays a major 

role in suppression of fungal plant pathogens and therefore is covered 

in a separate chapter (Chap. 3 by Kloepper and Ryu). 

4.3.5 

Plant Growth 

Plant growth is a factor that is indirectly involved in pathogen defence. 

Plants with vigorous growth, such as cucumber, can sometimes outgrow 

disease by fungal pathogens such as powdery mildew. Therefore, plant 

growth promotion by endophytic bacteria indirectly affects the pathogenicity 

of fungal pathogens. Nejad and Johnson (2000) described isolates of endophytic 

bacteria that significantly improved seed germination and plant 

growth of oilseed rape and tomato. Plant growth promotionmediated by endophytic 

bacteria may be exerted by several mechanisms, e.g. production of 

plant growth hormones, synthesis of siderophores, nitrogen fixation, solubilisation 

of minerals such as phosphorous, or via enzymatic activities, for 

example suppression of ethylene by 1-aminocyclopropane-1-carboxylate 

(ACC) deaminase (Chernin and Chet 2002). Strains of Pseudomonas, Enterobacter, 

Staphylococcus, Azotobacter and Azospirillum produce plant 

growth regulators such as ethylene, auxins or cytokinins, which are assumed 

to promote plant growth (Arshad and Frankenberger 1991; Leifert 

et al. 1994). However, in the past, most interest has focussed on the fixation 

of atmospheric nitrogen by free-living endophytic bacteria, especially of 

diazotrophs (Döbereiner and Pedrosa 1987; Hecht-Buchholz 1998; Estrada 

et al. 2002; Hurek and Reinhold-Hurek 2003). 

Overall, mechanisms of fungal control by endophytic bacteria may 

act synergistically, and individual endophytes quite often exhibit several 

modes of action. However, the expression of antifungal mechanisms is 

strain-specific (Neiendam-Nielson et al. 1998; Berg 2000; Berg et al. 2002) 

and most likely under the control of several biotic as well as abiotic factors. 

A better understanding of the underlying mechanisms has significant 

relevance for the optimisation of biocontrol strategies (see Sect. 4.5). 

4.4 

Control Potential of Endophytic Bacteria 

A broad spectrum of endophytic bacteria has been described to control 

fungal plant pathogens on different plant species (Table 4.3). The major-


ity of antagonistic endophytic bacteria are Gram-negative and belong to 

the group of fluorescent pseudomonads, which are effective BCAs (Bloemberg 

and Lugtenberg 2001; Whipps 2001). Fluorescent pseudomonads are 

common members of the endorhiza, making them ideal candidates for biological 

control measures. Antagonistic isolates have been reported to occur 

in Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas 

graminis, Pseudomonas putida, Pseudomonas tolaasii and Pseudomonas 

veronii (Table 4.3). Control potential in terms of reduced disease severity 

can approach 80% under greenhouse conditions (Pleban et al. 1995). Besides 

pseudomonads, other Gram-negative species with biocontrol activity 

against fungal pathogens are Phyllobacterium rubiacearum, Burkholderia 

solanacearum (Chen et al. 1995), Sphingomonas trueperi (Adhikari et al. 

2001) and Serratia plymuthica (Faltin et al. 2004). 

Gram-positive bacterial isolates with antagonistic properties are commonly 

found within the genus Bacillus (Emmert and Handelsman 1999). 

Bacillus spp. occur mainly in the soil/rhizosphere but have also been reported 

as endophytes of several plant species (see Chap. 2 by Hallmann 

and Berg). As summarized in Table 4.3, endophytic isolates of Bacillus,and 

the closely related genus Paenibacillus, havebeenshowntosignificantly 

control many fungal diseases. Gram-positive biocontrol bacteria other than 

bacilli include actinomycetes such as Streptomyces, Microbispora or Nocardioides 

(Coombs et al. 2004). For example, the streptomycete Actinoplanes 

missouriensis gave excellent control of Plectosporium tabacinum, the causal 

agent of lupin root rot (El-Tarabily 2003). 

Most studies have been carried out under greenhouse conditions. However, 

a few studies have achieved similar biocontrol effects under field 

conditions (Tjamos et al. 2004; Faltin et al. 2004). For example, the endophytic 

isolate Pseudomonas trivialis 3Re2-7 significantly reduced disease 

incidence of Rhizoctonia solani onlettuceandsugarbeetby40%and86%, 

respectively (Faltin et al. 2004). 

These examples illustrate the high potential of endophytic bacteria in 

fungal pathogen control. However, further fieldwork is required to confirm 

their control efficacy in different climatic regions and under different 

growth conditions. Formulations and applications that meet common 

farming practice still need to be developed. For promising BCAs, strategies 

that enhance overall control efficacy should be explored. 

4.5 

Enhancing Biocontrol Efficiency 

It is a well-accepted observation that biological control under field conditions 

is often inconsistent (Weller 1988). Therefore, enhancing the efficacy


Table 4.3. Examples of antagonistic 

Pathosystem BCA Trial Application Results Reference 

Pleban et al. 

1995, 1997 

Cotton, Bean 

(Rhizoctonia solani) 

Pleban et al. 

1995 

Chen et al. 

1995 

Bean 

(Sclerotium rolfsii) 

Cotton 

(Fusarium oxysporum 

f. sp. vasinfectum) 

Sharma and 

Nowak 1998 

Downing and 

Thomson 

2000 


Greenhouse Root incubation in Reduction of disease incidence 

Bacillus subtilis 

bacterial suspension approx. 50% 

Bacillus pumilus 


Greenhouse Root incubation in Reduction of disease incidence 


bacterial suspension of 70–80% 

Aureobacterium saperdae Pot experiment Pierced with a needle Reduction of disease severity 

Bacillus pumilus 

and plant growth promotion 

Phyllobacterium rubiacearum 

Pseudomonas putida 

Burkholderia solanacearum 

Pseudomonas Growth Bacterization of tissue Reduction of disease severity 

chamber plantlets or seedlings and plant growth promotion 

Pseudomonas fluorescens Plant growth Soil drenching 

Rif1::tc4 (with chiA gene) chamber 107 cfu ml−1 Reduction of disease incidence 

up to 48%, only if introduced as 

Introduction into the endophytes 

Tomato 

(Verticillium dahliae) 

Bean 


plant 

Adhikari et al. 

2001 

Reduction of disease incidence 

by 50–73%; plant growth 

promotion (plant height and 

Pot experiments Rice seeds were 

soakedfor1hin 

a bacterial suspension 

108 cfu ml−1 Pseudomonas fluorescens 

Pseudomonas tolaasii 

Pseudomonas veronii 

Rice 

(Achlya klebsiana, 

Pythium spinosum) 

dry weight) 

Sphingomonas trueperi 

Krechel et al. 

2002 

Seed treatment Reduction of fungal growth in 

vitro, of nematode infestation 

ad planta; plant growth 

promotion up to 78% 

Growth 

chamber 


Kitasatosporia cystargenia 

Streptomyces galilaeus 

Streptomyces griseus 

Pseudomonas graminis 

Potato 

[Rhizoctonia solani, 

Verticillium dahliae, 

Meloidogyne incognita 

(nematode)]



Pathosystem BCA Trial Application Results Reference 

Bacillus sp. Pot experiments Soil inoculation Reduction of disease incidence Cho et al. 2003 

Ahmed et al. 

2003 

Reduction of disease incidence 

of R. solani up to 55% and of 

Greenhouse Seed treatments 

Root drenching 


Bacillus licheniformis 

Balloon flower 


Pepper 

(Rhizoctonia solani, 

P. capsici up to 55% 

Phytophthora capsici) 

Tjamos et al. 

2004 

Reduction of disease severity by 

40–70%; yield increase 25% 

Root dipping 

Soil drenching 

107 cfu ml−1 talc-gum 

xanthan formulation 

Greenhouse 

Field 

Bacillus spp. 

Paenibacillus alvei 

Bacillus amiloliquefaciens 

Eggplant, potato 

(Verticillium dahliae) 

Faltin et al. 

2004 

Reduction of disease severity up 

to 76% 

Soil drenching 

Seed bacterization 

108 cfu ml−1 Greenhouse 

Field 

Serratia plymuthica 

Pseudomonas reactans 

Potato 


Coombs et al. 

2004 

Reduction of black lesions 

up to 71% 

Greenhouse Seed treatments 109 to 10 10 cfu ml−1 Streptomyces spp. 

Microbispora spp. 

Nocardioides 

Wheat 

(Gaeumannomyces 

graminis var.tritici)


and consistency of control by endophytic bacteria is a major factor in 

determining their future success as BCAs. Current strategies to enhance 

biocontrol efficiency include (1) optimised formulation and application 

technologies, (2) integrated biocontrol strategies, (3) management of the 

indigenous endophytic microflora, and (4) genetic engineering. 

The formulation of BCAs has undergone major progress in recent years 

(Burghes 1998). Most work has been done on rhizosphere bacteria and 

the acquired know-how now needs to be transferred to endophytic bacteria. 

Basically, two types of formulation seem to be preferable for endophytic 

bacteria: dry products and liquid suspensions. For Gram-positive 

endophytes, dry formulations with a long shelf life are already standard 

(Emmert and Handelsmann 1999; Whipps 1997) whereas stable formulations 

for Gram-negative bacteria are more difficult, due mainly to the lack 

of resting spores that can withstand the formulation process. Optimum 

formulations should ensure bacterial survival, but also promote bacterial 

activity after application. Microcapsules that protect the incorporated bacteria, 

while also providing nutrient sources, are an interesting alternative 

(Burghes 1998). 

The delivery of BCAs into the host plant is a prerequisite for successful 

biocontrol. Musson et al. (1995) studied eight methods for delivering 

endophytic bacteria into cotton stems and roots: stab-inoculation of bacteria 

into stems, soaking seed in bacterial suspensions, soil drench, methyl 

cellulose seed coating, foliar spray, bacteria-impregnated granules applied 

in-furrow, vacuum infiltration, and the pruned-root dip. Whereas each of 

the methods effectively established most of the endophytic bacteria based 

on re-isolation studies, none of the methods successfully delivered all 15 

strains tested, indicating that the optimum method is strain-specific. For 

practical reasons, soil drench, root dipping, and seed coating are the most 

promising methods. Standard technology can be used and the bacterium 

is delivered directly into the root system where it can immediately colonise 

the root and protect the plant against invasion by fungal pathogens. 

Fahey et al. (1991) described a seed inoculation technique in which 

pressure is applied to infuse the bacterial suspension into imbibed seeds, 

followed by redrying of the seeds. The inoculated seeds met the requested 

shelf-life requirements of several months, and application of commonly 

used fungicides had no adverse impact on bacterial survival or efficacy of 

the bacterial inoculation. This leads to another interesting option for biocontrol 

enhancement: the combination of endophytic bacteria with other 

BCAs or even chemicals. Integrated biological control strategies against 

fungal pathogens using a combination of antagonistic endophytes with 

complementary modes-of-action and/or colonisation sites, or of endophytes 

with synthetic control agents take advantage of synergies and should 

be exploited further.


The naturally occurring bacterial antagonistic potential within a plant is 

influenced by several biotic and abiotic factors (see Chap. 2 by Hallmann 

and Berg). By changing these factors, the antagonistic potential can be 

managed. For example, chitin application not only increased total numbers 

of antagonistic Burkholderia cepacia (Hallmann et al. 1999) but, as shown 

by Ahmed et al. (2003), also improved control of root rot disease in pepper 

by endophytic Bacillus subtilis and B. licheniformis. Furthermore, the chitin 

derivate chitosan was successfully used in combination with the endophytic 

bacterium Bacillus pumilus to enhance plant resistance towards Fusarium 

wilt of tomato (Benhamou et al. 1998). It is hypothesised that chitin as well 

as chitosan stimulate the establishment of a chitinolytic microflora, which 

then decomposes fungal cell wall chitin. 

Biocontrol efficiency might also be improved by breeding plant genotypes 

to support a high level of antagonistic endophytes. For example, 

plants expressing high levels of N-acylhomoserine lactones, or which are 

capable of degrading this important signal molecule of bacterial communication, 

have been shown to also influence plant-associated bacteria (Fray 

2002). On the other hand, endophytic bacteria might also be promoted 

by transgenic means. Promising biocontrol targets for genetic engineering 

are gene-regulated factors of endophytic bacteria involved in modes-ofaction, 

colonisation potential, survival, fitness, and adaptation to environmental 

conditions. For example, the endophytic bacterium Pseudomonas 

fluorescens, originally isolated from micropropagated apple plantlets, was 

genetically modified by Downing and Thomson (2000) to harbour a gene 

encoding the major chitinase of Serratia marcescens. ThegenechiA was 

cloned under the control of the tac promoter in the broad-host-range plasmid 

pKT240 and the integration vector pJFF350. P. fluorescens carrying 

tac-chiA either on the plasmid or integrated into the chromosome significantly 

controlled Rhizoctonia solani on beans. Although a promising 

approach, genetically modified BCAs still face many restrictions, making 

broad-scale application in the near future improbable and difficult. 

4.6 

Conclusions 

Most plants are colonised by a broad spectrum of endophytic bacteria that 

are potentially antagonistic towards fungal plant pathogens. This enormous 

potential needs to be further explored for its use in modern plant disease 

control strategies. This requires not only a better understanding of the 

underlying mechanisms and their regulation in response to environmental 

factors, but also a more comprehensive picture of what triggers endophytic 

colonisation as well as of the population dynamics of antagonistic bacterial


endophytes within the plant. Continuing research in this area will hopefully 

lead to new and innovative concepts for biological control of fungal 

pathogens. 


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5 

Role of Proteins Secreted by Rhizobia 

in Symbiotic Interactions 

with Leguminous Roots 

Maged M. Saad, William J. Broughton, William J. Deakin 

5.1 

Introduction 

Rhizobia are Gram-negative soil inhabitants with the ability to induce 

the formation of highly specialised organs called nodules on the roots or 

stems of leguminous plants. Some rhizobial species provoke nodule formation 

on a limited number of legume genera and are said to have narrow 

host ranges, e.g. Rhizobium meliloti, which nodulates only three genera 

of legumes. Other (broad host-range) rhizobia provoke the formation of 

nodules on many different legumes, e.g. Rhizobium sp. NGR234 (hereafter 

called NGR234), which nodulates more than 112 genera of legumes as well 

as the non-legume Parasponia andersonii (Pueppke and Broughton 1999; 

Trinick 1980). 

To form root nodules, legume roots undergo several new developmental 

changes. Initially, rhizobia attach to root hairs, causing deformation and 

then curling of the root hair. Rhizobia invade the root through newly formed 

tubular structures, called infection threads, which grow toward the root cortex. 

During invasion, rhizobia cause the induction of division of cortical 

cells, thus forming nodule primordia (Relić et al. 1994). Infection threads 

travel inter- and intra-cellularly toward the primordia. Wall-degrading enzymes 

help the passage of infection threads from cell to cell (van Spronsen 

et al. 1994). Rhizobia are released from the infection threads into the cytoplasm 

of host cells by a process resembling endocytosis (Stacey et al. 1991). 

Extensive cell division in the primordia leads to functional nodules containing 

rhizobia, in which the bacteria differentiate into their endosymbiotic 

form, known as the bacteroids (Franssen et al. 1992). Bacteroids, together 

with the surrounding plant-derived peribacteroid membrane (PBM) are 

Maged M. Saad: Université de Genève, Laboratoire de Biologie Moléculaire des Plantes 


William J. Broughton: Université de Genève, Laboratoire de Biologie Moléculaire des Plantes 

Supérieures, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland, E-mail: 

william.broughton@bioveg.unige.ch 

William J. Deakin: Université de Genève, Laboratoire de Biologie Moléculaire des Plantes 






72 M.M. Saad et al. 

called symbiosomes. At this stage, the bacteria synthesise nitrogenase, an 

enzyme complex that catalyses the reduction of atmospheric nitrogen to 

ammonia, which is subsequently assimilated into amino acids. In return, 

the plant reduces carbon dioxide to sugars during photosynthesis and 

translocates these compounds to the roots, where the bacteria use them 

as an energy source (Atkins et al. 1982; Fisher and Long 1992). Depending 

upon the plant species, at least two types of nodules are formed. Indeterminate 

nodules have a persistent meristem that grows continuously giving 

the nodules an elongated shape. Determinant nodules lack the persistent 

meristem, and are round in shape as the meristematic activity is limited to 

the early stages of nodule development. 

Coordination of this complex developmental programme requires the 

exchange of many signals between the two symbiotic partners and it is 

the response to (and synchronisation of) these signals that controls nodule 

formation. Amongst the first signal molecules are phenolic compounds, 

mainly flavonoids that are secreted by roots into the rhizosphere. The rhizobial 

protein NodD functions first as an environmental sensor of these 

phenolics, and later as a transcriptional activator of a series of rhizobial 

genes that encode proteins responsible for the production of rhizobial 

signals. NodD proteins belong to the LysR family of transcriptional regulators, 

which have the ability to bind to specific, highly conserved DNA 

sequences (nod-boxes) present in the promoter regions of many nodulation 

genes/loci (Perret et al. 2000). The number of nod-boxes varies in different 

rhizobia. For example, in NGR234 there are at least 19 nod-boxes that help 

regulate transcription of genes involved in a range of different signalling 

compounds (Kobayashi et al. 2004). The first rhizobial signal molecules 

to be synthesised and secreted are encoded by the nodulation genes (nod, 

noe,andnol), which are responsible for the synthesis of host-specific lipochito-oligosaccharide 

molecules called Nod-factors. Nod-factors provoke 

deformation and curling of the root hair and allow rhizobia to enter roots 

through infection-threads (Relić et al. 1993, 1994; D’Haeze et al. 1998). 

All invasive rhizobia produce Nod-factors, and the addition of purified 

Nod-factors alone causes root-hair deformation and cortical-cell division 

(Downie 1998; Perret et al. 2000). Nod-factors consist of a backbone of three 

to six β-1.4-linked N-acetyl-d-glucosamine residues. A fatty-acid chain of 

variable length and structure (depending on the Rhizobium species) is attached 

at the C-2 position of the non-reducing sugar residue. Synthesis 

of the backbone is brought about by the products of the nodABC genes, 

known as the core enzymes, as they are found in all rhizobia. NodB and 

NodC are responsible for the synthesis of the backbone while NodA is 

an acyl-transferase, which adds the fatty-acid side chain. Nod-factors are 

further modified by the action of other Nod enzymes that add various 

chemical groups to the backbone (Hanin et al. 1999; Broughton et al. 2000).

5 Protein secretion by Rhizobia 73 

The development of the infection threads needs other sets of rhizobial 

signals, including surface polysaccharides (SPS) as well as secreted proteins. 

SPSs include extracellular polysaccharides (EPS), capsular polysaccharides 

(CPS), lipo-polysaccharides (LPS) as well as cyclic β-glucans. Most of these 

polysaccharides function during infection thread development, where they 

possibly help suppress plant defence reactions. SPSs have highly diverse 

structures and may contribute to rhizobial host-range (for reviews, see 

Broughton et al. 2000; Perret et al. 2000). In this chapter we will concentrate 

on protein secretion by rhizobia, which are thought to play a role in 

the infection process leading to nodule formation and, thus, in successful 

nitrogen fixation. 

5.2 

Bacterial Protein Secretion Systems 

Gram-negative bacteria possess at least four systems (type I–type IV) to 

secrete proteins into the external environment (see Fig. 5.1) (Thanassi 

and Hultgren 2000). Examples of all four systems have been found in 

rhizobia. Type II secretion is said to be sec-dependent as it requires the 

sec system to export proteins into the periplasm prior to secretion across 

the outer membrane. Proteins secreted by the type II system thus possess 

a classical amino-terminal hydrophobic signal sequence, which is cleaved 

during sec-export. In contrast, type I and type III secretion systems are secindependent; 

proteins are secreted across the bacterial inner- and outermembranes 

in a single step process. Such proteins do not possess cleavable 

amino-terminal signal sequences. A number of proteins secreted by type I 

or type III secretion systems are able to directly affect nodulation in a variety 

of legume-rhizobia associations. 

5.2.1 

Type I Secretion Systems 

Many Gram-negative bacteria utilise type I [or ATP-binding cassette (ABC)] 

secretion systems (T1SS). Generally, the substrates of ABC transporters are 

toxins, proteases or lipases. All T1SS secreted proteins possess a carboxyterminal 

secretion signal of approximately 60 amino acids, which is not 

cleaved during export. The secretion machine consists of multimers of 

three proteins: an inner membrane exporter, an outer membrane pore, and 

an inner-membrane anchored protein that spans the periplasm linking the 

proteins found in the inner and outer membranes. Proteins can thus be 

directly secreted from the cytoplasm to the external environment without

74 M.M. Saad et al.


◮ Fig.5.1.Diagrammatic scheme describing four different types of protein secretion systems 

in Gram-negative bacteria. OM Bacterial outer membrane, IM bacterial inner membrane, 

PP periplasmic space, PM host plasma membrane. A number of proteins are involved in 

the assembling of the different secretion apparatus (indicated by spheres and ellipsoids). 

T2SS (type II secretion system) and T4SS (type IV secretion system) are Sec-dependent, 

thus proteins to be exported from the bacterial cell are first transported to the periplasm by 

the Sec system. The direction of this two-step secretion is shown by the black arrow, from 

the periplasm to the external environment. T4SS can also export other protein substrates 

directly from the cytosol, which does not require the Sec system. T1SS (type I secretion 

system) and T3SS (type III secretion system) are Sec-independent, transporting proteins 

directly from the bacterial cytoplasm to the external environment or into eukaryotic cells 

(for T3SS). Extracellular appendages are known to be components of several T3SS and 

T4SS. Secreted proteins are represented by black circles if they are exported from the 

periplasm (Sec-dependent) or black squares if they originate in the bacterial cytoplasm 

(Sec-independent) 

the formation of periplasmic intermediates (Hueck 1998; Thanassi and 

Hultgren 2000). 

T1SSs Involved in Symbiosis 

T1SSs have been found in a number of rhizobia, including Rhizobium 

leguminosarum bv. viciae and Rhizobium sp. BR816 (van Rhijn et al. 1996). 

NodO was first identified as a secreted protein in R. leguminosarum bv. 

viciae by de Maagd et al. (1989). The nodO gene is flavonoid inducible 

in a nod-box- and nodD-dependent manner (de Maagd et al. 1989; van 

Rhijn et al. 1996). NodO exists as a dimer in its native form, with an 

approximate molecular mass of 67 kDa (Sutton et al. 1994, 1996). The 

amino acid sequence of NodO contains putative repeated Ca 2+ -binding 

domains in the amino-terminal region, and has homology to a number 

of pore-forming bacterial toxins known as RTX proteins (Economou et 

al. 1990). Studies in vitro have shown that purified NodO forms cationselective 

pores in plasma-membrane lipid bilayers (Sutton et al. 1994). By 

analogy, NodO may thus form a pore in the root cell membrane, causing an 

influx of Ca 2+ ,whichcouldactasasecondmessengertherebystimulating 

the cytoskeletal changes required for infection thread growth. 

Mutation of the nodO gene has little effect on nodulation (Downie and 

Surin 1990), although double mutants of nodO and those involved in Nodfactor 

synthesis display clear Nod − phenotypes. This was unexpected as 

NodO is not involved in the biosynthesis or export of Nod-factors (Spaink 

et al. 1991; Sutton et al. 1994). Yet on Pisum sativum and Vicia sativa 

for example, NodO and a functional nodE are necessary for nodulation 

(Downie and Surin 1990; Economou et al. 1994). As infection threads appear 

to abort in the nodO/nodE doublemutant,itispossiblethatNodE(an


α-keto-acyl-synthase) is also involved in acylation of some components of 

the infection thread. Furthermore, mobilising nodO into different rhizobia 

extends the host range of the trans-conjugant (e.g. nodO into a nodE 

mutant of R. leguminosarum bv. trifolii allows transconjugants to nodulate 

V. sativa; Economou et al. 1994), thus perhaps pointing to a host-specific 

role for NodO. Another example of nodO complementing a defect in Nodfactor 

synthesis was shown when a nodO homologue of Rhizobium sp. 

strain BR816 was used to complement a nodS mutant of NGR234 for the 

nodulation of Leucaena leucocephala (van Rhijn et al. 1996). Again, NodO 

and NodS have distinct biochemical functions: NodS is a N-methyl transferase 

that methylates Nod-factors (Geelen et al. 1995; Jabbouri et al. 1995). 

The BR816 nodO gene was also shown to suppress the nodulation defect 

of the nodU mutants of NGR234 and R. tropici CIAT899 on L. leucocephala 

and of the nodE mutant of R. leguminosarum bv. trifolii ANU842 on white 

clover (Vlassak et al. 1998). 

Based on the observation that over-expression of nodO rescues nodulation 

by the multiple mutant nodFEMNTLO (of R. leguminosarum bv. 

viciae), Walker and Downie (2000) proposed a role for NodO in the complementation 

of Nod-factor defects. This mutant produces Nod-factors that 

are devoid of decorations and results in abortion of infection-thread development 

in Vicia sativa. They suggested that NodO stimulates ion flow 

across the cell membrane, thereby amplifying a weaker-than-normal signal 

transmitted by the undecorated version of Nod-factor. 

NodO is not the only T1SS Protein Involved in Symbiosis 

Although the phenotype of a R. leguminosarum nodO mutant was Fix + 

on P. sativum, inactivation of the type I system that secretes NodO results 

in Fix − nodules (Economou et al. 1994). It is thus possible that the T1SS 

is capable of secreting other proteins that play important roles in nodulation. 

Two such proteins were identified as PlyA and PlyB, two similar 

enzymes that function as extra-cellular glycanases, which are involved in 

processing rhizobial EPS (Finnie et al. 1998). ExpEI is secreted in a type 

I-dependent fashion by Rhizobium meliloti. Like the PlyAB proteins of 

R. leguminosarum, ExpEI is thought to be involved in the extra-cellular 

processing of EPS (Becker et al. 1997; Moreira et al. 2000). Thus it seems 

that proteins secreted via T1SSs in rhizobia play indirect roles in symbiosis, 

perhaps by amplifying or modifying other signal molecules. In this 

way they could increase ion flux following pore formation in plasma membranes, 

as proposed for NodO, or by augmenting the amounts of the active 

forms of EPS, as is possibly the case with ExpE1, and the PlyAB enzymes.


5.2.2 

Type II Secretion Systems 

A wide variety of Gram-negative bacteria utilise type II secretion systems 

(T2SSs) as a stepwise process to export proteins from the periplasm across 

the outer membrane. The amino-terminal signal peptides of the secreted 

proteins are first recognised and then translocated by a sec-dependent 

mechanism through the inner membrane. The signal peptide is cleaved, 

releasing the protein into the periplasm (Pugsley 1993; Sandkvist 2001). The 

T2SS is also called the general secretory pathway (GSP), and is responsible 

for the secretion of a large variety of degradative enzymes and toxins. T2SSs 

are composed of a core of between 12 and 15 proteins (Fig. 5.1), not all of 

which are present in every T2SS, as some appear to be dispensable for 

secretion (Sandkvist 2001). The core proteins are thought to form a multiprotein 

complex, spanning the periplasmic compartment that is specifically 

required for the translocation of any secreted proteins across the outer 

membrane (Sandkvist 2001; Peabody et al. 2003). In rhizobia, there is no 

clear evidence that any T2SSs play a role in symbiosis or nodule formation. 

Interestingly, the type II secretion machine shares many features with 

the type IV pilus biogenesis system found in many Rhizobium species, e.g. 

Bradyrhizobium japonicum USDA110, Mesorhizobium loti MAFF303099, 

R. meliloti and NGR234 (Kaneko et al. 2000, 2002; Galibert et al. 2001; Streit 

et al. 2004). Type IV pili are found on the surface of many Gram-negative 

bacteria, where they play an important role in bacterial adhesion to host 

cells, bio-film formation and conjugative DNA transfer (Wolfgang et al. 

2000). Nitrogen fixing bacteria of the genus Azoarcus utilise type IV pili to 

colonise grasses (Dörr et al. 1998). It remains to be seen whether rhizobia 

usetypeIVpiliduringthesymbioticinteractionwithlegumes. 

5.2.3 

Type III Secretion Systems 

Type III secretion systems (T3SSs) are characteristic of pathogenic Gramnegative 

bacteria, where their function is to inject proteins into the cytoplasm 

of eukaryotic cells, so facilitating the onset of disease. The T3SS 

is composed of a complex of about 20 proteins that spans both bacterial 

membranes (Fig. 5.1). Ten of these proteins are highly conserved in all 

T3SS-possessing bacteria and even show similarities to components of the 

flagella assembly apparatus, from which the pathogenic T3SS are thought 

to have evolved (Hueck 1998). Proteins that are secreted by T3SSs can be 

separated into four classes based on their functions. Some of them polymerise 

into extra-cellular components of the secretion apparatus forming


pili. There are also effector proteins that are actually injected into the 

cytosol of host cells, which then modulate cellular functions of the host 

by interfering with signalling cascades or disrupting the cytoskeleton. The 

third class of secreted proteins is termed translocators, and they polymerise 

to form a pore in the membrane of eukaryotic cells that allows the effectors 

to pass into the cells (Hueck 1998; Feldman and Cornelis 2003). Finally, in 

certain T3SS-possessing bacteria, secreted regulatory proteins that control 

cell contact-dependent secretion have also been identified (He 1998; Hueck 

1998). Proteins secreted by a T3SS do not require the sec system for their 

transit from the bacterial cytoplasm to the eukaryotic cell, although the sec 

pathway might be required for assembly of the type III secretion apparatus 

within the bacterial membranes. (Several components of the apparatus 

carry sec-characteristic amino-terminal signal sequences; Hueck 1998). 

Identification and Function of T3SSs in Rhizobia 

Given their importance in pathogenicity, it was a surprise to find T3SSs in 

symbiotic rhizobia. A complete T3SS was first identified in Rhizobium sp. 

NGR234 (Freiberg et al. 1997). All ten genes encoding the conserved components 

of T3SSs were found and named rhc (Rhizobium conserved) but 

thesamefinalletterasusedtodescribepathogenicT3SSgeneswasmaintained 

(Viprey et al. 1998). Sequencing other large replicons of B. japonicum 

USDA110 (Göttfert et al. 2001; Kaneko et al. 2002) and M. loti MAFF303099 

(Kaneko et al. 2000) also revealed the existence of loci encoding T3SSs. 

T3SSs are not ubiquitous in rhizobia, however, as no such system was 

found within the completed genome of R. meliloti (Galibert et al. 2001). 

Random mutagenesis also identified mutants of R. fredii strains that were 

affected in nodulation. Subsequent analysis of the insertion sites showed 

them to be within T3SSs (Marie et al. 2001; Krishnan et al. 2003). In fact, 

mutagenesis of T3SSs of rhizobia proved that although they are not absolutely 

essential for nodulation of all legumes, they have cultivar-specific 

effects and thus can be viewed as determinants of rhizobial host-range 

(Marie et al. 2001). This is exemplified by studies on the T3SS of NGR234 

(Viprey et al. 1998). In NGR234, the T3SS genes are grouped within a 30-kb 

region of the symbiotic plasmid pNGR234a (Freiberg et al. 1997). Knockout 

mutants in the T3SS machine of NGR234 cause three host-dependent 

effects. As compared to the wild-type rhizobia, there can be a dramatic impairment 

of nodule development e.g. on Tephrosia vogelii, resulting in the 

formation of a majority of non-fixing pseudo-nodules. Second, a dramatic 

enhancement of nodulation is often seen, as with Crotalaria juncea and 

Pachyrhizus tuberosus, while a third group of legumes seems to be unaffected 

by the presence/absence of a functional T3SS (e.g. Lotus japonicus 

and Vigna unguiculata; Marie et al. 2003; Viprey et al. 1998).


Regulation of Rhizobial T3SSs 

In rhizobia, transcription of the T3SS-related genes requires the presence 

of flavonoids and two bacterial regulatory elements: NodD1 and TtsI 

(Krishnan et al. 1995; Viprey et al. 1998; Krause et al. 2002). TtsI shares 

homology with transcriptional activators of the two-component sensorregulator 

family (Viprey et al. 1998). The gene encoding TtsI is found within 

the T3SS loci of rhizobia and is preceded by a nod-box. It has been shown 

that, after flavonoid activation, NodD1 induces ttsI transcription, which in 

turn activates genes within T3SS loci (Viprey et al. 1998; Kobayashi et al. 

2004). Induction of the T3SS genes occurs after induction of genes involved 

in Nod-factor synthesis, implying that the T3SS functions after Nod-factors 

in the symbiosis. A conserved promoter motif called the tts-box has been 

identified upstream of most T3SS regulons (Krause et al. 2002). Although 

it has not been demonstrated experimentally, it is thought that TtsI may 

bind to tts-boxes to induce transcription of the downstream genes. Transcriptional 

studies suggest that the T3SS of NGR234 does not function 

throughout the symbiosis, as the majority of T3SS gene transcripts could 

not be detected in nodules of V. unguiculata and Cajanus cajan (Perret et 

al. 1999). 

Functions of Proteins Secreted via Rhizobial T3SSs 

Protein secretion by T3SSs of rhizobia has been demonstrated in vitro 

for R. fredii USDA257 and NGR234. Proteins secreted in a T3SS-dependent 

manner are called Nops (nodulation outer proteins) (Marie et al. 2001). Five 

Nops are known to be secreted by USDA257 (Krishnan and Pueppke 1993) 

and at least eight by NGR234 (Marie et al. 2003). Several of these Nops have 

been identified and subsequently characterised. Functional studies have 

shown that rhizobial Nops can be placed into three of the general classes 

of type-III-secreted proteins. Figure 5.2 summarises the role of the Nops 

within a rhizobial T3SS. 

External Components of the Machinery 

Some type-III-secreted proteins of phyto-pathogenic bacteria have been 

shown to polymerise and form pili. These pili serve to connect the bacterium 

to the plant cell and allow proteins to pass through the hollow pili. 

As protein secretion is abolished in mutants of pili-genes, their phenotype 

resembles that produced by knock-outs of the T3SS machine (He and 

Jin 2003). In the presence of flavonoids, USDA257 produces extra-cellular 

appendages (pili), and requires a functional T3SS (Krishnan et al. 2003). 

Preliminary results indicate that NGR234 also produces pilus-like structures 

on its surface in a flavonoid and T3SS-dependent manner. These appendages 

were purified and shown to be composed predominantly of NopA


Fig.5.2. Hypothetical structure of the T3SS of Rhizobium sp. NGR234, adapted from Viprey 

et al. (1998) and Bartsev et al. (2004b). The conserved components (Rhc proteins) of the 

T3SS form a channel through the bacterial inner and outer membranes. The known roles 

of the Nops are also illustrated. NopA and NopB are thought to be the major components 

of a T3SS-dependent pilus that links the rhizobial cell to the plant cell. Nops are secreted 

through the pilus and can thus cross the plant cell wall. NopX may polymerise to form 

a pore in the plant root cell plasma membrane and, finally, NopL and NopP are possible 

effector proteins that function within the plant root cell. NGR234 probably secretes many 

other effector proteins 

(W.J. Deakin, unpublished data), the smallest protein secreted by NGR234 

(Marie et al. 2003). Furthermore, homologues of nopA have been identified 

in all T3SS-possessing rhizobia, suggesting that NopA is an essential 

component of the secretion machinery. Mutation of nopA also blocks the secretion 

of all the other Nops. This phenotype resembles that of mutations in 

other genes that encode external components of the T3SS. Although NopA 

is the major component of the rhizobial T3SS-pili, mutations in other genes 

that encode Nops also block Nop secretion, suggestion that other, minor 

components of the external secretion apparatus might exist (unpublished 

data). 

Translocators 

NopX, one of the first Nops to be identified in NGR234 (Viprey et al. 1998), 

has significant homology to a number of proteins secreted by T3SSs of


phytopathogens. Perhaps the best studied of these is HrpF of Xanthomonas 

campestris pv. vesicatoria (Huguet and Bonas 1997). HrpF is secreted in 

a T3SS-dependent manner and may function as a translocator of the effectors 

proteins into host cells (Rossier et al. 2000). Indeed, HrpF has 

been shown to form pores in lipid bilayers (Büttner et al. 2002) We have 

thus proposed that NopX could perform the role of translocator for rhizobial 

T3SSs (Marie et al. 2003). NopX of USDA257 has been localised to 

the infection threads, where it could play a role in the infection thread 

growth (Krishnan 2002). Although nopX homologues are present in most 

T3SS-possessing rhizobia, there does not appear to be a homologue in 

B. japonicum USDA110 (Krause et al. 2002). This is extremely puzzling as 

the translocon is thought to be essential for the transport of T3SS proteins 

into eukaryotic cells. 

Effectors 

This group of secreted proteins are thought to function within the root 

cells. So far, only one example of a rhizobial T3SSs effector has been identified 

(NopL), although it is suspected that there could be many more. 

Homologues of nopL are found only in T3SS-possessing rhizobia, although 

M. loti MAFF303099 does not appear to have a copy. Mutations in genes 

encoding effector proteins do not affect the secretion of any other T3SS proteins, 

and this was shown to be the case for a mutation in nopL of NGR234 

(Marie et al. 2003). A nopL mutant has a similar nodulation phenotype 

toNGR234onthemajorityoflegumestested.Thisisanothercharacteristicofeffectorproteinsofphytopathogens,fortherearemanyofthem 

and they are thought to be redundant in function. NopL is important for 

the efficient nodulation of Flemingia congesta (Marie et al. 2003), suggesting 

that it is a rhizobial “virulence factor” for this plant. Functional 

characterisation of NopL revealed that it can be phosphorylated by plant 

kinases (Bartsev et al. 2003). Furthermore, expression of nopL in Nicotiana 

tabacum inhibited this plant’s ability to accumulate pathogenesisrelated 

(PR)-proteins in response to pathogen attack. We thus suggest that 

NopL could suppress root-cell defence responses by disrupting the intracellular 

signalling cascades required for activation of PR-genes (Bartsev et 

al. 2004a). 

Sequence analyses of NGR234 and USDA110 revealed the presence of 

geneshomologoustosecretedeffectorproteinsfromotherT3SS-possessing 

pathogenic bacteria. The proteins encoded by these rhizobial genes are thus 

good candidates for secretion in a T3SS-dependent manner, and possibly 

function as effectors within legume root cells (Marie et al. 2001, Krause 

et al. 2002). These proteins have been studied extensively in pathogenic 

bacteria, where they act to suppress host defence responses, and it will be


interesting to determine whether their rhizobial counterparts function in 

a similar manner. 

The Role of Rhizobial T3SSs 

At this stage, explanations for the roles of T3SSs in rhizobia can only be 

hypothetical. It is thought that the T3SS functions during infection thread 

development and perhaps while the rhizobia are being released from infection 

threads into cortical cells. Similarities between attempts by rhizobia 

to colonise roots and the attack by pathogens of other plant cells are obvious. 

Undoubtedly, legumes mount defences against rhizobia. NGR234 

and similar rhizobia may have acquired a T3SS to suppress such defence 

responses, and effectors like NopL are the inter-cellular messengers in this 

process. Legumes, like most other plants have probably evolved sophisticated 

methods for detecting T3SS-containing pathogens. To some plants, 

NGR234 (and similar rhizobia) would reveal themselves as “pathogens” 

provoking defence-responses that block nodulation (e.g. P. tuberosus). In 

this scenario, other legumes would have evolved immunity to T3SS proteins 

(L. japonicus, V. unguiculata), while still others would positively welcome 

them (e.g. T. vogeli). Furthermore, it is possible that rather than responding 

in one of three ways to T3SS-proteins, a continuum of responses exists, 

some too slight to be detected. Other possibilities include that the T3SS is 

for some reason not activated by certain plants, or that some plants possess 

other signalling systems that over-ride or complement the T3SS. 

5.2.4 

Type IV Secretion Systems 

Type IV secretion systems (T4SSs) were initially defined on the basis of 

the homologies between components of three different macromolecular 

complexes: the Agrobacterium tumefaciens T-DNA transfer system that is 

required for exporting oncogenic T-DNA to susceptible plant cells; the conjugal 

transfer (Tra) system of the conjugative IncN plasmid pKM101; and 

the Bordetella pertussis toxin exporter (Ptl) machine (Winans et al. 1996; 

Christie 1997). Like T2SSs, T4SSs use a stepwise process to translocate 

macromolecular substrates first across the inner membrane, prior to transport 

across the cell envelope (Christie 2001). Some symbiotic nitrogenfixing 

bacteria also possess genes that could encode a T4SS e.g. R. etli, 

M. loti strain R7A and R. meliloti (Galibert et al. 2001; Sullivan et al. 2002; 

Gonzalez et al. 2003). It is interesting to note that in M. loti R7A the location 

of the genes that may encode a T4SS is exactly at the T3SS locus of 

M. loti MAFF303099 strain, although each strain possesses only one type of 

secretion machine. The role of T4SSs in symbiosis is not known, but there


is a suggestion that it could affect the nodulation process, as a putative 

nod-box is located in the promoter region of one of the genes of the R7A 

T4SS (Sullivan et al. 2002). 

5.3 

Conclusions 

Successful symbiotic associations between rhizobia and legumes require 

the exchange of many signal molecules. Both partners secrete these signals 

and it is the timing of their emission and perception as well as the quantity 

that are probably important. Symbiotic harmony depends on the precise 

meshing of these signals. Plant flavonoids are the first important group of 

signalling molecules and they act as inducers of nodulation genes (nod, 

noe and nol) (Broughton et al. 2000; Perret et al. 2000). The regulatory 

networks of flavonoids, the NodD family of transcriptional activators, and 

their conserved promoter sequences (nod-boxes) guarantee the timing of 

expression of downstream genes that are responsible for the synthesis of 

diffusible lipo-chito-oligosaccharidic Nod-factors – early symbiotic “master 

keys”. Once the legume “doors” have been opened to allow rhizobia in, 

different morphological and cytological changes in the roots occur. Nodfactors 

play only secondary roles in the later steps of invasion, at which 

time other signal molecules occupy centre stage. Bacterial SPSs and secreted 

proteins contribute to the infection process, where they assist in 

infection thread development within the root hair, and help modify hostdefence 

mechanisms. 

Acknowledgements. We thank D. Gerber for general support and encouragement. 

Research in LBMPS is financed by the Founds National Suisse 

de la recherché Scientifique (Project 31-63893.00) and the Université de 

Genève. 


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6 

Research on Endophytic Bacteria: 

Recent Advances with Forest Trees 

Richa Anand, Leslie Paul, Chris Chanway 

6.1 

Introduction 

Plantscanbeconsideredascomplex microecosystemsthatprovidedifferent 

habitats to a variety of microorganisms. These habitats are represented 

by the plant external surfaces as well as internal tissues (McInroy and 

Kloepper 1994). Whereas the importance of microbial colonisation of plant 

surfaces in plant growth promotion has been well understood for a long 

time, interior tissue colonisation was, until recently, largely perceived as 

being related only to the perpetuation of systemic diseases. It is now well 

known that tissues of healthy plants are also colonised internally by various 

microorganisms that establish neutral or, more interestingly, beneficial 

interactions with their host plants. The term “endophyte” is commonly 

used to describe such microorganisms. 

Although a variety of definitions have been applied to the term “endophyte”, 

it refers mainly to bacteria and fungi that live inside plant tissues 

without causing disease (Wilson 1995; see Chap. 1 by Schulz and Boyle). 

Whether or not latent pathogens can be considered endophytes has been 

a major topic of debate in the general acceptance of this definition (Misaghi 

and Donndelinger 1990; James and Olivares 1997; see Chap. 1 by Schulz 

and Boyle). 

The best-characterised microbial endophytes are fungi of the Balansiaceae, 

for which the most compelling evidence of plant–microbe mutualism 

has been provided (Clay 1988; Schardl et al. 2004). Some of the 

non-balansiaceous endophytic fungi are also mutualistic with their hosts 

(Carroll 1988; see Chap. 15 by Schulz), and produce compounds that render 

plant tissues less attractive to herbivores, while other strains may increase 

Chris Chanway: Faculty of Land and Food Systems, Faculty of Forestry, University 

of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4, E-mail: 

cchanway@interchg.ubc.ca 

Richa Anand, Leslie Paul: Faculty of Land and Food Sciences, Systems, University of British 

Columbia, Vancouver, British Columbia, Canada V6T 1Z4 

Leslie Paul: Department of Forest Mycology and Pathology, Swedish University of Agricultural 

Sciences (SLU), Box 7026, 750 07 Uppsala, Sweden (Current Address) 





90 R. Anand et al. 

host plant drought resistance. In return, fungal endophytes are thought to 

benefit from the comparatively nutrient rich, buffered environment inside 

plants. 

Apart from fungi, bacteria belonging to various genera have also been 

shown to exist inside plants without causing apparent disease symptoms. 

Some of these bacteria are known to impart benefits to their host plants 

by the same mechanisms as their soil- or rhizosphere-colonising counterparts. 

The primary mechanisms thought to lead to beneficial effects for 

the plant are nitrogen fixation (Boddey and Döbereiner 1995) and biocontrol 

of pathogenic and detrimental microorganisms, either through 

direct antagonism of pathogens or by inducing systemic resistance to such 

organisms (Hallman et al. 1997). Other known mechanisms by which beneficial 

bacteria can have a positive influence on plant performance are the 

production or stimulation of plant growth hormones and facilitation of 

nutrient uptake [see Chaps. 3 (Kloepper and Ryu) and 4 (Berg and Hallmann)]. 

Since their first reported isolation from potato plants (Tervet and Hollis 

1948; Hollis 1951), all the information available on endophytic bacteria has 

been derived from studies on plant species of agricultural and horticultural 

importance. The endophytic bacteria of rice (Reinhold-Hurek and Hurek 

1998), corn (Triplett 1996) and sugarcane (Döbereiner et al. 1995) are by 

far the best studied so far. In contrast to these crop species, very much 

less is known about bacterial endophytes of trees. Some trees survive and 

grow well in very difficult terrain under extreme conditions, for example 

lodgepole pine (Pinus contorta Dougl. var. latifolia)indryinteriorregions 

of British Columbia and western Alberta, Canada, as well as Tecomella 

undulata (Bignoniaceae) in the extremely arid deserts of northwestern 

India. It is possible that endophytic bacteria that enhance host survival and 

growth in exchange for protection in the relatively buffered environment of 

internal plant tissues may be involved under such extreme environmental 

conditions (Law and Lewis 1983). 

Although the realisation of this possibility has led to occasional reports 

of endophytic bacteria in asymptomatic angiosperm and gymnosperm tree 

species, little is known about their diversity and influence on plant growth. 

The earliest report of bacterial endophytes from trees was from Gardner 

et al. (1982), who isolated representatives of 13 genera from xylem fluid of 

rough lemon rootstock, and found population sizes ranging from 10 2 –10 4 

colony forming units (cfu) g −1 xylem fluid. Only 48 of the 850 isolates 

turned out to be phytopathogenic, but the role of the other 802 isolates was 

not determined. Similarly, several strains of Pseudomonas syringae were 

isolated and characterized from inside pear seedlings by Whitesides and 

Spotts (1991), but their exact role could not be determined.

6 Research on Endophytic Bacteria: Recent Advances with Forest Trees 91 

The procedures of isolation and identification of endophytic bacteria 

fromtreesisthesameastheirisolationfromagronomiccrops,andsuffers 

from the same limitations observed in crop species, e.g. the difficulty, or 

perhaps impossibility, of absolute surface sterilisation of external plant 

tissues as well as our inability to culture many bacteria we know to exist. 

The impact of these problems can be reduced by the use of standardised 

protocols and molecular techniques (James 2000; see Chap. 17 by Hallmann 

et al.). The major difficulty, therefore, lies in the evaluation of the effects of 

these bacteria on their host trees, owing to the long tree life-cycle and a lack 

of detailed physiological information on trees, particularly forest trees. 

The following sections provide a detailed review of our current knowledge 

about bacterial endophytes of forest trees, the mechanisms by which 

they benefit their host plants, and their potential application in the practice 

of sustainable forestry. 

6.2 

Bacterial Endophytes of Forest Trees 

Although limited, the results of research on endophytic bacteria and their 

role in growth promotion of forest trees so far are very encouraging and 

will hopefully draw more attention to this developing area of study. Brooks 

et al. (1994) conducted an extensive study in which endophytic bacteria 

were isolated from surviving live oak (Quercus fusiformis)inTexas,where 

oak wilt is epidemic, and evaluated as potential biological control agents 

for the disease. Of the 889 bacterial isolates tested, 183 showed in vitro inhibition 

of the pathogen Ceratocystis fagacearum. Six isolates were further 

evaluated for colonisation of containerised Spanish oak (Quercus texana) 

and live oak. Interestingly, in containerised live oaks inoculated with the 

oak wilt pathogen, preinoculation with 15 isolates of Pseudomonas denitrificans 

reduced the number of diseased trees by 50% and decreased the 

percentage of crown loss by 17%. In a subsequent trial, no reduction in 

numbersofdiseasedtreeswasobservedbutpreinoculationwiththesame 

isolates of P. denitrificans or a strain of Pseudomonas putida significantly 

reduced crown loss. These results clearly established the potential of such 

endophytic bacteria as pre-plantation nursery treatments for wilt control. 

Several endophytic aerobic heterotrophic bacteria belonging to the genera 

Bacillus, Curtobacterium, Pseudomonas, Stenotrophomonas, Sphingomonas, 

Enterobacter, andStaphylococcus, havealsobeenisolatedfrom 

phloem tissue of roots and branches of elm trees (Ulmus spp.: Mocali 

et al. 2003). An attempt was also made to determine the correlation between 

the seasonal fluctuations in the structure of the endophytic bacterial


community and phytoplasma disease infection of these trees; however, no 

consequential effect of the bacterial community on phytoplasmosis of elm 

trees could be demonstrated (Mengoni et al. 2003). 

Apart from these studies on bacterial endophytes of deciduous trees, 

most other reports of endophytic bacteria and their role in tree growth promotion 

are from studies on various conifer tree species conducted mostly 

by our research group. Our interest in endophytic bacteria of conifers has 

been largely inspired by the immense commercial, social, and environmental 

importance of forestry in Canada and the rest of North America and the 

fact that conifers are the most dominant trees in the temperate forests of 

this region. 

6.3 

Endophytic Bacteria of Conifers 

Conifers are members of the plant division Coniferophyta (2 Domain classification), 

which are characterised by naked seeds borne in specialised 

sporophylls or cones. Their vascular tissues differ from angiosperms in 

not having xylem vessels, and companion cells in phloem. The division is 

comprisedof550speciesspreadoversevenfamilies,eachdatingbacktothe 

Mesozoic era. Distributed throughout the world with extensive latitudinal 

and longitudinal ranges, conifers are of great commercial and ecological 

value. 

Traditionally, fungi, particularly mycorrhizae, were considered to be the 

only microorganisms that could exert a positive influence on the growth 

and survival of forest trees. The continuity of this trend until now is evident 

from the results of keyword searches for “endophytic bacteria + conifers” 

in all well known scientific databases. 

Although some confirmed reports of conifer tree growth promotion by 

naturally occurring soil and rhizosphere bacteria were available (Pokojska- 

Burdziej 1982; Chanway and Holl 1992, 1993, 1994; O’Neill et al. 1992), the 

mechanisms employed by these bacteria for growth promotion could not be 

determined. It was generally believed that the primary mechanism of plant 

growth promotion by these bacteria was only indirect, i.e. by facilitating 

the establishment and growth of mycorrhizae (Fitter and Garbaye 1994). 

The focus of research on endophytic microflora of conifers thus remained 

on fungi, even after the importance of endophytic bacteria had been well 

established in agronomic crop species. 

In an initial study of conifer root-associated bacteria, O’Neill et al. (1992) 

isolated 22 strains from surface-sterilised roots of naturally regenerating 

white x Engelmann (Picea glauca x P. engelmannii) hybrid spruce seedlings. 

A range of effects on seedling growth in a greenhouse-screening assay


using spruce were found: three strains were inhibitory, five strains were 

stimulatory and the remaining strains had no significant effect on seedling 

growth (O’Neill et al. 1992). Based on the magnitude and consistency of 

seedling growth effects, the two best plant growth-promoting endophytes 

were identified and selected for further study: one isolate was Pseudomonas 

putida and the other belonged to Staphylococcus. While the positive effect 

of both of these strains on plant growth was reproducible in the greenhouse, 

a field trial with two ecotypes of 1-year-old spruce seedlings planted at three 

different reforestation sites yielded mixed results (Chanway and Holl 1993). 

For example, P. putida enhanced seedling growth of only one of two spruce 

ecotypes planted at two of three reforestation sites. In addition, it had 

inhibitory effects in three of the spruce ecotype x planting site treatment 

combinations. 

Evaluation of gymnosperm bacterial endophytes was only a small part 

of a larger project designed to characterise gymnosperm root-associated 

bacterial (i.e. external and internal) colonists (O’Neill et al. 1992; Chanway 

and Holl 1992, 1994). Therefore, our group undertook a subsequent bacterial 

isolation and screening program emphasising endophytic bacteria 

as possible tree seedling growth-promoting agents (Chanway et al. 1994, 

1997). As seen in our earlier work (O’Neill et al. 1992), several bacterial 

strains isolated from surface-sterilised roots of white x Engelmann hybrid 

spruce seedlings caused reproducible spruce seedling biomass increases of 

up to 36% 2 months after seed was sown and inoculated in greenhouse 

trials (Chanway et al. 1994). Three of these strains belonged to Paenibacillus, 

three were actinomycetes, most likely Streptomyces spp., and one was 

a Phyllobacterium. An additional strain that performed well in greenhouse 

assays could not be identified with certainty. 

In addition, the seedling growth promotion efficacy of some of these 

strains was altered significantly when assays were conducted in the presence 

of a small amount (2% v/v) of forest soil known to contain seedling growth 

inhibiting organisms (i.e. minor pathogens). One of the endophytic actinomycetes 

(isolate W2) as well as the Phyllobacterium isolate (W3) clearly 

stimulated spruce seedling growth only in the absence of forest soil. In its 

presence, seedling growth was inhibited, as it was when forest soil alone 

was used. These results suggested that growth promotion by W2 and W3 

occurred via a mechanism unrelated to biocontrol of minor pathogens, and 

mayhaveinvolvedoneofthedirectplantgrowthpromotionmechanisms, 

possibly production of plant growth regulators (Kloepper 1993; Glick 1995; 

Chanway 1997). Interestingly, actinomycete isolate N1 and Bacillus isolate 

N4 stimulated seedling growth only in the presence of forest soil, which 

suggested that these strains acted through a biocontrol mechanism, either 

by direct antagonism or by inducing systemic resistance in the host plant. 

Elucidation of these possibilities requires further experimentation.


We have also looked for bacterial endophytes in lodgepole pine. After isolation 

of several bacterial strains and screening trials for effects on seedling 

growth, we identified a plant-growth-promoting Bacillus polymyxa (now 

Paenibacillus, Ash et al. 1993) strain (Pw2) that originated from internal 

root tissues of a naturally regenerating 2- to 3-year-old pine seedling 

(Shishido et al. 1995). Our studies indicate that Pw2 can colonise external 

and internal pine and spruce root tissues after seed or root inoculation. 

Colonisation of internal root tissues may depend on lateral root development, 

and results in endophytic bacterial population sizes approaching 

10 6 cfu g −1 fresh root tissue (Shishido et al. 1995; Chanway 1997; Shishido 

1997). In addition, using a surface-sterilisation, dilution plating assay as 

well as immunofluorescence microscopy, a rifamycin-resistant derivative 

of this strain, Pw2-R, was shown to be capable of colonising internal pine 

and white x Engelmann hybrid spruce stem tissues after soil or root inoculation 

(Chanway et al. 2000). Five months after root inoculation, internal 

stem bacterial populations reached 10 5 cfu g −1 fresh stem tissue (Shishido 

1997). 

In order to examine the effects of endophytes on conifer plant growth 

and to investigate the host specificity of bacterial endophytes in terms of 

the ability to promote growth of inoculated host plants other than the ones 

from which they were initially isolated, initial field trials with P. polymyxa 

strain Pw2-R and Pseudomonas chloroaphis strain Sm3-RN, another bacterial 

endophyte capable of stimulating white x Engelmann hybrid seedling 

growth in the greenhouse (Chanway et al. 1997), were also performed. 

Two years after bacterial inoculation and planting at nine sites in British 

Columbia and Alberta, Canada, white x Engelmann hybrid spruce treated 

with strain Pw2-R (initially isolated from pine) showed mean biomass increases 

up to 33% above controls at seven of the nine sites, but increases 

were significant at only one site. In contrast, Pseudomonas strain Sm3-RN 

(isolated from white x Engelmann hybrid spruce) caused significant white 

x Engelmann hybrid spruce biomass increases of up to 57% at three of 

the nine sites but a significant decrease in spruce biomass at one site. Site 

productivity was not correlated with plant growth promotion or inhibition. 

Population sizes of Pw2-R and Sm3-RN were generally below the assay 

detection limit of ca. 10 2 cfu g −1 plant tissue, which led us to question how 

effectively internal plant tissues were colonised at the onset of the experiment. 

Therefore, we also evaluated seedlings that were inoculated with 

strainsPw2-RandSm3-RNandgrowninthegreenhousefor4months 

before planting at four of the reforestation sites described above (Shishido 

and Chanway 2000). The period of growth in the greenhouse facilitated 

internal tissue colonisation by these microorganisms so that mean internal 

root populations reached ca. 10 3 –10 4 cfu g −1 tissue in the greenhouse. As


expected, mean seedling biomass also increased due to bacterial inoculation 

in the greenhouse. Because seedling growth responses in the field 

would be inseparable from those that occurred in the greenhouse, simple 

measurement of biomass accumulation after a period of growth in the 

field would yield spurious results. We evaluated plant growth using relative 

growth rates (RGRs), in which plant growth increments over time are expressed 

as a proportion of the biomass that existed at some previous time 

in the plant’s life (Hunt 1982). 

In general, after the first growing season, RGRs of seedlings containing 

endophytic bacteria were greater than those of control seedlings at all four 

planting sites (Shishido and Chanway 2000). In some cases, RGRs of inoculated 

plants were double the control value. This was particularly interesting 

inviewofresultswithseedlingsthatweinoculatedandplantedimmediately 

at the same sites. At two of the four sites, seedlings inoculated at the time 

of planting (i.e., with no greenhouse growth period) did not respond to 

bacterial treatment, and in one case responded negatively. However, shoot 

androotRGRsofseedlingspretreatedinthegreenhousebeforeplantingat 

the same sites were 23–132% greater than controls, and endophytic populations 

in root tissues of between 10 2 and 4×10 4 cfu g −1 plant tissue were 

detected in seedlings at three of the four sites. Similar effects on establishment 

and functioning of bacterial endophytes were observed by Brooks 

et al. (1994) in wilt-infested oak trees. These results suggest that a period 

of growth under a controlled environment to facilitate establishment of 

endophytic bacterial populations may be an important step in successful 

application of plant-growth-promoting bacterial endophytes in forestry. 

It has also been demonstrated that the benefits of pre-outplanting inoculation 

of seedlings with bacterial endophytes can be maximised by careful 

matching of the inoculant bacterial strain with outplanting sites (Chanway 

et al. 2000). However, much research into site quality and plant growth 

responseswillberequiredbeforereliablerecommendationscanbemade. 

In addition, much more research is warranted to answer the many questions 

regarding the entry and operation of endophytic bacteria in conifers, 

some of which are being actively pursued by our group. 

6.4 

ModesandSitesofEntry 

Endophytic bacteria have been shown to be able to gain entry in plants 

through wounded as well as intact tissues (Sprent and James 1995; see 

Chap. 18 by Bloemberg et al.). In an attempt to understand the modes and 

sites of entry of endophytic bacteria, Timmusk and Wagner (1999) followed 

the colonisation of a green fluorescent protein (gfp)-tagged endophytic


strain of Paenibacillus polymyxa in Arabidopsis thaliana; theyobserved 

aslightdegradationoftheroottipswithin5hofinoculation.Itwasfound 

that P. polymyxa hadtwopreferredzonesofinfection.Thefirstislocated 

at the root tip in the zone of elongation, which sometimes results in the 

loss of the root cap. The other colonisation region was observed in the 

differentiation zone. Similar colonisation zones have been reported for 

other endophytes, e.g. Azoarcus by Hurek et al. (1994), who suggested 

that plant cells were destroyed after bacteria had penetrated cell walls. 

Perhaps this is the reason why most endophytic bacteria are limited to 

the intercellular spaces inside tissues. However it is not clear how they are 

stopped from entering cells and causing necrosis. 

To determine which microbial characteristic(s) facilitate entrance of 

bacterial endophytes into plant tissues, we compared the biochemical capabilities 

of the endophytic Paenibacillus polymyxa strain Pw2 with those 

of another plant-growth promoting, non-endophytic strain, P. polymyxa 

L6-16R. Interestingly, strain L6-16R is unable to enter plant tissues even 

when co-inoculated with an endophytic microorganism (Shishido et al. 

1995; Bent and Chanway 1997). According to Biolog analysis, both strains 

possessed similar metabolic capabilities with some potentially important 

exceptions (Shishido et al. 1995). For example, strain Pw2-R was able to 

metabolise sorbitol, but strain L6-16R was not. Mavingui et al. (1992) found 

that, in general, P. polymyxa strains isolated from the rhizoplane of wheat 

(Triticum aestivum L.) were capable of metabolising sorbitol while rhizosphere 

and non-rhizosphere isolates were not. They hypothesised that 

intense competition for oxygen would occur on the root surface due to root 

respiration, which would result in selection pressure for bacteria capable 

of anaerobic growth on highly reduced, scarce substrates, such as sorbitol. 

In addition, strain Pw2 was able to metabolise d-melezitose, a sugar that 

has been detected in the sap of conifers (Lehninger 1975). However, the 

occurrence of sorbitol and d-melezitose in lodgepole pine root tissues and 

their utilisation by other Paenibacillus root endophytes must be demonstrated 

before a role for these substrates in internal root colonisation by 

Paenibacillus can be postulated with greater confidence. 

To facilitate root colonisation, it is logical to suspect that root endophytic 

bacteria may also possess the ability to metabolise structural components of 

plant cells. In particular, the ability to metabolise pectin (polygalacturonic 

acids), a major component of the middle lamellae of plant cell walls, has 

been proposed to at least partly explain why bacterial root endophytes 

are often found in the root cortex intercellularly (Balandreau and Knowles 

1978; Baldani and Döbereiner 1980). Both strains L6 and Pw2 possessed 

pectolytic activity in vitro, but only strain Pw2 was able to metabolise dgalacturonic 

acid (Shishido et al. 1995), the primary monomeric component 

of pectin (Paul and Clark 1989).


It is not clear whether the capability of strain Pw2 to metabolise monomeric 

galacturonic acid after breakdown of the pectin polymer was related 

to its ability to enter root tissues. However, breakdown products of plant 

cell walls are known to induce systemic disease responses in plants (Brock 

et al. 1994), which leads to the possibility that Pw2 avoids plant defence 

mechanisms by metabolising cell wall components before they elicit a defence 

response by the host plant. This possibility also requires further 

investigation. 

If, in fact, the entry of Pw2 in plant roots is facilitated by its capability to 

metabolise the primary components of the cell wall, the question as to why 

it does not cause necrosis of interior tissues, also needs to be answered. 

6.5 

Mechanisms of Plant Growth Promotion 

Unlike symbiotic rhizobia, mechanisms of plant growth promotion by 

plant growth-promoting rhizobacteria (PGPR) vary greatly, and have been 

broadly categorised into two groups, direct and indirect (Kloepper et al. 

1989; see Chap. 3 by Kloepper and Ryu). Direct plant growth mechanisms 

may involve nitrogen fixation (Cavalcante and Döbereiner 1988), production 

of plant growth regulators and antibiotics, or increased availability of 

plant growth-limiting nutrients. Indirect mechanisms may involve suppression 

of deleterious microorganisms as well as enhancement of mutualisms 

between host plants and other symbionts such as mycorrhizae (Kloepper et 

al. 1989). Similar to other aspects of studies on endophytic bacteria, there is 

a great deal of information on the mechanisms of plant growth promotion 

employed by these bacteria in agronomic crops (Lodewyckx et al. 2002). 

In the case of conifers, it was generally believed that these plants could derive 

benefits from bacteria only indirectly through their mycorrhizal symbionts 

(Fitter and Garbaye 1994). However, growth studies on lodgepole 

pine seedlings (Chanway and Holl 1991; Shishido et al. 1996) and hybrid 

spruce (Picea glauca x P. engelmannii) (Shishido et al. 1996) co-inoculated 

with PGPR and mycorrhizal fungi have clearly shown that growth promotionoftheseconifersbyPGPRisindependentofthemycorrhizalstatusof 

the seedlings. 

Despite many efforts, determination of the exact mechanisms of conifer 

growth promotion by PGPR has not been possible. Paenibacillus polymyxa 

strain L6-16R was shown to produce cytokinins (Holl et al. 1988), and this 

propertywasadvancedasalikelyexplanationofpinegrowthpromotion 

mediated by this strain. 

A detailed study was also conducted to determine the mechanisms of 

growth promotion of spruce by six Paenibacillus and Pseudomonas strains,


including the endophyte, B. polymyxa Pw2 (Shishido 1999). It could only 

be concluded that more than one mechanism was responsible for growth 

promotion. Production of plant growth promoters and enhancement of 

nutrient uptake were designated as the most likely of these mechanisms. 

Strain Pw2 isolated from lodgepole pine (Shishido 1996) possessed diazotrophic 

properties. This led to the intriguing possibility that lodgepole 

pine harbours a systemically endophytic nitrogen-fixing bacterial population, 

similar to that found in sugar cane (Boddey et al. 1995), which would 

explain its ability to grow, and even thrive, under nitrogen-deficient conditions 

in the absence of significant rhizospheric nitrogen fixation (Binkley 

1995). Indeed, the 15 N/ 14 N ratio of pine foliage in a central coast forest 

in British Columbia devoid of nitrogen-fixing species was observed to be 

low enough to suggest that biological nitrogen fixation supplies plant N 

(F.B. Holl, personal communication). 

However, nitrogen fixation could not be shown to be the primary mechanism 

of growth promotion by P. polymyxa strain Pw2-R, since seedlings inoculated 

with it failed to support sufficient rhizosphere acetylene reduction 

activity (ARA) even after 48 h of incubation with acetylene (Shishido 1997). 

Interestingly, similar limitations were encountered by Rhodes-Roberts 

(1981) and Achouak et al. (1999) while working with other strains of 

P. polymyxa. However, they were able to measure the nitrogen gains of 

seedlings by microkjeldahl analysis, which led them to suggest that the 

acetylene reduction assay is not always able to provide positive results for 

the nitrogen-fixing ability of P. polymyxa. Therefore, conclusions on the 

occurrence of N2 fixationinvivoshouldbedrawnfromanumberoflines 

of evidence, including a positive nitrogenase activity test (acetylene reduction 

assay), 15 N dilution and detection of conserved nif genes in the 

purported diazotrophic endophyte. 

We have also observed nitrogen-fixing bacteria inside what can only be 

describedasauniqueandenigmatictypeofmycorrhizaeonlodgepolepine, 

first described by Zak (1971) on Douglas-fir (Pseudotsuga menziesii) roots. 

These mycorrhizal structures, often referred to as tuberculate mycorrhizae, 

look more like leguminous root nodules than mycorrhizae (Fig. 6.1). They 

are fully enclosed subterranean “nodules” or tubercles attached to the tree 

root system, with hundreds more typical mycorrhizal root tips crowded 

inside the outer covering, or peridium (Fig. 6.2). Nitrogen-fixing bacteria 

have been previously detected on the peridium (Li et al. 1992), but more 

recently, in our laboratory, a limited number of strains representing four 

diazotrophic bacterial species have been detected inside the peridium, 

colonising the fungal hyphae within the tubercle (unpublished data). It is 

yet to be demonstrated that these endophytic diazotrophs fix N2 in situ, let 

alone transfer it to the host plant, but these intriguing possibilities remain 

to be evaluated.


Fig.6.1. External morphology of tuberculate ectomycorrhizae on Pinus contorta roots. Bar 

5mm 

Fig.6.2. Cross section through a mature tubercle from P. contorta revealing mycorrhizal 

root tips (brown) and interstitial hyphae (arrow). Note pinnate radiated fan form and 

dichotomous branching of root tips within the tubercle. Bar 2mm 

Recently, we tested the hypothesis that diazotrophic endophytes isolated 

from internal tissues of immature and mature, naturally regenerated, 

lodgepole pine produce biologically significant amounts of N through


N-fixation under controlled environmental conditions. Entire lodgepole 

pine seedlings, as well as root, stem, and needle samples from trees were 

collected from 40- to 140-year-old stands near Williams Lake, British 

Columbia (52 ◦ 05 ′ N, 122 ◦ 54 ′ W, elevation 1,300 m) and Chilliwack Lake, 

British Columbia (49 ◦ 10 ′ N, 121 ◦ 57 ′ W, elevation 600 m). In addition, western 

red cedar (Thuja plicata Donn ex D. Don) samples were collected from 

a similar aged stand near Boston Bar, British Columbia (49 ◦ 50 ′ N, 121 ◦ 31 ′ W, 

elevation 600 m). Cedar samples were obtained in the same manner in 

which pine samples were collected except cedar stem samples from trees 

were obtained by cutting small wedges from stems using a pruning knife 

wiped down with 6% NaOCl prior to each sampling. 

Stem samples from mature trees were obtained by taking cores with 

a surface disinfested increment borer after shaving a thin layer of bark from 

the stem with a sterile scalpel. Root samples were surface-sterilised and 

triturated (Chanway et al. 2000) before endophytic bacteria were isolated 

by plating the resulting slurry. 

Four diazotrophic endophytes were isolated using this sampling procedure 

and 16S rDNA sequencing identified them as belonging to the genus 

Paenibacillus (strains P2b-2R, P18b-2R and C3b) as well as to the Flexibacter 

group (strain P19a-2R). The three strains with names beginning with 

“P”wereoriginallyisolatedfrompinetissuesfromtheWilliamsLakesite. 

Strain P2b was isolated from within the surface-sterilised stem of a pine 

seedling, strain P18b was isolated from within surface-sterilised needles 

of another pine seedling, and strain P19a was isolated from the internal 

stem tissue of a third pine seedling. Strain C3b was isolated from within the 

surface-sterilised stem of a western red cedar tree at the Boston Bar site. 

These microorganisms were then used to inoculate pine seed sown in 

glass tubes (150 mm × 25 mm in diameter) filled with a severely nitrogen 

deficient seedling growth medium to which a small amount (0.0576 g/l) 

Ca( 15 NO3)2 (5% 15 N label) was added to facilitate identification of foliar nitrogen 

originating from the growth medium versus the atmosphere. Other 

nutrients were added in amounts sufficient to support healthy plant growth. 

Bacterial inoculum was prepared by streaking frozen cultures onto plates 

of combined carbon medium (Rennie 1981). Following growth on plates, 

aloopfulofeachstrainwasseparatelyinoculatedintoitsown1lflask 

containing 500 ml CCM broth. Flasks were then secured on a rotary shaker 

(150 rpm; room temperature) and agitated for up to 2 days. All bacterial 

cultures were harvested by centrifugation (10,000g for 30 min), and resuspended 

in sterile phosphate buffer (SPB). Strains P2b-2R and C3b were 

resuspended to a density of ca. 10 7 cfu/ml and strains P18b-2R and P19a-2R 

were resuspended to a density of ca. 10 6 cfu/ml. For inoculation, 5.0 ml of 

each bacterial suspension was pipetted into separate tubes. Control seeds 

received 5.0 ml SPB. Tubes were placed in a growth chamber (Conviron


CMP3244, Conviron Products Company, Winnipeg, MB). Photosyntheticallyactiveradiation(PAR)atcanopylevelwasca.300µmols 

−1 m −2 during 

an 18-h photoperiod, and 20 ◦ C/14 ◦ C day/night temperature cycle. 

Of the four diazotrophic endophytes, only inoculation with strain P2b- 

2R resulted in large, statistically significant increases of 27–66% in pine 

foliar nitrogen derived from the atmosphere in all three plant growth 

trials we have conducted to date (Table 6.1). Interestingly, strain P18b- 

2R, which was found to be phylogenetically very similar to P2b-2R, had no 

such effect on seedling foliar nitrogen. Clearly, minor genetic differences 

betweenendophyticstrainscanresultinprofoundlydifferenteffectson 

plant growth and foliar nitrogen derived from the atmosphere (NDFA). 

While large, statistically significant amounts of foliar NDFA were detected 

in all three trials with pine, there was no corresponding positive 

growth response in the first two experiments. Indeed, pine seedling growth 

was inhibited by strain P2b-2R compared to noninoculated controls in the 

first two trials (Bal 2003). That inoculated seedlings may derive nitrogen 

primarily from the atmosphere is an intriguing phenomenon; poor growth 

of inoculated seedlings may be an indication of the energetic cost of supporting 

nitrogen-fixing bacteria in the plant. Seedling growth reduction 

during the establishment of symbiosis is not uncommon, due to the energy 

diverted from the plant, and has been reported previously (Chanway 

and Holl 1991). This idea is supported by results from the third growth 

trial. By the end of the trial, control seedlings had a restricted growth rate, 

presumably due to N limitation, compared to the inoculated seedlings. 

P2b-2R-inoculated seedlings had greater biomass (47%) and total nitrogen 

(38%) compared to noninoculated controls, and derived 27% of foliar 

Nfromtheatmosphere.Intrials1and2,controlseedlingswereapparentlyscavengingthesmallamountofsoilNthatwasinitiallyprovided, 

and indeed outgrew the inoculated seedlings, which may have been in 

a “symbiosis development” mode. This tenet is hypothetical at this time 

and requires further investigation. 

Table 6.1. Percentages of nitrogen derived from the atmosphere (NDFA) in pine seedlings 

after inoculation with Paenibacillus polymyxa strain P2b-2R in three separate growth trials 

Growth Trial Duration (months after planting and inoculation) %NDFA a 

1 8 30 

2 9 66 

3 9 27 

aCalculated according to Rennie et al. (1978), where: 

� 

atom % 15N excess(inoculated plant) 

%NDFA = 1 − 

atom % 15 � 

× 100 

N excess (uninoculated plant)


In addition, strain P2b-2R was detected inside root, stem and needle 

tissue using a surface-sterilisation-trituration plating technique as well as 

with confocal laser scanning microscopy after insertion of green fluorescent 

protein into the bacterium. These results leave us with little doubt that pine 

can derive biologically significant amounts of nitrogen from endophytic 

diazotrophs. 

Two conclusions can be drawn from our work so far. Nitrogen-fixing bacteria 

can be isolated from internal root, stem and needle tissues of lodgepole 

pine seedlings and mature trees, some of which can contribute biologically 

significant amounts of fixed nitrogen to lodgepole pine seedlings under 

controlled environments. It is possible that a significant amount of plant 

nitrogen originates from diazotrophic endophytes in lodgepole pine, but 

additional research is required to elucidate the role of these bacteria in 

forest ecosystems. 

6.6 

Future Work 

We are currently involved in detecting in planta expression of strain P2b-2R 

nif genes to demonstrate that internal pine stem, root and needle tissues 

provide a suitable environment for nitrogen fixation, as we have already 

confirmed the existence of nif genes in this bacterial strain. These experiments 

will allow localisation of P2b-2R within the plant tissues as well 

as provide evidence that the observed nitrogen gains were in fact derived 

from the endophytic P2b-2R. In addition, we need to understand the effect 

of soil nitrogen availability on growth promotion by strain P2b-2R and the 

extent to which other mechanisms are responsible for growth promotion. 

Plant growth studies involving a non-diazotrophic mutant of strain P2b- 

2R, under various levels of available nitrogen will provide data to answer 

these questions. 

Systemically colonising endophytic bacteria such as P. polymyxa strain 

Pw2-RandP2b-2Rcouldalsobeusedasvectorstodeliverspecificgene 

products to plants. Such an approach may be more feasible than attempting 

to genetically alter the plant host directly. In addition, diazotrophic 

endophytic bacteria hold great potential for reducing application of fertilisers, 

especially of mineral N. Inoculation of forest seedlings with effective 

diazotrophic or plant growth promoting endophytic bacteria could 

enhance growth and yield of trees significantly, especially at nutrient-poor 

sites.However,thereismuchmoreworktobedoneifwearetounderstand 

these plant/microbe associations to the degree that we can manage 

them effectively for more efficient and sustainable nursery and field treatments.


More knowledge of the population dynamics and activity of endophytic 

bacteriaintheirhostplantsisrequired.Aconsiderableresearcheffortisalso 

required to design strategies for the reinoculation of endophytic bacteria. In 

order to guarantee reproducibility, reliable methods of inoculum delivery 

should be developed. This is especially the case for the inoculation of 

trees with endophytic bacteria. Intense testing of different delivery systems 

has indicated that the efficacy of the application method for introducing 

endophytic bacteria into plant tissue is strain specific (Musson et al. 1995). 

The development of successful application technologies would fully depend 

on improving our understanding of how bacterial endophytes enter and 

colonise plants. This is just one aspect of the study of bacterial endophytes 

that needs to be undertaken in order to fully realise their potential use in 

forestry as in agriculture. 

Acknowledgements. The authors acknowledge the excellent help of Ms.S.E. 

Chanway in reference management and typing this manuscript. 


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7 

Biodiversity of Fungal Root-Endophyte 

Communities and Populations, 

in Particular of the Dark Septate Endophyte 

Phialocephala fortinii s. l. 

Thomas N. Sieber, Christoph R. Grünig 

7.1 

Introduction 

The peripheral root tissues form a morphologically, physically and chemically 

complex microcosm that provides different habitats for diverse communities 

of microorganisms. This microcosm is not stable, and changes 

over space and time because the boundaries between soil, rhizosphere, and 

living roots are continually shifted as a result of root growth and the constant 

modification of nearby soil by root mechanical and metabolic activity 

(Foster et al. 1983). Microorganisms colonise the rhizoplane, epidermis and 

outer cortex in a nonrandom patchy manner and contribute to the modification 

of the soil-rhizosphere-root continuum. Microorganisms affect 

their plant hosts, and hosts reciprocally affect their symbionts, leading to 

a feedback that drives changes in both the microbial and plant communities 

(Bever et al. 1997). Many soil bacteria and fungi are able to colonise epidermal 

and outer cortical cells of healthy roots inter- and intra-cellularly. 

A comparatively small number of organisms, e.g. mycorrhizal fungi, endophytic 

and pathogenic fungi and bacteria, possess, however, the ability 

to cross the inner boundary of the rhizosphere and to penetrate deeper 

into the root (Bazin et al. 1990). The interaction of host and endophyte 

depends on the disposition of host and fungus or bacterium and the environmental 

conditions, but may be neutral, mutualistic or antagonistic and 

may change over time. Some endophytic fungi adopt mycorrhizal functions 

and/or place plants at a competitive advantage against herbivores, insect 

pests or pathogens (Carroll 1988; Hawksworth 1991). Other endophytes 

can switch to a pathogenic behaviour when conditions are unfavourable 

for the host (Schulz et al. 1999). The biodiversity of root endophyte communities 

varies in relation to environmental factors, type of vegetation, 

Thomas N. Sieber: Swiss Federal Institute of Technology, Department of Environmental 

Sciences, Institute of Integrative Biology, Forest Pathology and Dendrology, 8092 Zürich, 

Switzerland, E-mail: thomas.sieber@env.ethz.ch 

Christoph R. Grünig: Swiss Federal Institute of Technology, Department of Environmental 

Sciences, Institute of Integrative Biology, Forest Pathology and Dendrology, 8092 Zürich, 

Switzerland 





108 T.N. Sieber, C.R. Grünig 

spatiotemporal patterns of the root microcosm and interactions among 

microorganisms. There is currently an urgent need to assess biodiversity 

in pristine ecosystems and to use these data as references to measure the 

effects of disturbances on diversity and to better enable informed decisionmaking 

on the fate of threatened natural habitats (Cannon 1997). Threats 

may come from a variety of sources, including exploitation by logging, 

machine-graded soils, urban development, pollution, climate change and 

input of pesticides and fertilisers. Biodiversity can be explored at several 

levels, i.e. in terms of communities, species and populations (Hawksworth 

1991). Here, we will explore current knowledge on the biodiversity of nonmycorrhizal 

fungal root endophytes at all levels. The first part of this review 

will be dedicated to biodiversity at the community level in relation to environmental 

factors. In the second part, special emphasis will be placed on the 

diversity of dark septate endophytes (DSE), in particular of Phialocephala 

fortinii s. l. 

Readers of this chapter should always bear in mind that the methods of 

detection are highly selective and, thus, the species list and species diversity 

derived for any habitat will be incomplete and will be biased in respect to 

physiological features selected for by the method used [Sieber 2002; Swift 

1976; see Chaps. 9 (Bayman and Otero), 18 (Bloemberg and Camacho 

Carvajal) and 19 (Van Overbeek et al.)]. 

7.2 

Species Diversity of Root Endophyte Communities 

“Species diversity” comprises two distinct components: the total number of 

species, which ecologists refer to as “species richness”, and “evenness” or 

equitability, which refers to how species abundances are distributed among 

the species present. An ecosystem is said to be more diverse if many species 

with equal population sizes are present and less diverse if some species 

are rare and a few are very common. Other helpful terms are “spectrum 

of species” or “community composition” to describe habitat or ecosystem 

differences with respect to the species found. The species diversity and the 

species spectrum of root-endophyte communities are related to various 

factors, which can tentatively be arranged into four groups: (1) geography 

and climate, (2) soil, (3) multitrophic interactions, and (4) natural and 

anthropogenic disturbances. This grouping is rather artificial and does 

not account for the intricate interplay among factors that often makes it 

impossible to determine the contribution of each factor. Another aspect 

obscuringtheeffectsofdifferentfactorsisthatofsitehistory,i.e.the 

dynamics of plant and endophyte communities. Nevertheless, the above 

grouping seems to be the most appropriate structure for this section.

7 Biodiversity of Fungal Root-Endophyte Communities and Populations 109 

7.2.1 

Geography and Climate 

Fungal species diversity is higher in tropical than in temperate regions 

owinginparttothegreatdiversityofhosts,butalsototheoptimalgrowth 

conditions for many fungi as a result of the hot and moist climate (Cannon 

and Hawksworth 1995). Whether this relationship is also valid for fungal 

root endophytes remains to be tested. Compared to habitats in the temperate 

or the tropical zones, species diversity is distinctly reduced in arctic-alpine 

environments, not only because of the lower number of available 

host species, but also with respect to the number of endophyte species in 

each host. For example, only seven root endophyte species were detected in 

Dryas octopetala in arctic Spitsbergen (Fisher et al. 1995) [Table 7.1(i)]. Correspondingly, 

species richness in Erica carnea was highest at an altitude of 

640 m and lowest at 2,140 m in Switzerland (Oberholzer-Tschütscher 1982). 

The species spectra differed greatly among sites, as expressed by very low 

between-site similarities [Table 7.1(ii)]. Evenness was lowest at the lowest 

altitude where the comparatively species-rich community was dominated 

by only four to five species. 

There is strong evidence for a shift from arbuscular mycorrhizal fungi 

(AMF) and ectomycorrhizal fungi (ECM) in temperate habitats towards 

symbioses of uncertain status, especially dark septate endophytes (DSE), 

in arctic-alpine ecosystems (Bledsoe et al. 1990; Christie and Nicolson 

1983; Read and Haselwandter 1981; Väre et al. 1992). Correspondingly, the 

frequency of roots colonised by Phialocephala fortinii s. l., a ubiquitous and 

dominant DSE in conifer roots (see Sect. 7.3.3), was positively correlated 

with the altitude in forest ecosystems (Ahlich and Sieber 1996). 

Weather and climatic conditions are assumed to have a weaker direct 

effect on species diversity of endophyte assemblages in root tissues than in 

aerial plant parts due to the insulating and compensating properties of soils 

(Fitter et al. 1985). Thus, changes in root endophyte assemblages become 

manifest only if the climatic conditions deviate from the “normal” over 

an extended period of time, i.e. if the climate changes. In fact, long-term 

changes in mean annual temperature, frequency and amount of precipitation,aswellasenhancedCO2 

may affect root endophyte diversity through 

shifts in the quantity and quality of photosynthates and secondary plant 

metabolites translocated to the roots, the rate of root turnover, and shifts in 

the competivity of endophytes and other soil microorganisms (Coûteaux et 

al. 1999; Rillig et al. 1999; Körner 2000). However, nothing is known about 

the direction and magnitude of effects on root-endophyte diversity.


Table 7.1. Influence of geographical, physical, chemical and biological factors on species diversity and similarity of communities of fungal root 

endophytes 

Reference 

Pairwise 

similarities of 

Sample 

sizee communities f 

Total 

number 

of 

isolates 

Evenness 

indexd Number 

of very 

abundant 

speciesc Adjusted 

species 

richnessb Hosts and factors Observed 

species 

richnessa (2) (3) (4) (5) 

(i) Dryas octopetala R Fisheretal. 

(1) Spitzbergen, site A 4 3.8 ± 0.4 2.4 0.81 42 50 0.73 

1995 

(2) Spitzbergen, site B 7 5.8 ± 0.4 3.8 0.83 24 50 – 

(ii) Erica carnea R Oberholzer- 

(1) Fläsch (640 m) 35 26.5 ± 2.2 4.2 0.51 296 333 0.26 0.21 0.30 Tschütscher 

(2) Näfels (760 m) 19 17.1 ± 1.2 4.7 0.71 219 120 – 0.20 0.19 1982 

(3) Davos-Wolfgang (1,640 m) 22 21.8 ± 0.4 7.0 0.72 173 298 – – 0.24 

(4) Davos-Schatzalp (2,140 m) 12 10.6 ± 1.0 2.7 0.68 235 300 – – – 

(iii) Picea abies R Kattnerand 

(1) Soil pH neutral 27 26.9 ± 0.3 11.9 0.70 153 480 0.57 

Schönhar 

(2) Soil pH acidic 29 28.8 ± 0.4 12.6 0.72 154 480 – 

1990 

(iv) Alnus glutinosa R Fisheretal. 

(1) Submerged roots 45 44.1 ± 0.8 17.4 0.66 114 40 0.37 

1991 

(2) Non-submerged roots 31 29.2 ± 1.2 13.8 0.75 126 40 – 

(v) Rhizophora mucronata R Ananda and 

(1) Low-tide level 6 5.3 ± 0.7 2.2 0.80 21 30 0.50 0.38 

Sridhar 2002 

(2) Mid-tide level 14 9.7 ± 1.2 3.6 0.87 34 30 – 0.58 

(3) High-tide level 10 9.4 ± 0.6 3.2 0.91 17 30 – –



Reference 

Pairwise 


Sample 


Total 

number 

of 

isolates 

Evenness 

indexd Number 

of very 

abundant 


species 


species 

richnessa (2) (3) (4) (5) 

(vi) Phragmites australis Wirsel et al. 

Location 1 R 

2001 

(1) Flooded site 10 9.5 ± 0.7 4.6 0.74 63 45 0.52 0.52 – 

(2) Dry site 13 12.9 ± 0.3 7.4 0.80 51 45 – – 0.75 

Location 2 

(3) Flooded site 13 11.9 ± 0.9 5.6 0.71 47 45 0.58 

(4) Dry site 11 10.0 ± 0.9 4.8 0.72 52 45 – 

(vii) Triticum aestivum Sieber et al. 

Development stage: R 

1988 

(1) One leaf – end of tillering 63 61.0 ± 1.3 15.3 0.59 547 5040 0.67 

(2) Inflorescence emerged – caryopsis hard 62 39.5 ± 2.9 6.3 0.53 1857 3360 – 

(viii) Triticum aestivum Sieber et al. 

Preceding crop: R 

1988 

(1) Sugar beet 51 44.7 ± 2.1 9.3 0.56 623 2400 0.58 0.57 0.67 

(2) Red clover 49 48.3 ± 3.3 10.9 0.60 413 1200 – 0.60 0.59 

(3) Maize 44 35.9 ± 2.2 4.7 0.45 773 2400 – – 0.65 

(4) Potatoes 39 33.5 ± 1.9 6.9 0.61 595 2400 – – –



Reference 

Pairwise 


Sample 


Total 

number 

of 

isolates 

Evenness 

indexd Number 

of very 

abundant 


species 


species 

richnessa (2) (3) (4) (5) 

(ix) Various vegetables R Narisawa 

(1) Eggplant 7 5.5 ± 0.9 3.2 0.71 35 45 0.67 0.55 0.67 0.86 et al. 2002 

(2) Tomato 5 4.7 ± 0.4 2.7 0.78 19 45 – 0.44 0.60 0.50 

(3) Melon 4 3.9 ± 0.3 2.6 0.83 17 45 – – 0.44 0.55 

(4) Strawberry 5 4.5 ± 0.6 2.1 0.71 21 45 – – – 0.67 

(5) Chinese cabbage 7 5.9 ± 0.8 4.4 0.79 29 45 – – – 

(x) Betula pendula T Görke 

(1) Plantation in cleared windthrow 30 12.5 ± 1.7 10.6 0.59 87 160 0.34 0.29 0.38 (1998) 

(2) Natural regeneration 

11 9.3 ± 1.0 5.7 0.73 27 75 – 0.41 0.50 

in untouched windthrow 


18 10.9 ± 1.5 7.4 0.60 48 100 – – 0.57 

in cleared windthrow 


in low density forestg 17 9.3 ± 1.5 6.3 0.70 58 100 – – – 

(x) Pinus sylvestris T Görke 

(1) Plantation in cleared windthrow 16 6.2 ± 1.2 6.8 0.72 56 160 0.42 0.40 0.38 (1998) 


8 5.5 ± 1.0 4.7 0.79 20 75 – 0.55 0.53 

in untouched windthrow 


14 7.0 ± 1.2 6.9 0.69 26 100 – – 0.38 

in cleared windthrow 

(4) Natural regeneration in low density forest 7 5.4 ± 0.9 4.8 0.80 19 100 – – –



Reference 

Pairwise 


Sample 


Total 

number 

of 

isolates 

Evenness 

indexd Number 

of very 

abundant 


species 


species 

richnessa (2) (3) (4) (5) 

(xi) Erica carnea R Cevniket 

(1) Control (Cd 1.4; Pb 171; Zn 61.8) al. 2000 

h 9 8.2 ± 0.7 3.9 0.78 104 240 0.50 0.60 0.63 

(2) Low pollution (Cd 6.9; Pb 667; Zn 177) 7 6.9 ± 0.2 3.3 0.81 72 240 – 0.67 0.71 

(3) High pollution (Cd 35.8; Pb 5422; Zn 582) 11 10.5 ± 0.6 8.0 0.87 107 240 – – 0.67 

(4) Highest pollution 

10 8.9 ± 0.8 3.8 0.72 142 240 – – – 

(Cd 87.7; Pb 31320; Zn 1330) 

a Diversity index N0 according to Hill (1973); number of species 

b Mean and standard error of the number of species adjusted 

to the lowest within-study number of isolates using rarefaction according to Hurlbert (1971) 

c Diversity index N2 according to Hill (1973) 

d Evenness index according to Hill (1973); this index converges towards 1 as one species tends to dominate 

e Number of root segments (R) or trees (T) examined 

f Soerensen index (Soerensen 1948); column numbers (in brackets) correspond to factor identifiers in the column “Hosts and factors”; 

0 ≤ Soerensen index ≤ 1, the index is 0 if two communities have no species in common, and it is 1 if all species occur in both communities 

g Low density forest = selectively logged forest stand; aim: increased solar radiation within the stand 

h Concentrations of heavy metals in micrograms per gram of soil


7.2.2 

Soil 

Soil and rhizosphere are highly variable habitats. Chemical properties such 

as pH or the availability of minerals and carbohydrates may vary significantly 

within a few centimetres of soil (Papritz and Flühler 1991). Similarly, 

differences in soil texture and water regime contribute to the variability 

of soils. In addition, roots constantly modify the nearby soil structure by 

depletion of minerals, ions and water and by the secretion of root exudates. 

Soils offer habitats for various communities of microorganisms including 

potential root endophytes. Plant and microbial metabolites may differentially 

influence the surrounding soil and change some of its properties, thus 

preparing the soil for the microorganisms of the next successional stage 

(Van Der Putten 2003). 

Physical and Chemical Soil Characteristics 

SoilpHhadaneffectoncommunitycompositionbutnotonspeciesdiversity 

of endophytic fungi in Norway spruce roots (Picea abies) (Kattner 

and Schönhar 1990) [Table 7.1(iii)]. The similarity of only 57% of the endophyte 

communities in roots from neutral and acidic soils reflects either 

the selectivity of soil pH or the historical presence/absence of certain endophyte 

species, e.g. endophytes with low dispersion and/or survival rates. 

For example, Phialocephala fortinii preferentially occurs in roots growing 

in acidic soils (Ahlich et al. 1998). 

Species richness was not related to soil texture in wheat roots (Triticum 

aestivum) (Riesen and Sieber 1985; Sieber et al. 1988). However, texture affected 

the frequency of Microdochium bolleyi and Periconia macrospinosa. 

M. bolleyi was more frequently isolated from roots originating from silty 

loam, whereas P. macrospinosa was isolated more often from roots growing 

in pure loam. 

Root endophytes differ in their ability to metabolise minerals and carbohydrates, 

making some endophytes more successful than others in a given 

habitat. DSE are thought to be excellent metabolisers of phosphorus (P) 

and to mediate P uptake for their hosts (Jumpponen et al. 1998; Barrow 

and Osuna 2002). In fact, DSE were more abundant in habitats poor in 

P (Haselwandter and Read 1982; Ruotsalainen et al. 2002). Similarly, differential 

utilisation of carbohydrates as well as which carbohydrates were 

available determined fungal species diversity and endophyte-community 

composition in the experiments of Hadacek and Kraus (2002).


Water Regime 

The water regime in soils and streams has a strong impact on species diversity 

and especially on the species spectrum of endophytic fungi (see 

Chap. 10 by Bärlocher). In roots of the same tree, 45 species were isolated 

from roots submerged in a river as opposed to only 31 species from nonsubmerged 

roots (Fisher et al. 1991) [Table 7.1(iv)]. The similarity of the 

community composition in submerged and non-submerged roots of the 

same individual black-alder tree was only 37%. Colonisation of submerged 

roots by aquatic hyphomycetes, together with the absence or scarcity of 

these specialists in non-submerged roots, emphasise the importance of the 

milieu in which roots grow in determining the composition and diversity 

of endophyte communities. For example, high water tables restricted the 

occurrence of P. fortinii in wetlands (Addy et al. 2000). The endophyte 

species diversity in roots of the mangrove Rhizophora mucronata strongly 

depended on the tidal level at which the roots were collected. Diversity 

was highest at the mid-tide level, i.e. the zone submerged in seawater approximately 

half of the time, and roots from the high-tide and the low-tide 

level had, on average, only 38% of species in common (Ananda and Sridhar 

2002) [Table 7.1(v)]. Flooding and site conditions affected endophyte 

species spectra but not species richness in roots of common reed (Phragmites 

australis) (Wirsel et al. 2001) [Table 7.1(vi)]. In contrast, species 

spectra in bracken rhizomes (Pteridium aquilinum) did not differ among 

wetland and woodland sites (Petrini et al. 1992). 

7.2.3 

Multitrophic Interactions 

The diversity of soil microorganisms is tremendous; 1 g soil can contain 

between 5,000 and 10,000 species of microorganisms (Torsvik et al. 1990). 

However, only 1,200 species of fungi have been isolated from soil (Watanabe 

1994), perhaps because, as estimates suggest, only 17% of known fungi can 

be readily grown in culture (Hawksworth 1991). If this percentage were 

applied to the 1,200 species as suggested by Watanabe (1994), this would 

give an estimate of approximately 7,000 species of soil fungi (Bridge and 

Spooner 2001). The total length of fungal hyphae varies greatly according 

to soil type and soil biology and has been reported to be as high as 66,900 m 

in 1 g dry soil (Bååth and Söderström 1979). The high number of species 

and the high amount of microbial biomass in such small volumes of soil 

suggest that multitrophic interactions among soil bacteria, soil fungi, soil 

microfauna and plants are frequent. Interspecific competition may be “the” 

factor that overrides all others in regulating species abundance of soil fungi 

(Gochenaur 1984). If a community is dominated by inter- and intra-specific


competition, the resources are more likely to be fully exploited. Endophyte 

speciesdiversityandspectrumwillthendependontherangeofavailable 

resources, including host tissues, the extent to which species are specialists, 

antagonism among competitors, their ability to overcome host defences and 

the permitted extent of habitat overlap. 

Microdochium bolleyi is a frequent and successful endophyte in cereal 

roots, where it functions as an effective antagonist of various root 

pathogens. For example, its presence in wheat roots was negatively correlated 

with the presence of Septoria nodorum, the causal agent of glume 

blotch disease of wheat (Riesen and Sieber 1985; Sieber et al. 1988). Similarly, 

M. bolleyi inhibited various Fusarium species and Gaeumannomyces 

graminisvar. tritici (KirkandDeacon1987;Reinecke1978).Whether M. bolleyi 

interacts with these pathogens indirectly by inducing systemic resistance 

in the host plant, or directly by either parasitising pathogens or 

producing inhibitory metabolites, remains to be examined. 

Thephenologicalstateoftherootsand/ortheseasonmayinfluenceendophyte 

species diversity by affecting the probability of interactions among 

endophytic thalli. For example, the number of dominant species was higher 

in young than in mature winter wheat, presumably because freshly established 

thalli were small. Growth was reduced due to the cold temperatures 

in winter, making hyphal interference less likely and/or weaker and, thus, 

also allowing less competitive fungi or fungi better adapted to cold temperatures 

to establish endophytic thalli [Table 7.1(vii)] (Riesen and Sieber 

1985; Sieber et al. 1988). This situation changed in spring and summer, 

when the growth rate of endophytic thalli increased, making intra- and 

inter-species hyphal interactions more probable, leading to the dominance 

of the few most competitive species. 

Similar to mycorrhiza, strict host specificity is the exception rather than 

the rule for fungal root endophytes (Bruns et al. 2002; Jumpponen et al. 

2004). However, the likelihood of occurrence of some endophyte species 

increases in the presence of particular host species, suggesting fungal host 

preference or shared habitat preferences. The diversity of the plant community 

in which the host species grows may, therefore, influence rootendophyte 

diversity similarly as it has been shown to affect diversity of soil 

microfungi (Christensen 1981, 1989). Ahlich and Sieber (1996) presented 

an example of the importance of the plant community in determining the 

spectra of fungi associated with the host. The dominant root endophytes 

of European beech (Fagus sylvatica), Cryptosporiopsis radicicola and Cylindrocarpon 

didymum, were rare or absent in roots of Scots pine (Pinus 

sylvestris) growing in monoculture. Likewise, P. fortinii,thedominantroot 

endophyte of Scots pine, was rare or absent in monocultures of beech. 

However, when the roots originated from mixed stands of Scots pine and 

beech, Scots pine roots showed a comparatively high rate of colonisation


by C. radicicola and C. didymum. Correspondingly, the roots of beech were 

frequently colonised by P. fortinii in mixed stands. In contrast, frequency 

of colonisation of Betula papyrifera and Pseudotsuga menziesii seedlings 

by DSE was not affected by whether or not the plants were grown in mixed 

culture or in monoculture (Jones et al. 1997). 

In agriculture, the preceding crop may significantly affect endophyte diversity 

of the current crop. For example, species richness and the number 

of dominant species were significantly higher when wheat (Triticum aestivum) 

followed red clover than when it followed potatoes [Table 7.1(viii)] 

(Sieber et al. 1988). On average, only 59% of the endophyte species were indifferent 

to whether the preceding crop was clover or tomatoes. The range 

of indifferent endophyte species lay between 57% and 67% for other pairs 

of preceding crops [Table 7.1(viii)]. This observation may be related to 

differences in the spectra of endophytes that had colonised the preceding 

crop. Specific secondary metabolites and debris produced by the preceding 

crop, as well as the type and amount of agrochemicals (fertilisers, biocides, 

leafage killers) applied to the preceding crops may be other factors 

influencing both diversity and stimulation/inhibition of endophytes. 

When different vegetables are grown in the same soil, some endophytehost 

associations occur more frequently than others, suggesting host preference 

or adaptation. The similarity of the spectra of endophyte species 

among host species was as low as 44% in an experiment performed by Narisawa 

et al. (2002) [Table 7.1(ix)]. It is not known whether plants are able 

to actively recruit endophytes and vice-versa. Plant defence compounds 

probably select for certain rhizosphere microorganisms. Some evidence 

for such mechanisms comes from nematode and mycorrhiza research. Secondary 

metabolites released by roots of Thuja occidentalis upon attack by 

weevil larvae attracted entomopathogenic nematodes (Van Tol et al. 2001). 

Dormant propagules of mycorrhizal fungi were stimulated to germinate 

by chemical messengers from the host (Bruns et al. 2002). Correspondingly, 

mycelia of AMF were inhibited by non-host metabolites (Oba et al. 

2002). Nothing is known about whether certain root endophytes release 

“pheromones” to attract roots of host plants. 

7.2.4 

Natural and Anthropogenic Disturbances 

Anthropogenic and natural disturbances affect the species spectrum of 

plant communities and consequently also the communities of cohabiting 

microorganisms. Forest-management practices such as planting of trees, 

selective cutting or clearing of windthrows had a distinct effect on the 

endophytic mycobiota in the roots of forest trees (Görke 1998). Maximally


42% of the endophyte species were common to both planted and naturally 

regenerated trees [Table 7.1(x)]. Considering naturally regenerated trees 

only, species richness and the number of dominant species was highest 

in the cleared windthrow. Probably, endophyte diversity and community 

composition would also change as a consequence of gap formation by man 

and/or wind storm, which eliminates some hosts but creates habitats for 

many other hosts, i.e. ruderal plant species. 

Mycorrhization and root-endophyte colonisation of naturally regenerated 

seedlings of Betula platyphylla var. japonica in soils of machine-graded 

ski slopes depended on the time elapsed since disturbance (Hashimoto 

and Hyakumachi 2000). Seedlings thrived well only in soil samples from 

soils disturbed more than 3 years previously and mycorrhization was significantly 

higher in these samples. In contrast, colonisation of roots by 

DSE was distinctly higher in seedlings sampled from soils disturbed only 

1–3 years before sampling. In another study, the majority of naturally established 

seedlings of bishop pine (Pinus muricata)werecolonisedbyDSE 

shortly after wildfire, indicating that a resident inoculum (chlamydospores, 

microsclerotia) survived the fire (Horton et al. 1998). Species richness of 

endophytes in roots of Erica carnea was highest at sites where soil pollution 

by heavy metals was high, but DSE occurred less frequently in the 

heavily polluted soils (Cevnik et al. 2000) [Table 7.1(xi)]. Endophytic fungi 

are either more competitive in disturbed or moderately polluted soils or 

better equipped to survive periods of adverse environmental conditions 

than mycorrhizal fungi. 

The use of fungicides for crop protection can alter species diversity. 

Seed treatment with the systemic fungicide benomyl had no significant 

influence on endophyte species richness in wheat roots, but the frequency 

of roots colonised by seed borne Septoria nodorum was significantly reduced 

(Riesen and Sieber 1985). None of the fungicides applied to Lolium 

perenne fields at 18 sites in New Zealand had a significant effect on the 

root-endophyte communities (Skipp and Christensen 1989). 

Fertilisation can affect fungal assemblages in roots. The frequency of 

P. fortinii in seedlings of potted Picea glauca was negatively correlated with 

the amount of nitrogen (N) applied (Kernaghan et al. 2003). Wilberforce 

et al.(2003) suspected N fertilisers to be one of the mechanisms by which 

management affects root endophyte communities in temperate grasslands. 

Emissions of air pollutants such as SO2 and especially NOx are thought to 

have a similar fertilising effect as fertilisers applied in agriculture. Adverse 

effects of these air pollutants on mycorrhizal fungi have been demonstrated 

in several studies (Cairney and Meharg 1999; Jansen and van Dobben 1987; 

Taylor and Read 1996).


7.3 

Dark Septate Endophytes 

Fungi with regularly septate and melanised hyphae probably constitute 

the most abundant and most widespread group of non-mycorrhizal root 

endophytes.Inthissection,wewillbrieflypresentthehistoryoftheterm 

“DSE”, outline the diversity of DSE and give an overview of current knowledge 

of the diversity and population genetics of the most prominent species 

complex of DSE: Phialocephala fortinii s. l. 

7.3.1 

History 

Melin (1922, 1923) introduced the form taxon Mycelium radicis atrovirens 

(MRA) for sterile, melanised, septate mycelia that emerged from mycorrhizae 

and roots of Picea abies and Pinus sylvestris. The tree-fungus symbiosis 

was characterised by dematiaceous intra- and intercellular hyphae in 

the epidermal and cortical cells, but neither a Hartig net nor a mantle were 

formed. Melin(1923) coined the term“pseudomycorrhiza” for this relationship 

and considered it to form an antagonistic symbiosis. MRA-like fungi 

have been detected during numerous studies since Melin’s pioneering work 

(Ahlich and Sieber 1996; Chan 1923; Freisleben 1934; Harley and Waid 1955; 

Jumpponen et al. 1998; Richard and Fortin 1973; Robertson 1954; Stoyke 

and Currah 1991). Since trinomials are not valid species names according to 

the International Code of Botanical Nomenclature, less stringent and more 

informal names are preferable. Read and Haselwandter (1981) introduced 

the term “DS hyphae” (DS = dark septate) for sterile, dark, septate hyphae 

and microsclerotia that occurred in roots of various alpine plants. Stoyke 

and Currah (1991) implemented the form taxon “dark septate endophyte” 

(DSE) and used it for fungi that form partly or entirely melanised, septate 

thalli within healthy root tissues. The taxon “DSE” serves primarily to differentiate 

these fungi from endophytes with septate, hyaline hyphae, and 

from fungi with sparsely septate, hyaline hyphae that are characteristic of 

AMF. 

7.3.2 

Biodiversity 

The roots of more than 600 plant species representing about 320 genera in 

more than 110 families have been reported to be colonised by DSE (Ahlich 

and Sieber 1996; Barrow and Osuna 2002; Jumpponen and Trappe 1998b;


Kovacs and Szigetvari 2002; Ruotsalainen et al. 2002; Schadt et al. 2001). 

Dematiaceous mycelia are regularly received in culture during censuses of 

root endophytes, but it is often not known whether the endophytic thalli of 

these fungi are hyaline or melanised. This being the case, we must assume 

that DSE are much more widespread than previously assumed. 

Species identity of some DSE is known because they readily sporulate 

in culture, e.g. Microdochium bolleyi and several Phialophora species in 

grasses and sedges. Many non-pathogenic Phialophora endophytes are related 

to the take-all fungi (Gaeumannomyces graminis var. tritici and var. 

avenae) ofcerealsandgrassesintemperateareasandtoG. graminis var. 

graminis, which causes crown sheath rot of rice in the tropics. Phialophora 

radicicola forms melanised sclerotia in cortical cells of maize roots without 

causing any apparent harm (Cain 1952). P. radicicola was also observed 

in the roots of three alpine grasses growing at the timberline in Bavaria 

(Blaschke 1986) or in roots of Lolium perenne in New Zealand (Skipp and 

Christensen 1989). The DSE abundantly observed in many alpine sedges 

in the Tyrolean Alps may also belong to P. radicicola (Haselwandter and 

Read 1980; Read and Haselwandter 1981). P. radicicola and P. zeicola,the 

maize take-all fungi from China, were recently shown to be the same species 

(Ward and Bateman 1999). P. graminicola, another non-pathogenic DSE of 

cereal and grass roots (Newsham 1999), provided significant control of the 

take-all disease by competition for senescing root tissues (Deacon 1981). 

Taxonomic assignment of many DSE is problematic because sexual and 

asexual reproductive structures are either absent, rare, or are produced 

onlyunderspecificconditions.Coldtreatmentforupto1yearwasshown 

to induce sporulation in some DSE isolates, e.g. in isolates of Chloridium 

paucisporum, Phialophora finlandica,andPhialocephala fortinii (Wangand 

Wilcox 1985). Unfortunately, even then many DSE strains remain sterile 

and classification is complicated. Many mycologists have tried to bring 

some order into this difficult group of DSE (Harney et al. 1997; Melin 

1923; Richard and Fortin 1973). Culture morphology is often used for an 

initial classification (Ahlich and Sieber 1996; Girlanda et al. 2002; Steinke 

et al. 1996; Stoyke et al. 1992). However, modern molecular biology offers 

a multitude of additional and potentially more reliable methods for the 

identification and typing of species, varieties and individuals (Carter et al. 

1997; Geiser et al. 1994; White et al. 1990; Zietkiewicz et al. 1994). Some of 

these methods have been used to type DSE. Restriction patterns of a region 

on the ribosomal RNA (rRNA) genes indicated that two-thirds of the DSE 

from roots of subalpine plants were closely related to or conspecific with 

P. fortinii (Stoyke et al. 1992). Similarly, in a study by Harney et al. (1997), 

restriction site mapping of the nuclear rDNA internal transcribed spacer 

(ITS) regions showed that the majority of the isolates was P. fortinii-like 

and only two isolates were Phialophora finlandica.


According to isozyme analysis, DSE from various woody plant species 

belonged to two distinct groups (Ahlich-Schlegel 1997; Grünig et al. 2001; 

Sieber 2002). Members of the larger group were conspecific with P. fortinii, 

whereas those of the other group represented the sterile Type 1, which has 

been recently described as Acephala applanata (Ahlich and Sieber 1996; 

Grünig and Sieber 2005). Phylogenetic analysis of the ITS regions showed 

that P. fortinii and A. applanata are closely related and have Phialocephala 

compacta, P. dimorphospora and P. scopiformis as closest relatives (Grünig 

et al. 2002b). These five species are more closely related to members of the 

Leotiales such as Gremmeniella abietina, the causal agent of sclerroderis 

canker on pines, than to other Phialocephala species. The “P. fortinii-group” 

was also positioned within the Leotiales by phylogenetic analyses of the 

sequence data of the 18S and 28S subunits of the nuclear rRNA genes 

(Jacobs et al. 2003). 

7.3.3 

Diversity of Phialocephala fortinii 

Phialocephala fortinii was shown to be the dominant DSE in coniferous 

and ericaceous roots in heathlands, forests and alpine ecosystems of the 

Northern temperate zones (Ahlich and Sieber 1996; Stoyke and Currah 

1991). There is strong evidence that the roots of every Norway spruce 

(Picea abies) tree in natural forest habitats of Central Europe are colonised 

by this fungus (Ahlich and Sieber 1996; Grünig et al. 2004). The nature 

of root–P. fortinii symbioses and their ecological significance are largely 

unknown. 

P. fortinii mayfunctionasamycorrhizalfungusandmediatenutrient 

uptake, synthesise secondary metabolites, stimulate plant growth and/or 

play an important role in plant defence against root pathogens (Fernando 

and Currah 1996; Jumpponen and Trappe 1998a; O’Dell et al. 1993; Yu et al. 

2001). Alternatively, it may behave as an opportunistic pathogen (Wilcox 

and Wang 1987). However, considering its widespread distribution and 

abundanceitisveryunlikelythatP. fortinii is a primary pathogen. 

We will provide a compilation of the newest findings on the genetic 

diversity within and among populations of P. fortinii and will conclude 

this section by forwarding some ideas and thoughts that could explain the 

observed diversity of this ecologically very successful species. 

Genetic diversity of P. fortinii strains was examined on different spatial 

scales using isozymes, PCR-fingerprinting and analysis of the rDNA 

ITS regions either by polymerase chain reaction -restriction fragment 

length polymorphism (PCR-RFLP) analysis or sequencing. Ahlich-Schlegel 

(1997) studied the allelic diversity at seven isozyme loci and detected 108


different allozyme phenotypes among 194 European and North-American 

DSE strains. Allozyme patterns were neither host- nor site-specific. Harney 

et al. (1997) found many polymorphisms in the rDNA ITS regions of 

P. fortinii strains from Europe and North America by restriction mapping. 

Similarly, variability among rDNA ITS sequences was high (up to 12 substitutions) 

among 18 strains of P. fortinii from Central and Northern Europe 

(Grünig et al. 2002b). In contrast, Addy et al. (2000) detected a high degree 

of homogeneity among the rDNA ITS sequences of six strains of P. fortinii 

from Canada and Japan. 

Strain-specific markers are necessary to study the genetic diversity at 

small spatial scales. In contrast to allozyme markers, ISSR-PCR markers 

were strain specific and allowed discrimination among isolates with identical 

allozyme phenotypes (Grünig et al. 2001). These markers were used to 

detect the population structure of DSE isolated from Norway spruce (Picea 

abies) roots collected within a 3 × 3 m plot of a 40-year-old plantation 

(Grünig et al. 2002a). Twenty-one unique ISSR-PCR genets were present 

among 144 strains. Identity of the isolated DSE as P. fortinii was confirmed 

by the morphology of the conidiogenous apparatus and by sequence comparisons 

of the rDNA ITS regions. Two genets dominated and were isolated 

from all sampling points within contiguous areas of at least 6.8 m 2 and 

5.3 m 2 that overlapped by 3.6 m 2 .Othergenetswererareandwereisolated 

only once or twice. 

Jumpponen (1999) employed the random amplified polymorphic DNA 

(RAPD) technique to determine the population structure of P. fortinii at 

a primary succession site on a glacier forefront. In one year, 23 genets of 

P. fortinii were detected in 34 strains, in the next year 10 genets were found 

in 40 strains, but none of the genets was isolated in both years. Diversity 

of P. fortinii canbehighevenwithinsinglerootpieces.Forexample,8to 

10-cm-long pieces of fine root of Picea abies were colonised by up to 

six different inter-simple sequence repeat (ISSR) phenotypes (N. Nüssli 

and C.R. Grünig, unpublished) (Fig. 7.1). In summary, genetic diversity of 

P. fortinii seems to be high at every level. This is surprising for a supposedly 

asexual fungus. Therefore, studies on population genetics were initiated to 

findthesourcesofthishighdiversity. 

ISSR-PCR and RAPD markers have many analytical drawbacks, such 

as dominance, and they cannot be used to infer population differentiation 

and recombination. In contrast, single-locus RFLP markers are codominant 

and supply robust data for precise population genetic analyses. In addition, 

data are comparable among studies and thus may be used for global analyses 

(Sunnucks 2000). Therefore, single-locus RFLP probes were developed 

for population genetic analysis of P. fortinii and used to find evidence for 

recombination, gene and genotype flow in P. fortinii (Grünig et al. 2003, 

2004). Strains collected from three Norway-spruce plots up to 10 km apart


Fig.7.1. Distribution of six inter-simple sequence repeat-polymerase chain reaction (ISSR- 

PCR) phenotypes belonging to three cryptic species of the root endophyte Phialocephala 

fortinii s. l. along a healthy fine root of Norway spruce (Picea abies). Identical symbols 

indicate positions on the root where the same phenotype was isolated. Symbols with identical 

shape represent the same cryptic species. C Positions on the root where Cylindrocarpon 

didymum wasisolatedasanendophyte 

from each other were studied using 11 single-locus RFLP probes. The average 

gene diversity was high and up to 96 multilocus haplotypes (MLH) were 

observed per study plot. Significant population subdivision was detected 

among groups of MLH within plots, suggesting that groups were reproductively 

isolated and should be considered cryptic species. The RFLP data of 

more than 1,000 European strains indicate that P. fortinii s. l. is a species 

complex of at least eight cryptic species (C.R. Grünig, unpublished). The 

index of association (IA) did not deviate significantly from zero within any 

cryptic species, suggesting that recombination occurs, or has occurred, 

within these species. Although evidence for recombination is strong for all 

cryptic species, it remains unclear whether sexual or parasexual processes 

are involved, and how often and where recombination occurs or when it last 

occurred (Taylor et al. 1999). Even a little sex is, however, already enough 

to give an organism the appearance of a recombining population (Brown 

1999). 

The sympatric occurrence of up to four reproductively isolated, cryptic 

species within a few square metres of forest floor, and sometimes even 

in the same root segment, is a highly interesting phenomenon and deserves 

a brief discussion (Grünig et al. 2004) (Figs. 7.1, 7.2). Reproductive 

isolation is essential for speciation. Geographically isolated populations 

are often reproductively isolated, and may experience allopatric speciation 

through genetic drift (Carter et al. 2001). On the other hand, niche or 

habitat specialisation may lead to sympatric speciation when local populations 

are confronted with heterogeneous habitats or several niches within 

habitats (Futuyma and Moreno 1988; Maynard Smith 1966). The patterns 

observed by Grünig et al.(2004) are clearly indicative of speciation. Possibly, 

the cryptic species were the products of allopatric speciation in the


Fig.7.2. Distribution of the four most frequently observed cryptic species (csp) ofPhialocephala 

fortinii s. l. within healthy fine roots of Norway spruce (Picea abies) collectedat 

the intersections of a 2 × 2 m grid superimposed on a forest plot (14 × 14 m) at Zürichberg, 

Switzerland. The four graphs represent the same study plot; the distributions of the four 

cryptic species are presented in separate graphs to maintain clarity. The number of isolates 

and the multilocus haplotypes (MLH) of each cryptic species are given in brackets 

past due to geographical isolation. The ranges of these species may have 

subsequently overlapped (Brasier 1987). In this respect it is interesting to 

study the role of Quaternary climatic changes (Hewitt 2000). The succession 

of several glaciations and warmer inter-glacial periods had profound 

effects on animals, plants, and, consequently, on fungi. During the Quaternary, 

each species experienced many contractions/expansions of range, 

leading to extinctions and foundations of populations, decreases and increases 

in diversity and, thus, also to speciation (Taberlet et al. 1998). 

Refugia of relevant hosts of P. fortinii were often geographically isolated,


making allopatric speciation of P. fortinii possible. Alternatively, habitat 

heterogeneities are certainly present even within very small compartments 

of root tissues, the rhizosphere, and the surrounding soil. These heterogeneities 

may be pronounced enough for ecological isolation and for the 

development of cryptic species. Some cryptic species may be interspecific 

hybrids. For example, most asexual Epichloë-related grass endophytes appear 

to be such hybrids (Scott 2001). Interspecific hybrids may be better 

adapted to new niches such as new hosts and can provide greater or more 

diverse benefits to host plants (Schardl and Craven 2003). However, such 

hybrids were never observed for P. fortinii using codominantly inherited 

single-copy RFLP markers. 

MLHwithidenticalISSRfingerprintingpatternswerecommontoatleast 

two of the sites in the study of Grünig et al. (2004). These results indicate 

that not only gene flow but also genotype flow most likely occurs in cryptic 

species of P. fortinii. Gene and genotype flow occur either naturally via 

conidia or microsclerotia transported by wind or micro- and macrofauna, 

or by silvicultural practices. Genotypes may be introduced by planting 

plants from nurseries located up to several hundreds of kilometers away 

(Bürgi and Schuler 2003), since nursery plants are frequently colonised 

by DSE including P. fortinii (Danielson and Visser 1990). Alternatively, 

machinery used during thinning and harvesting could be responsible for 

the import of genotypes. 

Nothing is known about the significance of mutations, the ultimate 

source of genetic variation, for speciation within P. fortinii s. l. If a population 

is large and the mutation rate high, it is likely that mutants with 

higher fitness, e.g. better mutualists, will emerge (McDonald and Linde 

2002). Non-lethal somatic mutations in the mitotic phase may affect the genetic 

diversity of a population since each nucleus has the capacity to be the 

founder genome of another, new mycelium (Burnett 2003). The diversity 

thus generated may supplement diversity generated by recombination. 

7.4 

Conclusions 

Colonisation of roots by fungal endophytes is a common feature in the plant 

kingdom. In contrast to classical mycorrhizae, endophytes are regularly 

present in roots undergoing secondary growth. Root-endophyte species 

diversity is affected by climatic, physical, chemical, biological and anthropogenic 

factors. DSEs are among the most abundant root endophytes. 

They constitute a taxonomically very heterogeneous group of fungi, mostly 

ascomycetes, that form melanised, septate hyphae, chlamydospores or microsclerotia 

within the roots of the host.


Phialocephala fortinii is the most prominent DSE, especially in woody 

plant species. P. fortinii s.l.isgenotypicallyverydiverseandformsacomplex 

of several cryptic species that can occur sympatrically. Cryptic species 

and selected genotypes of P. fortinii s. l. can now be used to test the ecological 

significance of these extremely abundant and successful organisms and 

to explain some of the contradictory results on fungus-host interactions 

reported in earlier studies. The elucidation of the mating mechanism(s) 

and the evolutionary forces that govern speciation in P. fortinii s. l. are 

other fascinating topics for future research. 

We have reviewed patterns of species diversity and within-species genotypic 

diversity and presented several plausible explanations for these patterns, 

although conclusive evidence for cause and effect are still virtually 

lacking. Nevertheless, we would like to conclude with a motivating citation 

by Begon et al. (1990): “This is not so much a disappointment as a challenge 

to ecologists and biologists of the future. Much of the fascination of ecology 

and biology lies in the fact that many problems are blatant and obvious for 

everybody to see, while the solutions have as yet eluded us”. 


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8 

Endophytic Root Colonization 

by Fusarium Species: Histology, 

Plant Interactions, and Toxicity 

Charles W. Bacon, Ida E. Yates 

8.1 

Introduction 

Fusarium species have adapted to a wide range of geographical sites, climatic 

conditions, ecological habitats, and host plants, and species of this 

polyphyletic genus have been documented to occur worldwide (Backhouse 

et al. 2001). In spite of the information available on the extremes in geographic 

distribution and climatic conditions, appropriate data to predict 

the center of origin(s) or the mode(s) of dispersion of this genus have 

not been obtained. Much of the information on distribution patterns has 

been determined from analyses of soil samples, a common habitat, in addition 

to colonization of many plant species. The diversity of plant species 

colonized by members of the genus Fusarium is amazing. A recent literature 

survey determined that Fusarium species have been isolated from 

plants belonging to the gymnosperms and the monocotyledonous and 

dicotyledonous angiosperms (Kuldau and Yates 2000). They are the primary 

incitants of root, stem, and ear rots in many agriculturally important 

crops. For example, F. verticillioides (= F. moniliforme) is capable of colonizing 

well over 1,000 plant species, including maize (Zea mays L.), one 

of the world’s most important food crops. Another species, F. oxysporum, 

is cosmopolitan; certain strains are usually host specific and pose a severe 

threat to most of the world’s supply of food crops. Furthermore, species 

such as F. graminearum, along with F. verticillioides and related species 

within the Liseola section, are notorious for the production of mycotoxins 

on wheat, maize, barley, rice and other cereal grains and foodstuffs 

(Marasas et al. 1984). Consequently, studies on the association of Fusarium 

species with plants are critical in order to develop control measures for 

this group of fungi that affects the quality and quantity of the world’s food 

supply. 

Charles W. Bacon: Richard B. Russell Research Center, ARS, United States Department of 

Agriculture, Toxicology and Mycotoxin Research Unit, SAA, P.O. Box 5677, Athens, GA 

30604, USA, E-mail: cbacon@saa.ars.usda.gov 

Ida E. Yates: Richard B. Russell Research Center, ARS, United States Department of Agriculture, 

Toxicology and Mycotoxin Research Unit, Athens, GA 30604, USA 





134 C.W. Bacon, I.E. Yates 

The discussion of Fusarium root endophytes in this chapter is based 

on, and will be discussed relative to, our knowledge of fungal endophytes 

of grasses. Noted examples of fungal endophytes include the species of 

the Balansieae that show various degrees of tissue specificity and often 

display evidence of infection by the production of sporulation structures 

on the adaxial or abaxial leaf surface of grasses (Diehl 1950), as well as the 

productionofcharacteristictoxicsecondary metabolites(Baconetal. 1986). 

For example, fungi of the genus Neotyphodium (teleomorph = Epichloë) 

arefoundonlyinthestems,leavesandseedofgrassesbutarenotfound 

in roots, while species of Myriogenospora are restricted to the leaves, and 

species of Balansia are restricted to stems or leaves, but all produce ergot 

alkaloids. 

Some research suggests that there are similar positive interactions of 

endophytic Fusarium species with plants (Damicone and Manning 1982; 

Hallmann and Sikora 1994a, 1994b; Blok and Bollen 1995). We use the 

definition of fungal endophytes as indicated by Stone et al. (2000) to include 

those Fusarium species that are associated with roots as intercellular, 

symptomless fungi (Fig. 8.1a–e), although the endophytic association may 

extend to above ground plant parts and there may be a differential expression 

of infection with different host tissue types (Bergman and Bakker-Van 

der Voort 1979; Fisher et al. 1992; Foley 1962; Yates and Jaworski 2000). 

In addition to grass endophytes, specific attention will be directed to our 

past and present toxicological, physiological, and morphological studies on 

Fusarium verticillioides [synonym F. moniliforme, teleomorphGibberella 

fujikuroi (Sawada) Ito in Ito & K. Kimura] and its association with maize. 

Thus, species of Fusarium endophytes include those fungi that occupy the 

intercellular spaces of plants, and the intercellular infections may be localized 

to roots. However, localization to roots is not mutually exclusive as 

some Fusarium species, while living as root endophytes, may also infect 

above ground plant organs, although the foliage origin of such hyphae 

from the endophytic infections in the roots has not been established for 

all species. Indeed, secondary infections from aerial spores are suspected 

of contributing to most foliage infection (Adams 1921; Boshoff et al. 1996; 

Kang and Buchenauer 2000) and, as discussed below, there is the possibility 

of different specific fungal strains that infect specific tissue types, especially 

the flower infections. 

8.2 

Plant and Fungus Interactions 

Contrary to the association found in Neotyphodium grass species, Fusarium 

species are not necessarily obligate endophytes. Indeed, as presented

8RootEndophyticFusarium Species 135 

Fig.8.1. a–e Light micrographs of the endophytic habit of a non-virulent isolate of Fusarium 

verticillioides, RRC 826, in roots of 2-week-old maize seedlings. a Hyphae (arrowhead) 

running parallel within intercellular spaces of the first internodes and junction of primary 

root of maize (54.4X). b Higher magnification of maize root showing a branching septate 

hypha (arrowhead) between two cell walls (272X, phase contrast). c Hyphae running parallel 

within intercellular spaces with a branching hypha at arrowhead (272X, phase contrast). 

d Cross section of a secondary root with hyphae (arrowhead) in the intercellular spaces 

(54.5X) (very dark spherical bodies within cells are artifacts of staining). e Cross section 

through the cortex of a primary root with groups of hyphae (arrowheads) in the intercellular 

spaces (109X, phase contrast) (from Bacon and Hinton 1996, with permission) 

below, their nutritional physiology, i.e., parasitic and saprophytic, predicts 

a transitory nature of the endophytic phase of Fusarium associations. 

Understanding to what extent species or isolates of this genus are endophytic 

is hampered by studies, histological or otherwise, that inadequately 

describe the qualitative and quantitative distribution of Fusarium within 

hosts. Nor are there studies to indicate any host requirements or benefits 

derived from such association that are indicated and characteristic of 

those derived from the Neotyphodium grassendophytes(forreview,see 

Bacon and White 2000). However, some studies are highly suggestive of 

benefits, at least to the fungus (Lee et al. 1995; Yates et al. 1997; Munkvold 

and Carlton 1997; Kuldau and Yates 2000; Pinto et al. 2000; Bacon et al. 

2004).


ReportsabouttheinteractionsofF. verticillioides with maize have been 

contradictory since the initial description of this fungus as both a pathogenic 

and a symptomless infection (Sheldon 1902; Voorhees 1934). Anatomical 

features of pathogenic infections by Fusarium species, including F. verticillioides, 

have been reviewed (Pennypacker 1981; Bacon and Hinton 1996), 

although some of the reports were concerned mainly with above ground 

plant parts. Voorhees (1934) described an initial infection of F. verticillioides 

intorootsthatoccurredfromthesoil.Infectiontookplaceviathe 

primary radicle by the fungus entering the epidermis, although it can also 

enter through ruptures produced in the cortex by emerging lateral roots. 

Since the endodermis of the young radicle acts as the barrier against penetration 

(Voorhees 1934), spread of infection into the stele is prevented. 

However, infection of the stele can occur from soil via the wounds produced 

by adventitious and lateral roots, suggesting that the degree and rate 

ofsuberizationinyoungseedlingscanserveasthekeytothenatureof 

disease development as opposed to development and the duration of the 

symptomless state. 

Modern day maize cultivars are more resistant and, thus, symptomless 

endophytic colonization by F. verticillioides is the rule today (Foley 1962; 

Kommedahl and Siggerirsson 1975; Thomas and Buddenhagen 1980; Bacon 

and Hinton 1988; Ayers et al. 1989; Leslie et al. 1990; Corell et al. 1992; 

Bentley et al. 1995; Ahmed et al. 1996; Anaya and Roncero 1996; Bacon and 

Hinton 1996; Bai 1996; Bakan et al. 2002; Anjaiah et al. 2003). Maize kernels 

are universally infected by F. verticillioides and related species, but disease 

symptoms are rarely exhibited. Possibly, plant breeding and selection may 

also form the basis for symptomless root infection in other agricultural 

species and cultivars. Further, species and strain genetics are important 

within the overall population of each species, and are important in the 

overall impact of a species on a host. Indeed, there is now information to 

indicate that a genetic change that will convert a Fusarium pathogen to 

a nonpathogenic endophytic mutualist can occur (Freeman and Rodriguez 

1993). The following are brief descriptions of both the symptomless and 

pathogenic infections. 

Molecular tools have also been utilized to study the association of F. verticillioides 

with maize and other plants. In situ studies of this species were 

made possible with avirulent isolates of F. verticillioides transformed with 

a plasmid containing the gusA gene coding for β-glucoronidase (GUS) 

and the hygr gene coding for hygromycin resistance (Yates et al. 1999). 

Subsequent GUS activity is detectable by histochemical and fluorometric 

enzymatic assays during the colonization period. The results indicated 

that F. verticillioides could be traced from the initial seed, to recovery 

from roots of plants produced from these seed, up through the stem, and 

finally isolated internally from seed (Bacon et al. 2001). Further, it was


Fig.8.2. a–e Scanning electron micrographs of a symptomless F. verticillioides-infected 

maize kernel. a A longitudinal section showing the location of the fungus at the tip cap 

(x13). b Magnified version of a, showing the location of the fungus in box (arrow) below 

the vascular tissues (v). c AhyphaofF. verticillioides (arrow) within the boxed area 

of b. d Growth of the fungus on culture medium showing chains of microconidia (arrow) 

characteristic of this species. e Growth of the fungus on the other half of the sectioned maize 

kernel in a, above, incubated aseptically for 2 days on damp filter paper, and showing the 

chains of microconidia, which established that the hypha in a was that of F. verticillioides 

(from Bacon et al. 1992, with permission) 

demonstrated that seed produced from these plants are sound, the fungus 

is internally seed borne (Fig. 8.2a–f), and produce seedlings that are infected 

with the transformed fungus (Bacon et al. 2001). Thus, this species is 

disseminated vertically, but horizontal dissemination is expected to occur 

through wounds due to the activity of insects (Munkvold and Carlton 1997), 

from soil to roots and other injured plant parts (Foley 1962), as well as via 

aerial borne spores. Most Fusarium species are also disseminated horizontally 

(Adams 1921), although vertical transmission is equally possible since


several species have been recovered from seed as endophytes (Gordon 1952; 

Fisher and Petrini 1992). 

8.2.1 

Hemibiotrophic Characteristics 

Hemibiotrophic fungi include those that infect living tissue, similar to 

biotrophs, but after an extended incubation period of days or weeks the 

infected tissue dies, within which the fungus now continues to develop as 

a saprotroph, usually resulting in sporulation (Luttrell 1974). The distinction 

between a hemibiotrophic parasite and a necrotrophic parasite is one 

of tissue infections. Necrotrophs kill host tissue in advance of penetration; 

while biotrophs penetrate, infect and obtain their food from living tissue, 

and this includes sporulation on living tissue. Having made the distinction 

between the basic terminologies used to distinguish the nutrition association 

of parasitic fungi with vascular plants, we now can apply this to 

endophytic Fusarium species. 

Following the definition above, Fusarium speciesshouldbeconsidered 

hemibiotrophs. That is, they are fungi that infect living tissue as biotrophs 

but after a latency period, which may last for a period of days to weeks, can 

cause host tissue to die, at which point the fungus becomes a saprotroph. 

Thus, endophytic Fusarium species, along with other fungal endophytes, 

are facultative biotrophic parasites, although this mode of nutrition can 

be a transient feature. Fungi belonging to Claviceps and Colletotrichum,as 

well as Ustilago maydis and Magnaporthe grisea,aretypicalhemibiotrophs. 

However, as we shall see below, not all isolates of the species F. verticillioides 

are hemibiotrophic and this might be due to either host and fungus genetics 

or environmental factors, or both. Certainly, in those situations where the 

saprotrophic stage is prevented, mycotoxin accumulation might be reduced, 

providing one point of reducing the concentration of these toxins. 

The parasitic association of Fusarium species with roots as endophytes 

may be viewed as a long-term event, perhaps confounded by plant breeding, 

and selection not for endophytic infection but for disease expression. Plant 

breeding might make it difficult to clearly define the degree of biotrophy 

characteristic for each Fusarium species relative to specific modern agricultural 

host cultivars that differ in disease resistance. In addition, the degree 

of biotrophy depends on the genetics of fungal strains and hosts, and the 

site of inoculation (Corell et al. 1992; Munkvold and Carlton 1997; Carter et 

al. 2000; Mesterhaszy et al. 2003). Thus, the association of Fusarium species 

with plants as symptomless endophytes cannot be explained entirely on 

the basis of the nutritional parasitic relationships described above. Complete 

understanding of Fusarium species as symptomless root endophytes


is hampered due to the complex of interactions, compounded by the fact 

that symptomless associations are altered by damaged portions of roots, 

due to predation by insects and wounds from emerging lateral roots. This 

results in a transformation of the symptomless state of the root-infecting 

species to the biotrophic and saprophytic state that infects dying and dead 

tissue. Most Fusarium rot diseases, i.e., root rots, are characterized during 

this phase. However, this appearance of biotrophic and saprophytic states is 

not necessarily obligatory and, indeed, a plant may have the symptomless 

state in roots while other parts of the same plant may be diseased. It is this 

aspect of the association that is of major concern in this review. 

8.2.2 

Histology 

Symptomless root infections are characteristic of many Fusarium species 

(Foley 1962; Pennypacker 1981; Wong et al. 1992; Leslie 1994; Bacon and 

Hinton 1996; Bowden and Leslie 1994; Gang et al. 1998; Nicholson et al. 

1998; Kedera et al. 1999; Kuldau and Yates 2000; Bai et al. 2002), and there 

are very aggressive or virulent strains of all species that serve as incitants 

of several plant diseases (Foley 1962; Malalasekera et al. 1973; Pennypacker 

1981; Manaka and Chelkowski 1985; Liddell and Burgess 1985; Wilcoxon et 

al. 1988; Fisher and Petrini 1992; Bacon and Hinton 1996; Bai 1996; Boshoff 

et al. 1996; Parry and Nicholson 1996; Kosiak et al. 1997; Nicholson et 

al. 1998; Wildermuth et al. 1999; Hysek et al. 2000; Ribichich et al. 2000). 

Disease development is considered to be a consequence of fungal and host 

genetics, while environmental biotic and abiotic factors negatively affect 

overall survival of the host (Schroeder and Christensen 1963; Bacon et al. 

1996; Ribichich et al. 2000). 

Detailed histological studies of specific Fusarium species are lacking 

since it is the disease state that is most often studied. Further, molecular 

analyses now suggest that these studies involved either different species or 

more than one species. For example, we now know that studies of scab or 

head blight of wheat consisted of several cryptic species. The flower-foliage 

disease or scab is caused primarily by F. graminearum and, to a lesser extent, 

by a new species F. pseudograminearum, while crown rot of wheat is caused 

by yet another new species, Gibberella coronicola (Aoki and O’Donnell 

1999; O’Donnell et al. 2000). Nevertheless, there are similarities and the 

following discussion takes these into account. 

Bacon and Hinton (1996) compared root and shoot infections by both 

virulent and non-virulent strains of F. verticillioides on maize using lightand 

electron-transmission-microscopy and concluded that this strain, and 

perhaps most others, should be considered as symptomless endophyte(s)


(Fig. 8.1a–e), especially since most isolates produce symptomless infections 

with most modern day cultivars of maize. It was also concluded that this 

species was not a vascular rot fungus, agreeing with the earlier observations 

of Pennypacker (1981) that F. verticillioides has the potential to be a cortical 

rot fungus (Fig. 8.1d–e). The hyphae of F. verticillioides run parallel within 

intercellular spaces, although branching hyphae are observed, especially 

in areas of branching roots (Fig. 8.1c). A study of the symptomless state 

in seedling roots suggests that the symptomless state persists beyond the 

seedling stage and contributes, without visual signs, to mycotoxin contamination 

of maize both before and during kernel development (Bacon and 

Hinton 1996). Symptomless root infections have been reported in plants 

infected by several species of Fusarium including F. graminearum (Gordon 

1952; Sieber et al. 1988), F. oxysporum f. sp. melonis (Katan 1971), F. oxysporum 

(Lemanceau et al. 1993), F. nivale, F. culmorum (Sieber et al. 1988), 

F. crookwellense (Boshoff et al. 1996), F. culmorum (Kang and Buchenauer 

1999); see additional species in the review of Kuldau and Yates (2000), and 

in Warren and Kommedahl (1973). 

Symptomless infection by Fusarium species varies and is related to the 

infection age of the hyphae (Baayen and Rykenbuerg 1999), and compartmentalization 

of the disease within resistant host tissues (Baayen et al. 1996) 

while others remain symptomless and biotrophic and are characterized as 

having interfacial membranes that separate fungal and plant plasma membranes 

(Fig. 8.3a–c) (Marchant 1966; Malalasekera et al. 1973; Baayen and 

Elgersma 1983; Bacon and Hinton 1996; Boshoff et al. 1996) The association 

is compatible over an extended time period, and non-specialized hyphae, 

which appear not to differ morphologically from the intercellular hyphae of 

symptomless infection (Bacon and Hinton 1996; Figs. 8.3a–c, Figs. 8.4a–f), 

are used for nutrient absorption. Endophytic hyphae of Fusarium spp. are 

not dormant (or quiescent), but are metabolically active throughout the 

association and within the intercellular spaces (Miller and Young 1985; 

Evans et al. 2000; Kang and Buchenauer 2000; Bacon et al. 2001). In such 

hyphae, there is a consistent lack of distinct nutrient absorbing structures 

characteristic of the usual pathogenic infections in rust and powdery 

mildews such as haustoria. Virulent strains of F. verticillioides do have intracellular 

hyphae that are not distinct haustoria (Fig. 8.4b–f) (Bacon and 

Hinton 1996). However, haustoria represent only one type of nutrient absorption 

structure. Fungal cell-wall-to-plant-wall appositions, as observed 

in most strains of F. verticillioides and other Fusarium speciesaswellas 

other fungal endophytes, are one of three fungus-plant cell nutrient absorbing 

structures (Honneger 1986). Thus, nutrient absorption by wall-to-wall 

contact and nutrients from the apoplasm are apparently just as efficient as 

intracellular hyphae since growth and colonization of the host is still accomplished, 

along with the production of secondary metabolites (Bacon et


Fig.8.3. a–c Transmission electron micrographs of the symptomless endophytic hyphae of 

F. verticillioides, RRC 826, as they appear in cross-section of 1- to 8-week-old plants of 

maize. a Two hyphae (f )inanintercellularspace(i)ofmaizecells(h); bar 1µm.b Another 

view of the intercellular nature of hyphae in roots of maize plants approximately 8 weeks 

old; bar 1µm.c Twogroupsofhyphaeinintercellularspacesoftherootcortex;bar 1µm 

(from Bacon and Hinton 1996, with permission) 

al. 2001). The accumulation of secondary compounds must require, a priori, 

a tremendous amount of energy. Certainly, the greater distribution 

of Fusarium hyphae in roots (Foley 1962; Warren and Kommedahl 1973; 

Kommedahl and Siggerirsson 1975; Blok and Bollen 1995; Bacon and Hin-


Fig.8.4. a–f Transmission electron micrographs of a virulent strain of Fusarium verticillioides, 

RRC pat, in maize plants during the early stages of seedling blight showing several 

variations of intracellular hyphae. a A noninfected plant showing intact chloroplast (ch); 

bar 1µm.b At 3 weeks, the fungus is primarily intracellular; chloroplasts, while intact, are 

becoming disorganized; one fungus (f ) is connected between cells with a hyphal penetration 

peg (arrowhead); bar 1µm.c Inter- and intra-cellular location of the fungus in leaves; 

intracellular fungus (f ) with an elongated hyphal penetration peg (arrowhead) within cell 

of maize (h); chloroplasts are no longer intact; bar 1µm.d Intercellular hyphae invading 

another cell in stem tissue of a 3-week-old plant; bar 1µm.e An intracellular hypha growing 

between two cells along and between the middle lamellae; bar 1µm.f Maize tissue showing 

extensive inter- and intra-cellular colonization; bar 1 µm (from Bacon and Hinton 1996, 

with permission) 

ton 1996) reflects the larger amounts of nutrients within the root apoplasm 

that accumulate as a sink from photosynthesis. 

Reports on symptomatic infections are numerous, although detailed histological 

studies of these types of infections tend to be restricted to the specific 

plant organs showing the effects (Kang and Buchenauer 2000; Boshoff 

et al. 1996; Ribichich et al. 2000). However, there are several histological 

similarities during the change from biotrophic phase to necrotrophic phase


(Adams 1921; Bennet 1931; Malalasekera et al. 1973; Wong et al. 1992; Bacon 

and Hinton 1996; Baayen and Rykenbuerg 1999; Kang and Buchenauer 

2000), reinforcing our concept that most Fusarium species are hemibiotrophic. 

Specialized intracellular infection and nutrient absorbing hyphae are 

absent during the biotrophic state of F. verticillioides infecting maize 

(Figs. 8.1a–e, 8.3a–c) and in other hosts as well (Malalasekera et al. 1973; 

Manaka and Chelkowski 1985; Honneger 1986; Boshoff et al. 1996; Kang 

and Buchenauer 1999, 2000), and in general these fall within the type 1 

category that ranges from the simple to the appressorium configuration 

(Honegger 1986). This type of fungus-plant cell interaction is characteristic 

of interactions that occur in different symbiotic systems (Honegger 

1986). We do not know if the lack of specialized nutrient absorption structures 

during this phase is characteristic of all Fusarium species. However, 

infection of host cells by specialized hyphae is described for F. culmorum 

and other species during the change to the intracellular stage of infection 

(Malalasekeraet al. 1973; Kang and Buchenauer 2000) and these would fall 

within the definition of one of the type 2 intracellular haustoria without 

sheath and papilla described by Honegger (1986) as indicative of mutualistic 

symbioses commonly found in lichens. However, the nature of the 

association with a particular host may vary, as some endophytic associations 

appear to be more epicuticular (on glumes) than endophytic and 

systemic (Kang and Buchenauer 1999). A lack of specialized intracellular 

absorbing structures assures less injury to the host, thus insuring compatibility, 

and presumably nutrients are obtained from the apoplasm of the 

intercellular spaces, although there may be a fungus-directed source and 

sink relationship. 

8.2.3 

Mycotoxins 

Fusarium species are a highly successful group of fungi that produce a variety 

of secondary metabolites, some of which might be important in both 

the long- and short-term strategies of the species. Most biochemical studies 

have concentrated on the production of, and factors leading to the accumulation 

of, mycotoxins and related compounds. However, there are sporadic 

and often observational reports on the positive and negative effects of 

symptomless infections by Fusarium species on hosts, and on competing 

organisms, which may be activities of secondary metabolites. Fusarium 

species produce a wide diversity of mycotoxins, resulting in a variety of 

effects on animals (Marasas et al. 1984, 1988; Voss et al. 1990; Norred et 

al. 1992; Riley et al. 1993). Mycotoxins have been speculated to play an


important role in the long-term survival strategy of the producing fungus. 

However, in some instances, some mycotoxins might play an even greater 

role in the day-to-day competitive fitness strategies of Fusarium species 

and these are presented briefly below. 

8.2.4 

Mycotoxins and Host Relationships 

Fumonisins are mycotoxins and are the subject of current concern due to 

their widespread occurrence in maize and maize products. F. verticillioides 

and other fungi of the Liseola section produce fumonisins in maize and 

other commodities. Fumonisins were shown to accumulate in colonized 

maize roots early during maize seedling development, and more fumonisins 

are isolated from roots than from shoots at this early stage of growth 

(Bacon et al. 2001). Fumonisin mycotoxins probably also occur in the roots 

of other plant species, and a rooting response has been shown for one cultivar 

of tomato (Bacon and Williamson 1992). A genetic and morphological 

study of conidiation mutants of F. verticillioides yielded interesting results 

concerning the role of fumonisins in plants (Glenn et al. 2004). Wild type 

isolates produced enteroblastic phialidic conidia, while mutants incapable 

of enteroblastic conidiogenesis produced undulating germ tube-like outgrowths. 

These mutants were not capable of infecting maize roots, and 

varied in their fumonisin production. Although they could not infect the 

plant, only those mutants that could produce the fumonisin were able to 

cause death of seedlings, but only in a small sampling of maize cultivars. 

This suggests that fumonisins play a role in the pathogenic processes of 

some Fusarium species but expression of toxicity on the host depends on 

the host genotype. 

Other Fusarium mycotoxins that might have a function in the physiology 

of the association include those produced by F. graminearum and 

related species. These include deoxynivalenol (DON or vomitoxin, a type 

B trichothecene), other related trichothecenes, and zearalenone. The mycotoxin 

DON is by far the most dominant of the trichothecenes, occurring 

on oats, rye, and maize, and occasionally on rice, sorghum, and triticale. 

In addition to the economic impact of this toxin in reducing animal performance, 

DON has adverse effects on plant performance. 

DON has been implicated as a virulence factor for some hosts (Bai 

et al. 2002; Desjardins and Hahn 1997; Harris et al. 1999), although the 

concentrations produced may or may not directly correlate with the degree 

of fungal virulence (Desjardins et al. 1996; Carter et al. 2000; Bai et al. 

2001; Mesterházy et al. 2003). DON was isolated from florets and grains of 

rye, wheat, and maize (Desjardins et al. 1996), but nothing is known about


its distribution and early toxin production within root tissue, or about 

how much of this toxin is produced during any endophytic stage of wheat 

or maize root colonization by F. graminearum, the main cause of blight 

disease of cereals. Nevertheless, this species also colonizes maize stalks and 

all tissues of wheat asymptomatically (Sieber et al. 1988; Dodd 1992; Fisher 

et al. 1992). McLean (1996) has reviewed additional information on the 

phytotoxicity of various Fusarium metabolites. 

8.2.5 

Physiological Interactions and Defense Metabolites 

Positive physiological interactions with maize were recorded for several 

strains of F. verticillioides These include increased rooting (Bacon and 

Williamson 1992), and earlier lignification of roots in seedling plants (Yates 

et al. 1997). Biocontrol uses of several Fusarium species not only indicate 

the utility of the genus but also suggest possible mutualistic interactions 

derived from the associations, including insecticidal (Abado-Becognee et 

al. 1998), nematocidal (Hallmann and Sikora 1994a, 1994b), and fungicidal 

(Damicone and Manning 1982; Lemanceau et al. 1993) activities. 

The endophytic association of F. verticillioides with maize has evolutionary 

and physiological implications since it has been established that 

all maize isolates of this fungus can detoxify the host’s native antimicrobial 

compounds, the benzoxazinoids (Glenn and Bacon 1998; Glenn et al. 

2001, 2002). The ability to detoxify these compounds, the concentrations 

of which may be high in roots (Xie et al. 1991), has been interpreted as 

one mechanism by which endophytic colonization is not prevented, since 

the benzoxazinoids are especially toxic to fungi (Xie et al. 1991; Schulz and 

Wieland 1999; Sicker et al. 2000; Glenn et al. 2001, 2002). Further, strains of 

F. verticillioides isolated from banana cannot detoxify the benzoxazinoids 

(Glenn et al. 2001, 2002). Several other species of Fusarium can detoxify 

the benzoxazinoids, suggesting the importance of this mechanism to this 

group, as well as to other maize pathogens (Schulz and Wieland 1999), for 

the endophytic association with grasses (Glenn 2000; Sicker et al. 2000). 

It is well known that several species of bacteria are endophytic in plants, 

also occupying intercellular spaces [for review, see Chanway 1998; see also 

Chaps. 2 (Hallmann and Berg), 3 (Kloepper and Ryu), and 6 (Anand et al.)]. 

Endophytic bacteria are ecological homologues for most species of endophytic 

Fusarium species, and compete for nutrients within the apoplasm. 

Several biocontrol strategies are based on the use of bacterial endophytes 

[Chanway 1998; Kobayashi and Palumbo 2000; see Chaps. 3 (Kloepper and 

Ryu), and 4 (Berg and Hallmann)]. However, all Fusarium species examined 

produce fusaric acid (Bacon et al. 1996), and possibly other unknown an-


tibiotics that serve to control bacteria in planta. Fusaric acid is inhibitory 

to most strains of endophytic and rhizosphere bacteria (Notz et al. 2002; 

Bacon et al. 2004). Fusaric acid is moderately toxic to mammals (Porter 

et al. 1995), but is produced by most isolates of Fusarium (Bacon et al. 

1996). However, its activity is broad and it has pronounced effects on microorganisms. 

Fusaric acid is an antibiotic, showing activity against both 

Gram-negative and -positive bacteria, especially those used for biocontrol. 

Fusaric acid was shown to interact with genes specifically used by the 

biocontrol bacterium Pseudomonas fluorescens by preventing expression 

of genes encoding specific inhibitors of fungi (Notz et al 2002). A similar 

mode of action has been established for DON, which prevents the production 

of the chitinase gene expressed by the biocontrol agent Trichoderma 

atroviride (Lutz et al. 2003). 

Knowledge of the complete spectra of activity for most Fusarium secondary 

metabolites is incomplete. Moniliformin is a weak mycotoxin and 

an even better phytotoxin for specific hosts (Cole et al. 1973), but any overall 

effects pertaining to Fusarium species are unknown. The beauvericins 

show strong selective insecticidal properties (Vesonder and Hesseltine 

1981; Plattner and Nelson 1994; Logrieco et al. 1998). The beauvericins 

are produced by at least 12 Fusarium species (Logrieco et al. 1998), and 

can protect Fusarium-infected plants from insect predation. The chemical 

isolation and some biological activities of numerous other metabolites 

have been reviewed by Marasas et al. (1984) and Thrane (1989) and include 

the enniatins, butenolide, wortmannin, diacetoxyscripenol, nivalenol, visoltricin, 

chrysogine, culmorin, aurofusarin, equisetin, fusoproliferatum, 

fusarochromanone, acuminatopyrone, fusamarin, chlamydosporol, additional 

derivatives of T-2 toxins, and several unidentified antibiotics. The 

identities of the Fusarium speciesproducingthesemetabolitesandamore 

comprehensive listing of the activities of these and other mycotoxins can 

be obtained from Thrane (1989, 2001) and Summerell et al. (2001). 

8.3 

Summary 

Fusarium is a very important genus from the point of view of food production 

and food safety. Fusarium species exist as intercellular root endophytes 

in both cultivated and wild plants and their role during the symptomless 

state of infection is ambiguously defined. However, many species are 

pathogenic, causing diseases such as root, stem, and ear rot on crop plants, 

thereby reducing plant productivity. F. verticillioides and other Fusarium 

species are unique endophytes, with similarities to other endophyte species 

such as the foliage endophytes of forage grasses, but are more versatile since


they are also hemibiotrophic in their associations with plants. The problems 

associated with this genus are global as their distribution is worldwide, 

and most host plants are susceptible to infection by one or more 

Fusarium species. Certain biotic and abiotic factors may alter Fusarium 

relationships with plants from a symptomless endophytic association to 

a hemibiotrophic and finally a saprotrophic association where mycotoxins 

might accumulate, leading to animal and human health concerns. To summarize, 

a major concern is that many Fusarium species produce mycotoxins 

that are harmful to humans and animals ingesting food or feed products colonized 

by the fungus. The mycotoxins are produced during the pathogenic 

and saprophytic states, but the infections caused by these two states can be 

initiated from the symptomless root endophytic biotrophic state. 

The dual characterization of F. verticillioides as both a pathogen and 

a symptomless endophyte indicates both the complex relationship of this 

species with plants as well as suggesting similar complexities for other 

Fusarium-plant interactions. Consequently, the development of appropriate 

control measures for virulent Fusarium isolates are expected to be 

difficult. For example, on the one hand, diseases and mycotoxins produced 

by F. verticillioides must be controlled, while on the other hand, the intimate 

association of the endophytic state of this species appears to confer some 

positive competitive fitness traits to certain plants. The extent to which this 

occurs due to present-day plant breeding efforts will be determined with 

more detailed studies. The association of this genus with roots as symptomless 

endophytes indicates a role for these fungi in nutrition, and suggests 

the importance of the root as an endophytic niche during the co-evolution 

of Fusarium species and plants. 


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9 

Microbial Endophytes of Orchid Roots 

Paul Bayman, J. Tupac Otero 

9.1 

Introduction 

Orchids are interesting on many levels. Their beautiful and bizarre adaptations 

for pollination have fascinated many people, including Darwin (1887). 

Interest in orchid pollination biology has overshadowed another, equally 

important symbiosis: mycorrhizal relationships. In turn, interest in orchid 

mycorrhizae has overshadowed the relationships between orchids and endophytes. 

In most cases, studies on orchid root fungi have ignored all fungi 

not thought to be mycorrhizal. In some cases, fungi in orchid roots have 

been presumed to be mycorrhizal when they may in fact be endophytes. 

Thus the frequency, diversity and importance of orchid root endophytes 

remain largely unexplored. 

The goal of this chapter is to review the incidence and importance of 

endophytes in orchid roots, and to disentangle the literature on mycorrhizal 

fungi from that on non-mycorrhizal endophytes. 

9.2 

Habits and Types of Orchid Roots 

The Orchidaceae is one of the largest families of plants, with 25,000 species – 

close to one-tenth of all known flowering plant species (Dressler 1990). 

Orchids are found in all except the most extreme terrestrial environments. 

Orchids are epiphytic (growing on plants), lithophytic (growing on 

rocks), terrestrial, or, in a few cases, some combination thereof. Epiphytic 

and lithophytic orchids are all tropical or subtropical; they comprise about 

75% of all species (Dressler 1990). Terrestrial orchids are found worldwide; 

all temperate orchids are terrestrial. Most of what is known about 

orchid-fungal associations comes from terrestrial orchids (perhaps because 

Paul Bayman: Departamento de Biologia, Universidad de Puerto Rico – Rio Piedras, PO Box 

23360, San Juan, PR 00931, USA, E-mail: pbayman@upracd.upr.clu.edu 

J. Tupac Otero: Universidad Nacional de Colombia-Palmira, Departamento de Ciencias 

Agrícolas, AA 237, Palmira, Valle del Cauca, Colombia 





154 P. Bayman, J.T. Otero 

most orchidologists live in temperate countries) even though they are less 

speciose than epiphytes. 

Roots of epiphytic/lithophytic and terrestrial orchids differ (Rasmussen 

1995). Epiphytic and lithophytic roots are ecologically equivalent because 

inbothcasestherootsareexposedtolightandair.Rootsofepiphytic 

and lithophytic orchids are photosynthetic, perennial, and fairly constant 

throughout the year. Roots of terrestrial orchids, in contrast, are usually 

non-photosynthetic, live ≤ 3 years, and often show marked seasonal differences 

in growth and composition. They are usually buried in soil or 

leaf litter. Some terrestrial orchids have two morphologically distinct types 

of roots, one of which is mycorrhizal (Rasmussen 1995). Non-mycorrhizal 

roots tend to have more xylem and more amyloplasts than mycorrhizal 

roots. 

Orchid roots have a velamen, a multiple epidermis of one to several layers 

of thin-walled cells. The velamen helps the root trap water and probably 

nutrients (Dressler 1990; Rasmusssen 1995). It has been suggested that the 

velamen evolved to facilitate colonization of roots by mycorrhizal fungi 

(Dressler 1990); this seems unlikely because pelotons are more common in 

the cortex than in the velamen. (Pelotons are coils of hyphae within root 

cells, and are characteristic of orchid mycorrhizae). Also, epiphytic orchid 

roots generally have more developed velamens than terrestrial orchid roots 

(Dressler 1990), even though the frequency of mycorrhizal infection is 

lower. 

Terrestrial orchids are usually obligately mycorrhizal, even as adults 

(Rasmussen 1995). Some species are non-photosynthetic (or more accurately, 

myco-heterotrophic) and depend on fungi for nutrition (see 

Sect. 9.8). Many epiphytic orchids are facultatively mycorrhizal, at least 

as adults, and there is wide variation in frequency of mycorrhizal colonization; 

the same is probably true of epilithic orchids (Hadley and Williamson 

1972; Lesica and Antibus 1990; Goh et al. 1992; Richardson et al. 1993; 

Zelmer et al. 1996; Currah et al. 1997; Bayman et al. 1997; Rivas et al. 1998; 

Otero et al. 2002; Rasmussen 2002). 

9.3 

Bacteria as Epiphytes and Endophytes of Orchid Roots 

In general, bacterial root endophytes have been studied in an agricultural 

context, rather than in an ecological or biodiversity context [Chanway 1995; 

see Chaps. 6 (Anand et al.) and 19 (van Overbeek et al.)]. Orchids are no 

exception: interest in bacteria in orchid roots has focused on pathogens 

of cultivated plants rather than on endophytes of wild plants (Hadley et 

al. 1987). This oversight is unfortunate, since endophytic bacteria may be


importantforthehealthofwildplantsaswellascropplants(Hallmannet 

al. 1997; Sturz et al. 2000). 

Most knowledge of endophytic bacteria in orchid roots comes from 

studies in Australia. Endophytic bacteria were isolated from 12 species of 

terrestrial orchids in Western Australia (Wilkinson et al. 1994). The most 

common genus isolated was Pseudomonas,whichvariedfrom23%to73% 

of isolates from each orchid species. Most isolates from Pterostylis spp. were 

also Gram-negative, while most isolates from all other orchids were Grampositive. 

This difference may reflect morphological differences among the 

orchids: in Pterostylis the main absorptive organs are underground stems, 

whereas the other orchids tested use adventitious roots. Some of these 

bacteria stimulated germination in vitro of seeds of P. vittata (Wilkinson 

et al. 1989). 

Epiphytic bacteria on orchid roots have also been studied, mainly to 

determine if nitrogen-fixing bacteria can help orchids obtain nitrogen. 

Arthrobacter, Bacillus, Mycobacterium, Pseudomonas, Oscillatoria and Nostoc 

were isolated from the surfaces of roots of the terrestrial orchid Calanthe 

vestita,andBacillus, Curtobacterium, Flavobacterium, Nocardia, Pseudomonas, 

Rhodococcus, Xanthomonas and Nostoc were isolated from the 

surface of roots of the epiphyte Dendrobium (Tsavkelova et al. 2001). Of 

these, the cyanobacteria Oscillatoria and Nostoc are capable of nitrogen fixation; 

they were not isolated from soil collected near the plants, suggesting 

some special affinity for the root surface. These and other cyanobacteria 

formed a biofilm on the surface of roots of epiphytic orchids in a greenhouse 

(Tsavkelova et al. 2003). Cyanobacteria have also often been observed 

within velamen cells of epiphytic orchids (Dressler 1990; Sinclair 1990). 

Nitrogen-fixing bacteria also occur on epiphytic Tillandsia plants, which 

often occur together with orchids (Brighigna et al. 1992). Despite the interesting 

implications of these studies, the transfer of nitrogen from bacteria 

to orchid roots has not been demonstrated (Dressler 1990; Sinclair 1990). 

9.4 

Orchid Endophytes or Orchid Mycorrhizal Fungi? 

The focus of this chapter and volume is on endophytes, not mycorrhizal 

fungi. However, it is impossible to review the literature on orchid root 

endophytes without also discussing mycorrhizae. The study of orchid mycorrhizae 

and endophytes are so inextricably linked that orchids are a good 

example of how hard it can be to disentangle the two [see also Chaps. 14 

(Cairney) and 16 (Brundrett)]. 

Mycorrhizae are mutualisms between plant roots and fungi, whereas endophytes 

are microorganisms growing inside plant tissues without causing


symptoms of disease (see Chap. 1 by Schulz and Boyle). This concept of 

mycorrhizae is functional and describes a relationship, whereas the concept 

of endophytes principally describes where an organism lives, without 

assuming or excluding the possibility of benefit for either party. This distinction 

is complicated by the fact that mutualisms are part of a continuum 

of symbiotic relationships, and a relationship that is mutualistic may become 

commensalistic or parasitic, or vice versa [Bronstein et al. 2003; see 

Chaps. 15 (Schulz) and 16 (Brundrett)]. It is further complicated by the 

fact that, unlike most mycorrhizae, orchid mycorrhizae are not known to 

provide any benefit to the fungal partner (Andersen and Rasmussen 1996, 

Taylor et al. 2002); in some cases and perhaps in all, the relationship is 

actually parasitic rather than mutualistic. Furthermore, fungal endophytes 

that are not mycorrhizal in the field may stimulate orchid seed growth 

in culture – ‘functional specificity’ as opposed to ‘ecological specificity’, 

as defined by Masuhara and Katsuya (1994). This means that seed germination 

and seedling growth tests in vitro may not be entirely accurate in 

distinguishing mycorrhizal fungi from non-mycorrhizal endophytes (Rasmussen 

2002). The most reliable criterion for mycorrhizae is the visual 

detection of pelotons in the root, but in tropical, epiphytic orchids, the 

pelotons observed are often degraded (Otero et al. 2002). 

The distinction between orchid mycorrhizal fungi and endophytes is 

further complicated by the inconsistent use of terminology in the literature. 

Most studies of Rhizoctonia-like fungi assume that the relationship 

is mycorrhizal without demonstrating any functional benefit to the plant. 

Since Rhizoctonia-like fungi can also be plant pathogens, endophytes or 

saprotrophs, in some cases this may be an unwarranted assumption (Alconero 

1969; Masuhara and Katsuya 1994; Carling et al. 1999; Rasmussen 

2002). A more precise (but less convenient) description would be ‘presumably 

mycorrhizal endophytes.’ On the other hand, some authors are aware 

of this problem and err on the side of caution, preferring to call their fungi 

‘endophytes’ for lack of functional evidence of a mycorrhizal relationship, 

even though they are almost certainly mycorrhizal (e.g., Hadley and Ong 

1978; Ramsay et al. 1986; Currah 1991; Currah et al. 1997; Richardson et 

al. 1993; Otero et al. 2002). So it is hard to say how many papers with 

‘mycorrhiza’ in the title really mean to say ‘endophyte’ and vice versa. 

However, when the papers that focus on mycorrhizal fungi are excluded, 

theexistingbodyofworkonorchidendophytesissurprisinglysmall 

(Currah et al. 1997). For example, an excellent, comprehensive treatise 

on orchids and their mycorrhizal relationships mentions non-mycorrhizal 

endophytes only in passing (Rasmussen 1995). 

To simplify things, in this chapter we will assume that Rhizoctonia-like 

fungi and their telomorphs (= sexual stages), including Ceratobasidium, 

Thanatephorus, Tulasnella, andSebacina, aremycorrhizalinassociations


with orchids, that other basidiomycete fungi may be mycorrhizal in mycoheterotrophic 

orchids, and that members of other groups of fungi are 

endophytes and not mycorrhizal. Known exceptions to this generalization 

are mentioned where relevant. 

9.5 

Problems with the Taxonomy of Orchid Endophytic Fungi 

Three problems complicate the study of endophytic fungi, including orchid 

root endophytes. First, many endophytic fungi do not sporulate in pure culture, 

and efforts to induce sporulation in vitro are often unsuccessful. Since 

traditionally fungi are classified by their spores and spore-bearing structures, 

nonsporulating fungi are very difficult to identify. For this reason, 

unidentifiable fungi are often grouped into ‘morphospecies’ on the basis 

of colony color, morphology and growth rate on agar media (Gamboa and 

Bayman 2001). DNA sequencing studies have shown that this technique is 

quite successful at grouping related fungi together (Arnold et al. 2000; Lacap 

et al. 2003) – but they remain unnamed, making comparisons between 

studies very difficult. 

Second, many endophytic fungi are undescribed, and do not fit well into 

previously described taxa (Hawksworth and Rossman 1997; Hawksworth 

1991, 2000). This complicates the job of studying them, but it also provides 

an incentive to do so: endophytes may be a largely unstudied reservoir of 

fungal biodiversity (Hawksworth and Rossman 1997; Arnold et al. 2001). 

Third, some endophytes do not grow in culture. Culturing of microorganisms 

from plant tissues provides a skewed picture of the organisms that 

grow there (see Chap. 17 by Hallmann et al.). One solution to this problem 

is to use PCR-based methods to amplify DNA directly from orchid roots 

using fungal-specific primers. So far, such techniques have been used to 

study orchid mycorrhizal fungi (see Sect. 9.8), but not non-mycorrhizal endophytes. 

This approach has revealed that some endophytes of grass roots 

belong to previously unknown major taxa of fungi (Vandenkoornhuyse 

et al. 2002); it is possible that orchids also harbor important undescribed 

lineages of fungi However, these PCR-based approaches are also biased: 

specific PCR primers are needed to amplify the fungal DNA but not the 

plant DNA, and primers can preferentially amplify certain groups of fungi 

(Bruns et al. 1998); using more than one set of primers and varying amplification 

conditions may help reveal additional taxa.


9.6 

Host Specificity of Orchid Endophytes 

Host specificity of endophytes is important for estimates of global fungal 

biodiversity. There are many fungal species associated with each plant 

species, and if these fungi are host-specific, the number of endophyte 

species will increase linearly with the number of plant species. Ratios of fungal 

species to plant species are used to extrapolate fungal biodiversity from 

plant species richness; the most commonly cited ratio is 6:1 fungi: plants, 

including pathogens and endophytes (Hawksworth 2000). Endophytes are 

an undersampled group in terms of fungal biodiversity (Hawksworth 1991; 

Hawksworth and Rossman 1997; Fröhlich and Hyde 1999).There are three 

reasons to expect that studying orchid root endophytes may make a significant 

contribution to this biodiversity: (1) orchids represent almost 10% of 

angiosperm species; (2) orchid roots are anatomically, morphologically and 

ecologically different from other roots (see Sects. 9.2, 9.3) most orchids are 

in the tropics, probably the most undersampled area for fungal biodiversity 

(Fröhlich and Hyde 1999; Hawksworth 2000; Arnold et al. 2001). 

There is century-old debate about the specificity of orchids for mycorrhizal 

fungi, (see Arditti et al.1990; Rasmussen 1995, 2002; Taylor et al. 2002 

for reviews), which applies to non-mycorrhizal endophytes as well. However, 

few attempts have been made to compare non-mycorrhizal endophytes 

among orchid species using quantitative methods. Given the diversity of 

endophyticfungiinorchidrootsandthevariationinmethodsamongstudies, 

it is difficult to determine the levels of specificity and preference in the 

interaction. Taxonomic problems (see Sect. 9.5) also complicate the issue of 

host specificity in orchid mycorrhizal fungi: since many endophytes cannot 

be identified to the species level with confidence, it is difficult to determine 

whether different orchid species have different communities of endophytes. 

9.7 

Endophytic Fungi in Roots of Terrestrial, 

Photosynthetic Orchids 

Non-mycorrhizal endophytes have been isolated from various terrestrial, 

photosynthetic orchids (Table 9.1), most extensively by Randall Currah and 

associates in Canada (Table 9.1; see Currah et al. 1997; Taylor et al. 2002). 

The most common and widespread endophyte isolated is Phialocephala, 

one of the ‘dark septate endophytes.’ These fungi are also common in many 

plants [Currah et al. 1987; Fernando and Currah 1995; see Chaps. 7 (Sieber 

and Grünig), 12 (Girlanda et al.) and 15 (Schulz)]. They may function as


Table 9.1. Non-Rhizoctonia fungireportedfromorchidrootsa . Basidiomycetes are in bold; those from myco-heterotrophic orchids have been 

assumed or shown to be mycorrhizal 

Orchids Fungi Location Reference 

Terrestrial, 

photosynthetic orchids 

Amerorchis rotundifolia Phialocephalab Canada Zelmer 1994 

Amerorchis rotundifolia Phialocephala fortinii Canada Currah et al. 1987 

Blettia striata Favolaschia thwaitesii Zambia Jonsson and Nylund 1979 

Calypso bulbosa Leptodontidium orchidicola, Phialocephala fortinii Canada Currah et al. 1987 

Coeloglossum viride Dactylella sp., Phialocephala Canada Zelmer 1994 

Coeloglossum viride Leptodontidium orchidicola, Trichosporiella multisporum Canada Currah et al. 1987 

Cymbidium sinense Mycena orchidicola sp.nov. China Fan et al. 1996 

Cypripedium calceolus Alternaria sp., Chaetomiumsp.Cylindrocarpon sp., Epicoccum purpureum, Canada Zelmer 1994 

Phialocephala, Phoma sp. 

Cypripedium candidum Acremonium killense, Humicola sp., Phialocephala Canada Zelmer 1994 

Cypripedium montanum Phialocephala Canada Zelmer 1994 

Cypripedium passerinum Phialocephala Canada Zelmer 1994 

Cypripedium reginae Fusarium sp. Canada Vujanovic et al. 2000 

Dactylorhiza majalis cf. Laccaria Denmark Kristiansen et al. 2001 

Epipactis microphyllac Tuber, other Pezizales France Selosse et al. 2004 

Epipactus helleborine Cylindrocarpon destructans, Humicola fuscoatra, Morchella sp., 

Finland Salmia 1988 

Sordaria fimicola 

Goodyera oblongifolia Humicola sp., Phialocephala, Phoma sp., Thermomyces verrucosus Canada Zelmer 1994 

Listera cordata Penicillium sp., Phialocephala Canada Zelmer 1994 

Piperia unalascensis Phialocephala Canada Zelmer 1994 

Piperia unalascensis Sistotrema sp. Canada Currah et al. 1990




Platanthera dilatata Phialocephala Canada Zelmer 1994 

Platanthera hyperborea Acremonium kiliense, Phialocephala, Sporormia minima, 

Canada Zelmer 1994 

Thielavia basicola 

Platanthera hyperborea Leptodontidium orchidicola, Trichocladium opacum Canada Currah et al. 1987 

Platanthera obtusata Acremonium killense, Phialocephala Canada Zelmer 1994 

Platanthera obtusata Sistotrema sp. Canada Currah et al. 1990 

Platanthera praeclara Fusarium oxysporum, Phialocephala Canada Zelmer 1994 

Spiranthes lacera Acremonium kiliense, Phialocephala Canada Zelmer 1994 

Spiranthes magnicamporum Cylindrocarpon sp., Papulaspora sp., Phialophora richardsiae, Canada Zelmer 1994 

Ulocladium sp. 

Spiranthes romanzoffiana Acremonium kiliense, Cylindrocarpon sp., Gliomastix murorum, Canada Zelmer 1994 

Phialocephala 

Myco-heterotrophic orchids 

Cephalanthera austinae Thelephora-Tomentella (14 spp.) United States Taylor and Bruns 1997 

Corallorhiza maculata Armillaria melea United States Campbell 1970a 

Corallorhiza maculata Russulaceae (20 spp.) United States Taylor and Bruns 1997 

Corallorhiza maculata Russulaceae (3 spp.) United States Taylor and Bruns 1999 

Corallorhiza maculata Cylindrocarpon sp., Phialocephala Canada Zelmer 1994 

Corallorhiza maculata Leptodontidium orchidicola Canada Currah et al. 1987 

Corallorhiza striata Cylindrocarpon sp., Phialocephala Canada Zelmer 1994 

Corallorhiza trifida Phialocephala Canada Zelmer 1994 

Corallorhiza mertensiana Russulaceae (22 spp.) United States Taylor et al. 2003 

Corallorhiza striata Thelephora-Tomentella United States Taylor 1997 

Corallorhiza trifida Mycena thuja New Zealand Campbell 1970a




Corallorhiza trifida yellow basidiomycete w/ clamps Canada Zelmer and Currah 1995 

Corallorhiza trifida Thelephora-Tomentella Scotland McKendrick et al. 2000 

Danhatchia australis 

Lycoperdon perlatum New Zealand Campbell 1970b 

(= Yoania australis) 

Didymoplexis minor Marasimius coniatus Paleotropics Burgeff 1959 

Galeola altissima Erythromyces crocicreas, Ganoderma australe, 

Hamada and Nakamura 

Loweporus tephroporus, Microporus affinus 

1963, Umata 1995 

Galeola (= Erythrorchis) ochobiensis Hymenochaete crocicreas Umata 1998 

Galeola (= Erythrorchis) ochobiensis Auricularia polytricha, Lyophyllum shimeji Umata 1997a, 1997b 

Galeola (= Erythrorchis) ochobiensis Lentinula edodes Umata 1998 

Galeola (= Erythrorchis) ochobiensis Lenzites betulinus, Trametes hirsuta Japan Umata 1999 

Galeola septentrionalis Armillaria mellea Japan Hamada 1939, 

Terashita 1985 

Galeola septentrionalis Armillaria jezoensis sp nov. Cha and Igarashi 1996 

Galeola sesamoides Fomes sp. New Zealand Campbell 1964 

Gastrodia cunninghamii Armillaria mellea Campbell 1962 

Gastrodia elata Armillaria mellea China Kusano 1911 

Gastrodia elata Armillariella mellea China Lan et al. 1994 

Gastrodia elata Mycena osmundicola New Zealand Lan et al. 1996 

Gastrodia minor brown basidiomycete w/clamps New Zealand Campbell 1962 

Gastrodia sesamoides Fomes mastoporus Campbell 1964 

Wullschlaegelia calcarata Acremonium, Colletotrichum, Curvularia, 

Puerto Rico J.T. Otero (unpublished) 

Cylindrocladium, Gliocladium, Paecilomyces, 

Penicillium, Trichoderma, Xylaria




Epiphytic & epilithic orchids 

Campylocentrum micranthum Calonectria kyotensis, Phomopsis cf orchidophila Costa Rica Richardson 1993 

Catasetum maculatum Acrogenospora sp., Codinaea parva, Colletotrichum crassipes, Costa Rica Richardson 1993 

Epicoccum andropogonis, Glomerella cingulata, Hadrotrichum sp., 

Lasiodiplodia theobromae 

Catasetum maculatum Troposporella sp. Costa Rica Richardson 

and Currah 1995 

Dichaea standleyi Hadrotrichum sp., Nectria alata, Phomopsis cf. orchidophila Costa Rica Richardson 1993 

Dichaea trulla Colletotrichum crassipes Costa Rica Richardson 1993 

Dimerandra emarginata Epicoccum andropogonis, Hadrotrichum sp. Costa Rica Richardson 1993 

Dryadella pusiola Chaetomium homopilatum Costa Rica Richardson 1993 

Encyclia fragrans Alternaria alternata, Colletotrichum crassipes, Dactylaria sp., Costa Rica Richardson 1993 

Epicoccum andropogonis, Glomerella cingulata, Hadrotrichum sp., 

Lasiodiplodia theobromae, Nodulisporum sp., 

Pseudallescheriaboydii 

Epidendrum difforme Lasiodiplodia theobromae, Pithomyces maydicus Costa Rica Richardson 1993 

Epidendrum difforme Troposporella sp. Costa Rica Richardson and Currah 1995 

Epidendrum isomerum Nodulisporum sp. Costa Rica Richardson 1993 

Epidendrum nocturnum Epicoccum andropogonis Costa Rica Richardson 1993 

Epidendrum octomerioides Lasiodiplodia theobromae, Nectria ochroleuca, 

Costa Rica Richardson 1993 

Pestalotiopsis papposa, Phomopsis cf. orchidophila 

Epidendrum porpax Fusarium oxysporum, Guignardia sp. Colombia Dreyfuss and Petrini 1984 

Epidendrum schlechterianum Nectria haematococca, Periconiella sp., Pestalotiopsis papposa, Costa Rica Richardson 1993 

Xylaria sp.




Epidendrum stangeanum Fusarium oxysporum, Hadrotrichum sp., Nodulisporium sp., Costa Rica Richardson 1993 

Pithomyces maydicus 

Epidendrum stangeanum Troposporella sp. Costa Rica Richardson and Currah 1995 

Epidendrum spp. Ascochyta sp., Colletotrichum gloeosporioides, Colletotrichum sp., Colombia/ Dreyfuss and Petrini 1984 

Cryptocline sp., Lasiodiplodia theobromae, Pestalotia cf. heterocornis, Brazil 

Phaeoseptoria cf. vermiformis, Phoma sp., Xylaria sp. 

Gongora unicolor Epicoccum andropogons, Hypoxylon cf. unitum, Lasmeniella sp., Costa Rica Richardson 1993 

Nodulisporium sp., Pestalotia poppola, Xylaria sp. 

Hexisea imbricata Arthrinium sp., Hadrotrichum sp., Pestalotiopsis aquatica Costa Rica Richardson 1993 

Jacquinella globosa Colletotrichum crassipes Costa Rica Richardson 1993 

Lepanthes caritensis Penicillium, Trichoderma, Xylaria corniformis Puerto Rico Tremblay et al. 1998 

Lepanthes rupestris Acremonium, Colletotrichum, Fusarium, Guignardia, Humicola, Puerto Rico Bayman et al. 2002 

Pestalotia, Phomposis, Trichoderma, Xylaria 

Lepanthes spp. Aspergillus, Colletotrichum, Penicillium, Pestalotia, 

Puerto Rico Bayman et al. 1997 

Xylaria arbuscula, X. corniformis, X. cf. cubensis, 

X. cf. curta multiplex, X. obovata, X. polymorpha, Xylaria sp. 

Maxillaria confusa Arthrinium sp., Malbranchea sp. Costa Rica Richardson 1993 

Maxillaria endresii Drechslera ellisii, Pestalotiopsis papposa Costa Rica Richardson 1993 

Maxillaria neglecta Chaetomium subspirale, Colletotrichum crassipes sp., 


Drechslera australensis, Glomerella cingulata, Hadrotrichum sp., 

Humicola sp., Nectria haematococca, N. ochroleuca, 

Phomopsis cf. orchidophila, Xylaria sp. 

Maxillaria nicaraguensis Pestalotiopsis gracilis Costa Rica Richardson 1993 

Maxillaria uncata Cryptosporiopsis sp., Hadrotrichum sp. Costa Rica Richardson 1993 

Maxillaria xylobiflora Epicoccum nigrum Costa Rica Richardson 1993 

Maxillaria sp. Colletrotrichum crassipes, Hadrotrichum sp., Xylaria sp. Costa Rica Richardson 1993




Maxillaria sp. Acremonium strictum, Fusarium sambucinum, Tubercularia, Colombia, Dreyfuss and Petrini 1984 

Xylaria sp., Pestalotia cf. heterocornis, Phomopsis sp. 

Brazil 

Myoxanthus scandens Colletotrichum crassipes Costa Rica Richardson 1993 

Nidema boothii Dactylaria sp., Epicoccum andropogonis, Lasiodiplodia theobromae, Costa Rica Richardson and Currah 1995 

Leptosphaerulina australis, Pestalotiopsis papposa, Phomopsis cf. 

orchidophila 

Nidema boothii Troposporella sp. Costa Rica Richardson et al. 1993 

Octomeria sp. Melanotus alpiniae Costa Rica Richardson 1993 

Oncidium stenotis Chaetomium subspirale, Colletotrichum crassipes, Humicola sp., Costa Rica Richardson 1993 

Nigrospora sphaerica 

Pleurothallis corniculata Cryptosporiopsis sp., Pestalotiopsis papposa Costa Rica Richardson 1993 

Pleurothallis guanacastensis Hypoxylon cf. unitum, Lasiodiplodia theobromae, 


Pithomyces maydicus, Xylaria sp. 

Pleurothallis pantasmi Hadrotrichum sp., Humicola sp., Nectria peziza Costa Rica Richardson 1993 

Pleurothallis periodica Chaetomium subspirale, Cladosporium cladosporioides, 


Geotrichopsis sp., Hadrotrichum sp., Xylaria sp. 

Pleurothallis phyllocardioides Lasiodiplodia theobromae, Nectria haematococca Costa Rica Richardson 1993 

Pleurothallis uncinata Nectria haematococca, Pithomyces maydicus Costa Rica Richardson 1993 

Pleurothallis verecunda Epicoccum andropogonis Costa Rica Richardson 1993 

Pleurothallis sp. Chaetomium funicola, Pyrenochaeta cf. rubi-idaei, 


Ramichloridium cf. subulatum 

Pleurothallis sp. Cryptocline sp., Xylaria sp. Colombia Dreyfuss and Petrini 1984 

Polystachya foliosa Hadrotrichum sp., Xylaria sp. Costa Rica Richardson 1993 

Psychilis kraenzlinii Colletotrichum, Curvularia, Mucor, Paecilomyces, Pestalotia, Puerto Rico J.T. Otero (unpublished) 

Xylaria




Psychilis krugii Colletotrichum, Curvularia, Mucor, Paecilomyces, Pestalotia, Puerto Rico J.T. Otero (unpublished) 

Xylaria 

Rodriguezia compacta Colletotrichum crassipes, Epicoccum andropogonis, 


Hadrotrichum sp., Leptosphaerulina australis, 

Pestalotiopsis aquatica 

Scaphyglottis cf. prolifera Nectria ochroleuca Costa Rica Richardson 1993 

Scaphyglottis gracilis Nectria haematococca, N. ochroleuca Costa Rica Richardson 1993 

Scaphyglottis minutiflora Arthrinium sp. Costa Rica Richardson 1993 

Sobralia cf. mucronata Arthrinium sp., Lasiodiplodia theobromae Costa Rica Richardson 1993 

Sobralia mucronata Pseudallescheria boydii Costa Rica Richardson 1993 

Sobralia powellii Xylaria sp. Costa Rica Richardson 1993 

Sobralia sp. Epicoccum nigrum, Glomerella cingulata, 


Lasiodiplodia theobromae, Nectria haematococca 

Stelis endresii Curvularia cymbopogonis, Nectria haematococca, 


Phomopsis cf. orchidophila 

Stelis sp. Alternaria alternata, Arthrinium sp., Chloridium virescens, Costa Rica Richardson 1993 

Colletotrichum crassipes, Cryptosporiopsis sp., 

Epicoccum andopogensis, Hadrotrichum sp., Hypoxylon cf. unitum, 

Nectria alata, Nectria ochroleuca, N. radicicola, 

Neoplaconema napelli, Pithomyces maydicus, Xylaria spp. 

Trichosalpinx blaisdellii Lasiodiplodia theobromae Costa Rica Richardson 1993 

Trichosalpinx orbicularis Chaetosticta cf. perforata, Colletotrichum crassipes, Nectria alata Costa Rica Richardson 1993 

Trichosalpinx sp. Hadrotrichum sp. Costa Rica Richardson 1993 

Trigonidium egertonianum Chaetosticta cf. perforata, Epicoccum andopogensis Costa Rica Richardson 1993 

Trigonidium riopalaquense Chaetomium aureum, Colletotrichum acutatum, C. crassipes, Costa Rica Richardson 1993 

Nectria haematococca, Nodulisporium sp., Periconiella sp.




Unidentified Acremonium pteridii, Anthostomella aracearum, Ascochyta sp., French Petrini and Dreyfuss 1981 

Aureobasidium caulivorum, Chaetosphaeria endophytica, Guayana 

Colletotrichum spp., Coniothyrium sp., Cryptocline spp., 

Cryptosporiopsis sp., Curvularia pallescens, Cytogloeum sp., 

Fusarium oxysporum, Gelatinosporium spp., Gliocladium roseum, 

Glomerella cingulata, Kaskaskia sp., Lasiodiplodia theobromae, 

Melanconium sp., Microascus cinereus, Microcyclus sp., 

Nodulisporium gregarium, Nodulisporium spp., Pestalotia adusta, 

Phialaspora sp., Phoma sp., Phomatospora berkeleyi, 

Phomopsis orchidophila, Phomopsis sp., Phyllosticta capitalensis, 

P. colocasiicola, Ramichloridium apiculatum, Verticillium lecanii 

aThe following fungal genera are presumed to be mycorrhizal and are not included in this table: Ceratobasidium, Oliveonia Sebacina, Serendipita, 

Thanatephorus, Tulanella and Ypsilonidium (teleomorphs); Ceratobasidium (anamorphs) (Roberts 1999). These fungi can be found in tables 

published by Currah et al. (1997), Rasmussen (2002) and Taylor et al. (2002) 

bAlso called Mycelium radicis atrovirens (MRA) 

c This species can be either myco-heterotrophic or photosynthetic


mycorrhizae in some plants (Fernando and Currah 1996; Jumpponen 2001), 

but their role in orchids is still unclear (Rasmussen 2002). 

One of the most ubiquitous and interesting groups of endophytes is 

Fusarium and its teleomorphs. Fusarium moniliforme (= F. verticilloides) 

wasisolatedfromleavesandrootsofCypripedium reginae (Peschke and 

Volz 1978). Inoculation of spores induced disease symptoms in hybrid 

orchids, suggesting that the fungus was a potential pathogen (Peschke and 

Volz 1978). This fungus is a ubiquitous endophyte and pathogen of all 

organs of various hosts, including maize (Leslie et al. 1990; Kuldau and 

Yates 2000; see Chap. 8 by Bacon and Yates). 

On the other hand, the ability of Fusarium to stimulate orchid seed 

germination has been known for 100 years (Bernard 1909). An unidentified 

Fusarium strain isolated from a germinating seed of Cypripedium reginae 

induced germination of C. reginae seeds in vitro (Vujanovic et al. 2000). 

Although this is functional rather than ecological specificity (Masuhara 

and Katsuya 1994), it raises the question of whether a single Fusarium 

isolate can be endophytic, pathogenic and mycorrhizal under different 

circumstances. 

9.8 

Endophytic Fungi in Roots of Myco-Heterotrophic Orchids 

Non-photosynthetic (or more precisely, myco-heterotrophic) orchids have 

been extensively studied because of their interesting relationships with 

fungi (Leake 1994). Unable to assimilate their own carbon, these orchids 

are parasitic on fungi. Several myco-heterotrophic orchids are very specific 

for certain fungi, which is interesting because orchid mycorrhizal relationships 

are generally considered to be non-specific (Taylor et al. 2002). In 

most cases the myco-heterotrophic orchids are parasitizing a mycorrhizal 

partner of a nearby photosynthetic plant, which means that they are indirectly 

parasitizing the plant as well. However, the amount of carbon taken 

by the orchid is probably insignificant to the plant host (McKendrick et 

al. 2000; Sanders 2003). An excellent review of mycorrhizal specificity in 

myco-heterotrophic plants is available (Taylor et al. 2002). 

Fungal DNA has been amplified directly from roots or pelotons of mycoheterotrophic 

orchids using fungal-specific (or in some cases, basidiomycete-specific) 

primers. This approach has been used on several orchids 

in North America: Cephalanthera (Taylor and Bruns 1997), Corallorhiza 

spp. (Taylor and Bruns 1997, 1999) and Hexalectris (Taylor et al. 2003). It 

has also been used on Dactylorhiza in Denmark (Kristiansen et al. 2001) 

and Neottia in the United Kingdom, Germany (McKendrick et al. 2002) 

and in France (Selosse et al. 2002). In all these plants, the only fungi


amplified from peletons belonged to taxa that are considered ectomycorrhizal. 

No other endophytic fungi were reported, which may suggest that 

secondary colonization of pelotons by non-mycorrhizal endophytic fungi 

is uncommon. No such studies have been done with the aim of identifying 

non-mycorrhizal fungi in orchids, e.g., using ascomycete-specific PCR 

primers. 

Myco-heterotrophic orchids tend to associate with ectomycorrhizal fungi 

rather than the Rhizoctonia-like fungi typical of orchids, presumably because 

ectomycorrhizal fungi are more reliable suppliers of photosynthate. 

In other cases, the presumed mycorrhizal fungi are basidiomycetes that are 

wood decomposers not known to form ectomycorrhizae, e.g., Armillaria, 

Fomes, Ganoderma and Phellinus sp. (for references, see Rasmussen 2002; 

Taylor et al. 2002). However, most such studies have not demonstrated 

functional relationships between the fungus and the myco-heterotrophic 

orchid, so this is another area where the boundary between mycorrhizal 

fungi and endophytes is unclear. 

Endophytes have been reported from only one tropical myco-heterotrophic 

orchid, Wullschlaegelia. Forty-one morphospecies of endophytes 

were isolated from plants of W. calcarata in Puerto Rico (J.T. Otero, unpublished; 

Table 9.1). Common endophytes included Xylaria, Trichoderma, Colletotrichum 

and various dematiaceous hyphomycetes. These genera were 

not randomly distributed among sites (χ 2 = 31.84, df = 17, P = 0.004). 

A morphospecies accumulation curve suggested that most of the culturableendophyticfungiintherootswereisolated(Fig.9.1).Itislikelythat 

temperate myco-heterotrophic orchids contain a mycoflora as diverse as 

W. calcarata, but the peloton isolation technique used in many of the above 

studies excluded most of the non-mycorrhizal endophytes. 

Fig.9.1. Species accumulation curve for endophytic fungi isolated from roots of 

Wullschlaegelia calcarata, a myco-hetrotrophic orchid. A total of 32 plants were collected 

from eight populations in El Verde, Puerto Rico. Morphospecies were identified by morphology 

in culture; the identified fungi are listed in Table 9.1


9.9 

Endophytic Fungi in Roots of Epiphytic 

and Lithophytic Orchids 

Reports on non-mycorrhizal endophytes in roots of epiphytic and lithophytic 

orchids have mostly come from the neotropics. These reports have 

focused on identifying the fungi rather than exploring their relationship 

with the orchids. Descriptions of some common endophytes that have been 

published will facilitate further studies (Richardson et al. 1993; Richardson 

and Currah 1995; Currah et al. 1997). 

South America Three taxa of Ascomycetes, 5 of Hyphomycetes and 13 of 

Coelomycetes were isolated from epiphytic orchid roots in French Guiana; 

many of these fungi were also isolated from roots of aroids and bromeliads, 

suggesting that the fungi are generalists (Table 9.1; Petrini and Dreyfuss 

1981). Some of the same fungi were also found in orchid roots from the 

Colombian Amazon (Dreyfuss and Petrini 1984). 

Costa Rica The most extensive sampling of epiphytic orchid roots for endophytes 

was done in La Selva, Costa Rica (Richardson et al. 1993; Richardson 

and Currah 1995). Of 59 species of epiphytic orchids sampled in La 

Selva, Costa Rica, mycorrhizal pelotons were observed in the roots of 23 

(= 39%) (Richardson et al. 1993), a fairly low infection frequency that 

agrees with other studies. Basidiomycetes comprised only 3% of the fungi 

isolated, suggesting that mycorrhizal fungi were much less common than 

non-mycorrhizal endophytes or pathogens. Hadrotrichum, Colletotrichum, 

Epicoccum, Lasiodiplodia and Phomopsis were the most common genera 

of deuteromycetes and ascomycetes (Richardson and Currah 1995). Relative 

proportions of ascomycetes, hyphomycetes and coelomycetes were 

comparable to those reported by Petrini and Dreyfuss (1981). 

Puerto Rico We have sampled endophytic fungi from roots of various epiphytic 

orchids in Puerto Rico (Table 9.1). The most common endophytic 

fungi have been fairly consistent from study to study. A variety of endophytic 

fungi were isolated from nine species of epiphytic orchids in Puerto 

Rico, including Xylaria, Pestalotia and Colletotrichum (Otero et al. 2002). 

Rhizoctonia-like fungi were isolated at lower frequency, from about 20% of 

the samples. 

Fifty-five fungi (in 26 morphospecies) were isolated from roots of Tolumnia, 

from both juvenile and adult plants (J.T. Otero, unpublished). 

Thirty-one of these strains (in 13 morphospecies) were tested for potential 

mycorrhizal activity with T. variegata seeds. Thirteen strains (in 6 morphospecies) 

had a positive effect on seed germination in vitro, all of which


were Rhizoctonia-like fungi; others were parasitic on seeds. These data 

suggest Rhizoctonia are the principal mycorrhizal fungi of T. variegata, but 

thesampledidnotincludeFusarium (see Sect. 9.9). 

A comparison of two sympatric populations of Psychilis and P. krugii 

found 26 morphospecies of endophytic fungi (Table 9.1; J.T. Otero, unpublished). 

There was no apparent difference between the fungal communities 

of the two orchids. The dominant species were Xylaria spp.; Rhizoctonialikefungiweremuchlesscommonthanintheorchidscitedabove,andfew 

pelotons were seen in root sections. 

Endophytic fungi were isolated from roots and leaves of six species of 

Lepanthes in Puerto Rico (Bayman et al. 1997). Xylaria and Rhizoctonia 

were the most common genera isolated from roots and leaves. At least nine 

species of Xylaria wereisolated,uptofourspeciesoccurringinasingle 

plant. Frequency of Xylaria species and Rhizoctonia did not differ significantly 

between roots and leaves, which is perhaps not surprising given that 

roots of epiphytes are exposed to light and air. Frequency of both Rhizoctonia 

and Xylaria differed significantly among Lepanthes species. However, 

there was also significant variation among different roots of a single plant, 

which means that studies that wish to compare levels of infection should 

sample intensively. In another study of L. rupestris,endophyticfungiwere 

isolated from roots of lithophytic plants (Bayman et al. 2002). The most 

common genera were Guignardia (isolated from 22% of root pieces), Colletotrichum 

(10%) and Xylaria (7%). Rhizoctonia-like fungi (which include 

the presumed mycorrhizal fungi) were isolated at much lower frequencies 

(3%). 

Is distribution of fungi a limiting factor in distribution of orchids? Tremblay 

et al. (1998) isolated fungi from roots of Lepanthes caritensis,anorchid 

species whose distribution is limited to a few trees along a single river. Fungi 

isolated from orchid roots were compared to fungi isolated from bark of 

host trees, and of conspecific trees without orchids (Tremblay et al. 1998). 

The most common fungi in L. caritensis roots were Xylaria spp, particularly 

X. corniformis. Penicillium, Trichoderma and Rhizoctonia were also 

isolated from roots, and were the most common fungi isolated from tree 

bark. However, Xylaria sp. were not common on tree bark, suggesting that 

they had a particular affinity for Lepanthes roots. The number of other, 

unidentified fungi was significantly higher on bark of trees without orchids 

than on trees with orchids. This may suggest that the presence of certain 

fungi inhibits the establishment of orchids. 

In general, there is little evidence that non-mycorrhizal endophytes in 

orchids are specific to orchids. The most common groups of orchid endophytes 

(Table 9.1) are fungi that are ubiquitous in soil and as endophytes 

of other plants. There are marked differences between terrestrial and epiphytic 

orchids: in terrestrials, Phialocephala is the most frequently isolated


group; in epiphytes, Hadrotrichum, Epicoccum, Lasiodiplodia, Xylaria and 

Pestalotiopsis are most frequent. However, these differences reflect differences 

in temperate vs. tropical mycofloras and are not particular to orchidassociated 

fungi. Several new species have been described from orchid 

endophytes (e.g., Mycena sp. nov. (Fan et al. 1996), Armillaria jezoensis sp. 

nov. (Cha and Igarashi 1996), Leptodontidium (Currah et al. 1987)), but in 

most cases their distribution is not sufficiently known to claim a special 

affinity for orchids. 

9.10 

Endophytic Fungi in Epiphytic Orchid Roots: 

Importance to Plant Hosts 

There are two reasons to believe that the presence of endophytes could 

affect mycorrhizal fungi, and vice versa. First, orchids may produce phytoalexins 

such as orchinol when challenged by a fungus; these phytoalexins 

may then limit the ability of other fungi to colonize the plant – a type of 

induced resistance. These phytoalexins inhibit growth of a broad range of 

fungi, though bacteria are less susceptible (Gäumann et al. 1960). According 

to Rasmussen (1995), “...all underground parts of terrestrial orchids 

must either accommodate the endophyte (i.e., mycorrhizal fungus) or actively 

reject it.” Second, endophytes and mycorrhizal fungi could compete 

for nutrients in the root, or could actively inhibit each other by production 

of secondary metabolites. Nutrient translocation from mycorrhizal fungi 

to orchids has been demonstrated repeatedly (see Rasmussen 1995; Bidartondo 

et al. 2004), but it is unknown how non-mycorrhizal endophytes 

might affect this transfer. 

Several studies have asked whether the presence of endophytic fungi 

was associated with the presence of other endophytes or of mycorrhizal 

fungi. Pieces of Lepanthes roots colonized by Colletotrichum had a significantly 

lower rate of infection with Xylaria thanwouldbeexpectedfrom 

the frequency of each genus alone (Bayman et al. 2002). Presence of Colletotrichum 

also showed a significant, negative correlation with Guignardia. 

This suggests there may be competition or antagonism between these 

genera;alternatively,theircolonizationorgrowthcouldbefavoredbydifferent 

environmental conditions. Also, mycorrhizal fungi in Vanilla often 

occur together with other, presumably pathogenic, fungi (Alconcero 1969; 

Porras-Alfaro and Bayman 2003). Roots of Vanilla plants in Puerto Rico 

were often colonized by both the pathogen Fusarium oxysporum and by 

Rhizoctonia solani, which was both mycorrhizal and pathogenic to Vanilla 

(Alconcero 1969).


Fungicides were applied to plants of L. rupestris to see if the costs of 

harboring fungi outweighed the benefits (Bayman et al. 2002). Propiconazole 

significantly reduced the number of total fungal colonies isolated from 

roots (but not from leaves), increased plant mortality, and increased loss 

of leaves, as compared to control plants. Benomyl significantly reduced 

the number of fungi isolated from leaves (but not from roots) and decreased 

plant mortality, but did not significantly effect plant growth. These 

data suggest that fungi have both positive and negative effects on growth 

and survival of orchid plants in the field: benomyl, which affects mainly 

ascomycete fungi, may have reduced pathogens and endophytes, not harming 

plants, whereas propiconazole, which also affects basidiomycetes, may 

have reduced mycorrhizal fungi as well. However, these results are very 

difficult to interpret: a single plant may have simultaneous infections by 

endophytes, mycorrhizal fungi and pathogens, and the positive effects of 

one may mask the negative effects of another. 

The interaction between plants and endophytic fungi may be viewed as 

a ‘balanced antagonism’ (Schulz et al. 1999). The plant produces secondary 

metabolites that are sufficiently toxic to restrict the growth of the endophyte 

without being able to kill it; the endophyte in turn produces enzymes and 

metabolites that allow it to colonize the plant but are not sufficient to cause 

pathogenicity. This balance becomes still more complex if the endophyte 

and its activities also affect other fungi, either endophytic or mycorrhizal, 

as these data from Lepanthes suggest. 

9.11 

Conclusions 

About 90% of the cells that comprise a human body are microbial – we are 

walking communities (Hamilton 1999). Most of these microorganisms are 

poorly understood: pathogenic microorganisms are intensively studied, 

but much less is known about non-pathogenic, commensal microorganisms, 

which are more common than pathogens. Their importance becomes 

obvious when an antibiotic designed to kill a pathogen affects the community 

of commensals as well, causing a secondary condition – for example, 

vaginal yeast infections in women who are taking antibiotics for bladder infections. 

Cross-talk between microorganisms and the host may have other 

roles as well; for example, it may be necessary for the proper development 

of the immune system (Hooper et al. 1998). Even well-studied pathogens 

may have complex ecological roles: Helicobacter was considered a serious 

pathogen, but recent studies suggest that it is a commensal that sometimes 

turns pathogenic (Pütsep et al. 1999). Our ignorance of the non-pathogenic 

microflora is a limiting factor in medicine.


The same situation exists with orchid root endophytes. Interest in orchid 

mycorrhizal fungi has overshadowed work on other orchid endophytes, 

which may have important interactions with mycorrhizal fungi and with 

the orchids themselves. In some cases the distinction between endophytes 

and mycorrhizal fungi is unclear, and it is likely that, as with H. pylori 

in humans, a single organism can behave as an endophyte, mycorrhizal 

symbiont or pathogen, depending on the environment and the health of 

the host plant. The study of orchid endophytes is not much more advanced 

than the study of orchid mycorrhizae was in 1900 – interesting observations, 

intriguing ideas, but little information about functional significance. 


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10 

10.1 

Introduction 

Fungal Endophytes in Submerged Roots 

Felix Bärlocher 

It has long been known that plants harbour fungal endophytes, and it was 

suspected that systemic grass endophytes, primarily clavicipitaceous fungi, 

are associated with toxicity to grazing livestock (Saikkonen et al. 1998). This 

connection was firmly established in the 1970s (Bacon et al. 1977). The early 

emphasis on grasses and their endophytes have led some authors to consider 

the term endophyte as being synonymous with mutualist. However, 

many fungal pathogens may be latent in grasses without causing disease 

or long before the outbreak of disease symptoms (Petrini 1991; Fisher and 

Petrini 1993). The first systematic surveys of plants other than grasses 

were stimulated by the observation that many common phyllosphere fungi 

invade stomatal cavities of Douglas fir needles within their first year. Bernstein 

and Carroll (1977) demonstrated that with increasing age, all needle 

segments become infected with endophytes. The presence of primarily 

non-balansiaceous endophytes was extended to other conifers (Carroll et 

al. 1977) and has since been documented in every tree, shrub and herb 

that has been examined (Carroll 1995; Sridhar and Raviraja 1995; Saikkonnen 

et al. 1998). Generally, a large number of species can be isolated from 

a given host, yet only four to five are common and likely to be host specific 

(Fisher and Petrini 1993). Community ordination analyses have generally 

shown that endophyte assemblages are specific at the host species level, 

and may be impoverished outside the host’s natural range. While a few of 

these associations provide clear benefits to the plant by fungal interference 

with herbivores or microbial pathogens, others eventually cause damage 

to the plant, while some are essentially neutral. A widely accepted definition 

of an endophyte is as an agent of a currently asymptomatic infection, 

without specifying the role of the agent in the host or its development 

at a later stage (Petrini 1991; Fisher and Petrini 1993; Schulz et al. 1998). 

However, Schulz et al. (1999) showed that even in infections without visible 

symptoms, colonisation led to the synthesis of higher concentrations of 

Felix Bärlocher: 63B York Street, Department of Biology, Mount Allison University, Sackville, 

New Brunswick, E4L 1G7, Canada, E-mail: fbaerlocher@mta.ca 





180 F. Bärlocher 

potentially antimicrobial compounds. In vitro, endophytic fungi produce 

more herbicidally active substances than soil fungi. Schulz et al. (1999) 

therefore hypothesise that the host-endophyte interaction is a case of balanced 

antagonism: pathogens overcome the host’s defences to the extent 

that they cause visible damage, whereas endophytic virulence is only sufficient 

to be able to infect and colonise without causing visible damage. If 

the balance shifts, the endophyte may turn pathogenic. 

Much of the current interest in endophytes is based on the hope of finding 

uniquesecondary metabolitesandenzymesaffectingplants,herbivoresand 

microbes, with potential applications in medicine and agriculture (Petrini 

et al. 1992). The continuum of endophyte interactions with plants also provides 

interesting case studies for the evolution of mutualism and pathology, 

and for understanding how environmental factors might favour one or the 

other (Carroll 1988). Until recently, it has commonly been believed that 

the first fungi were saprotrophs from which necrotrophs and biotrophs 

evolved, but there are convincing arguments supporting an alternative 

view (Parbery 1996). A recent comparison of 1,551 ribosomal sequences 

of the two sister groups of chitinous fungi, the Glomeromycota and the 

Dikaryomycota, both of which have symbiotic life-styles, suggests that the 

symbiosis between fungi and green plants was present before the colonisation 

of land by plants (Tehler et al. 2003). Finally, endophytes may not have 

been seriously taken into account when assessing diversity (Hawksworth 

1991, 2001); Dreyfuss and Chapela (1994) concluded that over 1.3 million 

species of fungal endophytes remain to be discovered and described. 

10.2 

Aquatic Hyphomycetes 

Up to 99% of the energy available to stream communities consists of 

terrestrial plant detritus (leaves, needles, twigs; Allan 1995). Aquatic hyphomycetes, 

a heterogeneous group of aquatic fungi, are an indispensable 

link in the food web between this detritus and stream invertebrates 

(Bärlocher 1992). The annual fungal production per stream bed area falls 

within the same order of magnitude as that of bacteria and invertebrates 

(Suberkropp 1997). Aquatic hyphomycetes disperse from leaf to leaf by producing 

conidia, whose shapes are predominantly tetraradiate (four arms) 

or sigmoid. Both types have been shown to increase the conidium’s likelihood 

of settling and germinating on new leaves; they are clearly the result 

of convergent evolution (Webster 1987). 

In temperate streams, the number of conidia in the water column declines 

from up to 30,000 l −1 in late fall to almost nil during summer, 

undoubtedly a response to the seasonal availability of terrestrial leaves


(Bärlocher 1992, 2000). Combined with the unidirectional displacement 

of substrates and spores in running water, this raises the question of how 

aquatic hyphomycetes can maintain themselves within a given reach of 

a stream and avoid being washed downstream. Potential solutions include 

(1) the fact that the fungi also colonise woody substrates, which can persist 

for several years in a stream, (2) the presence of teleomorphs in some 

species with ascospores that may be dispersed aerially, (3) dispersal of 

fungal-colonised leaves or conidia by animals, (4) the occurrence of the 

fungi in terrestrial habitats, e.g. as plant pathogens or endophytes (Bärlocher 

1992). For example, Hartig (1880) first described a parasite of maple 

seedlings as Cercospora acerina. Later, its identity as Centrospora acerina 

was established, and it is now known as Mycocentrospora acerina (Hartig) 

Deighton. It is a remarkably widespread and versatile species: it is 

a well-known plant pathogen, has been implicated in human infections, 

andisacommonstreamfungus.Morphologically,thereisnodifference 

among various strains. Iqbal and Webster (1969) showed that strains they 

isolated from a stream were pathogenic to carrots and parsnips. Nemec 

(1969) isolated Anguillospora longissima (Sacc. & Syd.) Ingold and Tetracladium 

marchalianum de Wild., which had been isolated from aqueous 

habitats, from the roots of diseased strawberry plants. Several other species 

have also been isolated from terrestrial root surfaces of apparently healthy 

plants (Waid 1954; Taylor and Parkinson 1965; Parkinson and Thomas 

1969; Watanabe 1975). These observations suggested that some aquatic 

hyphomycetes might be root endophytes. 

10.3 

Fungi in Submerged Roots 

Fisher and Petrini (1989) were the first to demonstrate an endophytic phase 

of two aquatic hyphomycete species. They examined terrestrial roots of Alnus 

glutinosa (L.) Gaertner on the banks of Exeter Canal (Exeter, Devon, 

UK). Only 1.7 and 0.7% of 300 root segments were colonised by the aquatic 

hyphomycetes Tricladium splendens and Campylospora purvula, respectively,comparedtothe19%thatwerecolonisedbythemostcommon 

endophyte Cylindrocarpon destructans In a later study, Fisher et al. (1991) 

compared aquatic and terrestrial alder roots along the banks of the River 

Dart (Devon, UK) They separated roots into bark and xylem (decorticated 

roots), and found more endophytic aquatic hyphomycetes in the former. 

Mean frequency of occurrence of aquatic species in submerged roots was 

as high as 30%, compared to 12% on terrestrial roots. In addition to typical 

aquatic hyphomycetes, they also found species of the genera Fusarium 

and Cylindrocarpon. Members of these two taxa are often found on leaves


in decaying streams. Cluster and correspondence analyses suggested that 

aquatic and soil root samples are colonised by two distinct endophyte 

populations, indicating that the external environment may have a greater 

influence on endophyte communities of roots than those of leaves (Fisher 

and Petrini 1993). Three previously unknown species (Fontanospora fusiramos, 

and two species belonging to Filosporella) were subsequently isolated 

and described from submerged alder roots (Marvanová and Fisher 1991; 

Marvanová et al. 1992, 1997). 

The host range of endophytic aquatic hyphomycetes was extended by 

Sridhar and Bärlocher (1992a). They found additional species in spruce 

(Picea glauca [Moench] Voss), birch (Betula papyrifera Marsh) and maple 

(Acer spicatum Lam.). Again, fungal endophytes were more common in the 

bark,suggestingthatrootsarecolonisedbyfungisettlingonsurfacesand 

growing toward the interior. In addition to plating out surface-sterilised 

root fragments, Sridhar and Bärlocher (1992a) aerated them in distilled 

water and were able to observe release of typical tetraradiate or sigmoid 

conidia. However, aeration had to continue for 4 days (compared to the 

usual 1–2 days) before spores were detected (any superficial mycelia that 

may have been present were killed by surface sterilisation). Spore production 

per unit mass was less than 1 mg −1 , compared to 100–150 mg −1 from 

dead submerged branches, and up to 8,000 mg −1 on dead leaves (Gessner 

et al. 2003). Nevertheless, the root biomass in streams is considerable and 

its turnover rapid (Waid 1974), suggesting that it may be an important secondary 

resource for aquatic hyphomycetes. Their existence as endophytes 

may provide them with a head start in the use of root detritus, a possible 

advantage of the endophytic life style that has also been suggested for 

leaf-decomposing saprobes (Fisher and Petrini 1993). 

On spruce roots, the number aquatic hyphomycetes in the xylem was 

highest in 4- to 5-year-old segments (Sridhar and Bärlocher 1992b). Total 

fungal biomass, estimated by ergosterol, amounted to 0.002 to 0.2% of 

root biomass. This compares to values exceeding 15% on decaying leaves 

(Gessner et al. 2003). 

Iqbal et al. (1994) reported 17 species of endophytic aquatic hyphomycetes 

from tree roots along canal banks in Pakistan (Mangifera indica L., 

Populus hybrida Reichb., Salix babylonica L.). Two plantation crops (Coffea 

arabica Linn., Hevea brasiliensis M.) and four ferns (Diplazium esculentum 

(Retz) Sw., Macrothelypteris torresiana (Gaudich.) Ching., Angiopteris 

evecta (Forst) Hoffin., Christela dentata Brownsey & Jermy), all from India, 

were also shown to harbour aquatic endophytes in their submerged roots 

(Raviraja et al. 1996). Again, their incidence was higher in the bark than in 

the xylem of tree roots. Conidium release per root biomass upon aeration 

was much higher from the fern C. dentata (12,900 g −1 ) than from the other 

plants (36–410 g −1 ).


Permanently or periodically submerged roots are common in mangrove 

swamps. Ananda and Sridhar (2002) examined fungal epiphytes in prop 

rootsorpneumatophores(whichcyclebetweenexposuretoairandimmersion 

in salt or brackish water) of Avicennia officinalis L., Rhizophora mucronata 

Lamk. and Sonneratia caseolaris (L.) Engl.. Aquatic hyphomycetes 

were represented by Mycocentrospora acerina in roots of A. officinalis L., 

and Triscelophorus acuminatus in roots of R. mucronata and S. caseolaris. 

In addition, various marine and terrestrial fungi were found. 

Overall, 35 aquatic hyphomycete species (plus seven taxa identified to 

genus) have been reported from submerged roots of 13 plants, including 

Angiosperms, Gymnosperms and ferns in eight studies (Table 10.1). 

This corresponds to roughly 10% of the total number of described species 

(L. Marvanová, personal communication). Clearly, submerged roots can 

provide a stationary refuge for aquatic hyphomycetes, which may help 

them maintain their presence in a given stream reach despite the unidirectional 

flow of water. 

Table 10.1. Endophytic aquatic hyphomycetes recovered from roots, submerged in saltwater 

(*) or freshwater (all others). Root sections: R Entire root, B bark, X xylem (decorticated 

root) 

Fungus Substrate 

Anguillospora filiformis 

Greath. 

A. longissima 

(de Wild.) Ingold 

Articulospora antipodea 

Roldán 

A. atra 

Descals 

A. tetracladia 

Ingold 

A. proliferata 

Jooste, Radon & Merwe 

Bacillispora inflata 

Iqbal & Bhatty 

Root 

section References 

Acer spicatum B, X Raviraja et al. 1996; 

Sridhar and Bärlocher 1992b 

Betula papyrifera B, X Sridhar and Bärlocher 1992a 

Picea glauca B Sridhar and Bärlocher 1992a 

Mangifera indica B, X Iqbal et al. 1995 

Populus hybrida B, X Iqbal et al. 1995 

Salix babylonica B, X Iqbal et al. 1995 

Picea glauca B, X Sridhar and Bärlocher 1992a 

Alnus glutinosa B Fisher et al. 1991 


Acer spicatum B, X Fisher et al. 1991 

Alnus glutinosa B, X Fisher et al. 1991; Sridhar 

and Bärlocher 1992a, 1992b 





Mangifera indica B Iqbal et al. 1995 

Populus hybrida B Iqbal et al. 1995 

Salix babylonica B Iqbal et al. 1995




Campylospora 

chaetocladia 

Ranzoni 

Clavariopsis aquatica 

de Wild. 

Root 


Salix babylonica B Iqbal et al. 1995 

Alnus glutinosa B, X Fisher et al. 1991 

Picea glauca X Sridhar and Bärlocher 1992a 



C. azlanii Nawawi Mangifera indica B Iqbal et al. 1995 

Cylindrocarpon aquaticum 

(Nils.) Marvanová & 

Descals 

Acer spicatum B, X Sridhar and Bärlocher 1992a 





Filosporella sp. Alnus glutinosa B Fisher et al. 1991 

F. fistucella 

Marvanová & Fisher 

Alnus glutinosa B Marvanová and Fisher 1991 

F. versimorpha 

Marvanová et al. 

Alnus glutinosa B Marvanová et al. 1992 

Flagellospora curvula 

Ingold 

F. fusarioides 

Iqbal 

F. penicillioides 

Ingold 










Fontanospora fusiramosa 

Marvanova et al. 

Alnus glutinosa R Marvanová et al. 1997 

Geniculospora sp. Picea glauca B, X Sridhar and Bärlocher 1992b 

Heliscus lugdunensis 

Sacc. & Therry 

Lunulospora curvula 

Ingold 

Acer spicatum B, X Sridhar and Bärlocher 1992a 

Alnus glutinosa B, X Iqbal et al. 1995 

Betula papyrifera B, X Sridhar and Bärlocher 1992a 

Picea glauca B, X Sridhar and Bärlocher 

1992a, 1992b 

Salix babylonica B,X Iqbal et al. 1995 


Angiopteris evecta R Raviraja et al. 1996 

Christela dentata R Raviraja et al. 1996 

Coffee arabica B, X Raviraja et al. 1996




Root 


Hevea brasiliensis X Raviraja et al. 1996 




Mycocentrospora sp. 1 Alnus glutinosa B Fisher et al. 1991 

Mycocentrospora sp. 2 Acer spicatum B, X Sridhar and Bärlocher 1992a 


Mycocentrospora sp. 3 Coffee arabica B Raviraja et al. 1996 

Diplazium esculentum R Raviraja et al. 1996 

Hevea brasiliensis X Raviraja et al. 1996 

Macrothelypteris 

torresiana 

R Raviraja et al. 1996 

M. acerina 

(Hartig) Deighton 

*Avicennia officinalis R Ananda and Sridhar 2002 

M. clavata Iqbal Betula papyrifera B, X Sridhar and Bärlocher 1992a 


M. iqbalii sp.ind.F.BareenMangifera indica B, X Iqbal et al. 1995 


Phalangispora constricta 

Nawawi & Webster 

Picea glauca B, X Sridhar and Bärlocher 1992b 

Pseudoanguillospora sp. Alnus glutinosa B Fisher et al. 1991 

Tetrabrachium elegans Acer spicatum B, x Sridhar and Bärlocher 1992a 

Nawawi & Kuthubutheen Betula papyrifera B, X Sridhar and Bärlocher 1992a 


Tetracladium sp Angiopteris evecta R Raviraja et al. 1996 

T. furcatum Descals Angiopteris evecta R Raviraja et al. 1996 

T. marchalianum Mangifera indica B Iqbal et al. 1995 

de Wild. 



T. setigerum 

(Grove) Ingold 

Picea glauca B Sridhar and Bärlocher 1992b 

Tricladium chaetocladium Alnus glutinosa B Fisher et al. 1991 

Ingold 


Tricellula aquatica 

Webster 


Triscelophorus acuminatus Angiopteris evecta R Raviraja et al. 1996 

Nawawi 


Coffea arabica B Raviraja et al. 1996 

Diplazium esculatum R Raviraja et al. 1996 

Hevea brasiliensis B, X Raviraja et al. 1996




T. konajensis 

Sridhar & Kaveriappa 

T. monosporus 

Ingold 

Tumularia aquatica 

(Ingold) 

Marvanová & Descals 

Varicosporium elodeae 

Kegel 

Root 


Macrothelypteris 

torresiana 


*Rhizophora 

mucronata 

R Ananda and Sridhar 2002 

*Sonneratia caseolaris R Ananda and Sridhar 2002 

Antipteris evecta R Raviraja et al. 1996 


Coffea arabica B Raviraja et al. 1996 

Macrothylpteris 

torresiana 


Angiopteris evecta R Raviraja et al. 1996 


Coffea arabica B, X Raviraja et al. 1996 

Diplazium esculatum R Raviraja et al. 1996 

Macrothelypteris R Raviraja et al. 1996 

torresiana 






Picea glauca B, X Sridhar and Bärlocher 

1992a, 1992b 

V. giganteum Crane Picea glauca B, X Sridhar and Bärlocher 

1992a, 1992b 

10.4 

Conclusions and Outlook 

Work on submerged roots, primarily in fresh water, has been dominated 

by a very specific objective: to evaluate their role as habitat for aquatic 

hyphomycetes. Other aspects of the plant-endophyte relationship include 

the potential production of unique secondary metabolites allowing the 

fungi to live within the plant without overt symptoms, and which might 

be toxic to potential pathogens or herbivores. Several observations suggest 

that aquatic fungi can produce diffusible antibiotics. For example, 

Massarina aquatica, the teleomorph of Tumularia aquatica, releases antifungal 

substances (Fisher and Anson 1983). Similar observations on other


species have been reported by Asthana and Shearer (1990) and Poch et 

al. (1992). Chamier et al. (1984) demonstrated inhibition of bacteria by 

aquatic hyphomycetes in field experiments. Isolation and characterisation 

of antimicrobial compounds from Anguillospora longissima and A. crassa 

resulted in the discovery of novel metabolites (Harrigan et al. 1995). Two 

surveys of aquatic hyphomycetes and ascomycetes demonstrated that antibacterial 

and antifungal substances are produced by about one-half of 

the species tested (Gulis and Stephanovich 1999; Shearer and Zare-Maivan 

1988). Lignicolous aquatic ascomycetes and hyphomycetes were generally 

more antagonistic than foliicolous species, possibly because long-lasting 

substrata,suchaswood,favourcolonisationbyspeciescapableofdefending 

captured resources (Shearer 1992). It is currently unknown how such compounds 

affect the root’s susceptibility toward pathogens or herbivores. 

Sexual and asexual reproduction of the endophyte are often initiated 

upon the death of the host tissue (Fisher et al. 1986). Sridhar and Bärlocher 

(1992a) reported that Heliscus lugdunensis produced a teleomorph upon 

subculturing; aquatic hyphomycetes endophytic in roots may therefore 

be useful in establishing additional anamorph-teleomorph connections 

(Webster 1992; Sivichai and Jones 2003). 

It is generally accepted that aquatic hyphomycetes and ascomycetes had 

terrestrial ancestors, and several have indeed close terrestrial relatives 

(Kong et al. 2000; Liew et al. 2002). Shearer (1993) suggested that when 

terrestrial plants invaded freshwater habitats, they brought with them fungalpathogens,endophytesandsaprobes.Alternatively,plantdetritus,precolonised 

by fungal biotrophs or saprotrophs may have fallen into streams. 

Some of these fungi may subsequently have adapted to dispersal and reproduction 

in water. The most comprehensive analysis of fungal gene sequences 

suggests that the biotrophic lifestyle was a synapomorphic trait 

(i.e. present in a common ancestor; Tehler et al. 2003). The first fungi that 

colonised the terrestrial habitat most likely did so while closely associated 

with plants. The question remains whether such fungi first evolved into 

terrestrial saprobes, and then into aquatic hyphomycetes, or whether there 

was a direct transition from terrestrial biotrophs to aquatic saprobes. Were 

the presumed ancestors restricted to specific plant organs, e.g. aerial twigs 

and leaves, or roots? Upon their death, roots submerged in streams may 

have released propagules of fungal endophytes, some of which settled on 

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may have favoured adaptation to life in running water. Or, terrestrial leaves 

infected with endophytes were shed and landed in streams. Even waterfilms 

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Widler and Müller (1984) isolated two undescribed species of Gyoerffyella 

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from submerged green leaves. 

It will be of considerable interest to investigate the common route of 

invasion by aquatic hyphomycetes: do they first colonise the roots, and 

then spread through the rest of the plant, or do some invade aerial parts? 

A thorough study of endophytes in aerial and subterranean plant parts at 

various ages, and molecular data of such strains may eventually allow us to 

reconstruct the origins of aquatic hyphomycetes. 


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11 

11.1 

Introduction 

Nematophagous Fungi 

as Root Endophytes 

Luis V. Lopez-Llorca, Hans-Börje Jansson, 

José Gaspar Maciá Vicente, Jesús Salinas 

Nematophagous fungi constitute a group of fungal antagonists to nematodes. 

The latter are small roundworms living in soil and water. Most 

nematodes are saprotrophic, but many species are parasites of plants and 

animals (Poinar 1983). The nematophagous fungi have been suggested as 

promising candidates for biological control of parasitic nematodes (Stirling 

1991), but so far no successful commercial products have been presented. 

Many of the previous studies on these organisms have been concerned 

with the ecology and physiology of interactions between nematophagous 

fungi and nematodes. More recently, molecular techniques have been employed 

(Jansson and Lopez-Llorca 2001). Nematophagous fungi also have 

the ability to infect and colonise other organisms, including other fungi 

and plant roots (Jansson and Lopez-Llorca 2004). In the current review we 

will briefly describe the nematophagous fungi, with special emphasis on 

their interactions with plant roots. 

11.2 

Nematophagous Fungi 

Nematophagous fungi, or nematode-destroying fungi, have the capacity 

to infect, kill and digest living stages of their nematode hosts (eggs, juveniles 

and adults). These fungi are ubiquitous soil inhabitants found in 

most parts of the world and in all climate types (Barron 1977). Many of the 

Luis V. Lopez-Llorca: Departamento de Ciencias del Mar y Biología Aplicada, Universidad 

de Alicante, Apdo. 99, 03080 Alicante, Spain, E-mail: lv.lopez@ua.es 

Hans-Börje Jansson: Departamento de Ciencias del Mar y Biología Aplicada, Universidad 

de Alicante, Apdo. 99, 03080 Alicante, Spain, E-mail: hb.jansson@ua.es 

José Gaspar Maciá Vicente: Departamento de Ciencias del Mar y Biología Aplicada, Universidad 

de Alicante, Apdo. 99, 03080 Alicante, Spain, E-mail: jgmv@ua.es 

Jesús Salinas: Departamento de Ciencias del Mar y Biología Aplicada, Universidad de Alicante, 

Apdo. 99, 03080 Alicante, Spain, E-mail: jesus.salinas@ua.es 





192 L.V. Lopez-Llorca et al. 

nematophagous fungi are facultative parasites and can grow saprophytically 

in soil. In the presence of hosts they can change from a saprophytic to 

a parasitic stage and form infection structures, e.g. trapping organs, hyphal 

coils or appressoria. These infection structures vary depending on the type 

of host–nematode, fungus or plant. 

Entomopathogenic fungi, e.g. Lecanicillium lecanii,havethecapacityto 

infect both nematode eggs (Meyer 1998) and other fungi. Furthermore, 

species closely related to nematophagous fungi, e.g. Arthrobotrys ferox, 

can infect other small soil animals like springtails (Rubner 1996), but are 

generally not known to infect nematodes. 

11.2.1 

Nematode Parasites 

The nematophagous fungi can be divided into four groups depending on 

their mode of attacking their hosts. The first three groups infect vermiform 

nematodes (juveniles and adults), whereas the fourth group infects 

nematode females and eggs (Jansson and Lopez-Llorca 2001). Nematodetrapping 

fungi use various types of trapping organs formed on their hyphae, 

e.g. adhesive networks, adhesive knobs (Fig. 11.1a) or constricting rings, 

and these fungi are facultative parasites to various extents. The nematodes 

are captured in the traps formed by the fungi either by adhesion 

or mechanical function. In the endoparasitic fungi, the spores (conidia, 

zoospores) function as infection structures, which either adhere to the nematode 

cuticle or are ingested. These fungi are generally obligate parasites 

of nematodes (Fig. 11.1b). The toxin-producing fungi, comprising for instance 

the common wood-decomposing oyster mushroom, intoxicate their 

nematode victims before penetrating them. The egg- and female parasitic 

attack mature females of cyst- and root-knot nematodes and the eggs they 

contain (Fig. 11.1c). Infection usually takes place via appressoria. Common 

to all types of nematophagous fungi is that after contact with the nematode 

cuticle, or egg shell, penetration takes place followed by digestion of the 

contents resulting in formation of new fungal biomass inside, and later 

outside, the nematode. 

Taxonomy 

The nematophagous fungi are found in most fungal taxa (Dackman et 

al. 1992). In the Basidiomycetes, nematophagous fungi such as the oyster 

mushroom (Pleurotus ostreatus) andHohenbuehelia spp. (teleomorph of 

Nematoctonus spp.) can be found. Many of the nematode-trapping fungi 

belong to the Deuteromycetes or mitosporic fungi, e.g. Arthrobotrys spp. 

and Monacrosporium spp., but the Arthrobotrys spp. have been found


Fig.11.1. a Adhesive knob trap of Monacrosporium haptotylum adhering to the nematode 

cuticle. Note adhesive pad between trap and nematode (arrow). b Conidia of the endoparasitic 

fungus Drechmeria coniospora adhering to the mouth region of a nematode. (From 

Jansson and Nordbring-Hertz 1983, courtesy of Society of General Microbiology). c A nematode 

egg infected by the egg-parasitic fungus Pochonia rubescens. (From Lopez-Llorca 

and Claugher 1990, courtesy of Elsevier). Bars a, b 5µm;c 4µm 

to be Ascomycetes with their teleomorph in Orbilia spp. (Pfister 1997). 

Other nematode-trapping fungi, e.g. Stylopage and Cystopage spp. are Zygomycetes, 

and some endoparasitic fungi, such as Catenaria anguillulae,are 

zoosporic Chytridiomycetes. The main egg-parasitic fungi are now placed 

in the new genus Pochonia (formerly Verticillium) (Gams and Zare 2003). 

Therefore, the various nematophagous fungi seem to have acquired their 

nematophagous ability independently during the course of evolution. 

The few phylogenetic studies that have been presented (Ahrén et al. 

1998; Hagedorn and Scholler 1999) show that the orbiliaceous nematodetrapping 

fungi are closely related, and that the type of trap, rather than 

traditional spore morphology, is more determinate on the species level. 

Biology 

We will focus on two types of nematophagous fungi: the nematode-trapping 

Arthrobotrys oligospora and the egg parasite Pochonia chlamydosporia. 

These fungi are common soil inhabitants living both saprophytically and 

parasitically and, as we will show, also endophytically. 

Arthrobotrys oligosporaforms three-dimensional adhesive network traps 

in the presence of nematodes (Nordbring-Hertz 1977). Apart from a low


nutrient status, small peptides, e.g. phenylalanyl-valine, can induce trap 

formation (Nordbring-Hertz 1973). When traps are formed, and even before 

traps are fully developed, nematodes can be captured in the adhesive 

covering the traps. The adhesive has been partially characterised and appears 

to be a polymer complex containing proteins, neutral sugars and 

uronic acids (Tunlid et al. 1991). The adhesive changes properties, from 

an amorphous stage to directed fibrils, after contact with the nematode 

cuticle (Veenhuis et al. 1985). This is in contrast to the endoparasitic fungus 

Drechmeria coniospora, wherethefibrilsappeardirectedwhethernematodes 

are present or not (Jansson and Nordbring-Hertz 1988). After 

adhesion, the fungus penetrates the nematode cuticle from the trap, probably 

using both mechanical and enzymatic means. Since the nematode 

cuticle contains mainly proteinaceous material (Bird and Bird 1991), extracellular 

proteolytic enzymes involved in cuticle penetration have been 

studied. The major protease appears to be subtilisin PII. This serine protease 

has been characterised and genomically cloned (Åhman et al. 1996). 

After penetration, an infection bulb is formed, from which trophic hyphae 

grow out and digest the contents of the nematode. New hyphae and traps 

arethenformedoutsidethenematodecorpustostartanewinfection 

cycle. 

Pochonia spp. adhere to nematode egg shells by means of an appressorium 

formed at the hyphal tip (Lopez-Llorca and Claugher 1990, Lopez- 

Llorca et al. 2002b). An extracellular material (ECM) probably functions as 

adhesive, but possibly also seals the perforation in the egg shell caused by 

the penetration hypha beneath the fungal appressorium. This extracellular 

material can be labelled with the lectin Concanavalin A, indicating that 

the ECM contains mannose/glucose moieties probably on the side chains 

of glycoproteins (Lopez-Llorca et al. 2002b). Most ECMs of fungal hyphae 

consist of proteins and carbohydrates (Nicholson 1996). The nematode egg 

shell consists mainly of proteins and chitin (Bird and Bird 1991) and therefore 

proteases and chitinases would be important for fungal penetration of 

the egg shell. Serine proteases have been isolated from Pochonia rubescens, 

P32 (Lopez-Llorca 1990), P. chlamydosporia, VcP1 (Segers et al. 1994) and 

Paecilomyces lilacinus, PL (Bonants et al. 1995). In some cases these have 

been immunolocalised in infected hosts, their peptides sequenced, and the 

coding genes cloned. Recently, the chitinolytic system of P. rubescens and 

P. chlamydosporia has been studied (Tikhonov et al. 2002). For both species, 

among other enzymes, a similar major 43 kDa endochitinase (CHI43) was 

purified and characterised. A combination of protease P32 and chitinase 

CHI43, or the enzymes individually, caused removal of egg shell layers of 

the potato cyst nematode Globodera pallida (Tikhonov et al. 2002). Following 

penetration of the egg shell, the fungus digests the contents of 

the egg, proliferates inside and later grows outside the egg to penetrate


neighbouring eggs in nematode cysts or egg masses; alternatively it can 

grow saprophytically. 

11.2.2 

Mycoparasites 

Mycoparasitism is a common feature of fungi (Jeffries 1997). The ability 

of nematophagous fungi to attack other fungi was first described by Tzean 

and Estey (1978). Nematode-trapping fungi such as A. oligospora attack 

their host fungi, e.g. Rhizoctonia solani, in a manner similar to that of the 

well known mycoparasite Trichoderma spp. (Chet et al. 1981). The mycoparasitic 

behaviour of A. oligospora takes place by coiling of the hyphae of 

the nematode-trapping fungi around the host hyphae, which, in contrast to 

Trichoderma spp., results in disintegration of the host cell cytoplasm without 

penetration of the host (Persson et al. 1985). It has been shown using 

radioactive phosphorous tracing that nutrient transfer takes place between 

the nematode-trapping fungus A. oligospora and its host R. solani (Olsson 

and Persson 1994). Although this phenomenon has never been observed in 

soil, it may increase the fitness of the nematode-trapping fungi in soil by 

reducing competition and providing nutrients. Moreover, it may extend the 

biocontrol capability of nematophagous fungi as biocontrol agents to fungal 

parasites as well as nematodes. Furthermore, P. chlamydosporia has been 

described as being able to infect propagules of important plant pathogens, 

such as uredospores of rust fungi (Leinhos and Buchenauer 1992), and 

oospores of Phytophthora and other Oomycetes (Sneh et al. 1977). 

11.2.3 

Root Endophytes 

Most work on the root biology of nematophagous fungi has concerned 

externalrootcolonisation(ectorhizosphere).Lately,colonisationintheroot 

tissues has also been reported (endorhizosphere). Some of these studies 

will be discussed in this chapter. 

Ectorhizosphere 

Since plant-parasitic nematodes generally attack plant roots it has been 

an important task to study the rhizosphere biology of nematophagous 

fungi – the root zone is an area with an abundant supply of the nematode 

prey. Not surprisingly, the nematode-trapping fungi have been found to 

be more frequent in the rhizosphere than in the bulk soil (Peterson and 

Katznelson 1965; Gaspard and Mankau 1986; Persmark and Jansson 1997).


Egg-parasitic fungi were also found to be more abundant in the rhizosphere 

(Bourne et al. 1996; Kerry 2000). 

External root colonisation varies between plant species. For instance, 

Persmark and Jansson (1997) studied the presence and frequency of nematode-trapping 

fungi in field soils planted with barley, pea or white mustard. 

The pea rhizosphere harboured by far the highest frequency of nematodetrapping 

fungi: 19 times higher than in the root free soil. The number 

of species of nematode-trapping fungi was also higher in the pea rhizosphere, 

with A. oligospora as being the most common species (Persmark 

and Jansson 1997). In an investigation on chemotropic growth towards 

roots of pea, barley and white mustard by seven species of nematophagous 

fungi, only isolates of A. oligospora were attracted to the roots of all plants, 

but this was confined to the 2 mm closest to the roots (Bordallo et al. 

2002). In a pot experiment, the colonisation of tomato roots by several nematophagousfungiwasfollowedfor3months.Monacrosporiumellipsosporum 

and Arthrobotrys dactyloides were especially competent in colonising 

the roots (Persson and Jansson 1999). Several nematode-trapping fungi are 

able to form so-called “conidial traps” in response to roots and root exudates 

(Persmark and Nordbring-Hertz 1997). The conidial traps are capture 

organs formed directly on the conidia without hyphal growth, and give the 

fungi an extra advantage by spreading the fungi and capturing nematodes, 

similar to most endoparasitic fungi, which infect nematodes entirely with 

adhesive conidia. 

External root colonisation by the egg-parasite Pochonia chlamydosporia 

also varied with plant species. For instance, kale and cabbage had a density 

of the fungus twice as high as that on soya bean and tomato (Bourne 

et al. 1996). Furthermore, rhizosphere colonisation by P. chlamydosporia 

was increased when plants were infected with the root-knot nematode 

Meloidogyne incognita. Thiseffectispossiblyduetoincreasedleakageof 

root exudates after damage to the root surface by the nematodes (Bourne 

et al. 1996). In none of the investigations mentioned above was the fungal 

colonisation of internal root tissues examined. 

Endorhizosphere 

Using axenic barley and tomato plants inoculated with the nematophagous 

fungi P. chlamydosporia or A. oligospora, we found that both fungi have the 

capacity to colonise epidermis and root cortex of barley and the epidermis 

of tomato (Lopez-Llorca et al. 2002a, Bordallo et al. 2002). 

In these experiments roots were sequentially sampled, cryo-sectioned, 

and observed under light- or cryo-scanning electron microscopes. Both 

fungi grew inter- and intra-cellularly and formed appressoria (Figs. 11.2a,b) 

whenpenetratingplantcellwallsofepidermisandcortexcells,butnever


Fig.11.2. a Formation of appressoria (arrow)byArthrobotrys oligospora during penetration 

of the epidermis of barley roots. (From Bordallo et al. 2002, courtesy of Blackwell Science). 

b Colonisation of tomato roots by Pochonia chlamydosporia. Note appressorium and cell 

wall protein apposition (arrow). (From Bordallo et al. 2002, courtesy of Blackwell Science). 

c Colonisation of barley roots by A. oligospora. Callose deposit in papillae (arrow) stained 

with Sirofluor. (From Bordallo et al. 2002, courtesy of Blackwell Science). d Colonisation 

of tomato roots by Pochonia chlamydosporia. Note externally produced chlamydospores 

(arrow). (From Bordallo et al. 2002, courtesy of Blackwell Science). Bars a, c 20 µm; b, 

d 30 µm 

entered vascular tissues. In contrast to Pochonia spp., appressoria had 

previously never been observed in A. oligospora. Using histochemical 

stains, we could show plant defence reactions, e.g. papillae, lignitubers 

and other cell wall appositions induced by nematophagous fungi, but these 

never prevented root colonisation. Callose deposits in papillae induced by 

A. oligospora in barley roots are shown in Fig. 11.2c. Nematophagous fungi 

grew extensively, especially in monocotyledonous plants, producing abundant 

mycelia, conidia and chlamydospores (P. chlamydosporia) (Fig. 11.2d). 

Necrotic areas of the roots were observed at initial stages of colonisation 

by A. oligospora, but were never seen at later stages even when the fungus 

proliferated in epidermal and cortical cells. Roots colonised by P. chlamydosporia 

displayed higher proteolytic activity than non-inoculated control 

roots using immunochemical techniques. The significance of this fact for 

biological control of root pathogens is under investigation in our laboratory.


The growth of the two nematophagous fungi in plant roots appears to resemble 

that of an endophyte, i.e. the host remains asymptomatic. Whether 

this endophytic growth induces systemic resistance to nematodes and/or 

plant pathogens in plants is as yet unknown, but worth further investigation. 

We have found that P. chlamydosporia could reduce growth of the 

plant-pathogenic fungus Gaeumannomyces graminis var. tritici (take-all 

fungus, Ggt) in dual culture Petri dish and in growth tube experiments. In 

pot experiments, P. chlamydosporia increased plant growth whether Ggt 

was present in the roots or not, suggesting a growth promoting effect by 

P. chlamydosporia (Monfort et al. 2005), as has also been found in the case 

of colonisation by other endophytic fungi (see Chap. 15 by Schulz). 

In a recent screening in our laboratory on the capacity of various types 

of nematophagous fungi to grow endophytically in roots, we have shown 

that fungi other than A. oligospora and P. chlamydosporia had this ability 

(Table 11.1). With these fungi, all four ecological groups of nematophagous 

fungi are represented. The results regarding root colonisation are shown in 

Table 11.2. 

Hirsutella rhossiliensis, whichinfectsnematodesbymeansofadhesive 

conidia (Jaffee and Zehr 1982), behaves ecologically as an endoparasitic 

fungus (“obligate” parasite), although it can grow in the laboratory on 

artificial media. The fungus reacts in a density-dependent manner (Jaffee 

et al. 1992) and, in spite of its low efficiency as a nematode antagonist, it 

suppresses plant-parasitic nematodes in agroecosystems with little human 

disturbance, such as old peach orchards in the United States (Stirling 1991). 

H. rhossiliensis, unlike A. oligospora and P. chlamydosporia,doesnotseem 

to colonise barley roots endophytically. Three weeks after inoculation, 

cortex and epidermal cells were free from hyphal colonisation (Table 11.2). 

However, the fungus seems to colonise the rhizoplane abundantly, where it 

forms viable conidiophores (Fig. 11.3a). 

Nematoctonus (teleomorphHohenbuehelia)isapeculiargenusofbasidiomycetous 

nematophagous fungi. It comprises about 15 species, some of 

Table 11.1. Endophytic root colonisation of barley by the four ecological groups of nematophagous 

fungi 

Fungus Type Colonisation Reference 

Pochonia chlamydosporia Egg-parasite + Lopez-Llorca et al. 2002a 

Arthrobotrys oligospora Nematode-trapping + Bordallo et al. 2002 

Arthrobotrys dactyloides Nematode-trapping + This chapter 

Nematoctonus robustus Nematode-trapping + This chapter 

Nematoctonus pachysporus Endoparasite – This chapter 

Hirsutella rhossiliensis Endoparasite – This chapter 

Pleurotus djamor Toxin producing + This chapter


Table 11.2. Semiquantitative endophytic barley root colonisation by nematophagous fungi 

(unpublished results). In these experiments barley seeds were sterilised, germinated and 

planted together with the appropriate fungus in culture tubes containing sterilised vermiculite 

and water according to Bordallo et al. (2002). After 21 days the roots were harvested, 

surface sterilised to remove external hyphal growth, cut into 1-cm fragments and plated on 

corn meal agar. Half of the roots were not surface sterilised. After approx 7 days the root 

fragments were examined and fungal growth was recorded. Data are based on three roots, 

with six fragments of each (n = 18) 

Fungus Type Root fragments with fungus (%) 

Non-surface Surface 

sterilised sterilised 

Arthrobotrys dactyloides Nematode-trapping 77.8 5.6 

Nematoctonus robustus Nematode-trapping 100.0 22.0 

Nematoctonus pachysporus Endoparasite 50.0 0 

Hirsutella rhossiliensis Endoparasite 100.0 0 

Pleurotus djamor Toxin producing 77.8 11.1 

which fit the endoparasitic behaviour, infecting nematodes by means of 

adhesive conidia, whereas others capture nematodes with adhesive traps, 

yet others share both types of behaviour (Thorn and Barron 1986). This 

diverse nematophagous behaviour was also reflected in root colonisation 

by the two species studied. Whereas the nematode-trapping N. robustus 

penetrated and colonised barley roots (Fig. 11.3b) and formed typical 

clamp-connections (Fig. 11.3c) as soon as 1 week after inoculation, the 

endoparasite N. pachysporus did not enter the roots but colonised the root 

surfaceasdidH. rhossiliensis (Table 11.2). It thus appears that, in general, 

the endoparasitic fungi may not be endophytic root colonisers, but this may 

also be a reflection of their slow growth rate and their mode of infecting 

nematodes. 

The genus Pleurotus also belongs to the Basidiomycetes.Itformsbasidiocarps 

and its standard means of living is saprophytic growth on decaying 

wood. The most common species, P. ostreatus, is a commercially cultivated 

edible mushroom. The fungus compensates for the lack of nitrogen 

in wood, its natural substrate, with its nematophagous habit (Thorn and 

Barron 1984). In fact, the nematophagous habit has been described for 

several species of Pleurotus (Thorn and Barron 1984). The fungus immobilises 

nematodes with a toxin (Kwok et al. 1992) prior to infection and 

digestion of its prey (Nordbring-Hertz et al. 1995). The strong relation with 

plant tissues, as a natural wood decomposer, was also confirmed in roots, 

when we found that, just as N. robustus, P. djamor penetrated early, and 

extensively colonised barley roots (Fig. 11.3d). The fungus was in fact very 

aggressive since some parts of the root appeared to be decorticated 3 weeks


Fig.11.3. a Rhizoplane colonisation of barley roots by the endoparasitic fungus Hirsutella 

rhossiliensis 3 weeks after inoculation, forming conidiophore with phialide (arrow). 

b Colonisation of cortex of barley roots by the nematode-trapping basidiomycete Nematoctonus 

robustus 2 weeks after inoculation. c Colonisation of cortex and epidermis of barley 

roots by the nematode-trapping basidiomycete Nematoctonus robustus 1 week after inoculation. 

Note clamp-connection (arrow). d Colonisation of cortex of barley roots by Pleurotus 

djamor 10 days after inoculation. e Colonisation of cortex of barley roots by the nematodetrapping 

fungus Arthrobotrys dactyloides 2 weeks after inoculation. f Colonisation of cortex 

of barley roots by the nematode-trapping fungus Arthrobotrys dactyloides 2weeksafter 

inoculation showing coiling structure. Bars a, b 30 µm; c–f 15 µm 

after inoculation, as was A. oligospora in previous experiments (Bordallo 

et al. 2002). 

Arthrobotrys dactyloides is a nematode-trapping fungus that captures 

nematodes by means of constricting rings (Barron 1977). The fungus was 

shown to form functional traps in soil (Jansson et al. 2000) and on the surface 

of tomato roots infected with root-knot nematodes (Riekert and Tiedt


1994), and has also been used in biological control experiments of plant 

parasitic nematodes (Stirling and Smith 1998). In our recent experiments, 

A. dactyloides was an active, and early, root coloniser. We found evidence 

of epidermal cell penetration and colonisation (Fig. 11.3e) 1 week after 

inoculation. Like P. chlamydosporia, the fungus formed coiling structures 

in barley root cells (Fig. 11.3f) and extensively colonised the roots. Such 

structures are also formed by other root endophytes, e.g. Piriformospora 

indica (Varma et al. 1999; Chap. 15 by Schulz), and presumably improve 

the exchange of metabolites. 

These preliminary studies lack the most important component, the nematode. 

With trapping and endoparasitic nematophagous fungi, such experiments 

are easy to perform axenically, since the nematophagous habit 

can easily be triggered by free-living nematodes. Such experiments are 

underway in our laboratory to address the question whether the root 

endophytic behaviour of nematophagous fungi is functional in their nematophagous 

habit, i.e. if the mycelium growing in roots is able to develop 

active trapping organs or adhesive spores. 

In the case of fungal nematode egg parasites the inclusion of a plant 

parasitic nematode is technically more difficult. This is because the natural 

targets of nematophagous Pochonia spp. are cyst and root knot nematodes. 

These phytopathogenic nematodes have an endoparasitic behaviour 

and a life span of at least a month – too long for our axenic system. Although 

these nematodes can be multiplied in axenic systems based on 

Agrobacterium-transformed roots on a tissue culture medium (Verdejo- 

Lucas 1995), these extremely rich conditions are incompatible for studying 

root colonisation by nematophagous fungi. An alternative, which we have 

explored (unpublished) is the use of migratory endoparasitic nematodes 

such as Pratylenchus spp., some species of which have broad host specificity 

and can infect cereals such as barley under our conditions. We have 

encountered two problems that have prevented further development. One 

is that Pratylenchus spp. is not a host of Pochonia spp. (or has not been 

describedyet).Sincethenematodelayseggsinroottissue,onecouldexpect 

to find egg infection. However, in our experiments we found that the axenic 

conditions are too favourable for the nematode, which multiplies much 

faster than the fungus. This may be a question of optimising inoculum. We 

are trying to adapt our methods to include endoparasitic nematodes such 

as Meloidogyne spp.,andthefinalgoalofourstudiesistodevelopbiological 

control strategies to such severe plant pathogens. Development of 

alternative control methods, e.g. biological control, is especially important 

since the phasing out of the most widespread method of plant parasitic 

nematode control, fumigation with methyl bromide, is to be enforced. 

Endophytic rhizobacteria that reduce plant-parasitic nematodes have 

also been described [Hallman et al. 2001; Chaps. 4 (Berg and Hallmann)


and 3 (Kloepper and Ryu)], as well as arbuscular mycorrhizal fungi that 

reduce root knot nematodes (Waecke et al. 2001). If this is also the case for 

nematophagous fungi this will open up a new area of biocontrol using these 

fungi. The internal root colonisation by egg-parasitic fungi, e.g. Pochonia 

spp.,maygivethefungianopportunitytoinfectnematodeeggsineggsacks 

of root-knot nematodes inside the roots and reduce subsequent spread and 

infection of roots by the second stage juveniles. Structures resembling trapping 

organs were observed in epidermal cells colonized by A. oligospora 

(Bordallo et al. 2002), and these may serve the purpose of trapping newly 

hatched juveniles escaping the roots. The ability to colonise plant roots 

may also be a survival strategy of these fungi and could explain soil suppressiveness 

to plant-parasitic nematodes in nature. Root inoculation of 

nematophagous fungi may help us to circumvent the lack of receptivity 

of soil (even sand) to inoculum of nematophagous fungi (Monfort et al. 

2006), which has hampered the capability of fungi such as P. chlamydosporia 

to control agronomically important nematodes such as Meloidogyne 

spp. (Verdejo-Lucas et al. 2003). This, despite the fact that the nematode is 

naturally found to be infected by P. chlamydosporia and other egg-parasitic 

fungi in similar Mediterranean agroecosystems (Verdejo-Lucas et al. 2002; 

Olivares-Bernabeu and Lopez-Llorca 2002). The root colonisation of plant 

roots is a new area of research that deserves in-depth investigation, particularly 

for biocontrol purposes. 

11.3 

Concluding Remarks 

The role of the host plant in the tritrophic relationship between nematophagous 

fungi, plants and phytopathogenic nematodes has largely been 

neglected. In this chapter we have collected and drawn conclusions from 

the data accumulated in the literature. We have also presented our own data, 

which indicate that the outcome of the interaction between nematophagous 

fungi and plants depends both on the host plant (mono- vs di-cotyledon) 

and the fungal species, but also on the ecological groups of nematophagous 

fungi (trapping, endoparasitic, egg-parasite, toxin producer). 

The plant host is, after all, the most important living entity in an agroecosystem 

and is, of course, the target of any approach to disease control. 

We would like to stress the biological – theoretical – importance of the 

endophytic behaviour of nematophagous fungi, for instance, as a likely 

means to explain soil suppressiveness to plant parasitic nematodes. Another 

theoretical component is the possible discovery of a new mechanism 

of the mode of action of nematophagous fungi, that of interaction with 

the plant host. This may function in two ways. We have initial evidence of


plant growth promotion. The other function could be modulation of plant 

defences. Similar activities have been found for antagonists (bacterial and 

fungal) of other plant pathogens, but also for other endophytic fungi that 

are not necessarily antagonists [see Chaps. 3 (Kloepper and Ryu), 4 (Berg 

and Hallmann) and 14 (Schulz)]. 

Basic discoveries in the field of pathogen–plant host interaction have 

led to new developments and approaches to plant disease control. For 

instance, the recent proliferation of compounds modulating plant defence 

responses as “magic bullets” to control a wide array of biotic and abiotic 

stresses in plants had its origin in the study of the molecular basis of 

virulence/avirulence in the plant pathogenic bacteria–plant interaction. 

There are similar examples, such as the effect of oligosaccharides or other 

molecules on plant defence systems. A future possibility for enhancing 

control of plant pathogens may be to devise a way to obtain synergies with 

non-chemical means of control. Perhaps we are in the dawn of a new era of 

biological control of nematodes that may circumvent the difficulties found 

in the mere application of nematophagous fungi to soil. 


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12 

12.1 

Introduction 

Molecular Diversity and Ecological 

Roles of Mycorrhiza-Associated Sterile 

Fungal Endophytes in Mediterranean 

Ecosystems 

Mariangela Girlanda, Silvia Perotto, Anna Maria Luppi 

Under natural conditions, plant roots sustain considerable fungal diversity 

(Vandenkoornhuyse et al. 2002). In healthy plants, colonisation of root 

tissuesisnotrestrictedtomycorrhizalsymbionts;otherfungicanalsogrow 

asymptomatically in the roots, occurring either in the presence or absence 

of ecto- or endo-mycorrhizal mycobionts. Isolation from surface-sterilised 

roots usually yields a great proportion of fungi with dematiaceous hyphae 

and ascomycetous septa, which are sterile in culture, and are referred to as 

“dark septate endophytes” (DSE) or “dark sterile mycelia” (DSM) (Schild 

et al. 1988; Summerbell 1989; Read 1991; Holdenrieder and Sieber 1992; 

Girlanda and Luppi-Mosca 1995; Ahlich and Sieber 1996; Ahlich et al. 1998; 

Jumpponen and Trappe 1998a; Schadt et al. 2001; see Chap. 7 by Sieber and 

Grünig). These mycelia can also be directly observed to grow inter- and 

intra-cellularly in the root cortex, producing peculiar structures, such as 

microsclerotia that fill cortical cells (Jumpponen and Trappe 1998a; Barrow 

and Aaltonen 2001; Yu et al. 2001; Kovacs and Szigetvari 2002; Ruotsalainen 

et al. 2002; Barrow 2003). By virtue of their constant association with roots, 

they can be qualified as true root symbionts (see Chap. 1 by Schulz and 

Boyle). 

Although the presence of DSE in roots was noted early in the last century 

(Melin 1922, 1923; Peyronel 1924), their abundant, regular, and ubiquitous 

occurrence was given prominence only recently, and is suggestive of 

a significant role in natural ecosystems. As a group, these fungi colonise 

a broad range of hosts, being reported from nearly 600 plant species, representing 

about 320 genera and 114 families (Jumpponen and Trappe 1998a). 

However, DSE represent a heterogeneous assemblage of ascomycetous taxa. 

Mariangela Girlanda: Dipartimento di Biologia Vegetale and IPP - Torino, Viale PA Mattioli 

25, 10125 Torino, Italy, E-mail: mariangela.girlanda@unito.it 

Silvia Perotto: Dipartimento di Biologia Vegetale and IPP - Torino, Viale PA Mattioli 25, 


Anna Maria Luppi: Dipartimento di Biologia Vegetale, Viale PA Mattioli 25, 10125 Torino, 

Italy 





208 M. Girlanda et al. 

Although these fungi are mostly sterile when brought into culture, sporulation 

has been occasionally induced under particular incubation conditions 

(Richard and Fortin 1973; Wang and Wilcox 1985; Fernando and Currah 

1995; Ahlich and Sieber 1996), leading to recognition of distinct conidial 

forms (Gams 1963; Deacon 1973; Richard and Fortin 1973; Wang and 

Wilcox 1985; Currah et al. 1987). In persistently sterile isolates, which are 

unidentifiable with conventional criteria, systematic heterogeneity is indicated 

by different cultural macro- and micro-scopic morphologies and by 

polymorphisms revealed by molecular markers (Stoyke et al. 1992; Harney 

et al. 1997; Jumpponen and Trappe 1998a; Schadt et al. 2001; Addy et al. 

2001; Grünig et al. 2001, 2002b; see Chap. 7 by Sieber and Grünig). For 

instance, sequencing of the 18S nuclear ribosomal DNA (rDNA) region has 

shown polyphyletic placement of DSE fungi within Ascomycetes, with representatives 

of distinct orders such as Pleosporales, Leotiales, and Pezizales 

(Lobuglio et al. 1996; Jumpponen and Trappe 1998a; Schadt et al. 2001; 

Girlanda et al. 2002). As a consequence of such heterogeneity, actual host 

specificity might vary within the DSE group. Specificity is an important attribute 

of fungus-plant associations, and understanding this aspect of the 

association is crucial to clarifying the functional nature of the interactions 

with the host plants, and hence the ecological role of DSE associates. Indeed, 

inoculation experiments of different plants with different DSE strains 

have indicated that plant growth response may depend on the particular 

combination of the fungus and plant being tested (Jumpponen and Trappe 

1998a; Jumpponen 2001). 

To date, most of the knowledge available on the specific identity of DSE 

fungi, as assessed by molecular analyses of internal transcribed spacer 

(ITS) regions of nuclear rDNA, derives from isolates obtained from subantarctic 

and northern temperate forests or from Arctic areas in Europe 

and Canada (Stoyke et al. 1992; Harney et al. 1997; Jumpponen 1999; Addy 

et al. 2001; Grünig et al. 2001, 2002a, 2002b; Schadt et al. 2001; see Chap. 7 

by Sieber and Grünig); other biomes remain largely unexplored in DSE 

research. Extending investigations to different environments offers the opportunity 

of assessing both host and habitat specificity patterns for these 

fungi. Mediterranean ecosystems are especially interesting in this respect. 

They develop in temperate-warm climates (mean annual temperature generally 

ranging from 14 ◦ to 20 ◦ C), with precipitation of 350–1,000 mm/year, 

characterised by hot, dry summers and mild, wet winters (“winter-rain 

and summer dry” climates). Such climatic conditions occur in five distinct 

regions of the world, i.e. the Mediterranean basin, California, Central Chile, 

the Cape Province in South Africa, and Southern Australia (Di Castri and 

Mooney 1973). Although other regions may exhibit similar mean annual 

temperatures and rainfall, they differ in rain distribution over the year 

(Japan, for instance, lacks summer drought). Vegetation in Mediterranean

12 DSE of Mediterranean mycorrhizal plants 209 

climate areas has different regional expressions (such as “maquis” and “garrigue” 

in the Mediterranean basin, “chaparral” in California, “matorral” 

in Chile, “fynbos” and “veld” in capensic flora, “kwongan” and “mallee” 

in Australia), which, however, display striking convergence in life forms 

as an adaptation to the distinctive climatic regime (Pignatti et al. 2002). 

A characteristic, shared feature of Mediterranean vegetation is a high diversity 

of plants and their associated mycorrhizal types: sclerophyllous 

and evergreen shrubs, and small trees bearing arbuscular, ericoid, arbutoid 

mycorrhiza and ectomycorrhiza, which coexist and may take up equal 

size and dominance (Allen 1991). These environments offer therefore an 

interesting scenario for comparative studies of root-fungus associations in 

plants with different mycorrhizal status. 

We have been investigating molecular diversity and the possible ecological 

roles of DSE associates of host pairs in Mediterranean ecosystems in 

Northern Italy (Liguria). Neighbouring, healthy-looking individuals of the 

ectomycorrhizal Pinus halepensis Mill. (Halep pine) and Quercus ilex L. 

(holm oak) and the endomycorrhizal Rosmarinus officinalis L. (arbuscular 

mycorrhiza; rosemary) and Erica arborea L. (ericoid mycorrhiza; Mediterranean 

heather) were selected for isolation of DSE fungi. Using both molecular 

approaches and synthesis experiments the fungi were characterised 

for their range of diversity and for possible ecophysiological traits involved 

in interactions with different plant hosts. 

12.2 

Diversity of DSE Associates of Ecto- and Endo-Mycorrhizal 

Plants in Mediterranean Ecosystems in Northern Italy 

To assess the diversity of DSE isolates obtained from surface-sterilised mycorrhizal 

roots of the two different host pairs (Pinus halepensis / Rosmarinus 

officinalis, Quercus ilex / Erica arborea), morphotypes were recognised 

based on macro- and micro-scopic somatic features and assessed for consistency 

through internal transcribed spacer-restriction fragment length 

polymorphism (ITS-RFLP); further molecular characterisation was carried 

out on morphotypes repeatedly obtained from both hosts (Girlanda et al. 

2002; Bergero et al. 2000, 2003). Such shared DSE groups occurred regularly 

and at high frequency. In the P. halepensis / R. officinalis study, for instance, 

such groups were isolated in all the collection periods, spanning the course 

of 11 years. Ribosomal DNA sequences obtained for representative isolates 

from each morphotype were used as queries for BLAST searches in publicDNAdatabasesaswellasforphylogeneticreconstructionscarriedout 

on datasets comprising both named alignable sequences retrieved from 

BLAST searches and sequences of closely related taxa.


In the investigation on the P. halepensis / R. officinalis host pair, carried 

out at Varigotti (Girlanda et al. 2002), taxonomic affiliation through 

sequence analysis of rDNA regions has revealed a peculiar spectrum of 

taxa, distinct from those recognised to date in other hosts. In some cases, 

ITS (ITS1–5.8S–ITS2) sequence matches with sequences in GenBank were 

poor (


(Shoemaker et al. 1990), causing a root and crown rot disease of lucerne 

in Australia, characterised by symptoms that are somewhat reminiscent of 

those induced by R. vagum on cucurbit hosts (Alcorn and Irwin 1987). 

None of the DSE morphotypes investigated, therefore, is identifiable with 

any of the taxa known to date as DSE sporulating forms (such as Phialocephala 

spp., Phialophora spp., Cadophora finlandia (Wang & Wilcox) Harrington 

& McNew, Chloridium paucisporum C.J.K. Wang & H.E. Wilcox, 

Leptodontidium orchidicola Sigler & Currah; Gams 1963; Deacon 1973; 

Richard and Fortin 1973; Wang and Wilcox 1985; Currah et al. 1987; Harrington 

and McNew 2003). Among these, Phialocephala fortinii C.J.K. Wang 

& Wilcox appears as the major component of DSE assemblages in northern 

alpine and subalpine forest ecosystems, occurring with no apparent host 

specificity in North America, Europe, and Japan (Wang and Wilcox 1985; 

Currah et al. 1987; Stoyke and Currah 1991, 1993; Stoyke et al. 1992; Currah 

and Tsuneda 1993; O’Dell et al. 1993; Ahlich and Sieber 1996; Dahlberg 

et al. 1997; Hambleton and Currah 1997; Harney et al. 1997; Addy et al. 

2001; Grünig et al. 2001, 2002a, 2002b; see Chap. 7 by Sieber and Grünig). 

Although the occurrence of these taxa in the Mediterranean ecosystem we 

have studied cannot be ruled out entirely, they certainly do not appear 

among the dominant DSE in such an environment, where P. halepensis and 

R. officinalis represent the most abundant plant species. 

In the Q. ilex (holm oak) /E.arborea(Mediterranean heather) studies 

(Bergero et al. 2000, 2003), investigations were carried out at different stages 

in the evolution of the plant community at a site near Borgio Verezzi. If 

left undisturbed, Mediterranean vegetation in Northern Italy develops into 

pure Q. ilex woodland, characterised by an extremely reduced understorey 

vegetation, due to the disappearance of several plant species of earlier 

stages of succession. Later stages are dominated by ectomycorrhiza, in 

contrast to maquis and garrigue vegetation, where ectomycorrhizal plants 

often co-dominate with endomycorrhizal hosts, such as E. arborea, atypical 

ericoid Mediterranean shrub. In the climax woodland, disturbances 

such as human activities and fire, which play a key role in shaping Mediterranean 

vegetation, open the way for recolonisation by pioneer plant species, 

thus initiating secondary successions. Investigations were carried out both 

within a pure woodland where Q. ilex establishment had caused the disappearance 

of most pioneer species, including E. arborea, and in post-cutting 

clearings where the latter species had become re-established. 

Two DSE morphotypes (Sd2 and Sd9) were isolated from both holm oak 

and heather roots and characterised in detail. When tested in dual axenic 

culture trials on E. arborea, isolates assigned to these morphotypes were 

found to be able to form typical intracellular hyphal coils characteristic 

of ericoid mycorrhizal infection (see below). Morphotype Sd2 was also 

isolated from soil of the thickest part of the mature holm oak woodland


(under adult trees), using E. arborea as a bait plant. No significant ITS 

sequence identity was found in GenBank for this morphotype. However, 

based on more conserved sequences such as the 5 ′ end of 28S rDNA, it 

was found to cluster with ericoid fungi from Gaultheria shallon in a clade 

comprising Capronia species (Chaetothyriomycetidae; Allen et al. 2003). 

In contrast, morphotype Sd9 displayed significant matches over the ITS region 

with nameless fungi of Helotialean affinities represented in GenBank. 

This morphotype apparently belongs to an undefined complex within the 

Helotiales, comprising DSE, ericoid endomycorrhizal and ectomycorrhizal 

fungi from ericoid (Gaultheria shallon), epacrid (Astroloma pinifolium, 

Woollsia pungens), tree and sedge hosts (Tsuga sp., Pinus sylvestris, Betula 

pubescens, Kobresia myosuroides) from Australia, Norway, Canada and Colorado 

(Monreal et al. 1999; McLean et al. 1999; Vrålstad et al. 2002a; Bergero 

et al. 2003; and unpublished sequences). In particular, Sd9 shared high sequence 

identity of approx. 99% over ca. 440 bp with an ectomycorrhizal 

fungus from the sedge Kobresia myosuroides from Colorado (Fig. 12.1). 

Genetic relatedness between sterile mycorrhizal isolates obtained from 

ericoid and epacrid hosts in the northern and southern hemispheres, respectively, 

has been shown by several authors, in accordance with the 

current view of these plants as members of the single family Ericaceae 

(Crayn and Quinn 2000), and suggests the monophyletic origin of their 

endomycorrhizal associations (see Chap. 14 by Cairney). Such mycorrhizal 

fungi of Ericaceae from both hemispheres exhibit phylogenetic affinities to 

the Helotiales. Their precise placement within this order, however, remains 

unclear: nameless mycorrhizal fungi are either part of a Hymenoscyphus 

ericae-relatedgrouporofseparate,unspecifiedgroupsoftenreceivingpoor 

bootstrap support (McLean et al. 1999; Monreal et al. 1999; Chambers et 

al. 2000; Sharples et al. 2000; Cairney and Ashford 2002; see Chap. 14 by 

Cairney). Genetic relatedness has also been demonstrated between Hymenoscyphus 

ericae and the fungal symbiont forming Piceirhiza bicolorata 

ectomycorrhizae, belonging to a monophyletic aggregate possibly comparable 

with a generic unit (Vrålstad et al. 2000). It is thus accepted that 

◮ Fig.12.1. Neighbour-joining tree for nuclear rDNA ITS (ITS1–5.8S–ITS2) sequences of 

DSE morphotypes Sd1, Sd9, Sm1, Sm2, Sm3, Sm5 and Sm8 and other alignable sequences 

from BLAST searches. Morphotypes were isolated from Erica arborea roots (Sd1, Sd9 and 

Sm1), Quercus ilex roots (Sd1 and Sd9), pure woodland soil where Q. ilex establishment had 

caused the disappearance of E. arborea (Sd1, Sm1, Sm2, Sm5 and Sm8), soil of post-cutting 

clearings where the latter species had re-established (Sd1, Sd9, Sm1, Sm2 and Sm3), and 

were found to be able to form typical mycorrhizal hyphal coils when tested in dual axenic 

culture trials on E. arborea (Bergero et al. 2000, 2003). The Kimura-2-parameter model was 

used for pairwise distance measurement. Bootstrap values above 50% are indicated (1,000 

replicates). The bar indicates 0.1 bp changes. The tree was rooted automatically

12 DSE of Mediterranean mycorrhizal plants 213


Helotiales encompass fungi with different strategies of association with 

roots, including ecto-, ectoendo-, ericoid endo-mycorrhizal and DSE fungi 

(LoBuglio et al. 1996; Monreal et al. 1999; Sharples et al. 2000; Vrålstad et 

al. 2000, 2002a). However, subgrouping within the order, and hence the 

possible occurrence of strict ericoid-, ecto-, and DSE lineages (Read 2000), 

is still unclear, since the boundaries between intra- and inter-specific ITS 

variation are presently uncertain for these fungi. More detailed molecular 

comparisons, using sequence data from further loci, as well as other genetic 

markers are needed before relationships within this group can be resolved 

with further detail (Cairney and Ashford 2002). In the case of morphotype 

Sd9, however, identical random amplified polymorphic DNA (RAPD) profiles 

were obtained for isolates from both E. arborea and Q. ilex, indicating 

that not only the same taxon, but also the same genotype of that taxon 

is shared between an ericoid and an ectomycorrhizal host (Bergero et al. 

2000). 

Database searches using ITS sequences from Mediterranean DSEs have 

allowed comparisons over geographically distinct areas and even biomes. 

Some taxa of Helotialean affinities appear to recur across unrelated biomes, 

suggesting that such fungi find a suitable niche within root tissues, independently 

of the precise host and environment. The apparent absence 

in the Mediterranean habitats investigated of Phialocephala fortinii, the 

dominating DSE inhabitant of roots in northern temperate forests, and, 

by contrast, the presence of Rhizopycnis vagum (see above) suggests the 

existence of some environmental selection on distribution of these fungi. 

Within a given biome, while ITS data leave the question of actual host specificity 

unresolved, there is evidence from RAPD data on Mediterranean DSE 

that some DSE genotypes may actually associate with both ecto- and endomycorrhizal 

plants. Such a capacity is a prerequisite for these fungi to play 

a role in interactions between different plant hosts. 

12.3 

Ecological Relationships with Conventional Mycorrhizal 

and Pathogenic Symbionts 

The functional significance of DSE associations is variously described in 

the literature. A range of enzymatic activities has been reported for DSE, 

conferring potential ability to utilise some of the major organic detrital 

nutrient pools (Bååth and Söderström 1980; Haselwandter 1983; Currah 

and Tsuneda 1993; Fernando and Currah 1995; Caldwell et al. 2000). By 

using axenically grown Earboreaas a bait plant, we have shown that soil 

from mature Qilexforest from which the ericoid host had disappeared 

at least 10 years previously, maintains a high and diverse inoculum of


DSE fungi capable of associating with the ericaceous plant (Bergero et al. 

2003). DSE fungi have also been isolated from soil from northern temperate 

forests (Jumpponen and Trappe 1998a). However, it remains uncertain 

whether DSE fungi are endowed with “competitive saprophytic ability” 

(Garrett 1950, 1956), i.e. are actually efficient at saprotrophically decomposing 

organic debris in the complex soil environment, or whether they 

exist primarily as root associates of different plants, including ectomycorrhizal 

hosts (see Chap. 13 by Rice and Currah). Enzymatic activity may 

assist penetration into root tissues (Jumpponen and Trappe 1998a; Schulz 

et al. 2002). 

Whatever their actual free-living and saprotrophic abilities, when in association 

with host roots DSE isolates have been shown to increase host 

foliar P and N concentrations and plant biomass under some experimental 

conditions (Haselwandter and Read 1982; Fernando and Currah 1996; 

Jumpponen and Trappe 1998b; Jumpponen et al. 1998; Newsham 1999). 

These results suggest that at least some strains of DSE may have, from 

a functional point of view, a relationship with their host plants not dissimilar 

from that of conventional mycorrhizal symbionts. It should also be considered 

that variation in host response to classical mycorrhizal fungi may 

represent a continuum, ranging from parasitism to mutualism [Jumpponen 

2001; see Chaps. 16 (Brundrett) and 15 (Schulz)]. Involvement in nutrient 

and possibly water acquisition could be especially relevant in unfavourable 

environments exposed to droughts (Sengupta et al. 1989; Jumpponen et 

al. 1998). DSE are found extensively in xeric cold and warm environments 

(Read and Haselwandter 1981; Haselwandter and Read 1982; Kohn and 

Stasovski 1990; Barrow et al. 1997; Ruotsalainen et al. 2002), where they 

maybeevenmoreprevalentthanconventionalmycorrhizalfungi,colonising 

roots extensively with active structures (Barrow and Aaltonen 2001; 

Barrow and Osuna 2002; Barrow 2003). This suggests special adaptations 

to the harsh conditions of dry soil, possibly providing, when they occur 

concurrently with mycorrhizal symbionts, a back-up system during periods 

when the latter are inhibited by environmental conditions (Jumpponen 

and Trappe 1998a). 

Inoculation experiments in Earboreawith DSE isolates obtained from 

either this plant or Qilexhave shown that, irrespective of the host of origin, 

some strains are capable of forming typical intracellular hyphal coils, 

suggesting ericoid mycorrhizal behaviour (Bergero et al. 2000, 2003). Morphotype 

Sd9 isolates could also produce an unusual phenotype in the form 

of an additional hyphal net surrounding root epidermal cells (Fig. 12.2). 

While ecto- and ectoendo-mycorrhizal potential was reported for fungi of 

the DSE complex (Wilcox and Wang 1987a, 1987b; Ursic and Peterson 1997; 

Vrålstad et al. 2002b), the capacity to form endomycorrhizal structures 

such as coils had thus far not been described for the latter fungi. Such


Fig.12.2. Effects of Rhizopycnis vagum isolates of different origins on melon roots. Roots 

were rated for disease severity on the scale of Aegerter et al. (2000) [0 = no symptoms; 

1 = few lesions (covering < 10% of root), secondary root rot slight; 2 = rot of secondary 

roots or lesions covering approximately 25% of the root; 3 = lesions covering at least 50% 

of the root and dead secondary roots; 4 = general root rot, most of the root affected] after 

inoculation with R. vagum (5,000 cfu/g soil) at 25–28 ◦ C for 40–50 days. Monosporascus 

cannonballus and Acremonium cucurbitacearum, two other vine decline pathogens, were 

inoculated for comparison [30 cfu/g soil and 20,000 cfu/g soil, respectively (Aegerter et al. 

2000)]. Error bars indicate the standard deviation of the mean of ten observations in one 

growth chamber experiment. Different letters indicate differences significant at P


gin. None of the tested isolates were able to form both kinds of mycorrhizal 

symbioses (Vrålstad et al. 2002b). Similarly, Sd2 and Sd9, the two morphotypesisolatedfromtheQ. 

ilex/ E. arborea host pair in Mediterranean plant 

communities, were only able to form typical ericoid mycorrhiza in the latterhostundertheconditionstested,despitethefactthatRAPDdatafor 

the Sd9 morphotype demonstrate the presence of the same genetic individual 

in both host plants (Bergero et al. 2000). Nonetheless, ectomycorrhizal 

behaviour on suitable hosts cannot be ruled out entirely, as suggested by 

high ITS sequence identity with an ectomycorrhizal symbiont of Kobresia 

myosuroides. 

Whatever their specific structural association with the root, in nature 

the multiple association potential of DSE fungi may favour inter-plant interactions,whichcaninturnaffectthediversityanddynamicsofplant 

communities. Mycelia of DSE fungi might interlink roots of different hosts, 

which could translocate nutrients via the hyphae of such shared fungal associates, 

similar to what has been shown in situations involving mycorrhizal 

fungi (Simard et al. 1997a, 1997b). Experiments with isotope tracers are 

obviously required to confirm such a hypothesis. A second possibility, not 

necessarily implying physical integrity and continuity of mycelia colonising 

different hosts, is that the ectomycorrhizal host provides a “reservoir” for 

mycorrhizal infection of other plants. Such a role as an efficient source of 

mycorrhizal inoculum for newly establishing seedlings would be especially 

relevant in highly disturbed habitats such as occur in Mediterranean environments. 

Results of isolation experiments from mature Q. ilex woodland 

soil have established survival of ericoid mycorrhizal fungi in the absence 

of the ericoid plant, which could facilitate secondary successions. 

Further experimentation is needed to verify the actual abilities of the 

Mediterranean DSE investigated thus far both in improving host growth 

and health and in forming typical mycorrhizal structures under natural 

conditions. In nature, these fungi face a variety of abiotic conditions as 

well as interactions with complex communities of microorganisms also 

interacting with the roots. 

A different behaviour was highlighted in the Halep pine/rosemary investigation 

(Girlanda et al. 2002). Identification of a DSE morphotype as 

Rhizopycnis vagum was unexpected since this fungus had thus far only 

been known as a root pathogen involved in cucurbit crop “vine declines” 

under agricultural conditions. R. vagum-specific PCR primers designed 

towards the ITS region have been developed to assist disease diagnosis 

(Ghignone et al. 2003). Association of R. vagum with unrelated hosts such 

as cucurbit crops and wild garrigue tree and shrub plants appears to differfromsituationsinwhichcroppathogensareendophyticinweedsin 

affected fields (Sinclair and Cerkauskas 1996). Pathogenic association has 

also been reported between Phialocephala fortinii and some host plants, as


revealed by resynthesis experiments under controlled conditions (Wilcox 

and Wang 1987b; Stoyke and Currah 1993; Fernando and Currah 1996). No 

disease, however, has so far been associated with DSE colonisation under 

natural conditions. Reports for R. vagum therefore raise several questions 

about the actual ecological plasticity of this fungus. Analyses of polymorphisms 

in single and multilocus genetic markers in pathogenic and 

endophytic isolates from cucurbit, P. halepensis and R. officinalis plants 

from Italy and Northern and Central America are currently underway to 

assess host-specific and geographical genetic variation within the fungus. 

Based on disease reaction in melon roots, the pathogenicity of endophytic 

R. vagum isolates from P. halepensis and R. officinalis wasconfirmedin 

greenhouses and growth chambers under different temperature regimes. 

Melon seedling inoculation demonstrated virulence on this host, establishing 

inherent pathogenic potential on a cucurbit plant, with at least some 

endophytic Italian isolates being more aggressive, under certain experimental 

conditions, than either of two pathogenic American isolates from 

diseased cucurbits (Fig. 12.2). 

Pathogenic behaviour has also been reported for Phomopsis / Diaporthe, 

another DSE morphotype from Halep pine and rosemary. Isolates of these 

genera are among the most common of the avirulent endophytes of both the 

above-ground organs and the roots of many plants, including trees, while 

others are known as plant pathogens (although mostly from epigeous plant 

organs) with a widespread occurrence (Sutton 1980; Alexopoulos et al. 

1996). Many fungal pathogens are often identified among the large numbers 

of fungi isolated from asymptomatic stems of woody hosts (Redlin and 

Carris 1996; Saikkonen et al. 1998). Disease symptoms were not apparent 

in the Halep pine and rosemary individuals sampled for DSE isolation. Endophytic 

fungi colonising asymptomatically plant parts may also include 

pathogens that have extended latency periods before development of disease, 

which occurs under conditions that induce host stress (Saikkonen et 

al. 1998, see Chap. 1 by Schulz and Boyle). Implicit in such a concept of 

latent pathogen is virulence, referring to the ability of the fungus to cause 

a disease in the particular hosts. Inoculation experiments in Petri dishes, 

containing mineral agar, of axenically cultured P. halepensis seedlings with 

DSE isolates identifiable as Phomopsis / Diaporthe did not result in disease 

symptoms, despite of experimental conditions not especially favourable 

to the plant, and the exposure to high fungal inoculum concentrations 

(unpublished data). Although the possibility of disease development in 

P. halepensis or R. officinalis under specific abiotic or biotic environmental 

conditions cannot be ruled out entirely, an alternative possibility is actual 

loss of virulence on these hosts and an inability to breach their defence barriers, 

while retaining some capacity of penetration into root tissues, which 

permits endophytic colonisation. Fungal endophytes of epigeous parts of


both grasses and woody plants are closely related to pathogenic fungi, and 

are thought to have evolved from them via an extension of latency periods 

and a reduction of virulence (Schardl and Clay 1997; Saikkonen et al. 1998). 

DSE fungi could thus fit a definition of “saprotrophic symbionts”, living in 

close association with their hosts but confined by saprotrophy to the dead 

host tissues, to diffusates or even refractory structural components of such 

tissues, or to exudates from living host tissues, or even to organic material 

made available by other root associates (Cooke and Rayner 1984; Cooke 

and Whipps 1987). 

The results described here suggest that the outcome of the interaction 

between the same DSE fungus and different plant hosts, determining a conventional 

mycorrhizal, endophytic or even pathogenic association, may 

result from differences in fungal gene expression in response to the plant 

or differences in the ability of a plant to respond to the fungus, as has been 

suggested for other plant-fungus associations (Redman et al. 2001). This 

would fit well to a model of a finely tuned equilibrium between fungal virulence 

and plant defence characterising the fungal endophyte-plant host 

interaction, which could be described as a “balanced antagonism” (Schulz 

et al. 1999, 2002; see Chap. 1 by Schulz and Boyle). 

12.4 

Conclusions 

Molecular studies based on rDNA sequences are beginning to unravel 

the heterogeneity of the assemblage of DSE endophytes associated with 

different plants in different ecosystems. Analyses of sequence data from 

isolates from Mediterranean environments in Italy have widened the spectrum 

of DSE taxa known to associate with ecto- and endo-mycorrhizal 

hosts, pointing to broad taxonomic boundaries of these endophytes. Sequencing 

of rDNA coding regions has indicated affiliation to distant taxa 

within Ascomycota (distinct subclasses); however, taxonomic placement 

of these fungi has been achieved with varying degrees of resolution. ITSbased 

species-level identification of sterile fungi remains elusive in most 

cases, either because of matches with sequences from other unidentified 

fungi, or because of poor matches with all available sequences in the 

EMBL/GenBank/DDBJ databases. In spite of the daily increase in fungal 

sequences available in public databases, less than 1% of the estimated 

1.5 million fungal species are represented in public databases (Vilgalys 

2003), with large gaping holes for most fungal groups – Ascomycota included, 

with many families and even orders having no sequenced members 

to date. Misidentifications of named published sequences (upwards of 20% 

of the named sequences may be attributed to incorrectly named organisms;


Vilgalys 2003), and hence their unreliability, may represent another problem 

restricting feasibility of sequence-based identifications (see e.g. Bridge 

et al. 2003, 2004; Hawksworth 2004). 

Since intraspecific variation in the ITS region may attain ca. 15% in some 

fungal taxa (Egger and Sigler 1992; Seifert et al. 1995), conspecificity may 

be supposed with confidence only in cases where ITS sequence identity is 

quite high. For this reason, the amount of nucleotide divergence in single 

loci cannot in itself be used to define taxonomic rank, and phylogenetic 

species recognition relying on concordance between several independent 

gene genealogies (Taylor et al. 2000) may be a more valuable diagnostic 

tool. However, fungal non-ITS sequences of systematic value at the species 

level are scarce in databases. 

Regardless of their precise identity, however, database searches for DSE 

ITS sequences indicate that while some DSE fungi are possibly restricted 

to specific environments, in other cases the same fungus, or very closely 

related taxa, may occur in different biomes and unrelated hosts. The possibility 

that taxonomically diverse fungi have evolved not only different 

patterns of biome and host specificity, but also diverse functional significance 

for the plant is not unexpected. Data from inoculation experiments 

under semicontrolled conditions suggest that the range of DSE associates 

may vary from fungi with conventional mycorrhizal potential to fungi with 

pathogenic attributes. Findings for Mediterranean DSE isolates adds credence 

to the view of a possible continuum of mycorrhizal, endophytic or 

pathogenic root associations, depending on phylogenetic and life history 

constraints, geography, interactions with other species in the community 

and prevailing abiotic factors (Saikkonen et al. 1998; Brundrett 2002; see 

Chap. 16 by Brundrett). Assessing the ecological significance of information 

derived from axenic culture work with isolated DSE and the influences 

of plant and fungal genotypes, as well as local abiotic and biotic environments, 

on endophyte-host interactions under natural conditions is a major 

challenge for the future. 

Acknowledgements. Stefano Ghignone, Roberta Bergero, Cristina Mariani 

and Giacomo Tamietti contributed to the experimental work described 

in this chapter. Grants were provided by IPP-CNR Torino, UNITO 60%, 

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13 

13.1 

Introduction 

Oidiodendron maius: 

Saprobe in Sphagnum Peat, 

Mutualist in Ericaceous Roots? 

Adrianne V. Rice, Randolph S. Currah 

Oidiodendron maius Barron is a hyphomycete species isolated from peat, 

soil, decaying organic matter, and plant roots throughout temperate ecosystems, 

including peatlands, forests, and heathlands (e.g. Barron 1962; Nordgren 

et al. 1985; Schild et al. 1988; Nilsson et al. 1992; Hambleton et al. 1998; 

Qian et al. 1998; Lumley et al. 2001; Thormann 2001; Thormann et al. 2001, 

2004; Tsuneda et al. 2001; Rice and Currah 2002; Rice et al. 2006). In pure 

culture, colonies are white due to the presence of abundant arthroconidia 

that develop in chains at the apex of thick-walled, melanized erect conidiophores 

30–500 µm tall (Fig. 13.1a). Conidia are thin-walled, subglobose to 

elongate or irregular, 2-5x1–2.5 µm, and have an asperulate perispore (Rice 

and Currah 2001) (Fig. 13.1b). 

A sexual state is unknown but morphological characters indicate a close 

affiliation to other taxa in the Myxotrichaceae (a cleistothecial family in 

the Helotiales; Tsuneda and Currah 2004): six teleomorph species within 

the Myxotrichaceae have Oidiodendron states (Hambleton et al. 1998; Rice 

and Currah 2005) and sterile ascomata with peridial elements resembling 

those formed by species of Myxotrichum canbeinducedwhenthespeciesis 

grown on autoclaved lichen (Rice and Currah 2002) (Fig. 13.1c). Molecular 

evidence confirms the position of O. maius among other species of Oidiodendron 

and their teleomorphic counterpart, Myxotrichum (Hambleton et 

al. 1998). 

The distribution of O. maius seems to parallel that of members of the Ericaceae 

(blueberries, cranberries, rhododendrons, etc.), a family that often 

dominates the vegetation in arctic and alpine meadows, temperate heathlands, 

the understory in boreal forests and peatlands (Hambleton 1998; 

Chambers et al. 2000; Hambleton and Currah 2000), and Mediterranean 

ecosystems (Perotto et al. 1995). This shared distribution pattern may be 

Adrianne V. Rice: Northern Forestry Centre, Canadian Forest Service, Natural Resources 

Canada, 5320-122 St., Edmonton, AB, T6H 3S5 Canada, E-mail: ARice@NRCan.gc.ca 

Randolph S. Currah: Department of Biological Sciences, University of Alberta, Edmonton, 

AB, T6G 2E9, Canada 





228 A.V. Rice, R.S. Currah 

Fig.13.1. a–c Morphology of Oidiodendron maius in axenic culture. a Tall, erect, melanized 

conidiophores and small, hyaline arthroconidia of O. maius (UAMH 9749) viewed under 

light microscopy. b Chains of arthroconidia of O. maius (UAMH 8920), showing asperulate 

ornamentation visible under scanning electron microscopy. c Sterile peridial elements 

produced by O. maius (UAMH 9749) on thalli of Cladonia mitis. The cage-like peridial 

elements resemble the cleistothecia of species of Myxotrichum. Bars a 40 µm, b 5µm,c 80 µm 

based on a predilection in both plants and fungus for acidic, nutrientpoor, 

organic soils; it is also possible that O. maius has some degree of 

dependency on a mycorrhizal or at least root-endophytic relationship with 

ericaceous plants. 

Thus, two hypotheses can be advanced to explain the distribution of 

O. maius. The first hypothesis, that it is a competitive and effective saprobe 

on acidic, organic soils, is supported by its optimal growth in culture on 

acidic growth media (Rice and Currah 2001, 2005), its ability to grow and 

sporulate readily on Sphagnum plants (Rice and Currah 2002), and its 

ability to produce enzymes that degrade the cell walls of Sphagnum leaves 

in vitro (Tsuneda et al. 2001; Rice et al. 2006). The second hypothesis, that 

O. maius is a mycorrhizal endophyte of ericaceous shrubs, is supported by 

its frequent isolation from ericaceous roots (e.g. Douglas et al. 1989; Perotto 

et al. 1995, 1996; Hambleton and Currah 1997; Currah et al. 1999; Monreal

13 Saprobe and mutualist 229 

et al. 1999; Chambers et al. 2000; Johansson 2001; Usuki et al. 2003) and 

by observations that it can form typical ericoid mycorrhizal infection units 

when reinoculated on Ericaceae grown in culture (e.g. Douglas et al. 1989; 

Xiao and Berch 1995, 1999; Monreal et al. 1999). 

Since Douglas et al. (1989) described the ericoid mycorrhizas formed 

by O. maius in Rhododendron, most reports of O. maius have been from 

ericaceous plants. Its role in these associations has been considered one in 

which the plant derives some nutritional benefit (e.g. Perotto et al. 1995, 

1996; Hambleton and Currah 1997, 2000; Johanson 2001). Oddly, the benefits 

accruing to the fungus in these relationships are rarely considered, 

possibly because they are considered secondary to the needs of the plant 

but perhaps also because they are difficult to determine (Douglas and Smith 

1989). Unlike arbuscular mycorrhizal fungi and many nutritionally fastidious 

ectomycorrhizal basidiomycetes that are difficult to grow in culture 

or which require the addition of complex compounds in artificial media, 

O. maius does not display stringent demands for host-derived sugars and 

or complex growth factors, and grows readily in culture on many types of 

natural materials, including lichen thalli, Sphagnum gametophytes, context 

tissues of polypores, and on artificial growth media. In the absence of 

nutritional dependency, the benefits to the fungus in a mycorrhizal or root 

endophytic relationship are usually speculative and a number of possibilities 

could be considered. For example, the relationship may provide the 

fungus with a carbon source, growth factors, habitat, a preemptive position 

as a consumer of senescent tissue, or a competitive advantage over other 

saprobes. Alternatively, the fungus may not benefit from the association, 

with host plants exploiting their fungal partners, as in orchid mycorrhizas 

(Rasmussen 1995). In summary, there are two possible explanations for 

the distribution of O. maius: i.e. it may be a saprobe adapted to acidic 

conditions and enzymatically equipped to digest the intractable materials 

that accumulate in these areas, or it may be a type of root endophyte, possibly 

a mycorrhizal one, that requires its host plants to thrive in its habitats. 

These two suggested ecological roles are not necessarily mutually exclusive, 

i.e. the species could occupy both mycorrhizal and saprobic niches within 

suitable ecosystems. 

OnetypeofecosystemthatmaybesupportingO. maius both as a saprobe 

and a mycorrhizal associate is acidic Sphagnum peatlands, found throughout 

the circumboreal region. These peatlands include bogs and fens with 

an understory of ericaceous shrubs, including Rhododendron, Andromeda, 

and Vaccinium species, and a thick ground layer of Sphagnum spp. (e.g. 

Svensson 1995; Vitt et al. 1996; Hoosbeek et al. 2001). In Canada, many peatlands 

have a canopy of coniferous trees rooted in the Sphagnum (Vitt et al. 

1996; Piercey et al. 2002). Bogs have a dense canopy of black spruce (Picea 

mariana) while poor fens are dominated by black spruce and larch (Larix


spp.) (Vitt et al. 1996). European peatlands tend to have open canopies with 

few trees but, as in Canada, the common tree species in European peatlands 

are spruce (Picea abies) and larch (e.g. Peteet et al. 1998). Ericoid mycorrhizal 

fungi, including O. maius, have been isolated from ectomycorrhizas 

of conifers growing in such peatlands (Summerbell 1987; Schild et al. 1988; 

Perotto et al. 1995; Qian et al. 1998; Vrålstad et al. 2000); in some cases, 

O. maius was the most abundant sporulating species isolated from the roots 

(e.g. roots of sitka spruce sampled from blanket bogs; Schild et al. 1988). It 

has been proposed that these fungi may form associations with the roots 

ofconifersandericaceousshrubsinpeatlands,anddegradetheSphagnum 

matrix (Piercey et al. 2002). 

In this chapter, we first review the evidence suggesting that O. maius is 

a saprobe and then the evidence that it is an ericoid mycorrhizal fungus. 

Finally, we discuss why isolation records of this Helotialean anamorph point 

towards its simultaneous occupation of two apparently distinct niches. 

13.2 

Oidiodendron maius as a Saprobe 

From 1962 to 1989, Oidiodendron maius was known only from scattered 

records from soils and other decaying organic debris (Barron 1962; Nordgren 

et al. 1985), where it was presumed to occupy a saprobic niche, and 

from the ectomycorrhizal root tips of sitka spruce (Schild et al. 1988). Although 

Schild et al. (1988) considered O. maius a cortical parasite of spruce, 

evidence of parasitism was not presented; instead, the evidence suggested 

that O. maius inhibited root pathogens, including Phytophthora cinnamomi 

and Heterobasidium annosum. The inhibitory activity of O. maius towards 

root pathogens was also suggested by Qian et al. (1998), who found that 

O. maius was a dominant inhabitant of the ectomycorrhizal root tips of 

Norway spruce under acidified conditions. 

ThefirstreportofO. maius from the roots of ericaceous shrubs appeared 

in 1989, when it was isolated from ericoid mycorrhizal roots of Rhododendron 

(Douglas et al. 1989). Since then most records of O. maius have been 

based on isolates from presumably healthy ericaceous roots (e.g. Hambleton 

and Currah 1997; Currah et al. 1999; Monreal et al. 1999; Chambers et 

al. 2000; Usuki et al. 2003), but records from other substrates continue to 

appear (Nilsson et al. 1992; Qian et al. 1998; Lumley et al. 2001; Thormann 

et al. 2001, 2004; Rice and Currah 2002; Rice et al. 2006). The fungus is relatively 

easy to isolate from roots because it grows rapidly on artificial media, 

showing increases in colony radius of up to 1 mm/day on cornmeal agar 

(CMA) at pH 3 during periods of maximal growth (Rice and Currah 2001). 

The fungus has been shown to degrade a variety of carbon and nitrogen


sources including tannic acid (a soluble phenolic compound), cellulose, 

starch (Rice and Currah 2001, 2005; Thormann et al. 2002), chitin, pectin 

(Rice and Currah 2001, 2005), and TWEEN 20, a lipid-based detergent (Rice 

and Currah 2005). 

The presence of chitinases suggests that O. maius may obtain nutrients 

(e.g. nitrogen) from polymeric glucosamines found in insects and fungi. 

Oidiodendron maius grows luxuriantly on lichen (Rice and Currah 2002) 

and on the context tissue of the basidiocarps (polypores) of larger wood 

decay fungi (e.g. Fomitopsis pinicola). Oidiodendron maius also grows and 

sporulates abundantly on a lipid-rich growth medium with TWEEN 20. 

There are no data suggesting that O. maius is a fungal or arthropod parasite, 

but it could be a saprobe on materials rich in lipids and chitin, such as the 

remains of fungi and microfauna. 

This suite of cultural characteristics is more indicative of a saprobic 

lifestyle rather than a mycorrhizal one that relies on biotrophically derived 

photosynthates (Hutchison 1990, 1991). Alternatively, because ecto- and 

ericoid-mycorrhizal fungi have also been shown to degrade a variety of 

organic substrates (e.g. Bajwa and Read 1985; Northup et al. 1995; Bending 

and Read 1996, 1997; Aerts 2002; Leake et al. 2002; Olsson et al. 2002; 

Simard et al. 2002), these abilities may enable the absorption and transfer 

of organic or non-mineralised nutrients directly from the substrate to the 

cytoplasm of a host plant, effectively short-circuiting the mineralisation 

steps in nutrient cycling (‘organicization’) (Northup et al. 1995; Aerts 2002; 

Leake et al. 2002). In this instance, it is the host that derives benefit, and 

reciprocity for the fungus is not evident. 

The ability to sporulate in pure culture is much less common for mycorrhizal 

fungi than saprobes, with all arbuscular and most ectomycorrhizal 

fungi unable to reproduce in the absence of their hosts (Read 2002). Endorhizal 

fungi allied to the Helotiales are notable exceptions (Addy et 

al. 2005). For example, Rhizoscyphus ericae, an ericoid mycorrhizal fungus 

that also forms ectomycorrhizas (Vrålstad et al. 2000, 2002), produces 

chains of arthroconidia in culture, and in rare instances has formed discocarps 

(Hambleton et al. 1999). Unlike O. maius, R. ericae is unknown 

from non-mycorrhizal sources, although this may be due to its variable 

cultural morphology, and the concomitant difficulties in making definitive 

identification of this fungus (Hambleton and Currah 1997; Hambleton and 

Sigler 2005). 

As with other microfungi, assumptions about distribution and habitat 

preferences are biased by isolation protocols, expertise in identification, 

and by the nature and objective of the reports in which taxa are listed. 

Nevertheless, peat is a likely substrate on which to find O. maius because 

it is acidic and rich in many of the organic compounds, including tannic 

acid, cellulose, pectin, and chitin, that O. maius is able to degrade. The


first record of O. maius was from “peat soil” (Barron 1962) and subsequent 

reports of this species from peat (e.g. Nilsson et al. 1992; Thormann et al. 

2001, 2004; Rice and Currah 2002, Rice et al. 2006) remain more common 

than reports from other organic debris, such as wood (Lumley et al. 2001). 

O. maius was more abundant in ectomycorrhizal root tips of spruce in 

blanket bogs than in mineral woodland soils (Schild et al. 1988) and in 

acidified rather than limed soils (Qian et al. 1998), further supporting an 

apparent preference for acidic substrates. 

The scant isolation data from other materials is possibly the result of 

the biases mentioned above. For example, when Rice and Currah (2002) 

compared the isolation frequency of this taxon using agar media and moist 

chambering, O. maius was the most abundant sporulating species appearing 

directly on Sphagnum peat,butitwasonlyrarelyencounteredwhenthe 

same peat was placed on agar media (Rice and Currah 2002). O. maius was 

not isolated from ectomycorrhizal root tips when benomyl was added to 

the isolation medium (Schild et al. 1988). O. maius is capable of growing on 

benomyl-amended media (Rice and Currah 2002) but may not have been 

able to compete with basidiomycetes and other fungi favoured by the addition 

of this selective antifungal agent. Growth and sporulation of O. maius 

is restricted on rich artificial media, including the potato dextrose and 

malt extract agars that are commonly used to isolate fungi from substrates, 

further biasing against its recovery. 

The isolation history of O. maius coupled with in vitro studies of its 

behaviour on Sphagnum peat strongly suggest that O. maius is abundant 

in this material and may degrade large quantities of the substrate under 

natural conditions (Thormann 2001; Piercey et al. 2002; Rice and Currah 

2002, Rice et al. 2006). Three studies have shown that O. maius can cause 

significant mass losses of Sphagnum in vitro, ranging from 2–3% (Thormann 

2001) to 10–12% (Piercey et al. 2002) and from negligible to almost 

50% (Rice et al. 2006). These mass loss data may differ because intact Sphagnum 

was used by Thormann (2001) and ground Sphagnum by Piercey et 

al. (2002). Differences may also be due to the strains of O. maius used, 

as suggested by the wide range observed by Rice et al. (2006) for three 

different strains. Piercey et al. (2002) compared mass losses caused by two 

isolates of O. maius (UAMH 8919, 8920) with other ericoid mycorrhizal 

fungi (R. ericae and a non sporulating white-to-grey fungus designated 

“VWT”) from the roots of peatland and heathland Ericaceae and found 

that the strains of O. maius caused the greatest losses. Thormann (2001) 

found that the mass losses caused by O. maius (UAMH 9749) were intermediate 

among five saprobic hyphomycetes [O. maius, Acremonium cf. 

curvulum (identified later as Pochonia bulbillosa, Thormann et al. 2004), 

Penicillium thomii, O. scytaloides (= O. chlamydosporicum sensu, Rice and 

Currah 2005), and Trichoderma viride] and greater than an unidentified


basidiomycete. Tsuneda et al. (2001) used scanning electron microscopy 

to compare the ultrastructural patterns of decay of Sphagnum caused by 

O. maius and P. bulbillosa. Sphagnum cell walls are analogous to wood, 

Fig.13.2. a–e Degradation of Sphagnum fuscum leaf cell walls by Oidiodendron maius 

(UAMH 9749; Tsuneda et al. 2001). a Affected cell wall showing finely wavy deformations 

(arrows). b Severely distorted leaf cell wall (arrow). Note autolysing hypha (arrowhead)and 

degraded leaf cell wall in the immediate vicinity. H Hypha, C conidia. c Localized voids 

(arrows)andhyphae(H) emerging through the leaf cell wall. d More or less simultaneous 

degradation of the leaf cell wall by a hypha (H). Arrows Localized voids. e Enlarged view 

of an area showing the simultaneous degradation (arrow). Arrowheads Autolysing hyphae. 

H Sound, turgid hyphae. Bars a 3µm,b 20 µm, c 5µm,d 10 µm, e 2 µm. Reproduced with 

permission from Tsuneda et al. 2001


because both consist of cellulose microfibrils embedded in an amorphous 

matrix of phenolic polymers and polysaccharides (Tsuneda et al. 2001). 

Both species degraded Sphagnum leaves but decay patterns differed, with 

O. maius (UAMH 9749) eroding all cell wall components simultaneously 

(Fig. 13.2) and P. bulbillosa degrading preferentially the amorphous matrix 

material (Tsuneda et al. 2001). This pattern, analogous to the simultaneous 

white rot of wood, was confirmed in O. maius and other members of the 

Myxotrichaceae isolated from peat (Rice et al. 2006). 

These in vitro enzymatic studies, mass loss experiments, and scanning 

electron microscopic examinations indicate that O. maius has the potential 

to degrade Sphagnum peat in nature. The abundance of O. maius conidia 

and conidiophores on peat (Rice and Currah 2002), and the relatively frequent 

isolation of O. maius from this material (Barron 1962; Nilsson et al. 

1992; Thormann et al. 2001, 2004; Rice and Currah 2002; Rice et al. 2006), 

support the hypothesis that O. maius is an active component of the saprobic 

microfungal community in peatlands. 

13.3 

Ericoid Mycorrhizas 

Cronquist (1988) recognised eight families within the globally distributed 

order Ericales that are integral components of many acidic, nutrient-poor 

ecosystems with organic soils. Four families, the Ericaceae, Empetraceae, 

Monotropaceae, and Pyrolaceae, are found in the northern hemisphere 

(Cronquist 1988). Molecular evidence suggests that these families, along 

with the Epacridaceae in the southern hemisphere, should be included 

together in the Ericaceae (Kron 1996; Kron et al. 2002). Ericoid and ectendomycorrhizas 

are common within the Ericaceae but there are also reports 

of ectomycorrhizas (Largent et al. 1980; Smith et al. 1995; Horton et al. 

1999) and arbuscular mycorrhizas (Koske et al. 1990). 

Most Ericaceae are dwarf shrubs adapted to harsh ecosystems including 

bogs, heaths, alpine and arctic regions, and boreal forests (Hambleton 

1998). These woody plants have leathery, perennial leaves that minimise 

nutrient loss. Ericoid mycorrhizal associations may enhance the success 

of host plants in nutrient-poor, acidic, phenol-rich, and heavy metalcontaminated 

soils (Perotto et al. 1995; Hambleton 1998; Hambleton and 

Currah 2000). The below-ground network consists of well-developed mats 

of rhizomes and “hair roots” that form in the surface layers of organic soil 

(Read 1991). “Hair roots” have a narrow stele surrounded by an endodermis 

and one to two layers of cells, representing the cortex and/or epidermis 

(Read 1991; Smith and Read 1997). Hyphae penetrate the cell walls of the 

outer cell layers, form an interface with cell membranes (Read 1991; Smith


Fig.13.3. a,b Oidiodendron maius (S. Hambleton personal collection, S-272a) colonising 

the roots of Vaccinium vitis-idaea in resynthesis studies (S. Hambleton, unpublished). 

a Longitudinal section of mycorrhizal root showing hyphal complexes formed in the outer 

layerofrootcells(arrow). b Close up of hyphal complex (arrow) formed in the root cell. 

Bars 10 µm. Images provided by Sarah Hambleton 

and Read 1997), and develop into complexes made up of densely intertwined, 

thin, lightly pigmented hyphae (Fig. 13.3). Hyphae also extend out 

of the root and absorb nutrients by decomposing organic matter; some 

of these nutrients, at least, are then supplied to the plant (Northup et al. 

1995; Smith and Read 1997). Nutrient and carbon exchange is believed 

to occur across the interfaces between plant cell membranes and hyphal 

complexes for about 5 weeks until both the plant cell cytoplasm and the 

fungal hyphae within the cell degenerate (Read 1991; Smith and Read 1997). 

Ericoid mycorrhizal fungi have also been shown to break down phenolic 

compounds and sequester heavy metal ions, detoxifying the soil for their 

plant partner (Read 1991; Perotto et al. 1995; Smith and Read 1997; Yang and 

Goulart 2000). While the benefits to the host plant are readily demonstrated 

in resynthesis studies, the benefits to the mycobiont (fungal partner) are 

more difficult to measure; but it is assumed that the mycobiont receives 

photosynthates from the host plant (Smith and Read 1997). 

Identification of mycorrhizal fungal symbionts requires isolation and 

identification of the fungi in culture, followed by resynthesis of the association 

(Smith and Read 1997; Hambleton 1998). Symbiont identification has 

relied upon morphological and cultural characteristics of the fungi; these 

sources of characters work well in conjunction with DNA fingerprinting 

and sequence techniques (e.g. Gardes et al. 1991; Simon et al. 1992; Egger 

1995; Clapp et al. 2002; Erland and Taylor 2002; Allen et al. 2003). The 

first fungi confirmed, through resynthesis experiments, to form ericoid 

mycorrhizas did not sporulate and hence were unidentifiable (Doak 1928; 

Bain 1937; Gordon 1937; McNabb 1961). In 1973, Pearson and Read reported 

that some of their ericoid mycorrhizal isolates produced zigzag chains of


arthroconidia in culture and one produced small apothecia in pure culture 

and in pots containing Calluna vulgaris. The conidial fungus was later 

named Scytalidium vaccinii (Dalpé et al. 1989) and the apothecial fungus 

was named Pezizella ericae (Read 1974). This species was transferred to 

Hymnenoscyphus (Kernan and Finocchio 1983) and recently to Rhizoscyphus,asR. 

ericae (Zhang and Zhuang 2004). Scytalidium vaccinii has been 

confirmed as the anamorph of R. ericae (Egger and Sigler 1993; Hambleton 

1998; Hambleton et al. 1999). Hambleton and Currah (1997) described 

a series of isolates under “variable white taxon” (VWT) that were common 

endophytes in ericaceous roots. This taxon was shown to have marked 

affinities to R. ericae but produced neither a teleomorph nor conidia in culture. 

Recently, three phylogenetically distinct species in the VWT complex 

have been recognised in a new anamorphic genus (Hambleton and Sigler 

2005). Comparison of fungi detected in the roots of Gaultheria shallon using 

culturing and molecular (DNA) methods revealed that an abundance of 

hyphae within hair roots belonged to an unculturable species of Sebacina 

(Allen et al. 2003). Fungi that were culturable included R. ericae and an 

isolate tentatively identified as a species of Capronia (Allen et al. 2003). As 

detection techniques and identification protocols improve, it is expected 

that additional fungal taxa will be described as ericoid mycorrhizal endophytes. 

Species of Oidiodendron other than O. maius have also been reported 

from ericoid mycorrhizas (Pearson and Read 1973; Couture et al. 1983; 

Dalpé 1986, 1989, 1991, Douglas et al. 1989; Xiao and Berch 1992; Currah et 

al. 1993, 1999; Johansson 1994, 2001; Perotto et al. 1995, 1996; Hambleton 

and Currah 1997; Monreal et al. 1999; Chambers et al. 2000; Usuki et 

al. 2003). While many of the early reports implicated O. griseum in the 

associations, DNA analyses indicate that most, if not all, of these reports 

werebasedonmisidentifiedstrainsofO. maius (Hambleton and Currah 

1997; Hambleton et al. 1998). 

Mycorrhizal resyntheses between host plants and fungi isolated from 

their ericoid mycorrhizas are generally assumed to be the definitive indicator 

that a fungus is mycorrhizal, but assumptions based on these 

data are tenuous at best. The Ericaceae is particularly problematic in 

this regard because, in axenic culture situations at least, the family appears 

to permit a wide range of fungi into the peripheral cells of hair 

roots where they form the typical coiled “infection units”. For example, 

Dalpé (1986, 1989, 1991) found that blueberries (Vaccinium angustifolium) 

would form ericoid mycorrhizas with Myxotrichum setosum, O. cerealis, 

O. chlamydosporicum, O. citrinum, O. flavum, O. griseum, O. periconioides, 

O. rhodogenum, O. scytaloides, Pseudogymnoascus roseus (all members of 

the Myxotrichaceae, Leotiomycetes) as well as the unrelated species Gymnascella 

dankalienses (a member of the Gymnoascaceae, Eurotiomycetes).


Salal (Gaultheria shallon) has formed in vitro mycorrhizal associations with 

Acremonium strictum, a fungus that was able to supply organic nitrogen 

and enhance host plant growth (Xiao and Berch 1999). Ericaceous plants 

may “prefer” some fungi over others in the field but when constrained, as 

in a culture situation, may form mycorrhizal relationships with, or exploit, 

a range of different fungal species. 

13.4 

Oidiodendron maius as an Ericoid Mycorrhizal Fungus 

While the widespread isolation of O. maius from the roots of ericaceous 

roots supports the hypothesis that it may be mycorrhizal and as such, 

either a mutualist or a commensalist, it does not confirm the nature of 

the association. Many resynthesis studies have attempted to assess the 

morphological and functional aspects of the relationship; these studies have 

used a range of ericaceous shrubs and have reported positive, neutral, and 

negative effects on host plant growth (Douglas et al. 1989; Xiao and Berch 

1995, 1999; Yang et al. 1998; Monreal et al. 1999; Bergero et al. 2000; Yang 

and Goulart 2000; Johansson 2001; Starrett et al. 2001; Piercey et al. 2002; 

Yang et al. 2002) using a range of Oidiodendron species (Couture et al. 1983; 

Dalpé 1986, 1989, 1991; Currah et al. 1993; Xiao and Berch 1995; Monreal 

et al. 1999). Characteristic hyphal complexes have been observed in roots 

of various ericaceous shrubs, including Vaccinium vitis-idaea, colonized 

by Oidiodendron species (Fig. 13.3) (Dalpé 1986, 1989, 1991; Douglas et 

al. 1989; Xiao and Berch 1995; Johansson 2001; S. Hambleton, personal 

communication). While the plants in these studies appeared healthy, the 

functional nature of the relationship between the fungi and the hosts was 

not determined. 

Physiological evidence to support the mycorrhizal nature of the association 

between O. maius and ericaceous plants has been obtained from 

resynthesis studies. Yang et al. (1998) found that O. maius (UAMH 9263) 

did not affect the growth of blueberries (Vaccinium corymbosum), but later 

studies (Yang and Goulart 2000; Yang et al. 2002) on the same isolate of 

O. maius and the same plant species found positive effects of the fungus 

on plant growth, indicating that the nature of the relationship between 

O. maius and ericaceous shrubs may vary within fungal strains. Inoculation 

of salal (Gaultheria shallon) withfourisolatesofO. maius increased 

plant biomass regardless of the nitrogen source supplied (Xiao and Berch 

1999). Inoculation with O. maius increased blueberry root and shoot dry 

mass as well as plant access to organic nitrogen (Yang et al. 2000). O. maius 

has also been shown to reduce aluminum uptake and increase the cation 

exchange capacity of blueberry (Yang and Goulart 2000).


In contrast, several resynthesis studies using O. maius (Dalpé 1991; Bergero 

et al. 2000; Piercey et al. 2002) have not resulted in the formation of 

distinctive infection units. These results may be due to the strong saprobic 

abilities of O. maius, strain specific effects, or to the sources of nutrients 

available to the plants. In these axenic culture systems, O. maius might have 

acquired sufficient carbon from the substrate (Sphagnum peat in the study 

by Piercey et al. 2002) and did not need the host plant for nutrition (Piercey 

et al. 2002). Other resynthesis studies have shown either neutral (Yang et 

al. 1998) or negative effects on the plants (Starrett et al. 2001). Starrett et al. 

(2001) inoculated microshoots of mountain andromeda (Pieris floribunda) 

with the “ericoid mycorrhizal fungi” R. ericae, O. maius (ATCC 66504), 

O. griseum and an unidentified species of Oidiodendron, and found that inoculation 

with all of these species caused shoot necrosis, but that this effect 

could be reduced by providing an alternative carbon source. Mitigation of 

shoot necrosis varied with carbon source and fungal isolate. Adding sucrose 

to the medium prevented R. ericae,butnottheOidiodendron species, from 

causing shoot necrosis while adding a peat-vermiculite mixture reduced 

the shoot necrosis caused by the Oidiodendron species. Additionally, the 

Oidiodendron species did not induce root formation by the microshoots to 

the same extent as R. ericae, leading Starrett et al. (2001) to conclude that 

the Oidiodendron species were not mycorrhizal symbionts. 

None of the preceding studies investigated possible benefits to the 

mycobiont, so the mutualistic nature of the association has never been 

demonstrated. It is possible that the relationship is physiologically similar 

to the dynamics in orchid mycorrhizas in which the orchid “exploits” 

its saprobic or ectomycorrhizal mycobiont (e.g. Rasmussen 1995; 

McKendrick et al. 2000) or to the epiparasitic relationship of monotropes 

(non-photosynthetic Ericaceae) to ectomycorrhizal trees via their shared 

ectomycorrhizal fungi (Bidartondo et al. 2000). Many Ericaceae, similar to 

the Orchidaceae and the monotropes, are microspermous (e.g. Rhododendron, 

Menziesia). Perhaps the adoption of microspermy is a consequence of 

the host plants’ ability to exploit fungi as a source of nutrients, and ericoid 

mycorrhizal associations may be a part of a mycoheterotrophic evolutionary 

trajectory. The discovery of Sebacina, a basidiomycete genus known to 

form orchid mycorrhizas (Currah et al. 1990; McKendrick et al. 2002), in 

ericoid mycorrhizal roots of Salal (Allen et al. 2003) is another similarity 

between ericoid and orchid mycorrhizal systems. 

The nature of the relationship between O. maius and members of the 

Ericaceae is clearly complex and varies from one report to the next. Future 

research involving physiological assessment in laboratory, greenhouse, and 

field conditions coupled with morphological and molecular assessment in 

roots (Hambleton and Currah 2000) is required to identify and quantify 

possible benefits to both partners and to elucidate the factors determining


the functional aspects of the relationship. For example, tracing the movement 

of radiolabeled carbon and nutrients between the partners could help 

determine whether O. maius obtains host photosynthate and if the plant 

receives any fungal-derived carbon or other nutrients. Additionally, in vitro 

studies could determine other potential benefits to either partner, such as 

competitive and growth advantages for O. maius and pathogen resistance 

for the plant. The potential role of ericoid mycorrhizal fungi in symbiotic 

seed germination should also be examined. 

13.5 

Significance and Relevance 

Theoccupationofmultiplenicheswithinagivenenvironmentcouldconfer 

survival and competitive advantages on O. maius by providing different 

refuges to the fungus. During periods of host plant dormancy, O. maius 

could thrive as a saprobe in the peat matrix, while host plant roots may 

serve as a refuge from competition with other saprobes and as a source of 

inoculum for colonisation of senescing surface peat. On the other hand, 

the prevalence of O. maius as a saprobe within the peat could ensure 

rapid colonisation of new ericaceous roots, perhaps at the expense of other 

species that are less able to degrade the surrounding substrate. While there 

is currently no experimental data to support either hypothesis (i.e. whether 

roots or peat serve as primary refugia for O. maius), testing both could 

provide information key to understanding of the ecological role of this 

species. 

Ericoid and ectomycorrhizal fungi can degrade a variety of complex 

organic substrates (Bajwa and Read 1985; Read 1991; Northup et al. 1995; 

Bending and Read 1996, 1997; Smith and Read 1997; Aerts 2002; Leake et al. 

2002; Olsson et al. 2002; Simard et al. 2002), with the abilities of ericoid mycorrhizal 

fungi possibly exceeding those of ectomycorrhizal fungi (Bajwa 

and Read 1985; Read 1991; Bending and Read 1996, 1997; Smith and Read 

1997). These abilities are thought to aid in host plant nutrition by allowing 

the plant direct access to organic nutrient sources (Bajwa and Read 1985; 

Xiao and Berch 1999; Yang et al. 2002). Inorganic sources of nitrogen are 

scarce and organic sources relatively abundant when decomposition is slow, 

as in peatlands and heathlands, and when leaching is common (Perotto et 

al. 1995). Ericoid mycorrhizal fungi often produce phosphatase, enabling 

them to access and transfer organic phosphorus to their hosts (Aerts 2002). 

Ericaceous shrubs in these environments may rely on their mycorrhizal 

partners to supply them with sufficient carbon (Yang et al. 2002) and nutrients 

obtained from the organic sources (Northup et al. 1995; Xiao and Berch 

1999; Yang et al. 2002). The abilities of ericoid mycorrhizal fungi, including


O. maius, to access carbon and nutrients from organic debris could reduce 

their reliance on host plant photosynthates for carbon (Piercey et al. 2002), 

while still supplying the host plant with nutrients. 

Transfer of nutrients from organic matter to ericaceous shrubs via ericoid 

mycorrhizal fungi, such as O. maius, has important implications for 

nutrient cycling in ecosystems such as peatlands, where decomposition is 

slow and carbon and nutrients are sequestered in organic debris (Northup 

et al. 1995). Sphagnum decomposes slowly with relatively few fungi having 

the ability to cause significant mass losses (Thormann 2001). Decomposition 

of Sphagnum by O. maius may release a significant amount of carbon 

and nutrients from the peat and, instead of releasing carbon into the atmosphere 

and nitrogen and phosphorus into the pool of plant-available 

nutrients, these organic forms of nitrogen and phosphorus can be supplied 

directly to the ericaceous shrubs, giving them a competitive advantage over 

their neighboring plants (Aerts 2002; Leake et al. 2002). Short-circuiting 

nutrient cycles, by reducing the amount of decomposition required before 

nutrient absorption, results in increased supplies of nitrogen and phosphorus 

to mycorrhizal host plants and a resultant decrease in nitrogen and 

phosphorus available to saprobes and non-mycorrhizal plants (Leake et al. 

2002). 

Other Oidiodendron species are able to form ericoid mycorrhizal associations 

in vitro (Dalpé 1986, 1989, 1991; Currah et al. 1993). Species 

of Oidiodendron are the asexual states of myxotrichoid ascomycetes, and 

some of these, e.g. Pseudogymnoascus roseus, and other anamorphs, including 

species of Geomyces, also produce ericoid mycorrhizal associations in 

vitro (Dalpé 1989). However, only two species of Oidiodendron have been 

reported from ericaceous roots in situ. Currah et al. (1993) isolated O. periconioides 

from Rhododendron brachycarpum growninpotculturescontaining 

peat. The remaining species are known only as saprobes but since 

they share morphological characters, including dendritic arthroconidia 

and cage-like cleistothecial ascomata, and ecological characters, including 

the ability to degrade a variety of plant-based polymers and a predilection 

for cool, acidic conditions (Rice and Currah 2005), it is possible that these 

taxa could play biologically similar and significant roles to O. maius in cool, 

acidic soils. Additional surveys of ericoid mycorrhizal endophytes should 

employ a broad range of isolation and detection protocols to maximise the 

recovery of as wide a variety of fungi as possible. 

The occupation of multiple niches by O. maius may parallel the situation 

observed for Phialocephala fortinii. Usuallyconsideredarootendophyte, 

P. fortinii is isolated most frequently from healthy roots of woody plants [e.g. 

see Chaps. 7 (Sieber and Grünig) and 15 (Schulz); Stoyke and Currah 1991; 

Menkis et al. 2004; Piercey et al. 2004] and, until recently, was unknown as 

a saprobe. However, Menkis et al. (2004) isolated P. fortinii from healthy


wood in pine stems suggesting a possible role as a systemic endophyte, and 

inbirchsnagsandbirchandpinestumps,whereitispresumablyoccupying 

a saprobic niche (Menkis et al. 2004), perhaps as an agent of soft rot (Sieber 

2002; Menkis et al. 2004). Given the scattered and somewhat incidental 

reports of O. maius from wood and soil, a concerted and wider search for 

O. maius may reveal habitats in addition to ericaceous roots, where this 

speciesisabundant. 

13.6 

Conclusions 

Oidiodendron maius formsassociationswiththerootsofericaceousshrubs, 

though the nature of the relationship remains uncertain. Is it a mutualistic 

mycorrhizal association, a preemptively colonised refugium for the fungus, 

a case of parasitism of the fungus by the plant, or some combination of 

the three? In vitro studies indicate that O. maius can improve host plant 

growth both by aiding plant nutrition and detoxifying the soil environment, 

although the benefits to O. maius are unclear. It remains necessary 

to investigate the benefits to both partners and demonstrate what environmental 

conditions determine the functional nature of the relationship. 

O. maius has the potential to degrade complex organic polymers within 

the soil, thus it is unlikely that it would rely on host photosynthate for 

survival. However, it is possible that O. maius receives some photosynthate, 

which could supplement saprobically derived carbon, potentially giving 

O. maius a competitive advantage over other soil fungi. The tendency towards 

microspermy in the Ericaceae and the saprobic abilities of ericoid 

endophytes suggests that ericoid mycorrhizal associations may represent 

another example of controlled parasitism of a fungal partner by the host 

plant, similar to the type that occurs with orchids. Entrapment of O. maius 

could confer a competitive advantage on the host plants by increasing the 

supply of organically bound nutrients-unavailable to plants that lack ericoid 

mycorrhizas, and by supplementing host plant photosynthesis with 

fungal-derived carbon. Given the wide taxonomic tolerance that ericaceous 

plants have for root endophytic fungi in vitro, the obvious need for more 

detailed studies of endophytic diversity of fungi growing in plants in situ, 

the enigmatic ecological roles of related Helotialean fungi (e.g. P. fortinii, 

Geomyces, etc.), much more exploratory and empirical research is needed 

before we will be able to answer the question posed at the outset of this 

chapter.


Acknowledgements. The authors thank S. Hambleton and A. Tsuneda for 

providing images. Comments on previous versions of this manuscript by 

H.D. Addy and the continuing support of the Natural Sciences and Engineering 

Research Council of Canada are gratefully acknowledged. 


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14 

14.1 

Introduction 

Mycorrhizal and Endophytic Fungi 

of Epacrids (Ericaceae) 

John W.G. Cairney 

Epacrids are a group of over 450 species of woody plants that were conventionally 

classified as the family Epacridaceae (Powell et al. 1996). Detailed 

phylogenetic analyses based on morphological and molecular data indicate 

that epacrids represent a lineage within Ericaceae, with a recent classification 

regarding epacrids as Styphelioideae, one of eight Ericaceae subfamilies 

(Kron et al. 2002). Although epacrids occur in several southern hemisphere 

locations, including New Zealand, south east Asia, Pacific Ocean 

Islands and Patagonia, they are primarily an Australian group (Copeland 

1954; Powell et al. 1996). Species richness is greatest in Western Australia 

(WA), with some 181 named species occurring in this state, 98% of which 

are endemic to WA (Keighery 1996). Seven epacrid tribes are currently 

recognised (Kron et al. 2002), most of which comprise small-to-medium 

sized shrubby heath-like plants. Some Richeeae taxa, however, resemble 

large arborescent monocots (Allaway 1996). Epacrids occupy a range of 

habitats that includes dry sandy heathlands and sclerophyll forests, along 

with wet alpine bogs and Magellanic tundra (Read 1996). They generally 

occur in acidic or neutral soils, but are occasionally found in more basic 

soils (Keighery 1996). Despite being geographically and hydrologically 

disparate, these habitats characteristically encompass soils in which availability 

of mineral nutrients is relatively poor (Read 1996). 

In common with many other Ericaceae taxa, epacrids produce extremely 

finelateralrootsknownashairroots.Uptothreeordersofhairrootsmaybe 

present, and their structure is broadly similar in all taxa: a stele, surrounded 

by two layers of suberised cortical cells and an epidermis. Hair roots lack 

root hairs, but the surface is generally covered by a mucilage layer (Cairney 

and Ashford 2002). When collected from the field, a proportion of the 

epidermal cells of epacrid hair roots invariably contains hyphal structures 

that are morphologically similar to those produced by ericoid mycorrhizal 

John W.G. Cairney: Centre for Plant and Food Sciences, Parramatta Campus, University 

of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia, E-mail: 

j.cairney@usw.edu.au 





248 J.W.G. Cairney 

fungi in roots of other Ericaceae subfamilies (Reed 1989, 1996). Up to 90% 

of the hair root length can display this type of colonisation (Davies et al. 

2003), but in most cases considerably less root length is colonised and there 

is evidence that colonisation varies on a seasonal basis (Reed 1996; Hutton 

et al. 1994; Davies et al. 2003; Kemp et al. 2003). 

Detailed analysis of mycorrhiza-like infections of epacrid hair roots generally 

reveals longitudinally oriented surface hyphae from which perpendicular 

branches arise. Penetration of individual epidermal cells is generally 

by a single hypha, sometimes involving an appressorium, and is followed 

by development of a hyphal coil in the periplasmic space (Cairney and Ashford 

2002). These features, together with the fact that the fungi are generally 

ascomycetes, as evidenced by the presence of simple septa and Woronin 

bodies (Allen et al. 1989; Steinke et al. 1996; Briggs and Ashford 2001), are 

shared with ericoid mycorrhizal infection of other Ericaeae, and are generally 

taken to indicate mycorrhizal infection. Aside from putatively ericoid 

mycorrhizal ascomycetes, arbuscular mycorrhiza-like infections, suggesting 

the presence of Glomales taxa, have also been observed as apparent 

endophytes of field-collected epacrid hair roots (Khan 1978; McGee 1986; 

Bellgard 1991; McLean and Lawrie 1996; Reed 1996; Davies et al. 2003), 

and there is a single report of basidiomycete hyphae in epidermal cells of 

an epacrid (Allen et al. 1989). As emphasised by Reed (1996), however, the 

presence of infection does not necessarily indicate a mutualistic association. 

In the case of apparent arbuscular mycorrhizal infection at least, it 

seems most plausible that this reflects opportunistic infection by arbuscular 

mycorrhizal fungal hyphae that grew from neighbouring non-Ericaceae 

hosts (Reed 1996). 

14.2 

Endophytes of Epacrid Roots 

To date, only some of the endophytes obtained from Australian epacrid 

roots have been tested for their abilities to form ericoid mycorrhizas with 

host plants and, where this has been conducted, mycorrhizal status has 

been inferred simply from production of mycorrhiza-like coils in epidermal 

cells. For this reason these endophytes are referred to as putative ericoid 

mycorrhizal fungi throughout this chapter. 

Many endophytes have been isolated from surface-sterilised epacrid 

roots into axenic agar culture. These are typically isolated as sterile mycelia, 

rendering their classification on the basis of morphology difficult. Nonetheless, 

studies that grouped isolates on the basis of gross morphological 

characteristics of cultured mycelia and/or by pectic zymogram patterns 

established that multiple ascomycete taxa are likely to form ericoid mycor-


rhizal associations with epacrids in their native habitats (Reed 1989; Hutton 

et al. 1994, 1996b; Steinke et al. 1996). Subsequent molecular analyses of the 

isolated endophytes suggest that they are broadly similar to those obtained 

from other Ericaceae taxa in northern hemisphere habitats. 

To date, molecular analysis of endophytes from epacrid roots has concentrated 

largely on the rDNA internal transcribed spacer (ITS) region. 

Assemblages of isolated endophytes have thus been grouped on the basis 

of gross morphological characteristics of cultures (McLean et al. 1999; 

Chambers et al. 2000) or as ITS-restriction fragment length polymorphism 

(RFLP) groups (Midgley et al. 2002, 2004a) and ITS sequences obtained 

for representative isolates of each morphological or RFLP group. Comparison 

of the ITS sequences with those available in the GenBank nucleotide 

database has confirmed that most endophytes isolated from epacrid roots 

are ascomycetes or their anamorphs. Indeed, there is only a single published 

account of isolation of a fungus from epacrid hair roots that appears, 

on the basis of ITS sequence identity, to be a basidiomycete (Midgley 

et al. 2004a). This isolate did not, however, form ericoid mycorrhiza in 

gnotobiotic culture with Woollsia pungens (see below) and seems most 

likely to represent a facultative root inhabitant. Recent analysis of epacrid 

endophytes by direct DNA extraction from hair roots has revealed the 

presence of other putative basidiomycetes. These, however, appear to be 

relatively uncommon and their mycorrhizal status is unclear (D. Bougoure 

and J.W.G. Cairney, unpublished data). 

ITS sequence data suggest that many of the endophytes are Helotiales 

ascomycetes, several of which have affinities with the Hymenoscyphus ericae 

aggregate (McLean et al. 1999; Chambers et al. 2000; Sharples et al. 

2000; Cairney and Ashford 2002). This aggregate encompasses H. ericae, 

Phialophora finlandia and related taxa, many of which form ericoid mycorrhiza 

with northern hemisphere Ericaceae (Vrålstad et al. 2002). These isolateshavebeenobtainedfromepacridsatalpine,drysclerophyllforest,sand 

mine and coastal heathland sites, indicating that they have a widespread 

distribution in a variety of Australian habitats (McLean et al. 1998, 1999; 

Midgley et al. 2002). Many of the other endophytes are part of a poorly 

defined Helotiales ascomycete group that probably incorporates a number 

of taxa and includes many ericoid mycorrhizal and other root-associated 

fungi from northern hemisphere Ericaceae (McLean et al. 1999; Chambers 

et al. 2000; Sharples et al. 2000; Cairney and Ashford 2002; Berch et al. 2002; 

Midgley et al. 2002, 2004a). Other isolates had closest ITS sequence similarity 

to Capronia (Chaetothyriales) and Thielavia (Sordariales) (Midgley 

et al. 2002, 2004a), and, while some of these are also endophytes of nonericaceae 

hosts, similar isolates are known to form ericoid mycorrhiza with 

Gaultheria shallon in Canada (Berch et al. 2002). Oidiodendron spp. are 

widespread ericoid mycorrhizal fungi of northern hemisphere Ericaceae


(Perotto et al. 2002; see Chap. 13 by Rice and Currah). Endophytes with 

strong ITS sequence similarity to O. maius have recently been isolated, and 

may represent common endophytes of certain epacrids (Chambers et al. 

2000; D. Bougoure and J.W.G. Cairney, unpublished data). 

In addition to the putative ericoid mycorrhizal fungi, many fungi isolated 

from surface-sterilised epacrid roots show closest ITS sequence matches to 

fungi that are not known to be mycorrhizal fungi. These include isolates 

that have closest sequence matches to taxa that are regarded as saprotrophic 

or pathogenic, but also with dark septate endophytes (DSE) (Midgley et 

al. 2002, 2004a; Davies et al. 2003; D. Bougoure and J.W.G. Cairney, unpublished 

data). Isolates with close sequence similarity to Phialocephala 

fortinii and other DSE taxa, although occasionally obtained from lower elevation 

habitats, have been obtained primarily from epacrids in Australian 

sub-alpine and alpine habitats (Davies et al. 2003; Midgley et al. 2004a). 

Furthermore, Davies et al. (2003) found that infection of some epacrid 

roots by DSE at alpine sites can be more prevalent than ericoid mycorrhizal 

infection. Since only limited sampling has so far been undertaken in alpine 

habitats, and systematic comparisons of endophyte abundance in different 

habitats have yet to be conducted, the ecological significance of these 

observations is difficult to assess. 

These observations are, however, based on fungi that have been isolated 

from epacrid hair roots and assume that all endophytic fungi are isolated 

with equal efficiency, or indeed are isolated at all. It is possible, for example, 

that fast-growing endophytes may mask slower growing endophytes that 

are present in the same root piece, resulting in only the faster-growing taxa 

being isolated (Hambleton and Currah 1997). Endophytes that are difficult 

or impossible to culture may also be present in epacrid hair roots. This 

may be the case in the North American Ericaeae taxon Gaultheria shallon, 

for which direct DNA extraction and subsequent cloning of ITS sequences 

revealed the presence of a Sebacina-like basidiomycete that was not present 

in cultured assemblages of fungi from the same roots (Berch et al. 2002; 

Allen et al. 2003). Furthermore, these sequences were obtained from root 

segments that appeared to have mycorrhizal infection in epidermal cells, 

yet did not yield culturable endophytes. In contrast, where culturable endophyte 

assemblages from the Epacris pulchella hair roots were compared 

to fungal sequences obtained following direct DNA extraction from roots, 

the most-commonly isolated fungi were also commonly represented in 

the directly extracted DNA (D. Bougoure and J.W.G. Cairney, unpublished 

data). This clearly suggests that in the case of these E. pulchella plants the 

most common endophytes were culturable and that the observations from 

G. shallon do not serve as a paradigm for Ericaceae in general.


14.3 

Diversity and Spatial Distribution 

of Endophyte Taxa in Epacrid Root Systems 

Wheremultipleisolationshavebeenmadefromrootsystemsofindividual 

epacrids in the field, an assemblage of endophyte taxa has invariably 

been recovered (McLean et al. 1999; Chambers et al. 2000). Midgley et al. 

(2002, 2004a) undertook intensive isolations from 5.0 mm long root pieces 

from throughout the hair root systems of two Woollsia pungens plants and 

a Leucopogon parviflorus plant at a sclerophyll forest site in temperate eastern 

Australia. Between 99 and 227 isolates were obtained from each plant 

and ITS-RFLP analysis suggested that the assemblage from each plant comprised 

5 to 17 taxa. Most isolates (76–85%) in each assemblage from a single 

plant, however, were of the same ITS-RFLP-type, with a single RFLP-type 

found to dominate the root system of the L. parviflorus and a neighbouring 

W. pungens plant. Mapping the distribution of the RFLP types according to 

their position in the root systems from which they were isolated, demonstrated 

that the isolates that dominated the assemblages were widespread 

throughout the hair root systems (Midgley et al. 2002, 2004a). These dominant 

RFLP-types were shown to form typical ericoid mycorrhiza-like coil 

structures in gnotobiotic mycorrhizal infection experiments with W. pungens 

as host (Midgley 2003; Midgley et al. 2004a), suggesting that each 

root system was dominated by a single putative ericoid mycorrhizal fungal 

taxon. Only a few other RFLP-types from each root system formed 

mycorrhiza-like coils in W. pungens hair roots, with the remainder failing 

to form typical mycorrhizal structures in epidermal cells. 

Genetic diversity in populations of the dominant putative ericoid mycorrhizal 

fungal taxa isolated from epacrid roots has been investigated using 

inter-simple sequence repeat (ISSR) PCR (Midgley et al. 2002, 2004a). These 

investigations revealed that, in the case of W. pungens and L. parviflorus 

in a dry sclerophyll forest habitat, the population of the dominant taxon 

within a root system comprised three to six genotypes. The population 

from each root system was, however, dominated (81–96% of isolates) by 

a single, spatially widespread genotype of the most abundant putative mycorrhizal 

taxon, with the remaining genotypes being relatively uncommon. 

In the neighbouring W. pungens and L. parviflorus plants dominated by 

the same putative ericoid mycorrhizal fungal taxon, each root system was 

dominated by a different genotype of that taxon (Midgey et al. 2004a). The 

existence of genotypes that are widespread within root systems indicates 

that there is a high probability that isolates from individual root segments 

in the same root system will be from the same mycelial individual. This will 

complicate investigations of the community ecology of ericoid mycorrhizal


fungi, particularly where comparisons of relative abundance of taxa between 

sites is concerned, and may ultimately inform the spatial scale at 

which future sampling should be conducted. 

As proposed for populations of the DSE Phialocephala fortinii associated 

with roots in a conifer stand, domination of the root system by a single 

genotype might result from early establishment and/or greater competitiveness 

of the dominant genotype (Grünig et al. 2002). In either case, local 

micro-scale edaphic conditions are likely to play a strong selective role in 

structuring the endophyte communities and populations. Putative mycorrhizal 

fungi from epacrids vary in their responses to water stress (Hutton et 

al. 1996b; Chen et al. 2003). Furthermore, the extent of ericoid mycorrhizalike 

hair root colonisation varies seasonally for W. pungens in eastern Australian 

sclerophyll forests (Kemp et al. 2003). Indeed, in epacrids that are 

subject to a Mediterranean-type climate in south-western Australia, few 

hair roots survive the summer and apparent ericoid mycorrhizal infection 

disappears during the driest months. Infection then increases progressively 

with decreasing temperature and increasing rainfall (Hutton et al. 1994). 

Although no investigations have so far been conducted, it is thus possible 

that the relative abundance of different taxa and/or genotypes within root 

systems will be found to show considerable seasonal variation. 

The same genotype of a single putative ericoid mycorrhizal fungal taxon 

was found to be present in the root systems of the neighbouring W. pungens 

and L. parviflorus plants (Midgey et al. 2004a). Interestingly, Liu et 

al. (1998) isolated identical genotypes of what, based on the >99% ITS 

sequence similarity between the two (Midgley et al. 2004a), appears likely 

to be the same taxon identified by Midgley et al. (2004a) from hair roots of 

neighbouring W. pungens plants at a different sclerophyll forest site. This 

implies that the phenomenon is not uncommon for putative mycorrhizal 

fungi of these epacrids, but whether it reflects the presence of single genets 

that were continuous between the two plants, or ramets that were confined 

to one or other root system remains unresolved. In contrast to ecto- and 

arbuscular-mycorrhizal fungi, ericoid mycorrhizal fungi are generally regarded 

as producing only limited mycelial growth in soil (Smith and Read 

1997). This tenet is, however, based primarily on observations of H. ericae 

in mor-humus heathland vegetation communities in the northern hemisphere. 

It is possible that some ericoid mycorrhizal fungi of epacrids are 

capable of growing further from a host root into soil. Even if this is not 

the case, it is possible that the root systems of neighbouring epacrids were 

juxtaposed and that hyphal bridges do in fact interconnect epacrid plants. 

Such interconnectedness might have significant physiological and ecological 

consequences for the symbiotic partners (Robinson and Fitter 1999), 

and further investigation in this area is clearly warranted.


14.4 

Mycorrhizal Status of Endophytes from Epacrids 

Due to problems of germinating seeds and maintaining epacrid seedlings 

under sterile conditions in the laboratory, some of these experiments 

have been conducted using northern hemisphere Ericaceae hosts such 

as Vaccinium macrocarpon or V. corymbosum (Liu et al. 1998; Davies et 

al. 2003). Recent success in sterile propagation of epacrids such as Epacris 

impressa by micropropagation and Woollsia pungens from seed, however, 

have facilitated the use of epacrids as hosts for mycorrhiza infection testing 

(Fig. 14.1a,b) (McLean et al. 1998; Midgley et al. 2004a). ITS sequence data 

indicate that those endophytes that have been shown to produce ericoid 

mycorrhiza-like infection have affinity with either the Hymenoscyphus ericae 

aggregate or two broad groups of Helotiales ascomycetes (equivalent 

to “Assemblage A” and “Unknown 2 & possible relatives” in Berch et al. 

2002) (McLean et al. 1998; Chambers et al. 2000; Berch et al. 2002; Davies 

et al. 2003; Midgley et al. 2004a). Endophytes in the H. ericae aggregate 

and both Helotiales groups have also been isolated from, and shown to 

form ericoid mycorrhizas with, northern hemisphere Ericaceae (Berch et 

al. 2002), suggesting that these groups are important mycorrhizal fungi of 

Ericaceae worldwide. 

While O. maius and some Thielavia-like and Capronia-like endophytes 

from northern hemisphere Ericaceae have been shown to be ericoid 

Fig.14.1. a,b Testing for ericoid mycorrhiza formation by endophytes isolated from epacrid 

hair roots in gnotobiotic culture. a Woollsia pungens seedling in gnotobiotic culture with 

ericoid mycorrhizal fungi. b W. pungens hair root showing dense hyphal coils produced by 

an ericoid mycorrhizal fungus in epidermal cells (arrows) Bars a 1cm,b 50 µm


mycorrhizal fungi (Berch et al. 2002), none of the isolates from epacrids that 

have close sequence identity to these fungi formed mycorrhiza-like structures 

in gnotobiotic infection experiments (Chambers et al. 2000; Midgley et 

al. 2004a). Certain Thielavia-like and Capronia-like isolates from northern 

hemisphere Ericaceae hosts also appear to be non-mycorrhizal, suggesting 

that further work is required to ascertain the extent of mycorrhiza-forming 

abilitiesofisolatesinthesegroups.Theonlybasidiomycetesofarisolated 

from an epacrid hair root system failed to form ericoid mycorrhiza-like 

structures in W. pungens roots (Midgley et al. 2004a), suggesting that it, 

and the basidiomycete hyphae observed in Dracophyllum secundum epidermal 

cells by Allen et al. (1989), was probably a saprotroph. Although 

they can infect Ericaceae epidermal cells, the coils formed by isolates from 

epacrids that have close ITS sequence identity to Phialocephala fortinii 

are typical of those formed by DSE rather than ericoid mycorrhizal fungi 

(Davies et al. 2003; Midgley et al. 2004a). Interactions between DSE and 

their plant hosts remain poorly understood, and it appears that they may, 

under different circumstances, have mildly parasitic, neutral, or mildly 

beneficial effects on their hosts [Jumpponen 2001; see Chaps. 7 (Sieber and 

Grünig) and 15 (Schulz)]. The significance of infection of epacrids by DSE 

thus remains unclear. 

The broad taxonomic congruence between the fungi that appear to form 

ericoid mycorrhizas with epacrids and those that form associations with 

Ericaceae from other continents is consistent with the hypothesis that ericoid 

mycorrhizal associations evolved in a common ancestral host plant 

group (Cullings 1996). Fossil evidence and extant biogeographical patterns 

suggest that the association probably arose in Gondwanaland during 

the Cretaceous period, and that hosts and associated fungi subsequently 

radiated northwards (Cullings 1996; Cairney 2000). 

14.5 

Saprotrophic Potential of Mycorrhizal Fungi 

The presence of propagules of putative ericoid mycorrhizal fungi in sclerophyll 

forest soil has been demonstrated by direct DNA extraction from soil 

from which roots were removed (Chen and Cairney 2002). Similarly, in the 

seasonally drought-affected soils of Western Australian heathlands, ericoid 

mycorrhizal inoculum appears to persist through summer in surface soil 

above the zone of most hair root growth, again suggesting the presence 

of host-free fungal propagules (Hutton et al. 1996a). Further evidence of 

the abilities of ericoid mycorrhizal fungi to persist in the absence of their 

Ericaceae hosts has recently been obtained for the northern hemisphere 

taxon Erica arborea, which became infected by ericoid mycorrhizal fungi


when planted in a mature Quercus ilex forest that lacked Ericaceae vegetation 

(Bergero et al. 2003). Unfortunately the techniques used in these 

studies cannot discriminate between actively growing mycelia and inactive 

propagules such as asexual spores. However, the activities of the fungi in 

axenic culture suggest that, to some extent at least, they are capable of 

saprotrophic growth in the absence of an Ericaceae host. 

It is well established that, via production of an array of extracellular 

enzymes, H. ericae has considerable ability to exist as a free-living saprotroph 

(reviewed by Cairney and Burke 1998), and there is evidence that 

other mycorrhizal fungi of northern hemisphere Ericaceae have similar 

abilities (Piercey et al. 2002; Varma and Bonfante 1994). Relatively little is 

known regarding the saprotrophic potential of endophytes from epacrids. 

Midgley et al. (2004c), however, have shown that two frequently isolated putative 

ericoid mycorrhizal fungal taxa from W. pungens can utilise a range 

of compounds as sole carbon sources during growth in axenic culture. 

Thus, along with hexoses, these taxa can derive carbon from xylan and 

cellulose, suggesting production of 1-4-β-xylanase and β-d-xylosidase, 

along with a complete cellulase complex (Midgley et al. 2004c). While 

these enzyme activities are doubtless important in the penetration of host 

epidermal cell walls during the establishment of mycorrhizal symbiosis, 

they probably also facilitate a degree of saprotrophic growth and may 

be functionally important in the process of symbiotic nutrient acquisition. 

14.6 

Symbiotic Functioning of Mycorrhizal Fungi 

The mutualistic nature of ericoid mycorrhizal infection of epacrids has not 

yet been confirmed by gnotobiotic culture nutrient transfer experiments 

(see McLean et al. 1998; Anthony et al. 2000; Cairney and Ashford 2002). 

Nonetheless, the putative ericoid mycorrhizal fungi can utilise a range of 

amino acids and simple proteins as sole nitrogen sources, along with inositol 

hexaphosphate and DNA as sole sources of phosphorus (Chen et al. 

1999; Whittaker and Cairney 2001; Midgley et al. 2004b). Their abilities to 

access nitrogen and phosphorus thus appear to be on a par with H. ericae 

and other ericoid mycorrhizal fungi from northern hemisphere Ericaceae, 

which are known to acquire nitrogen and phosphorus from these substrates 

and effect transfer of the elements to plant hosts (see Smith and 

Read 1997; Xiao and Berch 1999). Given the relatedness of the putative 

ericoid mycorrhizal fungi of epacrids to these known mycorrhizal fungi, 

the similarities in the infections formed in host epidermal cells and their 

abilities to utilise organic nitrogen and phosphorus sources, however, it


seems probable that they function in a manner similar to their northern 

hemisphere counterparts. 

In addition to utilising organic nitrogen substrates, Midgley et al. (2004b) 

found that, as is the case for H. ericae, putative mycorrhizal fungi from 

epacrids are efficient users of nitrate. This contrasts with ectomycorrhizal 

fungi from the same eastern Australian sclerophyll forest habitats, which 

have limited abilities to utilise nitrate (Anderson et al. 1999; Sawyer et 

al. 2003). Although the soils in such forests are likely to be ammonifying 

(Connell et al. 1995), the ability to utilise nitrate may be advantageous to 

the mycorrhizal fungi and their epacrid hosts following fire, when nitrate 

may be relatively abundant (Stewart et al. 1993). Many epacrids are killed, 

but regenerate rapidly from seed, following fire, while others may survive 

fire by resprouting (Bell et al. 1996). There is also evidence that epacrid 

abundance increases following low intensity fires (Morrison 2002) and 

that putative ericoid mycorrhizal fungi can be present in some sclerophyll 

forest soils within a few days of a low intensity fire (Chen and Cairney 2002). 

Since non-mycorrhizal epacrids have only limited abilities to utilise NO − 3 

for growth (Stewart et al. 1993), these observations suggest that the fungi 

may be of particular importance to the success of epacrids in fire-prone 

vegetation communities. 

It has been postulated that different mycorrhizal fungal taxa or genotypes 

might differentially access nutrient sources in soil, and that this might 

be important in increasing overall nitrogen and/or phosphorus uptake by 

their plant hosts (Cairney et al. 2000; Koide 2000). Midgley et al. (2004b) 

investigated the relative abilities of six genotypes of an H. ericae-like putative 

mycorrhizal fungus from W. pungens and six isolates of an unknown 

Helotiales putative mycorrhizal fungal taxon from W. pungens and L. parviflorus 

to utilise a range of simple inorganic and organic nitrogen and 

phosphorus sources for growth. All isolates were found to utilise inorganic 

forms of the elements, acidic, neutral and basic amino acids, along with 

protein, phosphomonoesterase and phosphodiesterase. Although some intraspecific 

variation was observed on all substrates, one taxon produced 

significantly more biomass on most substrates than the other. For all substrates, 

however, the difference between the two taxa was considerably less 

than an order of magnitude, leading Midgley et al. (2004b) to conclude 

that both taxa would confer broadly similar nutritional benefits to their 

hosts. This, and the fact that screening of single isolates of a range of putative 

ericoid mycorrhizal fungal taxa from W. pungens suggests that all 

are broadly similar in their abilities to utilise organic nitrogen and phosphorus 

substrates (Chen et al. 1999; Whittaker and Cairney 2001), might 

appear to contradict the proposal that different isolates/genotypes vary in 

their abilities to enhance host nutrition. This, however, may not be the 

case. The efficiency with which the various fungi transfer nitrogen and/or


phosphorus from the substrates to the host plant remains to be tested, and 

different taxa/genotypes may differ considerably in this respect. 

14.7 

Conclusions 

Although research to date has been confined to a small number of epacrid 

taxa from a limited range of habitats, it appears that most endophytes that 

have been isolated from epacrid hair roots are probably ericoid mycorrhizal 

fungi. An array of mainly Helotiales ascomycetes forms putative ericoid 

mycorrhizal associations with epacrids, but root systems of individual 

plants in the field are dominated by a small number of taxa, and populations 

of these dominated by single genotypes. Studies of mycorrhizal fungal 

diversity in natural habitats are at an early stage and in the future will 

need to take into account the spatial distribution of mycelial individuals 

if meaningful comparisons of communities in different habitats are to be 

made. Functional aspects of the symbiotic relationships between putative 

ericoid mycorrhizal fungi and their epacrid hosts remain to be investigated, 

however it is likely that these will follow the established paradigm for ericoid 

mycorrhizal associations of northern hemisphere Ericaecae. 

Acknowledgements. I thank D.J. Midgley for his permission to use Fig. 14.1b. 


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15 

15.1 

Introduction 

Mutualistic Interactions 

with Fungal Root Endophytes 

Barbara Schulz 

With few exceptions, colonisation of a plant host is beneficial for the fungus, 

assuring it a supply of nutrients and shelter from most abiotic stressors. 

Previously, within plant roots only the symbioses of mycorrhizal fungi 

were considered to be mutualistic. Recently, it has been recognised that 

many other fungi, and in particular endophytic fungi, can participate in 

mutualistic root symbioses (e.g. Sieber 2002; Brundrett 2002; Schulz and 

Boyle 2005). 

There are various potential benefits for the host in mutualistic interactions 

with endophytic fungi, for example induction of defence metabolites 

potentially active against pathogens (Schulz et al. 1999; Mucciarelli et al. 

2003; Arnold and Herre 2003), endophytic secretion of phytohormones 

(Holland 1997; Rey et al. 2001; Römmert et al. 2002; Tudzynski and Sharon 

2002), mobilisation of nutrients for the host from the rhizosphere (Jumpponen 

et al. 1998; Caldwell et al. 2000; Usuki et al. 2002) and/or an alteration 

of host metabolism (Jallow et al. 2004). Colonisation by fungal root endophytes 

may lead to induced disease resistance (Picard et al. 2000; Benhamou 

and Garand 2001), improved growth of the host (Kimura et al. 1992; Jumpponen 

2001; Mucciarelli et al. 2002; Ernst et al. 2003), abiotic stress tolerance 

(Barrow and Aaltonen 2001; Redman et al. 2002; Barrow 2003) or protection 

from pathogenic competitors and insect predators of the host through 

synthesis of antagonistic fungal secondary metabolites (Schulz et al. 1995; 

Hallmann and Sikora 1996; Schulz et al. 2002; Miller et al. 2002; Selosse et 

al. 2004; see Chap. 8 by Bacon and Yates). This chapter reviews results dealing 

with mutualistic interactions between non-mycorrhizal root-colonising 

endophytic fungi with their plant hosts. When reading this chapter it is important 

to bear in mind that within the realms of our present knowledge, 

far from all of the interactions with fungal root endophytes are beneficial 

for both partners. 

Barbara Schulz: Technical University of Braunschweig, Institute of Microbiology, Spielmannstraße 

7, 38106 Braunschweig, Germany, E-mail: b.schulz@tu-bs.de 





262 B. Schulz 

15.2 

Colonisation and Histology 

To the extent that histological studies have been undertaken, growth of 

fungal endophytes in roots is usually extensive and can be inter- and/or 

intra-cellular (Boyle et al. 2001; Sieber 2002; Schulz and Boyle 2005). Most of 

the histological studies have dealt with endophytic colonisation by Fusarium 

and dark septate endophytes (DSE; Jumpponen and Trappe 1998; Sieber 

2002). 

Histological studies of colonisation by DSE have focussed on Phialocephala 

fortinii, but also on other DSE including Phialophora spp., other 

Phialocephala spp., Chloridium paucisporum, Heteroconium chaetospira 

and Leptodontidium orchidicola. The hyphae of DSE vary from thin and 

hyaline to melanised, sometimes forming black microsclerotia. The hyaline 

hyphae grow both intercellularly and within the vascular cylinder and, 

upon favourable nutrient status of the host, contain lipid vacuoles (Barrow 

and Aaltonen 2001; Barrow 2003). Barrow (2003) suggests that polymorphic 

fungal structures, which are found primarily within the sieve elements and 

accumulate massive quantities of lipids, are protoplasts. Not only DSE, but 

also other endophytes, e.g. Cryptosporiopsis sp., colonise the vascular cylinder 

(Schulz and Boyle 2005). The hyphae of root endophytes often develop 

intracellular coils, e.g. H. chaetospira and Oidiodendron maius (Usuki and 

Narisawa 2005), as does the basidiomycete, Piriformospora indica (Varma 

et al. 2000). These presumably increase the absorption of assimilates or the 

exchange of nutrients. 

In axenic culture, the DSE Phialocephala colonised the surface and the 

inner cortex of the roots of seedlings of Norway spruce and Scotch pine 

(Sieber 2002) and Larix decidua (Schulz et al. 1999; A.K. Römmert unpublished) 

with mats of mycelia growing within the roots both inter- and 

intra-cellularly. From roots of Norway spruce and Scotch pine collected in 

the field, the density of mycelial and intracortical development was much 

lower (Sieber 2002), suggesting that, under natural conditions, plant defence 

limited the density of colonisation. However, in arid habitats, Barrow 

(2003) found a highly branched extraradical hyphal network of hyaline 

vacuolated hyphae in a mucilaginous gel covering roots of Bouteloua.The 

polysaccharide mucilage, which was apparently produced by the fungi, 

stores moisture under arid conditions (see Sect. 15.6). 

Depending on the host being colonised, some DSE can develop mycorrhizal 

structures. For example, in the roots of at least 19 plant species, 

the dematiaceous hyphomycete H. chaetospira grew intra- or intercellularly 

within the cortical cells (Narisawa et al. 1998; Usuki et al. 2002; Ohki 

et al. 2002). However, within the roots of Rhododendron obtusum var. 

kaempferi, it developed typical ericoid mycorrhizas (Usuki and Narisawa


2005). P. fortinii has even been found to form a Hartig net and a thin patchy 

mantle, considered the anatomical hallmarks of ectomycorrhizae, both in 

axenic culture of seedlings of Salix glauca (Fernando and Currah 1996) and 

Betula platyphylla (Hashimoto and Hyakumachi 2001), with the roots of 

some nursery stocks of Pinus banksiana, Pinus contorta and Pinus glauca 

(Danielson and Visser 1990) and with the roots of Populus tremula x Populus 

tremuloides grown in an experimental field plot (Kaldorf et al. 2004). 

ThisisalsothecasefortheDSEPhialophora finlandia,whichformedectendomycorrhizal 

associations with the roots of Pinus resinosa (Lobuglio and 

Wilcox 1988) and ectomycorrhizal associations with the roots of several 

trees (Vralstad et al. 2002). 

The growth modus of avirulent strains of Fusarium spp. is variable, 

but often differs from that of virulent strains. Bacon and Hinton (1996) 

found that only pathogenic strains of F. moniliforme colonised maize both 

inter- and intracellularly; growth of the avirulent isolate was intercellular. 

However, other avirulent strains of Fusarium spp. colonised the roots of 

barley (Boyle et al. 2001) and pea (Benhamou and Garand 2001) both 

inter- and intra-cellularly. Whereas active growth of the avirulent strain 

of Fusarium oxysporum was restricted to the root surface, epidermis and 

outer cortex of the pea roots, the pathogenic strain of F. oxysporum rapidly 

colonised epidermis, cortex, endodermis and paratracheal parenchyma 

cells (Benhamou and Garand 2001). 

The colonisation modus of Stagonospora spp. in Phragmites australis is 

exemplary for fungi that initially colonise the roots, but then apparently 

grow systemically within the entire plant following vertical transmission 

(Ernst et al. 2003). Similarly, Sieber et al. (1988) isolated Stagonospora nodorum 

as an endophyte from both roots and shoots of wheat. Another fungus 

that colonises both roots and shoots is a non-sporulating endophyte of 

Mentha piperita. Theendophyteformedanexternalenvelopingmycelial 

sheet around the root with only little penetration of the root cortex (Mucciarelli 

et al. 2003), but was detected in the parenchymatic cells of mature 

leaves, especially during senescence (Mucciarelli et al. 2002). 

The histology of fungal root colonisations in mutualistic interactions is 

extremely variable, in part because any one species can exhibit extreme phenotypic 

plasticity. Colonisation can be inter- and/or intra-cellular, limited 

to the roots or apparently systemic within the entire plant. The morphologies 

vary from apparent protoplasts to very fine undifferentiated hyaline 

hyphae to highly differentiated ectomycorrhizas. So, it seems that neither 

fungal morphology nor mode of colonisation is decisive for determining 

whether or not an interaction is mutualistic. But perhaps the quantity of 

colonisation is a decisive factor, since extensive colonisation is common to 

all of the investigated mutualistic interactions.

264 B. Schulz 

15.3 

Secondary Metabolites 

A strikingly high proportion of endophytic fungi (80%) produce biologically 

active metabolites in vitro in tests for antibacterial, fungicidal and 

herbicidal activities (Schulz et al. 2002). Most of the investigations dealing 

with the synthesis of endophytic metabolites active against phytopathogens 

(Schulz et al. 1995; Tan and Zou 2001; Schulz et al. 2002; Fang-ting et al. 

2004) and against predators (Azevedo et al. 2000) have not specified from 

whichorgantheendophyteswereisolated. 

Fungal secondary metabolites may play a role within the host, and/or 

have an ecological significance. As hypothesised by Demain (1980): “If 

a fungus can produce metabolites in vitro, they must also have a function 

in nature” Fungi would not retain the multienzyme reaction sequences required 

for the synthesis of secondary metabolites without some beneficial 

effect for survival. As virulence factors, we have hypothesised that secondary 

metabolites are involved in maintaining a balance of antagonisms 

in the interaction with the host (Schulz et al. 1999; Schulz and Boyle 2005; 

see Chap. 1 by Schulz and Boyle). However, they may also have antagonistic 

functions. 

Some endophytes, for example Fusarium spp., that colonise both the 

shoots and roots of numerous hosts, synthesise a number of toxins including 

beauvericin, a cyclic hexadepsipeptide with insecticidal properties 

(see Chap. 8 by Bacon and Yates; Kuldau and Yates 2000; Miller 2001). 

Beauvericins are produced by at least 12 Fusarium species, and protect 

infected plants against herbivoric insects (see Chap. 8 by Bacon and Yates). 

Fungal root endophytes also synthesise metabolites toxic to the nematode 

Meloidogyne incognita. For example, both phomalactone, synthesised 

by Verticillium chlamydosporium (Khambay et al. 2000), and metabolites 

present in the culture filtrate of a non-pathogenic F. oxysporum reduced 

mobility of the nematode within 10 min of exposure (Hallmann and Sikora 

1996). 

Many root endophytic fungi produce antimicrobial metabolites (Schulz 

et al. 2002), e.g. the antibacterial and antifungal metabolite(s) produced in 

vitro by Cryptosporiopsis sp. from Larix decidua (Schulz et al. 1995). These 

included the antifungal metabolite mycorrhizin, which in situ could protect 

the host from phytopathogenic fungi (Schulz et al. 1995). Hallmann and 

Sikora (1996) found that the culture filtrate of F. oxysporum strain 162 was 

not only toxic to M. incognita, but was also antifungal, inhibiting soil-borne 

plant pathogens. 

Bultman and Murphy (2000) suggested that endophytic Neotyphodium 

spp. are stimulated to increase production of mycotoxins in shoots of 

grasses after damage to the host has occurred, the adaptive significance


being apparent. A similar effect could occur when roots colonised by endophytic 

fungi are injured. Fungal secondary metabolites may also play roles 

in signalling and growth enhancement (see Sect. 15.4). 

In conclusion, since most of the fungal root isolates can produce biologically 

active secondary metabolites in vitro, it seems probable that they are 

also produced in planta. They can be antagonistic against plant predators 

and microbial antagonists, in both cases suppressing disease. But they may 

also play roles within the host, e.g. in maintaining a balance of antagonism 

between endophyte and host. 

15.4 

Growth Enhancement 

The non-mycorrhizal fungal root colonisers that have been reported to 

improve growth of their hosts have various growth modi and belong 

to diverse genera, including Chaetomium, Cladorrhinum, Cryptosporiopsis, 

Fusarium, Heteroconium, Oidiodendron, Phialocephala, Piriformospora 

and Stagonospora. 

Stagonospora spp., and a non-sporulating endophyte of Mentha piperita, 

are exemplary for endophytes that grow systemically in roots and shoots of 

their hosts (see Sect. 15.2). When isolates of three species of Stagonospora 

were reinoculated into axenic host seedlings of Phragmites australis, all 

increased growth significantly (Ernst et al. 2003). The authors note that 

this is only the third reported case, besides those dealing with Neotyphodium, 

in which a seed-transmitted fungus enhanced the biomass of its 

host. Mucciarelli et al. (2002, 2003) speculated that improved growth of 

M. piperita might be due to a better nutrient supply or, alternatively, to 

the synthesis of plant growth hormones by a non-sporulating endophytic 

fungus. 

In contrast, colonisation in most of the studied interactions is limited 

totheroots,e.g.thatbyPhoma in Vulpia ciliata spp. ambigua, whichincreased 

shoot biomass, root lengths, and tiller numbers of the host (Newsham 

1994), and that of barley by Chaetomium or Chaetomium globosum, 

which increased root fresh weight (Vilich et al. 1998). Gasoni and Stegman 

de Gurfinkel (1997) suggested that increased phosphorous uptake, as observed 

in cotton roots colonised by Cladorrhinum foecumdissimum, was 

responsible for promoting growth of the host. Similarly, results obtained 

by Jumpponen and Trappe (1998) led Jumpponen (1999) to the conclusion 

that growth enhancement by the DSE may be due to improved phosphorous 

and nitrogen uptake (Jumpponen et al. 1998) or, in a closed system, 

to increased availability of carbohydrates and/or CO2, both resulting from 

fungal metabolism (Jumpponen and Trappe 1998).

266 B. Schulz 

It is also possible that growth enhancement is due to indirect acquisition 

of nutrients obtained saprophytically from the endophyte from the 

rhizosphere. Caldwell et al. (2000) suggested that due to their hydrolytic 

capabilities, DSE are able to grow both biotrophically and saprophytically. 

Of note: they found no evidence for lignolytic enzymes. They hypothesised 

that the fungi may access litter and detrital carbon, nitrogen and phosphorous, 

making this available to the host. On the basis of finding a continuous 

hyphal network extending from the rhizosphere to the vascular cylinder, 

Barrow (2003) also suggested that there is potential for bidirectional carbon 

transport. The situation could be similar to that hypothesised for Oidiodendron 

maius, which not only develops ericoid mycorrhizas within the 

host, but also can grow saprophytically in peat (Rice and Currah 2002, 

see Chap. 13 by Rice and Currah) and perhaps supplies its host with organic 

nutrients from the rhizosphere. Inter-plant connections may also be 

involved in growth enhancement, as suggested by Sen et al. (1999), who 

found evidence that a common population of Ceratobasidium cornigerum 

occupies roots of orchid and pine. DSE may even replace arbuscular mycorrhiza 

(AM) and ectomycorrhizal fungi at sites with extreme environmental 

conditions (Sieber 2002; see Chap. 7 by Sieber and Grünig). Co-inoculation 

of tomato with a non-pathogenic strain of Fusarium oxysporum and the 

AM fungus Glomus coronatum resulted in better mycorrhization, but did 

not improve growth more than mono-inoculation (Diedhiou et al. 2003). 

Colonisation by the DSE Phialocephala does not always exert positive effects 

on the host: in axenic culture it significantly retarded growth of Betula 

platyphylla var. japonica seedlings. Nevertheless, P. fortinii formed typical 

ectomycorrhiza with B. platyphylla, underlining the plasticity of the interaction 

(Hashimoto and Hyakumachi 2001). Whether or not colonisation by 

DSE improves growth of the host depends on environmental conditions and 

on the metabolic status of the host. Usuki et al. (2002) found that variance 

of the growth medium affected the mode of colonisation by Heteroconium 

chaetospira in seedlings of Chinese cabbage. Colonisation and growth enhancement 

were best, and intracellular, in peat moss amended with 0.1% 

glucose. At higher glucose concentrations both frequency of colonisation 

and plant growth decreased, and fungal colonisation was restricted mostly 

to the intercellular regions of epidermal and cortical cells, leading to the 

formation of microslerotia. 

Not only colonisation, but also culture extracts of the endophyte can 

improve growth. Colonisation of the roots of seedlings of Larix decidua by 

the endophytes P. fortinii and Cryptosporiopsis sp., both of which had been 

isolated from roots of the host, significantly improved lengths (Schulz et 

al. 2002) and dry weights (Römmert et al. 2002) of both roots and shoots 

(Fig. 15.1). Additionally, disease symptoms resulting from the stress of axenic 

culture decreased (Schulz et al. 1999; Römmert et al. 2002). Similarly,


Fig.15.1. Influence of colonisation on dry weights of shoots (a) and roots (b) of Larix 

decidua, cultivated for 3 months axenically in a synthetic medium in expanded clay and 

inoculated at day 0 with either an endophyte, Cryptosporiopsis sp. (Cs) or Phialocephala 

fortinii (Pf), or a pathogen, Heterobasidion annosum (Ha). n = 29−55, ∗P

268 B. Schulz 

Table 15.1. Addition of methanolic culture extract of Phialocephala fortinii grown in MMNA 

liquid medium (Schulz et al. 1999) for 14 days to surface-sterilised seedlings of Larix decidua 

(approx. 3 months old) and Triticum aestivum (approx. 1 week old) on filter paper, previously 

germinated on biomalt agar medium (5% w/v). Evaluation of L. decidua after 28 days, of 

T. aestivum after 7 days of incubation. Methanolic extracts of the sterile culture medium 

were used as the controls 

Average shoot length (cm) Average dry weight of 

root+shoot (mg) 

L. decidua 

+Cultureextract(n = 8) 1.0* 44.0* 

Control (n = 10) 0.08 16.9 

T. aestivum L. 

+Cultureextract(n = 20) 0.84 1.6 

Control (n = 20) 0.82 2.4 

∗P


Methylobacterium spp. that synthesise cytokinin in vitro, are found endophytically 

in actively growing tissues of many or even all plants. They 

are present in the apoplast and could degrade metabolic wastes, producing 

e.g. ammonium ions, which could be metabolised by the host. Holland 

concluded that since endophyte-free plant tissue has not been conclusively 

shown to synthesise cytokinins, “microbial symbionts are not accidental 

visitors. They are co-evolved participants in plant physiology.” Similar signals 

seem to be generated by fungal endophytes, which not only synthesise 

the metabolites reported above (Kimura et al. 1992; Yates et al. 1997; Rey et 

al. 2001; Römmert et al. 2002), but also cytokinins (Petrini 1991; Tudzynski 

1997; Tudzynski and Sharon 2002). 

The plasticity of fungal root endophytes is demonstrated by the multiple 

options they have for regulating plant growth: synthesis of secondary 

metabolites and fungal phytohormones; directly providing nutrients, i.e. 

nitrogen and phosphate from the rhizosphere; but also bidirectional carbon 

transport. These results suggest that fungal endophytes are not only 

involved in a balanced antagonism with their hosts, but also may be responsible 

for a balanced regulation of growth. Thus, in at least some interactions, 

fungal root endophyte and host seem to be highly adapted to one 

another, resulting in a balanced interaction, as has also developed during 

the co-evolution of fungus and algae in lichens. 

15.5 

Disease Suppression 

Theideaofendophytemediatedinducedresistanceisanextensionof 

the defensive mutualism hypothesis, i.e. plants may acquire protection 

through constitutive and induced resistance (Bultman and Murphy 2000). 

As defined by Schönbeck et al. (1993), “induced resistance describes the 

improvement of natural resistance of a plant without alterations of the 

genome”. It is a non-specific general response that precludes absence of 

toxic effects for the parasite of the inducing agent as well as the absence of 

a dosage-response correlation, and it necessitates a time interval between 

application and response. Colonisation with fungal root endophytes has 

been reported to negatively influence growth of nematodes, insects and 

microbial pathogens (Selosse et al. 2004). However, it is not always clear 

whether induced resistance, altered plant nutrition, metabolism and/or 

sink effects are responsible for the increased mortality of predators when 

systemic effects are observed and mycotoxins are not involved. 

Systemic fungal root infections have been reported to lead to acquisition 

of induced resistance in both roots and/or shoots (Bargmann and Schönbeck 

1992; Hallmann and Sikora 1994; Sieber 2002). For example, root

270 B. Schulz 

inoculations of cabbage plants with soilborne endophytic Acremonium alternatum 

decreased damage due to larvae of the diamondback moth on the 

leaves (Raps and Vidal 1998). However, the authors suggested that competition 

for phytosterol, which both fungi and insects obtain from their host, 

rather than induced resistance could account for reduced larval growth on 

inoculated cabbage plants. They furthermore hypothesise that a disjunction 

of fungal colonisation and predation is responsible for the observed 

effect, the roots serving as a sink for the plant metabolite. 

Non-pathogenic Fusarium spp. have been found to induce resistance 

or afford the host cross-protection against pathogenic fungi (Kuldau and 

Yates 2000; Sieber 2002). For example, colonisation of the roots of pea and 

tomato by non-pathogenic strains of Fusarium oxysporum reduced disease 

by fungal root pathogens (Benhamou and Garand 2001) and predation 

by nematodes (Diedhiou et al. 2003), respectively. Fungal growth within 

the roots of pea was restricted to the outermost cell layers. Benhamou and 

Garand (2001) and Narisawa et al. (2004) found that resistance was induced, 

at least in part, by a massive elaboration of cell wall appositions and the 

deposition of electron-opaque material surrounding hyphae in the roots of 

pea and Chinese cabbage, respectively. This might be due to the accelerated 

deposition of lignin, a mechanical barrier for invading organisms, as was 

observed in shoots of maize when the roots were colonised by an avirulent 

isolate of F. moniliforme (Yates et al. 1997). Such host defence responses are 

initially produced to limit colonisation of the fungal endophytic invader, 

presumably resulting in a balance of antagonisms between host and fungus 

and in an asymptomatic endophytic infection. However, the host mechanical 

defence response also limits colonisation by subsequent pathogens. 

Only a few of the endophytic isolates from a given host are effective protectants 

when reinoculated into the host. Of 322 endophytic fungi isolated 

from Chinese cabbage (Brassica campestris), only 16 isolates, including Heteroconium 

chaetospira, Mortierella elongata, Westerdykella sp. and three 

non-sporulating isolates, almost completely suppressed disease caused by 

Plasmodiophora brassicae when reinoculated into the host in sterile soil 

(Narisawa et al. 1998; Usuki et al. 2002). The authors suggest that disease 

suppression by DSE may be strain specific, as also seems to be the case with 

growth enhancement (see Sect. 15.4). Other DSE have also been reported 

to protect roots against phytopathogenic fungi (Sieber 2002; Narisawa et 

al. 2002). For example, three endophytic fungi reduced disease caused by 

Verticillium longisporum in Chinese cabbage: two were Phialocephala and 

one an unidentified DSE (Narisawa et al. 2004). The latter DSE, which extensively 

colonised root cells of the cortex (in contrast to the P. fortinii 

isolates), was very effective in preventing disease even in field experiments. 

In addition, of 16 isolates of H. chaetospira that suppressed disease in axenically 

cultured seedlings, only 2 were also effective in non-sterile soil


(Narisawa et al. 2002). This could be due to the fact that in non-sterile soil 

the roots are naturally colonised by endophytes. 

Root colonisation with fungal endophytes can activate not only mechanical 

defence, but also induce the synthesis of defence metabolites in 

the roots, i.e. induce resistance. For example, colonisation of the roots of 

seedlings of Larix decidua with either the endophyte Cryptosporiopsis sp. 

or P. fortinii increased concentrations of soluble proanthocyanidins, and 

colonisation of barley roots with Fusarium sp. led to higher concentrations 

of phenylpropanoids in the roots than in those of the controls (Schulz et 

al. 1999). Similarly, Mucciarelli et al. (2003) found that the concentration of 

total phenolics increased significantly when Mentha piperita was colonised 

by a non-sporulating endophyte, though they found no significant change 

in the concentrations of total terpenoids. 

Picard et al. (2002) investigated what fungal factors were responsible 

for inducing systemic resistance when the mycoparasite Pythium oligandrum 

asymptomatically colonised the roots of tomato without inducing 

extensive cell damage. They demonstrated that colonisation led to cell wall 

appositions and to the deposition of phenolic metabolites (Benhamou et 

al. 1997). Picard et al. (2000) found that oligandrin, an elicitin-like protein 

produced by P. oligandrum, migrates within the vascular system of the host 

and induces systemic resistance. 

Improving plant resistance without the use of pesticides is one goal 

of biocontrol in agriculture. Only a small proportion of the fungal root 

endophytes of any one host seem capable of inducing systemic resistance. 

Are these endophytes present under natural environmental conditions at 

a sufficient colonisation density to induce resistance? In most cases, too 

little is presently known about what factor(s) induce resistance in the field. 

This is a broad and important field for future investigations. 

15.6 

Stress Tolerance 

Whereas mycorrhizal fungi can alleviate abiotic stresses, e.g. salt (Rabie 

2005) and osmotic stress (Ruiz-Lozano 2003), there have been only a few 

investigations studying the influence of endophytic colonisation on abiotic 

stress tolerance. One example strikingly demonstrates induction of 

stress tolerance to abiotic stress: a novel endophytic Curvularia sp., which 

colonised both roots and shoots of the host, increased host tolerance to 

temperatures of up to 65 ◦ C (Redman et al. 2002). Another abiotic stress 

is transplantation to polluted sites or micropropagation; stresses that can 

be counteracted by hardening. This was achieved not only by mycorrhization, 

but also by colonisation by the non-obligate biotrophic endophyte

272 B. Schulz 

Piriformospora indica (Sahay and Varma 1999). An arid climate also poses 

an abiotic stress, which DSE may help alleviate. Barrow and Aaltonen 

(2001) suggest that DSE may play such a role, because they found that 

under xeric, in contrast to mesic, conditions, colonisation by DSE was 

more prevalent than that by aseptate hyphae (AM). It is also possible that 

lipid accumulation within hyaline hyphae of DSE serves as an energyrich 

carbon reserve to sustain plants during extended drought (Barrow 

2003). 

The potential of fungal root endophytes to alleviate other abiotic and 

biotic stresses is a field that has not been adequately investigated. Their 

capacity to induce tolerance to other individual stressors, as well as to accumulated 

stress, should be tested both with individual and with a mixture 

of stressors at sublethal levels to determine the limits and potentials for 

induction of stress tolerance by fungal root endophytes, but also for future 

use in sustainable agriculture. 

15.7 

Factors Determining the Status of the Interaction 

Establishment of any interaction is always a multifactorial process. One 

factor that may contribute to the interaction being mutualistic is the pure 

extent of colonisation, since in the reported mutualistic interactions with 

fungal root endophytes, growth has been extensive (see Sect. 15.2.; Stone 

et al. 2000; Sieber 2002; Schulz and Boyle 2005). However, other factors e.g. 

biotic and abiotic stressors, nutrient support, and ontogenetic status, may 

also be relevant. As hypothesised by Schulz and Boyle (2005), mutualistic 

interactions have more frequently developed between microorganisms and 

the roots, because (1) the roots are a natural carbon sink of the plants and 

can supply dual and multi-organism symbioses with nutrients, (2) infection 

is less limited by xeromorphic structures, and (3) roots are in close contact 

with an environment harbouring many different, mainly degradatively 

active, micro-organisms. 

In spite of the examples of mutualistic interactions with fungal root 

endophytes presented in this chapter, it is important not to draw the conclusion 

that all interactions with fungal root endophytes are mutualistic. 

Only a small percentage of these endophytes seem to have the capability 

to interact mutualistically with their hosts (see Sects. 15.5, 15.6), perhaps 

because interactions with their hosts depend on the genetic predisposition 

and momentary metabolic status of the host, as well as on environmental 

factors. The same endophytes that under certain conditions interact mutualistically 

with their hosts may become pathogenic, for example when the 

host is stressed and the balance of the antagonism is tilted “in favour” of


the fungus (Kuldau and Yates 2000; Jumpponen 2001; Sieber 2002; Schulz 

and Boyle 2005). 

15.8 

Conclusions 

Fungal endophytic colonisation of the roots of plants is very variable (Table 

15.2), reflecting the plasticity of individual fungal endophytes, but 

also of endophytes as a whole. The hyphae can be hyaline and very thin, 

melanised, or develop microsclerotia. Colonisation is often extensive, may 

be inter- and/or intra-cellular, and is sometimes limited to the epidermis 

or cortex. The hyphae may extensively colonise the vascular cylinder and 

in particular the sieve elements, but also grow on the root surface and into 

the rhizosphere. Some DSE have even been observed to develop ericoid, 

ectendo- and ectomycorrhizal-like structures. 

Benefits for the fungal partner are a stable nutrient source and some 

protection from abiotic stresses. Factors that fungal root endophytes may 

contribute to a mutualistic interaction include secondary metabolites, phytohormones, 

nutrients, elicitins and colonisation (Fig. 15.2). These factors 

may have more than one benefit for the host. Potential advantages of the interactions 

for the host are summarised in Table 15.3 and include improved 

growth, induced resistance, stress tolerance, and protection from microbial 

and insect predators by mycotoxins. 

Morphologically and physiologically, endophytic root colonisations mirror 

the variability and thus the plasticity of endophytic interactions, but also 

Table 15.2. Endophytic plasticity 

Average shoot length (cm) Average dry weight of root+shoot (mg) 

Attribute In planta 

Colonisation Intercellular, intracellular, on/in: root surface, root hairs, 

epidermis, cortex, and/or vascular cylinder (xylem and 

phloem), systemic within roots, systemic within roots and 

shoots, local colonisation (?) 

Fungal morphologies Lipid-filled hyaline hyphae and protoplasts (?), melanised 

hyphae, microsclerotia, coiled hyphae, appressoria 

Fungal-plant associations No specialised structures Ericoid, ectendo-, 

and ectomycorrhizas 

Secondary metabolites Growth enhancement, growth inhibition, elicitation, balanced 

antagonism, microbial antagonism 

Phytohormones Modulate host growth 

Nutrient source Parasitically from host assimilates, saprophytically

274 B. Schulz 

Table 15.3. Endophytes/metabolites known to be involved in mutualistic interactions. DSE: 

Dark septate endophyte 

Advantage for host Endophyte / metabolite Host Reference 

Growth 

Cryptosporiopsis sp. Larix decidua Schulz et al. 2002 

enhancement Chaetomium, C. globosum Barley Vilich et al. 1998 

Cladorrhinum 

Cotton Gasoni and Stegman 

foecumdissimum 

de Gurfinkel 1997 

Fusarium Maize Yates et al. 1997 

Heteroconium chaetospira Chinese cabbage Usuki et al. 2002 

Oidiodendron maius Ericaceous plants Rice and Currah 2002 

Phialocephala fortinii Pinus contorta Jumpponen 

and Trappe 1998; 

Jumpponen et al. 

1998 

P. fortinii Larix decidua Schulz et al. 2002 

P. fortinii Carex firma, Haselwandter and 

C. curvula Read 1982 

Phomafimeti Vulpiaciliate Newsham 1994 

Stagonospora spp. Phragmites 

australis 

Ernst et al. 2003 

n.i. a Mentha piperita Mucciarelli et al. 

2002, 2003 

Altechromones A and B Lettuce Kimura et al. 1992 

Fumonisin Maize Yates et al. 1997 

Disease 

Acremonium Tomato Raps and Vidal 1998 

suppression/ Fusarium moniliforme Maize Yates et al. 1997 

induced resistance Fusarium oxysporum Pea Benhamou 

and Garand 2001 

F. oxysporum Tomato Diedhiou et al. 2003 

Fusarium spp. Various crops Kuldau and Yates 

2000 

H. chaetospira, Mortierella Chinese cabbage Narisawa et al. 1998; 

elongata, Westerdykella sp. 

Usuki et al. 2002 

P. fortinii Chinese cabbage Narisawa et al. 2004 

Pythium oligandrum Tomato Benhamou et al. 

1997; Picard et al. 

2002 

Oligandrin Tomato Picard et al. 2000 

Stress tolerance Curvularia sp. Dicanthelium 

lanuginosum 

Redman et al. 2002 

Piriformospora indica Tobacco Sahay and Varma 

1999 

DSE Atriplex Barrow and Aaltonen 

canescens 2001



Advantage for host Endophyte / metabolite Host Reference 

Mycotoxins Cryptosporiopsis sp. Schulz et al. 1995 

Fusarium oxysporum Khambay et al. 2000 

Fusarium spp Chap. Bacon and 

Yates; Kuldau and 

Yates 2000; 

Miller 2001 

Verticillium 

Hallmann and Sikora 

chlamydosporium 

1996 

a Not identified 

Fig.15.2. Potential contributions of fungal root endophytes to mutualistic interactions, 

assuming extensive colonisation of the root. “Colonisation” indicates that it is unknown 

what aspect of fungal colonisation induces the response 

the evolutionary developmental stages from ubiquitous endophyte to mutualistic 

endophyte to specialised mycorrhizal fungus. Not only the exploitive, 

but also the mutualistic life history strategy becomes more prevalent with 

increasing specialisation (Brundrett 2002; see Chap. 16 by Brundrett). 

Acknowledgements. My thanks go to Anne-Kathrin Römmert and Miruna 

Oros-Sichler for permission to use unpublished results and to Christine 

Boyle for constructive discussions that helped develop the manuscript and 

ideas presented above.

276 B. Schulz 



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16 

Understanding the Roles 

of Multifunctional Mycorrhizal 

and Endophytic Fungi 

Mark C. Brundrett 

16.1 

How Mycorrhizal Fungi Differ from Endophytes 

16.1.1 

Definitions 

The most appropriate definition of endophytism is that of symptomless 

associations by organisms that grow within living plant tissues (see other 

chapters in this book). Many fungi colonise the cortex of living roots without 

causing disease, including pathogenic or necrotrophic fungi with latent 

phases as well as beneficial fungi that offer protection against pathogens, 

but it is difficult to precisely categorise these fungi [Saikkonen et al. 1998; 

Sivasithamparam 1998; see Chaps. 1 (Schulz and Boyle) and 8 (Bacon and 

Yates)]. A new definition of mycorrhizal associations was required to adequately 

separate them from other root-fungus associations (Brundrett 

2004). According to this definition, endophytic associations differ from 

mycorrhizas primarily by the absence of a localised interface of specialised 

hyphae, the absence of synchronised plant-fungus development, and the 

lack of plant benefits from nutrient transfer – the three key defining features 

of mycorrhizas. However, plants may benefit indirectly from endophytes 

by increased resistance to herbivores, pathogens or stress, or by other unknown 

mechanisms, as described elsewhere in this book (see in particular 

Chap. 15 by Schulz). 

There are a number of distinct categories of mycorrhizal fungi, all of 

which differ from other fungi primarily because they are dual soil-plant 

inhabitants that are efficient at growth and nutrient uptake in both soils and 

plants (Brundrett 2002). Conversely, endophytes and pathogens are plant 

inhabitants, which do not require efficient means of acquiring nutrients 

from soils to supply plants (Table 16.1). The examples in this chapter 

primarily concern root-fungus associations, but mycorrhizal fungi and 

Mark C. Brundrett: School of Plant Biology, Faculty of Natural and Agricultural Sciences, 

The University of Western Australia, Crawley, WA 6009, Australia, E-mail: 

mark.brundrett@environmnent.wa.gov.au 





282 M.C. Brundrett 

Table 16.1. Comparison of mycorrhizal, parasitic and endophytic root associations (Brundrett 

2004). VAM Vesicular arbuscular mycorrhizas, ECM ectomycorrhizas 

Criteria Mycorrhizal Parasitic Endophytic 

Morphology Specialised hyphae in 

specialised plant organ 

Specialised hyphae Relatively 

unspecialised hyphae 

Development Synchronised Often synchronised Not synchronised 

Impact on fungus Obligate requirement 

for plant supplied 

nutrients (VAM and 

ECM) 

Fungus obligately or 

facultatively 

dependent on plant 

Fungus moderately or 

weakly dependent 

Impact on plant Strong or weak benefit Strong or weak harm Weak harm or benefit 

Nutrient transfer Synchronised transfer, 

fungusastrongsink 

Active or passive 

transfer, fungus 

astrongorweaksink 

Passive transfer, 

fungus not a strong 

sink 

endophytes also occupy other substrate-contacting organs (e.g. rhizomes, 

stems, scale leaves, etc.). 

16.1.2 

Roles of Endophytes and Mycorrhizal Fungi 

It is harder to separate mycorrhizal fungi from other functional groups 

of fungi than it is to separate mycorrhizal associations from other plantfungus 

relationships, as mycorrhizas are defined by morphological criteria 

(Brundrett 2004). Differences between endophytic associations and mycorrhizal 

associations are summarised in Table 16.1. The most important 

of these differences are the absence of substantial fungus-to-plant nutrient 

transfer in endophytic associations and the lack of synchronised development 

between endophytes and plants. Endophytic associations lack 

the specialised interface, formed by complex hyphal growth synchronised 

with substantial cytoplasm synthesis by host cells, that is diagnostic of 

mycorrhizas. The complex host-fungus interface in mycorrhizal associations 

allows rapid bi-directional transfer of nutrients over a relatively short 

time (a few weeks). However, nutrient transfer to fungi in endophytes is 

still likely to be important to the fungus if it continues over much longer 

time-spans (months or years) than the active phase of mycorrhizal associations. 

We would also not expect plant-fungus coevolution in endophytic 

associations if plants do not receive substantial direct benefits from these 

organisms. However, the results of plant evolution to become more efficient 

at forming mycorrhizas (e.g. roots that evolved to house fungi, Brundrett 

2002) would ultimately have also benefitted endophytes.

16 Roles of Endophytic and Mycorrhizal Fungi 283 

While we can safely say that endophytes are not mycorrhizal (as defined 

above), it seems likely that mycorrhizal fungi have endophytic phases 

involving long-term coexistence with plants without active growth or nutrient 

transfer. For example, old roots are an important source of inoculum 

for Glomeromycete fungi, which can survive as endophytes in living roots 

for up to 10 years after arbuscules collapse (Brundrett and Kendrick 1988). 

These fungi can also have a necrotrophic phase in dying roots, providing 

thefunguswithfirstaccesstonutrientsthatcanbetransferredtohyphae 

within other plants (Eason et al. 1991). Examples of endophytic activity by 

mycorrhizal fungi are summarised in the next section. 

16.2 

Endophytic Activity by Mycorrhizal Fungi 

Endophytic growth of mycorrhizal fungi in plants is common and differs 

from mycorrhizal associations primarily by the absence of defining 

morphological features, as discussed above. It is not difficult to explain 

the endophytic competence of mycorrhizal fungi in non-host plants, as 

we would expect that they would normally have the highest inoculum potentials 

of soil organisms and these fungi require the capacity to rapidly 

and efficiently colonise plant roots. It has been suggested that plants must 

employ effective defences if they are to remain free of mycorrhizal fungi 

(Brundrett 1991). 

Another difference between mycorrhizal and endophytic fungi is that the 

nutritional resources provided by endophytic activity (access to endophytic 

exudates) do not seem to be sufficient to sustain mycorrhizal fungi, which 

generally cannot persist in soils without forming mycorrhizas with host 

plants (Brundrett 1991). It has been suggested that mycorrhizal fungi may 

endophytically colonise roots and other soil organisms for shelter rather 

than food, to avoid the many soil organisms that prey on them (Koske 

1984). Such use of roots as shelters by mycorrhizal fungi likely would be 

of limited immediate significance to the plant in which this occurs, but 

should ultimately benefit other plants in the same ecosystem, by maintaining 

a reservoir of inoculum. Examples of suggested endophytic associations 

by mycorrhizal fungi and other fungi that frequent mycorrhizas are listed 

in sections below, with fungi classified by their primary roles. 

16.2.1 

Glomeromycotan (Vesicular-Arbuscular Mycorrhizal) Fungi 

Glomeromycotan fungi, forming vesicular arbuscular mycorrhizas [VAM, 

or arbuscular mycorrhizas (AM)], are ubiquitous soil organisms that


proliferate within patches of soil organic material (St John et al. 1983; Joner 

and Jakobsen 1995; Azcón-Aguilar et al. 1999). As shown in Fig. 16.1A, B, 

these fungi commonly grow in non-host plants (Brundrett and Kendrick 

1988; Imhof 2001). They also occupy plant organs other than roots, dead 

soil animals and spores of other VAM fungi, presumably to acquire nutri- 

Fig.16.1. A–D Roots cleared in potassium hydroxide and stained with Chlorazol Black E in 

lactoglycerol. A, B Endophytic growth of a Glomeromycotan fungus in the nonmycorrhizal 

plant Hydrophyllum virginianum consisting of vesicles (v)andhyphae(arrow)inarhizome 

scale A and nonmycorrhizal root B. C, D Dense growth by unknown fungi (arrows)onthe 

epidermis of roots of Scaevola crassifolia C and Acer saccharum D. These plants also have 

vesicular-arbuscular mycorrhizas. E, F Unstained roots of Eucalyptus globulus grown in 

pasteurised soil colonised by an unidentified opportunistic conidial fungus. E Hyphae and 

conidia (arrows) formed in soil. F Hyphae on the surface of long roots forming a patchy 

Hartig-net-like structure (arrow)


ents or avoid predation (Rabatin and Rhodes 1982; Warner 1984; St John 

et al. 1983; Koske 1984; Brundrett and Kendrick 1988). Mycorrhizal colonisation 

of some mutants of normally mycorrhizal species also resembles 

endophytic activity by these fungi, as they form hyphae and vesicles but 

not arbuscles (Demchenko et al. 2004). This suggests that VAM fungi will 

switch between endophytic and mycorrhizal activity in plants in response 

to signals provided by the plant. 

Nonmycorrhizal plants are defined as those that normally exclude mycorrhizal 

fungi from their healthy young roots (Brundrett 1991). As implied 

by this definition, endophytic colonisation of nonmycorrhizal plant roots 

byVAMfungiiscommoninolderroots,butisconsideredtobeoflimited 

functional significance because it does not result in plant growth responses 

(Ocampo 1986; Muthukumar et al. 1997; Giovannetti and Sbrana 1998). 

However, there may be a fine line between nonmycorrhizal and facultatively 

mycorrhizal species in which VAM associations are present or absent 

as a result of soil conditions (see Brundrett 1991). These issues are best illustrated 

by the debate about the role of mycorrhizal associations in sedges 

(Cyperaceae and allied families) that has been ongoing for decades (Powell 

1975; Tester et al. 1987; Brundrett 1991; Muthukumar et al. 2004). The 

statement by Muthukumar et al. (2004) that the Cyperaceae is a mycorrhizal 

family, is in disagreement with the opinion of earlier reviewers (e.g. 

Powell 1975; Tester et al. 1987; Brundrett 1991) and is not as clear-cut as it 

seems. Half of sedges examined in studies summarised by Muthukumar et 

al. (2004) contained mycorrhizal fungi. However, they could not clearly distinguish 

endophytic from mycorrhizal associations using the information 

provided in many studies, and they suspected that these associations were 

often non-functional. Detailed studies of sedges such as Carex spp. have 

found them to be nonmycorrhizal throughout the year with endophytic 

VAM hyphae in older roots (Brundrett and Kendrick 1988; Cornwell et 

al. 2001). There may be a continuum from mycorrhizal to nonmycorrhizal 

species in plant families such as the Cyperaceae. In facultatively mycorrhizal 

species, VAM fungi may sometimes function more as endophytes than as 

mycorrhizal associates, as they can contribute to disease suppression in the 

absence of growth responses (Newsham et al. 1995; Cordier et al. 1998). 

It is often assumed that sparse or inconsistent mycorrhizal formation is 

of limited benefit to plants, but this has rarely been tested in experiments 

using appropriate soil conditions. 

A second common example of cases where the hyphal growth of Glomeromycotan 

fungi is difficult to interpret is their colonisation of roots of 

ectomycorrhizal (ECM) plants in species with dual mycorrhizal associations. 

In plants with both ECM and VAM, their relative importance can 

vary due to the age of plants and the habitats in which they grow (Chen 

et al. 2000; van der Heijden 2001). Most trees with dual ECM/VAM asso-


ciations are considered to benefit primarily from ECM, but large growth 

responses to VAM occur in experiments, especially when plants are young 

(Brundrett et al. 1996; Chen et al. 2000). A further complication is the fact 

that Glomeromycete fungi are commonly present as endophytes in roots of 

ECM plants (e.g. Harley and Harley 1987; Cázares and Trappe 1993; Smith, 

et al. 1998). This endophytic activity is distinguished from functional dual 

ECM/VAM associations by the absence of arbuscules. Glomeromycotan 

fungi can also grow endophytically in orchids (Hall 1976). 

16.2.2 

Ectomycorrhizal Fungi 

Ectomycorrhizal associations are defined by a key morphological criterion: 

labyrinthine Hartig net hyphae in an interface between cells of the primary 

root cortex or epidermis. However, designation of ECM without a welldefined 

Hartig net is not always clear cut, as “superficial” ECM roots with 

a thin or patchy Hartig net occur in synthesis experiments using hostfungus 

combinations that are not fully compatible (Burgess et al. 1994; 

Peterson and Massicotte 2004). These also occur in nature, where they are 

believed to benefit plants (Malajczuk et al. 1987). When initiated by spores 

or other limited sources of inoculum in experiments, ECM fungi weakly 

colonise root surfaces before they have sufficient energy to form typical 

mycorrhizas (Chilvers and Gust 1982). This establishment phase may equate 

to a transition from endophyte-like to mutualistic activity. Some fungi form 

associations with a mantle but no Hartig net on non-host roots, such as 

those of Morchella sp. on Pinaceae (Dahlstrom et al. 2000), Cortinarius sp. 

on Carex (Harrington and Mitchell 2002) and Tricholoma sp. on Pinus (Gill 

et al. 1999). The opportunistic growth of fungal hyphae on roots is common 

in nature and could be considered a form of endophytism in which fungi 

feed on root exudates without penetrating cells (Fig. 16.1C, D). 

Other examples of fungi that seem to occupy intermediate positions 

on an ECM-endophyte continuum are opportunistic fungi that weakly 

colonise seedling roots in the nursery (see Fig. 16.1E, F). These nursery 

fungi fill an empty niche in potting mixes that lack mycorrhizal fungus 

propagules or that are not conducive to growth by mycorrhizal fungi. 

These nursery fungi are probably of limited functional significance, as 

suggested by similar fungal growth on both long and short roots, a patchy 

Hartig net and the absence of morphological responses by short roots 

(root swelling). Ectendomycorrhizas, a variant of ECM, also tend to occur 

in substrates lacking other fungi and where nutrient levels are high, and so 

are considered to be of limited benefit to plants (Yu et al. 2001).


16.2.3 

Fungi in Orchids 

Most fungi that form orchid mycorrhizas are basidiomycetes belonging to 

a diverse assemblage of fungi assigned by morphological features to the 

asexual form genus Rhizoctonia (see also Chap. 9 by Bayman and Otero). 

These fungi are also more precisely defined using anastomosis reactions, 

enzyme assays and DNA-based methods (Currah et al. 1997; Taylor et al. 

2002; McKormick et al. 2004). Mycorrhizal associates of orchids occur in all 

three clades of the polyphyletic Rhizoctonia complex (Sebacinaceae, Tulasnellales, 

Ceratobasidiales), but are rare in sister clades of basidiomycetes 

(Taylor et al. 2002). As shown in Table 16.2, Sebacina members include 

ECM fungi, orchid mycorrhizal associates, and bryophyte fungi, and also 

occur in ericoid plants, but there is some doubt about how much roles 

overlap between separate clades within this genus (Warcup 1981; Selosse et 

al. 2002a, 2002b). Another multifunctional orchid fungus is Thanatephorus 

gardneri, the mycorrhizal associate of the underground orchid (Rhizanthella 

gardneri), which also forms ECM with Melaleuca uncinata and is 

capable of endophytic activity (Table 16.2). This multifunctional fungus is 

the only known member of the Rhizoctonia alliance outside of Sebacina 

that forms ECM. 

Attempts to isolate mycorrhizal fungi from orchids often produce cultures 

of bacteria, actinomycetes and common endophytes such as Fusarium 

and Verticillium,aswellasECMfungiandericoidfungi(Richardsonand 

Currah 1995; Currah et al. 1997; Kristiansen et al. 2001; Bayman et al. 2002; 

Otero et al. 2002, Bidartondo et al. 2004). Most of these other fungi seem 

to be endophytes, but Fusarium isolates are also capable of forming orchid 

mycorrhizas (Vujanovic et al. 2000; Abdul Karim 2005). We have observed 

that the frequency of isolation of non-Rhizoctonia fungi decreases substantially 

when fungi are isolated from single pelotons rather than from 

surface-sterilised tissue blocks, providing further evidence that many fungi 

isolated by the latter method are endophytes. It is recommended that only 

isolates that germinate orchids to an advanced seedling stage with a green 

leaf be designated as orchid mycorrhizal fungi (Batty et al. 2002). 

Kottke et al. (2003) found members of the Rhizoctonia complex (Tulasnella 

and Sebacina) closely related to orchid fungi that formed mycorrhizalike 

associations with hepatics. These bryophytes also contained ascomycetes, 

and the nature of these associations requires further investigation. 

Members of the Rhizoctonia complex include major pathogens of seedlings 

of crop and horticultural species (Sivasithamparam 1998), but the degree 

of overlap between pathogens and orchid associates is unknown (Batty et 

al. 2002). In general, fungi associating with orchids differ substantially 

from other mycorrhizal fungi, because they do not belong to discrete


Table 16.2. Examples of fungi with multifunctional roles. Fungi within a row have been shown to be closely related by molecular identification 

Orchid mycorrhizas Other role 

Fungus Endophyte Pathogen Ectomycorrhizal Ericoid mycorrhizas 

Decomposition of 

wood? 

Myco-heterotrophic 

orchids 

(McCormick et al. 2004; 

Primary role 

(Köljalg et al. 2000) 

Tomentella spp. 

(Thelephoraceae) 

Taylor et al. 2002) 

Mycorrhizas in hepatics 

(Kottke et al. 2003) a . 

See below Many orchid associates 

(see text) 

Major role: 

(see text) 

Rhizoctonia complex S.L. Common: e.g. 

Pinus sylvestris 

Saprophytic 

(Sen et al. 1999) 


orchids (Selosse et al. 

2002b; Taylor et al. 

2003) a .Saprophytic? 

Some orchids (Warcup 

1981; Y. Bonnardeaux, 

personal 

Suspected 

role? (Allen 

et al. 2003) 

(Glen et al. 2002; 

Selosse et al. 2002a; 

Urban et al. 2003) a 

Ericaceae? 

(Allen et al. 2003) 

Sebacina spp. 

(in Rhizoctonia complex) 

communication) 

Saprophytic? 


orchid Rhizanthella 

gardneri (Warcup 1985; 

Mursidawati 2003) 

Melaleuca uncinata 

and other plants 

(Warcup 1985; 

Mursidawati 2003) 

ECM of trees 

(Harney et al. 1997) 

Some endophytic 

activity 

(Mursidawati 

Thanatephorus gardneri 

(in Rhizoctonia complex) 

Saprophytic phases 

Tropical orchids 

(Bayman et al. 2002; 

Abdul Karim 2005) 

e.g. 

Pamphile 

and Azevedo 

2002 

2003) 

Phialocephala spp. Widespread 

(DSE fungi) 

(see text) 

Fusarium spp. Common (Kuldau 

and Yates 2000; 

Redman et al. 

2001)



Other role 

Orchid 

mycorrhizas 

Mycorrhizas of 

bryophytes (Duckett 

and Read 1995) a 

Fungus Endophyte Pathogen Ectomycorrhizal Ericoid 

mycorrhizas 

Hymenoscyphus ericae Picea mariana roots 

Six tree spp. 

(Piercey et al. 2002), 

(Vrålstad et al. 2000) 

Bryophyte 

a 

Primary role 

(see text) 

(Chambers et al. 1999) 

Some isolates are 

strong saprophytes 

(Piercey et al. 2002) 

Primary role? 

(see text) 

Oidiodendron spp. ECM roots of Quercus ilex 

(Bergero et al. 2000) 

Trees 

(Sakakibara et 

al. 2002) a 

In ECM of Betula platyphylla 

(Hashimoto and 

Hyakumachi 2001) 

Mycelium radicis 

atrovirens 

a Role not fully-established


evolutionary or taxonomic groups, and orchid mycorrhizal associations 

are not their primary ecological role (Brundrett 2002). These fungi are 

efficient plant colonisers where each has multiple roles as endophytes, parasites, 

ECM fungi and saprophytes. We require knowledge about the biology 

of these fungi in natural ecosystems to help us understand and control their 

plant parasitic activities, as well as their beneficial mycorrhizal associations 

with orchids. 

16.2.4 

Ericoid Mycorrhizal Fungi 

Mycorrhizal fungi that associate with members of the Ericaceae (including 

the Epacridaceae) include several discrete groups of ascomycetes (McLean 

et al. 1999; Monreal et al. 1999; Sharples et al. 2000). Ericoid fungi with 

dual roles include Hymenoscyphus ericae, which forms ECM and can also 

associate with bryophytes (Table 16.2). However, ericoid fungi also endophytically 

colonise roots of ECM hosts without forming mycorrhizas 

(Bergero et al. 2000; Piercey et al. 2002). Some strains of the ericoid fungus 

Oidiodendron maius, which do not form mycorrhizal associations (Piercey 

et al. 2002), appear to be efficient saprophytes (see Chap. 13 by Rice and 

Currah). The primary role of ericoid fungi is not clear, as their occurrence 

in soils is independent of their host plants [Sharples et al. 2000; Bergero 

et al. 2003; see Chaps. 12 (Girlanda et al.) and 14 (Cairney)] and they also 

have a high degree of endophytic, or saprophytic competence, or have ECM 

associations with trees (Table 16.2). 

16.2.5 

Endophytic Fungi in Mycorrhizal Roots 

Dark septate fungi (called DSF or DSE fungi) are common root endophytes 

in many ecosystems – in some cases with dual roles as ECM fungi (Stoyke 

and Currah 1991; O’Dell and Trappe 1992; Ahlich and Sieber 1996; see 

Chap. 7 by Sieber and Grünig). The DSE root endophytes may provide benefits 

to plants (Jumpponen and Trappe 1998). They include Phialocephala 

spp. with close relatives that are ECM or ericoid fungi (Vrålstad et al. 2002). 

However, DSE fungi do not form mycorrhizal associations as defined by 

morphological criteria. Phialocephala fortinii,anECMfungusthatmaynot 

benefit host plants, is closely related to other dematiaceous fungi, which 

are common root endophytes (Harney et al. 1997). Root endophytes can 

act as antagonists of ECM fungi, apparently by competing for space in roots 

(Hashimoto and Hyakumachi 2000, 2001). The role of fungi such as DSE 

and MRA [Mycelium radicis atrovirens; see Chaps. 7 (Sieber and Grünig)


and 12 (Girlanda et al.)], which commonly share roots with mycorrhizal 

fungi, has not been well established. 

16.3 

Issues with the Identification 

and Categorisation of Fungi in Roots 

Distinguishing endophytic activity from mycorrhizal associations caused 

by the same fungi is often problematic, especially for multifunctional fungi 

that are both endophytes and mycorrhizas. The examples discussed above 

illustrate how it is essential to use consistent definitions of mycorrhizal 

associations based on the structure and development of associations. A key 

defining feature of mycorrhizal associations is that they develop in young 

roots (Brundrett 2004). Consequently, mycorrhizal formation requires root 

growth while endophytic activity is not confined to young healthy roots. 

Despitethecommonoccurrenceofendophyticactivitybymycorrhizal 

fungi, we have very little knowledge of its ecological significance. Damage 

to roots of non-hosts plants caused by attempted colonisation by VAM 

and ECM fungi can lead to substantial growth reduction (Allen et al. 1989; 

Plattner and Hall 1995; Muthukumar et al. 1997). However, cases of antagonistic 

interactions caused by the endophytic activity by mycorrhizal fungi 

in non-host plants seem to be rare. 

The common occurrence of endophytic fungi in roots may cause confusion 

with mycorrhizal fungi, especially when they are identified from DNA. 

In most cases, DNA extraction will detect several different fungi within 

a root and it may not be clear which are most abundant. Examples of cases 

where roles of fungi are uncertain include the study by Allen et al. (2003), 

where Sebacina spp. dominated DNA sequences from Gaultheria shallon 

(Ericaceae) roots, but did not form ericoid mycorrhizas. Another study 

by Bidartondo et al. (2004) found ECM fungi in addition to endophytes 

and orchid associates in five orchid species. They concluded that the ECM 

fungi were mycorrhizal associates of these orchids, but did not confirm 

this by mycorrhizal synthesis. There are numerous other examples in the 

recent literature of fungi identified in roots that have been designated as 

mycorrhizal without providing sufficient evidence. 

Molecular methods used to detect fungi in roots or soils are still relatively 

new and more research is required to test the relative efficiency with 

which they detect different categories of fungi. In contrast, identification of 

mycorrhizalassociationsbymorphologyallowsamuchhigherdegreeof 

replication and can be more accurate than DNA-based studies, which sometimes 

fail to detect the most important fungi in roots. The interpretation 

of fungal presence data provided by molecular tools that allow miniscule


traces of fungi to be detected from almost any substrate will require further 

testing of key questions. These questions include: (1) Are the most common 

fungi in roots amplified most often? (2) Which of the detected fungi occur 

on the surface of roots or are endophytes? (3) How often are traces of DNA 

that contaminate samples detected? These questions can be answered by 

increasing the replication of sampling strategies, or combining isolation 

attempts with DNA extractions (e.g. Allen et al. 2003), but it may be even 

better to develop integrated approaches combining molecular and microscopic 

techniques. One such study by Sakakibara et al. (2002) found that 

microscopic identification of fungi by morphotypes and molecular methods 

(PCR-RFLP) were in close agreement, but multiple fungi were obtained 

from many mycorrhiza types. 

Mycorrhizologists often use the term “endophytes” to refer to both mycorrhizal 

and non-mycorrhizal fungi in roots. However, due to the increasing 

recognition of the importance of “endophytes” as a specific category of 

specialised plant-inhabiting fungi, it is no longer appropriate to call mycorrhizal 

fungi endophytes. Types and categories of mycorrhizas are defined 

by morphological criteria (see Brundrett 2004), so mycorrhizal fungi need 

to be linked to a properly identified mycorrhizal structure. We should designate 

fungi as mycorrhizal only if they are isolated from a mycorrhiza by 

reliable means and belong to known groups of mycorrhizal associates. It 

will be necessary to confirm the mycorrhizal status of fungi by resynthesis, 

if it is not clear if they are endophytic or mycorrhizal. 

16.4 

Evolution of Root-Fungus Associations 

An understanding of the capacity for mycorrhizal fungi to grow endophytically 

has led to a new theory of how these associations evolved (Brundrett 

2002). This theory suggests that the switch to a new mycorrhizal association 

type starts with endophytic occupation of roots by fungi, and culminates 

in fully functional associations with coordinated development and synchronised 

nutrient transfer. The high degree of endophytic competence of 

Glomeromycotan fungi apparently has resulted in reacquisition of VAM by 

some plant lineages such as the Ericaceae in Hawaii (Koske et al. 1990). 

However, the reverse trend, where plant families become nonmycorrhizal 

is more common (Brundrett 2002). Other examples of new mycorrhizal 

associations, which probably started by endophytic fungal activity, include 

plants with dual ECM and VAM associations. These are common in some 

plant families that have ancestors with VAM (Brundrett 2002). There also 

seem to be intermediate types of ECM associations that involve new lineages 

of fungi that are not fully functional, as described above.


The Orchidaceae contain the most examples of plants acquiring new 

lineages of symbiotic fungi, in both myco-heterotrophic and green species 

(Taylor et al. 2002; Bidartondo et al. 2004). The fungal associates of these 

orchids are amazingly diverse, and the only theme that unifies them is that 

most are known to be efficient plant colonists as pathogens, endophytes or 

ectomycorrhizal associates of other species (Brundrett 2002). This suggests 

that most of these fungi were endophytes within orchids before the orchid 

evolved means to exploit them for its own purposes. All orchids seem to 

require is a fungus that can efficiently invade their roots or stems, but 

they seem to have much more success controlling fungi in the Rhizoctonia 

alliance than other fungi with similar endophytic competence for reasons 

we do not understand. 

16.5 

Conclusions 

As demonstrated by the examples described above, fungi often have several 

roles and the primary roles of multifunctional fungi may not be certain. 

However,thenumberofrolesfungihavedoesseemtobelimited,presumablybecausetheycannotbeproficientatalloftheseroles.Consequently, 

we would expect multifunctional fungi to lose out due to competition from 

more efficient fungi that are more highly specialised. There also seem to be 

advantages to versatility in fungi. For example, multifunctional fungi seem 

to colonise new habitats or substrates more rapidly than specialised fungi, 

as shown by studies of mycorrhizal fungal succession after disturbance and 

the associations of nursery-grown plants (Jumpponen and Trappe 1998; Yu 

et al. 2001). Mycorrhizal fungi take part in a continuum of association types 

starting with endophytic associations and concluding with mycorrhizal associations. 

However, the endophytic and intermediate roles of these fungi 

seem to be less common than mycorrhizal associations, suggesting that 

there are clear advantages to fungi from their primary roles with plants. 

Recent advances in molecular biology have revealed a much more diverse 

and complex picture of the multifunctional nature of mycorrhizal fungi. 

Further research is required to determine the relative importance of fungi 

detected in roots, as it is not always clear which are endophytes and which 

are mycorrhizal associates, or how these roles change with time. 


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17 

17.1 

Introduction 

Isolation Procedures 

for Endophytic Microorganisms 

Johannes Hallmann, Gabriele Berg, Barbara Schulz 

There are good reasons for isolating endophytic microorganisms, e.g. for 

their characterisation, for studying population dynamics and diversity, use 

of microbial inoculants to improve plant growth and plant health, and 

as sources of novel biologically active secondary metabolites (Schulz et 

al. 2002; Strobel 2003; Schulz and Boyle 2005). The isolation procedure 

is a critical and important step in working with endophytic bacteria and 

fungi. It should be sensitive enough to recover endophytic microorganisms, 

butatthesametimebestrongenoughtoeliminateepiphytesfromtheroot 

surface. In practice, this is often difficult, because microorganisms attach to 

plant cells/surfaces or hide in intercellular niches, thus evading the isolation 

procedure. Besides, there is no sharp delineation between the external and 

internal plant environment. Some bacteria constantly move between these 

two microhabitats, e.g. fungal mycelia grow from the root surface into the 

roots. Therefore, in order to be effective, the isolation procedure must 

be adapted to the respective tissues and microorganisms. Some isolation 

procedures are suitable only for certain plant tissues, whereas others favour 

local or systemic colonisers. 

Themostcommonlyusedisolationprocedurescombinesurfacesterilisation 

of the root tissue with either maceration of the plant tissue and streaking 

onto nutrient agar, or plating small sterilised segments onto nutrient 

agar. Methods that avoid surface sterilisation are also available, e.g. vacuum 

or pressure extraction. In the past, isolation procedures focused primarily 

on culturable microorganisms. However, increasing interest in nonculturable 

endophytic microorganisms has recently led to the application 

Johannes Hallmann: Federal Biological Research Centre for Agriculture and Forestry, Institute 

for Nematology and Vertebrate Research, Toppheideweg 88, 48161 Münster, Germany, 

E-mail: j.hallmann@bba.de 

Gabriele Berg: Technical University of Graz, Department of Environmental Biotechnology, 


Barbara Schulz: Institute of Microbiology, Technical University of Braunschweig, Spielmannstraße 

7, 38106 Braunschweig, Germany 





300 J. Hallmann et al. 

of molecular methods for their identification. This chapter summarises isolation 

procedures for culturable and detection methods for non-culturable 

endophytic bacteria and fungi of plant roots and discusses the strengths 

and weaknesses of each procedure. Examples are given on how the choice 

of the procedure can affect the microbial spectrum being isolated. The information 

provided here should enable the researcher to specifically select 

the method most suitable to meet their research objectives. The reader is 

also referred to the review by Sieber (2002), who summarised the available 

sterilisation and isolation procedures for fungi in plant roots. 

17.2 

Surface Sterilisation 

Theoretically, the sterilising agent should kill any microbe on the plant 

surface without affecting the host tissue and the endophytic microorganisms. 

However, this is difficult to achieve because in time the agent may 

penetrate the root tissue. Conditions required to kill the last microbe on 

the surface may already be lethal for some endophytic microorganisms. In 

general, surface sterilisation of root tissue consists of the following steps: 

(1) thorough washing of the root tissue under tap water to remove adhering 

soil particles and the majority of microbial surface epiphytes and incidentals, 

(2) pre-treatment (optional) to eliminate hydrophobic substances on 

the plant surface and to provide better access for the sterilising agent to 

the root surface, (3) surface sterilisation to eliminate remaining microbial 

colonisers from the root surface, (4) several rinses under aseptic conditions 

in a sterile washing solution and (5) sterility check to confirm complete 

sterilisation of the root surface. It is very important that sterility is guaranteed 

for all tools and during all steps of this procedure, and that the 

researcher optimises the procedure for each plant tissue, since sensitivity 

varies with species, age and surface properties. 

All steps in the sterilisation procedure should be conducted with sterilised 

tools, e.g. pincers, and preferably under a laminar flow hood. Between 

steps the tissue should be blotted on filter paper to avoid dilution of the 

sterilising agents. 

17.2.1 

Pre-treatment 

Roots usually do not require special pre-treatment, since their surfaces do 

not contain hydrophobic substances like, e.g., the waxes of leaves. Washing 

with tap water and/or soft brushing is usually adequate. However, sonification 

may be used to dislodge soil and organic matter from the roots prior


to surface sterilisation (Holdenrieder and Sieber 1992; Coombs and Franco 

2003a). 

17.2.2 

Sterilising Agents 

Commonly used sterilising agents are sodium hypochlorite (Gardner et al. 

1982; Quadt-Hallmann et al. 1997; Schulz et al. 1993; Sieber 2002), ethanol 

(Dong et al. 1994; Gagné et al. 1987), and hydrogen peroxide (McInroy 

and Kloepper 1994; Misahgi and Donndelinger 1990; Sieber 2002). For 

health reasons, mercuric chloride should be used only very carefully and 

exclusively for plant tissues that cannot otherwise be sterilised (Gagné et al. 

1987; Hollis 1951; O’Dell and Trappe 1992; Sriskandarajah et al. 1993). Note 

that sodium hypochlorite, an excellent oxidant, decomposes spontaneously 

in storage. Other, less frequently used, agents include propylene oxide 

vapour (Sardi et al. 1992) and formaldehyde (Schulz et al. 1993; Cao et 

al. 2002). Woody root tissues may also be dipped for 15 s in 95% ethanol 

and flame sterilised as employed for alfalfa roots by Gagné et al. (1987). In 

addition, the outer layer of woody root tissue can be aseptically peeled and 

the internal plant tissue can be excised using a sterile cork borer (Maifeld 

1998; Reiter et al. 2002; Zinniel et al. 2002). 

Combinations of agents can improve the effectiveness of surface sterilisation. 

For example, a combination of physical and chemical sterilisation has 

given good results with lignified roots (Table 17.1; Görke 1998). A common 

protocol involves a three-step procedure with ethanol, sodium hypochlorite, 

and then ethanol again (Schulz et al. 1993; Bills 1996; Sieber 2002). 

For example, for wheat roots, Coombs and Franco (2003b) applied 99% 

ethanol for 1 min, 3.1% sodium hypochlorite for 6 min and 99% ethanol 

for 30 sec. Sieber (2002) suggested omitting the initial soak in ethanol because 

it leads to contraction of the plant tissues, perhaps entrapping some 

epiphytic conidia, spores or mycelia. All the conidia of Penicillium sp. on 

roots of Norway spruce (artificially contaminated) were killed when surface 

sterilisation with hydrogen peroxide was used without the initial soak in 

ethanol (Sieber 2002). Examples of commonly used sterilising agents and 

conditions for their application are given in Table 17.1. 

In general, a more stringent sterilisation procedure can be used with 

older and lignified plant tissue. Incubation times as well as concentrations 

of the sterilising agents directly affect the results. For example, an increase 

in the concentration of sodium hypochlorite from 3 to 6% active chlorine 

under otherwise constant conditions increases the number of root samples 

with complete sterilisation by 40%, but at the same time decreases 

the population densities of endophytic bacteria from 4.36×10 3 cfu/ml to


Table 17.1. Protocols for surface sterilisation of root tissue 

Procedures and sterilising agents Incubation Root type, age and/or Plant Reference 

time diameter 

Bacteria 

a) Wash under running tap water 5- to 7-week-old roots Cucumis sativus, Gossyium Hallmann et al. 1998; 

b) 1–3.1% Sodium hypochlorite hirsutum, Pinus contorta McInroy and Kloepper 1995; 

var. latifolia, Zea mays Shishido et al. 1995 

a 1–2 min 

c)Twotofourrinsesinsterilewater 

a) 99% Ethanol 1 min 1- to 3-month-old roots Triticum aestivum Coombs and Franco 2003a 

b) 3.1% Sodium hypochlorite 6 min 

c) 99% Ethanol 0.5 min 

a) Wash with soapy water 4-to 28-month-old roots Medicago sativa Gagné et al. 1987 

b) Wash thoroughly with tap water 

c) 0.2 M HgCl2 in 50% ethanol 4 min 

d) Three rinses in sterile water 1 min 

a) Wash under running tap water 3 min 1- to 4-year-old roots Citrus jambhiri Gardner et al. 1982 

b) Dip in 95% ethanol 

c) Flame 

Sardi et al. 1992 

Various herbaceous 

and woody plants 

d)Rinseinsterilewater 

a) Wash under running tap water Roots 1- to 5 mm in 

b) Exposure to propylene oxide vapor 1 h diameter 

20 min 13- to 14-week-old roots Solanum tuberosum Sessitsch et al. 2002 

Physical sterilisation 

a) Shake vigorously in 0.9% NaCl solution 

containing 0.3 g acid-washed glass beads 

b) Five Rinses in sterile water 

Fungi 

36% Formaldehyde Musa acuminata Cao et al. 2002



Plant Reference 

Oberholzer-Tschütscher 1982; 

Sieber et al. 1988; 

Crous et al. 1995b Triticum aestivum, 

Erica carnea 

Skipp and Christensen 1989; 

Kattner and Schönhar 1990; 

Hambleton and Currah 1997; 

Stoyke and Currah 1991b . 

Hallmann and Sikora 1994 

Lolium perenne, Picia abies 

various species of Ericaceae, 

various alpine plant species 

Lycopersicon esculentum 

Procedures and sterilising agents Incubation Root type, age and/or 

time diameter 

a) Wash under running tap water Nodule roots, fine hair 

b) 96% Ethanol 0.5–1.0 min roots, nonlignified 

c) 2–2.5% Sodium hypochlorite 1–4 min roots, lignified roots 

d) 96% Ethanol 0.5 min 

a) Wash under running tap water Adventitious roots, fine 

b) 0.3–2% Sodium hypochlorite 1–3 min hair roots 

c)Rinseinsterilewater 

Boyle et al. 2001 

Petrini et al. 1992; 

Werner et al. 1997b O’Dell and Trappe 1992 b 

a) Wash under running tap water Roots Hordeum vulgare, 

b) 70% Ethanol 0.5 min 

Phaseolus vulgarum 

c) 1–3% Sodium hypochlorite 1–3 min 


a) Wash under running tap water 1 min Rhizomes; 

Pteridium aquilinum; 

b) 75% Ethanol 3 min 6- to 9-month-old roots Aphelandra tetragona 

c) 3–5% Sodium hypochloritea 0.5 min 

d) 75% Ethanol 

0.01% Mercuric chloride 5–15 min Fine roots Lupinus spp., 

Oxytropis campestris 

O’Dell and Trappe 1992 b 

5% Hydrogen peroxide 5–15 min Fine roots Lupinus spp., 

Oxytropis campestris



Procedures and sterilising agents Incubation Root type, age and/or Plant Reference 

time diameter 

a) Wash under running water Adventitious roots Oryza sativa Fisher and Petrini 1992b b) 75% Ethanol 1 min 

c) 20% Sodium hypochlorite 3 min 


a) Wash under running water Non-ectomycorrhizal Abies alba Ahlich and Sieber 1996 

roots, 0–5.3 mm in 

diameter 

b 

b) 99% Ethanol 1 min 

c) 35% Hydrogen peroxide 5 min 


Picea abies Maifeld 1998 b 

Görke 1998 b 

Fagus sylvatica, Picea abies, 

Pinus sylvestris, Betula 

pendula 

Physical sterilisation 

a) Cut core into pieces Boring cores from which 

b) Move pieces quickly through 

bark has been removed 

flame of Bunsen burner 

Physical + chemical sterilisation 

a) Wash under running tap water 2- to 9-year-old roots 

b) 70% Ethanol 1 min undergoing secondary 

c) 10% Sodium hypochlorite 3 min growth 


e) Rinse twice in sterile water 

f) Discard bark 

g) Move pieces quickly through 

flame of Bunsen burner 

aConcentrations of sodium hypochlorite represent percent active chlorine 

bAdapted from Sieber 2002


5.75 × 10cfu/ml (A. Munif, personal communication). The effect of the 

sterilising agent penetrating the plant tissue on the bacterial cells was visually 

demonstrated by treating the root tissue with a tetrazolium-phosphate 

buffer solution (Patriquin and Döbereiner 1978). While metabolically active 

bacterial cells reduced tetrazolium and became stained, cells killed 

by the sterilisation remain unstained. Alternatively, apoplastic dyes such 

as 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) or 4.4 ′ -bis (2-sulfostyryl) biphenylcanbeusedtomonitortherelativeprogressionoftheappliedagent 

into the plant tissue (Petersen et al. 1981). 

17.2.3 

Surfactants 

Surfactants such as Tween 20 (Mahaffee and Kloepper 1997), Tween 80 

(Sturz 1995) or Triton X-100 (Misaghi and Donndelinger 1990) added to 

the sterilising agent reduce the surface tension and allow the agent to reach 

into niches and grooves beyond the epidermal cells (Bills 1996; Hallmann 

et al. 1997a). 

17.2.4 

Rinsing 

After each treatment, the plant tissue should be rinsed repeatedly in sterile 

water. 

17.2.5 

Sterility Check and Optimisation 

Only if complete surface sterilisation of the root tissue is confirmed, can the 

isolated microorganisms be assumed to be endophytes. Validation of the 

surface sterilisation procedure can be done by (1) imprinting the surfacesterilised 

plant tissue onto nutrient media (Pleban et al. 1995; Shishido et 

al. 1995; Schulz et al. 1998), (2) culturing aliquots of water from the last 

rinsing onto nutrient media (McInroy and Kloepper 1994) or (3) dipping 

the roots into nutrient broth (Gagné et al. 1987). All three methods gave 

comparable results (Musson et al. 1995). If no microbial growth occurs on 

themedium,surfacesterilisationisconsideredcomplete.Afurthercheckis 

to test the effect of the sterilising agent directly on fungal or bacterial cells. 

Rootsaredippedintoafungalorbacterialsuspensionofknowndensity, 

slightly dried, surface sterilised and subjected to a sterility check (Petrini 

1984; Coombs and Franco 2003a).


17.3 

Culture of Tissue and Plant Fluid of Sterilised Roots 

on Nutrient Medium 

17.3.1 

Segments 

The surface-sterilised root is cut aseptically into segments, which are plated, 

i.e. pressed directly, onto an appropriate nutrient medium (see below). It 

is very important to cut the tissue into very small and thin segments, since 

colonisation may be very limited (Carroll 1995; Boyle et al. 2001). For fungal 

isolation, the nutrient medium should be supplemented with antibiotics to 

suppress bacterial growth. When isolating fungi, antifungal substances may 

also be added to retard the growth of fast-growing fungi; when isolating 

bacteria to inhibit growth of fungi (see below). Endophytic microorganisms 

that emerge from the root fragments are subsequently transferred to 

freshmedia.Thismethodiscommonlyusedforendophyticfungi(Schulz 

et al. 1993; Hallmann and Sikora 1994; Bills 1996) and actinobacteria (Sardi 

et al. 1992; Coombs and Franco 2003a), but is rarely appropriate for endophytic 

bacteria (Araújo et al. 2001). This method selects for fast-growing 

microorganisms, therefore representing more a qualitative than a quantitative 

approach. When studying microbial diversity, slow growing microorganisms 

may be under-represented. In addition, concomitant growth 

of two or more microorganisms necessitates subsequent separation of the 

isolates and may also result in a lower number of colonies being recovered 

(Elvira-Recuenco and van Vuurde 2000). 

17.3.2 

Maceration of Root Tissue 

Maceration is the preferred method for the isolation of endophytic bacteria 

from surface sterilised tissue, but may also be employed for isolating endophytic 

fungi (Sieber 2002), particularly slow growing ones. Theoretically, 

it captures all endophytic colonisers of the root tissue, i.e. colonisers of 

the root cortex as well as of the vascular tissue, systemic as well as local 

colonisers, and intercellular as well as intracellular colonisers. Maceration 

is useful for determining the broad spectrum of culturable bacterial and 

fungal endophytes; however, it excludes obligate biotrophs. To improve 

maceration and form a homogenous suspension, sterile water or sterile 

buffer solution is usually added to the surface-sterilised root tissue prior to 

maceration. Maceration of the surface-sterilised tissue can be performed


with mortar and pestle or with mechanical devices such as a Klecco tissue 

pulveriser (Mahaffee and Kloepper 1997), a Polytron homogeniser (Zinniel 

et al. 2002) or a blender (Araújo et al. 2001), depending on sample size 

and the hardness of the plant material. Especially for woody plant tissues, 

processing by hand can be laborious and exhausting and mechanical devices 

are advisable. For the latter, optimal time and intensity of maceration 

need to be empirically determined. Following maceration of the surfacesterilised 

root tissue, the suspension is streaked onto nutrient medium. 

Sterility has to be guaranteed during all steps of this procedure. Since 

plant enzymes and toxins released during maceration can inactivate or 

kill endophytic microorganisms, temperature should not increase to lethal 

levels,necessitatingcooling;thesampleshouldbeimmediatelydilutedto 

decrease the concentrations of toxic compounds to ineffective levels. Alternatively, 

substances can be added to buffer the toxic compounds, e.g. 

polyvinylpyrrolidone (PVP) or EDTA. To minimise the time required for 

the entire procedure from surface sterilisation to maceration, Musson et al. 

(1995) described a microtitre plate method in which all steps are included 

in one plate. This method resulted in a higher detection limit and improved 

sterility, but was limited to small sample sizes. 

17.3.3 

Centrifugation of Root Tissue 

Centrifugation is commonly used to collect the intercellular (apoplastic) 

fluid of plant tissue (De Wit and Spikman 1982; Boyle et al. 2001), but 

may also be used to extract endophytic microorganisms. This technique 

has been successfully applied for the isolation of endophytic bacteria from 

sugarcane stems (Dong et al. 1994), but supposedly is also suitable for the 

isolation of endophytic microorganisms from root tissue. Depending on 

sample size, sterile Eppendorf tubes or larger glass test tubes are used for 

centrifugation of the surface-sterilised tissues. Most of the apoplastic fluid 

will be removed at 3,000g (Dong et al. 1994). The collected plant fluid is 

then streaked on nutrient medium for bacterial and fungal recovery. This 

approach avoids maceration of the root tissue, as demonstrated by cryoscanning 

electron microscopy, which showed that the cells were still intact 

and thus no contamination with symplastic fluid had occurred (Dong et al. 

1994). Although this technique seems to be time-consuming, requiring both 

surface sterilisation and centrifugation, several samples can be centrifuged 

atthesametimeandtheoveralltimerequirementmayineffectbenomore 

than for other methods. 

Whereas surface sterilisation, followed by subsequent plating of macerated 

tissue segments or of centrifuged fluid, is a simple and valuable method


that produces consistent results, it has its limitations: (1) it is laborious; (2) 

microorganismscanhideinnichesthatarenotreachedbythesterilising 

agent or “stick” to the host tissue, resulting in false identifications of endophytes; 

and (3) the sterilising agent may penetrate the root tissue and kill 

endophytes, so that microbial densities will be underestimated. These disadvantages 

can be avoided by using techniques to isolate microorganisms 

that do not involve surface sterilisation. 

17.4 

Vacuum and Pressure Extraction 

To bypass the above mentioned problems, in particular involving surface 

sterilisation, alternative methods have been developed. These techniques 

use vacuum or pressure to collect plant fluid from the root tissue. Both 

approaches have in common that plant fluid of the conducting elements 

and adjacent intercellular spaces is collected, as discussed for centrifugation, 

thus reaching two niches often considered favourable for systemic 

colonisers (Hallmann et al. 1997a, 1997b). However, it does not isolate 

those endophytes that grow intracellularly. Bell et al. (1995) and Gardner et 

al. (1982) used an aseptic vacuum extraction technique to isolate bacteria 

from the xylem of grapevine and citrus roots. Cohen (1999) described an 

oversized vacuum filtration unit for large scale isolation of fungi from leaf 

tissue, which might also be adaptable for root tissue. The Scholander pressurebombcommonlyusedformeasuringplantwaterstatusisalsoeffective 

for the isolation of endophytic microorganisms (Fig. 17.1; Hallmann et al. 

1997b). Von Tiedemann et al. (1983) described a technique for lignified root 

tissue. Sterile water is washed through the vascular system at low pressure 

to collect the endophytic microorganisms. To prevent damage of the plant 

Fig.17.1. Scholander pressure bomb. From left to right: Pressure bomb apparatus, inserting 

therootintothepressurecylinder,collectionofplantsapfromtherootwithasterilePasteur 

pipette


tissue, vacuum or pressure should be carefully increased to the maximum 

level tolerated by the respective tissue. In all cases, the plant extract is 

streaked onto nutrient media to allow recovery of the endophytic microorganisms. 

As with the surface sterilisation procedure, sterility checks need 

to be included to exclude the possibility that surface colonisers have been 

forced from the plant surface through the vascular system by the applied 

vacuum or pressure. This can be done by dipping the plant tissue immediately 

before extraction into a suspension with an indicator bacterium 

and checking for this bacterium after extraction. Additional sterility can 

be assured by surface sterilising the cut surface before applying pressure or 

vacuum. A sterile working environment can be easily achieved by treatment 

with ethanol. 

Comparison of the pressure bomb method with maceration of surface 

sterilisedrootsresultedinslightlyhigherbacterialnumbersforthelatter 

technique (Hallmann et al. 1997b). However, the pressure bomb technique 

recovered a higher number of less commonly detected genera, resulting in 

higher indices for bacterial richness and diversity. Gram-positive species, 

such as Bacillus spp., were more frequently isolated with the maceration 

method than with the pressure method, but Gram-negative genera such as 

Pseudomonas and Phyllobacterium were recovered at similar levels using 

both methods. These results confirm the previously made assumptions that 

the pressure bomb method recovers predominantly colonisers of the vascular 

tissue, while the maceration method recovers bacteria of the vascular 

as well as the cortical root tissue. An advantage of vacuum and pressure 

extraction methods is the time saved by avoiding the time-consuming surface 

sterilisation procedure. However, limitations of the pressure bomb 

technique were encountered for young, fleshy root tissue of cucumber and 

tomato, which collapsed under the applied pressure. Those limitations did 

not apply to young roots of cotton, soybean and bean (Hallmann et al. 

1997b). 

17.5 

Media 

The recipes for most commonly used microbial growth media are listed 

for example on the web page of the German National Resource Centre for 

Biological Material: http://www2.dsmz.de/media/media.htm.


17.5.1 

Media for Isolating Bacteria 

The choice of the growth medium is crucial as it directly affects the number 

and type of endophytic microorganisms that can be isolated from the root 

tissue. For endophytic bacteria, commonly used media include tryptic soya 

agar (TSA), which supports growth of a broad range of bacteria (Gardner 

et al. 1982), R2A for bacteria requiring low levels of nutrients (oligotrophs; 

Reasoner and Geldreich 1985), nutrient broth-yeast extract medium for 

growth of less selective bacteria (Zinniel et al. 2002), King’s B medium 

for growth of Pseudomonads (King et al. 1954; Misaghi and Donndelinger 

1990) or SC – originally designed for coryneform bacteria, but also useful 

for fastidious endophytic bacteria (McInroy and Kloepper 1995). Comparing 

the different media, Elvira-Recuenco and van Vuurde (2000) obtained 

significantly higher bacterial densities on 5% TSA than on R2A and SC, 

while McInroy and Kloepper (1995) found higher bacterial densities on 

R2A and SC than on full strength TSA. However, full strength TSA seems to 

be less suitable, due to the fact that its high nutrient concentration allows 

fast growing bacteria to overgrow slower growing ones. Compared with 

SC medium, plate counts from R2A were more accurate due to less colony 

overgrowth and smaller colony size (McInroy and Kloepper 1995). 

17.5.2 

Media for Isolating Fungi 

For isolating endophytic fungi, standard media include PDA (potato dextrose 

agar; Philipson and Blair 1957), malt extract–peptone–yeast extract 

(Schulz et al. 1995) and biomalt agar (Schulz et al. 1995), but minimal media 

such as SNA (synthetic nutrient agar; Boyle et al. 2001) may also be used. To 

isolate host-specific fungi, host tissue or extracts thereof may be included 

in a minimal medium (Arnold and Herre 2003). To increase the diversity 

of fungi isolated, it is wise to use a number of different media for each 

host plant, varying factors such as pH, temperature of cultivation, and also 

aeration. 

17.5.3 

Supplements 

Antibiotics, fungicides or specific nutrients are commonly added to media 

to stimulate or suppress growth of certain microbial groups. In spite of 

the fact that fungal growth media are usually at slightly acidic pH values,


antibacterial agents, e.g. oxytetracycline, streptomycin sulfate, penicillin, 

and/or novobiocin (Tsao 1970, Schulz et al. 1993), should always be included 

in the isolation media. Sublethal doses of fungicides restrict radial 

growth of fungal colonies, preventing overgrowth (Bills 1996). For example, 

1−2 mg/l Cyclosporin A (active component of Sandimmune; Novartis, 

Basel, Switzerland) may be added to the growth medium to suppress fastgrowing 

fungi (Dreyfuss and Chapela 1994). Even at low concentrations 

in agar (1−10 mg/l), it causes ascomycetous fungi to become unusually 

compact, and restricts growth of mucoraceous fungi (Bills 1996). Another 

technique is to “weed” out the ubiquitous fungi with a (hot) inoculating 

needle (Dreyfuss and Chapela 1994). 

17.5.4 

Selective Media 

Selective culture techniques are used for microorganisms with specialised 

physiological capabilities. For example, nitrogen-free enrichment media is 

used for the isolation of endophytic diazotrophs (Hartmann et al. 2000), 

while media containing chitin, pectin or cellulose as sole nutrient source 

are used to select for fungi and bacteria with chitinolytic, pectinolytic or 

cellulytic activity, respectively (Chernin et al. 1995; Bills 1996; Berg et al. 

2002). 

17.6 

Cultivation-Independent Methods 

Cultivation-dependent methods such as plate counts underestimate microbial 

numbers as they do not record viable but non-culturable cells, e.g. 

obligate biotrophs, and microorganisms with unknown growth requirements. 

Non-culturable microorganisms can be detected using molecular 

methods. Their DNA sequences can be compared with those in databanks 

to identify the microorganisms. Alternatively, RNA may be used to identify 

the active genes. In most cases target sequences are amplified using 

the polymerase chain reaction (PCR). The 16S rRNA gene (rDNA) has become 

a frequently employed phylogenetic marker with which to describe 

bacterial diversity in natural environments (Vossbrinck et al. 1987). Similarly, 

18S rDNA is frequently employed for determining fungal diversity 

(Kowalchuk et al. 1997; Smalla 2004). However, since it is not very variable, 

it may lead to amplification of metazoa (Zuccaro et al. 2003), oomycetes 

and diatoms (Nikolcheva et al. 2003). Thus, it is usually more reliable to 

amplify 28S rDNA or the internal transcribed spacer (ITS) regions (Schulz 

and Boyle 2005).


Fingerprinting techniques on the basis of PCR-amplified 16S rDNA (bacteria) 

or 18S and 28S rDNA (fungi) genes allow for analysis of the structure 

of the whole microbial community and their spatial and temporal 

variations in relation to environmental factors (Smalla 2004; Zuccaro et 

al. 2003; Nikolcheva et al. 2003; Nikolcheva and Bärlocher 2005). Such 

techniques include denaturing or temperature gradient gel electrophoresis 

(DGGE/TGGE; Kowalchuk et al. 1997; Heuer and Smalla 1997; Zuccaro 

2003), terminal restriction fragment length polymorphism (T-RFLP; Liu 

et al. 1997; Nikolcheva et al. 2003), and PCR-single-strand-conformationpolymorphism 

(SSCP; Schwieger and Tebbe 1998). All these techniques 

have been successfully applied to the analysis of endophytic communities, 

e.g. T-RFLP (Reiter et al. 2002; Krechel et al. 2002), SSCP (A. Krechel et al. 

unpublished data) and DGGE (Garbeva et al. 2001) to characterise endophytes 

of potato, DGGE for those from marrum grass (Kowalchuk et al. 

1997), citrus plants (Araújo et al. 2002), and fungi associated with decaying 

leaves (Nikolcheva and Bärlocher 2005) and algae (Zuccaro et al. 2003). 

Using eubacterial primers, it has been estimated that a population must 

representabout1%ofthetotalcommunitytobedetectableinafingerprint 

(Smalla 2004). Group-specific primers are available for Burkholderia 

(Salles et al. 2001), Proteobacteria (Sessitsch et al. 2002), actinomycetes 

(Heuer et al. 1997) and many other taxa, allowing a sensitive analysis of the 

microbial community. Prominent bands can be excised, cloned and used 

for sequence determination in order to obtain further information as to 

the identity (Zuccaro et al. 2003) and phylogeny of dominant ribotypes, as 

shown for endophytic bacteria of citrus rootstocks by Araújo et al. (2002). 

Another cultivation-independent approach is cloning of 16S rDNA, 18S 

rDNA or 28S rDNA fragments amplified from community or environmental 

DNA, with subsequent sequencing. This approach was applied by Sessitsch 

et al. (2002) to the analysis of diversity of potato-associated endophytes. 

However, DNA analysis does not generally allow conclusions regarding the 

metabolic activity of members of the microbial community or their gene 

expression to be drawn. This information may be obtained only through 

analysis of RNA (Griffith et al. 2000). Different methodological approaches 

have therefore been developed to link information on metabolic activity 

to certain ribotypes, such as the incorporation of bromide oxyuridine 

(BrdU) or 13 C into the nucleic acids of growing cells (Borneman 1999). Furthermore, 

fluorescence in situ hybridization (FISH) as well as marker and 

reporter genes are valuable tools with which to study the metabolic activity 

of endophytes (Martinez-Inigo et al. 2003; see Chap. 18 by Bloemberg et al.). 

However, problematic with molecular results is that: (1) the databanks 

are far from complete, and (2) the entries are not always accurate; in the case 

of fungi, 20% of entries are estimated to be false (Bridge et al. 2003). Thus, in 

order to determine the taxon of a particular microorganism it is important


not to rely only on one method (e.g. Zuccaro et al. 2003; Nikolcheva and 

Bärlocher 2005), but to evaluate multiple characteristics, i.e. morphology 

and physiology as well as molecular results. 

17.7 

Quantification of Colonisation 

None of the methods for quantifying the degree of colonisation of endophytic 

microorganisms within their hosts is optimal. Colonisation can 

be quantified using visual methods, e.g. by direct counts of infections 

(Stone 1987); however, this is difficult for bacteria and yeasts. With fungi, 

biomass can be correlated with the concentration of fungal specific ergosterol 

(Newell et al. 1988; Weete and Ghandi 1996). This method may give 

variable results because ergosterol concentration varies with the age of the 

mycelium (Olsson et al. 2003). Phospholipid fatty acids have been used for 

quantification, but their concentration varies between fungal (Olsson et al. 

2003) and bacterial (Olsson and Persson 1999) genera. Fungal biomass can 

also be measured using monoclonal antibodies. Here, the difficulty lies in 

developing an antibody specific enough to accurately quantify single endophytic 

taxa. Real-time PCR (Vaitilingom et al. 1998; Schena et al. 2004) is 

presumably the most accurate method for quantifying microbial colonisation 

within the host and has been successfully employed, e.g. by Winton et 

al. (2002), to quantify the density of fungal colonisation by Phaeocrytopus 

gaeumannii within the needles of Douglas-fir and by Oliveira and Vallim 

(2002) to quantify colonisation of the bacterial pathogen Xylella fastidiosa 

in the leaves of citrus trees. 

17.8 

Conclusions 

The decision as to which isolation procedure is best for a given host depends 

on its physical niche, on factors that are plant-dependent, e.g. species, age 

and tissue, but also on the available resources and the total number of 

samples to be processed (Hallmann et al. 1997a). A polyphasic approach 

as suggested in Fig. 17.2 is recommended for the analysis of endophytic 

communities. Combinations of different techniques, e.g. surface sterilisation, 

vacuum and pressure extraction, cultivation independent (molecular) 

methods and cultivation dependent (isolation and morphological) identification, 

increase the likelihood of completely analysing microbial diversity, 

and consequently also enhancing our understanding of the interactions of 

microorganisms with the roots of their plant hosts.


Fig.17.2. Analysis of endophytic communities using a polyphasic approach 


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18 

18.1 

Introduction 

Microbial Interactions with Plants: 

a Hidden World? 

Guido V. Bloemberg, Margarita M. Camacho Carvajal 

Endophytic microorganisms, e.g. bacteria and fungi, are not only present 

within the plant, but may also colonise epiphytically before and during 

infection. Some of these organisms have important functions for the plant, 

such as nitrogen fixation (e.g. Rhizobiaceae, Herbaspirillum, Azoarcus), 

protection against pathogens (e.g. Pseudomonas), and provision of essential 

nutrients from the soil (e.g. mycorrhizae), but they can also be parasitic 

(e.g. Agrobacterium). Stages in the development of an interaction between 

endophyte and plant include attachment to the plant surface, infection 

and invasion, colonisation within the plant’s tissues, proliferation (including 

sporulation), expression of various phenotypic traits, and spreading 

to other plant individuals. Several approaches can be taken to unravel the 

complex interactions between the plant and its endophytes. Genetics and 

measurement of enzymatic activities are powerful tools with which to discover 

the genes and traits involved in these interactions. However, these 

processes can only be understood in detail if the microorganisms and the 

expression of relevant genes can be visualised on and in the plant, where 

the endophytes encounter different tissues and conditions. Visualisation 

of the interactions between plant and microorganism on the cellular level 

provides a more detailed understanding of the basic principles of how endophytes 

function. In this chapter, methods of microscopy and techniques 

useful for the analysis of the spatio-temporal behaviour of endophytes 

on and in the plant will be evaluated, with emphasis on root-colonising 

microorganisms. 

Guido V. Bloemberg, Margarita M. Camacho Carvajal: Leiden University, Institute 

of Biology, Wassenaarseweg 64, 2333AL Leiden, The Netherlands, E-mail: 

bloemberg@rulbim.leidenuniv.nl 

Margarita M. Camacho Carvajal: Laboratory for Physiological Chemistry and Centre for 

Biomedical Genetics, Utrecht 3584CG, The Netherlands (Present Address) 





322 G.V. Bloemberg, M.M.C. Carvajal 

18.2 

Microscopic Techniques for Studying 

Plant-Microbe Interactions 

Various techniques of microscopy are available for visualising microorganisms 

in the plant environment. We will briefly discuss some of the available 

techniquesandgiveexamplesofhowthesetechniqueshavebeenvaluable 

in studying the interactions of microorganisms with the plant and its 

microflora. 

18.2.1 

Light Microscopy and Enzymatic Reporters 

Light microscopes belong to the standard equipment of every microbiology 

laboratory and are relatively inexpensive. Several reporter genes with 

enzymatic functions such as β-glucuronidase (GUS) encoded by gusA and 

β-galactosidase encoded by lacZ are used to visualise bacterial cells and 

gene expression (Sambrook and Russel 2001). The advantages of these 

reporters are their comparatively low costs and high sensitivity. A clear 

disadvantage of using gusA or lacZ as reporters is that plants have to be 

fixed, and staining reagents have to penetrate into the plant to reach the 

bacteria, which is time consuming, can make detection less efficient, and 

is artefact-prone. Another disadvantage can be background staining of the 

plant tissue, which should be checked for before using these reporters. 

We have used lacZ as a marker to facilitate visualisation of colonisation 

by single bacterial cells and bacterial microcolonies of the rhizosphere 

of both tomato and Arabidopsis thaliana, and to monitor gene expression 

(Fig. 18.1). No background β-galactosidase activity from tomato or 

Arabidopsis cells was observed. In particular we focused on the differences 

between the rhizosphere colonisation pattern of the biocontrol strain 

Pseudomonas fluorescens WCS365 on the model plant Arabidopsis and on 

tomato. WCS365 cells preferentially colonised the interjunctions between 

root cortex cells and the sites where lateral roots arise (Fig. 18.1A, B). At 

such sites, cells are disrupted by the emerging lateral root and nutrients 

are released. Cracks in the outer root cortex or plant surface are also very 

suitable sites for entering the plant and infecting the internal plant environment. 

The colonisation patterns of P. fluorescens WCS365 on tomato 

and Arabidopsis are more similar at early (first 3 days after inoculation) 

than at later stages, although the number of colony forming units (cfu) per 

centimetre of root tip is comparable. It seems that the bacteria around the 

root of a 7-day-old tomato plant are embedded in a “mesh” of root hairs 

and possibly plant and/or bacterial extracellular material, to which root


Fig.18.1. A–D LightmicroscopyanalysisoftomatoandArabidopsis thaliana root colonisation 

by Pseudomonas fluorescens strain WCS365 constitutively expressing the lacZ reporter 

gene encoding β-galactosidase. Sterile seedlings were inoculated with WCS365 2 days after 

germination and placed in a gnotobiotic growth system (tomato) or on solid plant nutrient 

solution (PNS) agar plates (A. thaliana) for 7 days. After staining for β-galactosidase activity, 

roots were examined using light microscopy. A, B Colonisation of the sites of lateral 

root emergence from tomato roots. C, D Microcolonies on the root surface of A. thaliana at 

the interjunctions of root cells. Bars 100 µm 

border cells (BRD) or their excreted proteins (Brigham et al. 1995) may be 

major contributors. In contrast, in Arabidopsis the bacteria are located on 

the root surface and in between the cell walls of adjacent epidermal cells 

(Fig. 18.1C, D). The difference in colonisation patterns between tomato and 

Arabidopsis is most likely due to the difference in the rhizosphere environment 

created by the two plants. Many plants release BRD from the root tip 

into the rhizosphere (Hawes 1990). By definition, BRD are cells that become 

dispersed into suspension in response to gentle agitation in water (Hawes 

and Lin 1990). The number and properties of BRD vary among different 

kinds of plants and are conserved among families (Hawes 1990) Most plants 

have BRD but A. thaliana does not (Hawes 1990). BRD are one of the main 

components of root exudate (Hawes 1990; Hawes and Lin 1990), which is 

the major nutrient source for rhizosphere micro-organisms. 

lacZ is not only a useful tool in determining the colonisation pattern of 

rhizobacteria in the rhizosphere of different plants and to observe single


cells within bacterial microcolonies, but can also be used to study bacteriaroot 

associations in which the bacteria penetrate deeper into the root tissue, 

as was shown for rhizobial cells in infection threads and root nodules (Gage 

et al. 1996). 

More sophisticated (and more expensive!) light microscopes can be very 

valuable for visualising cells without staining, for example endophytic 

colonisation and bacteria-fungus interactions. We successfully used phasecontrast 

microscopy and differential interference microscopy (DIC) to visualise 

the effects of the antifungal phenazine-1-carboxamide on the growth 

and hyphal morphology of the plant pathogen Fusarium oxysporum f.sp. 

radicis lycopersici (Bolwerk et al. 2003). 

18.2.2 

Scanning Electron Microscopy 

Scanning electron microscopy (SEM) has the advantage of high resolution 

and is, therefore, a powerful tool with which to follow the process of seed 

and root colonisation by microorganisms. Single bacterial cells and bacterial 

microcolonies can be visualised. Using SEM, bacteria were shown 

to preferentially colonise grooves on the seed coat and the root surface, 

where they formed densely packed microcolonies. Microcolonies are ideal 

environments for quorum sensing, which is used by bacteria as a regulatory 

process controlling, for example, the production of antifungal metabolites 

in the rhizosphere (Fig. 18.2, Chin-A-Woeng et al. 1997). Interestingly, 

Fig. 18.2D suggests that the microcolonies are covered by a mucous layer, 

possibly consisting of exopolysaccharides, which could form an additional 

diffusion barrier. 

SEM is also very suitable for showing the morphological differences 

within endogenous communities of microorganisms, including obligately 

biotrophic bacteria and fungi that cannot be cultured on standard growth 

media. A nice example of such a study was given by Fett and Cooke (2003), 

who visualised a population of bacteria with different morphologies on 

mung bean sprouts. 

SEM is ideally suited to visualising the total microflora since it does not 

require the tagging of microorganisms with reporters. However, it has the 

disadvantage that (1) the living material has to be fixed, and (2) bacterial 

species of similar shape and size cannot be differentiated. In addition 

the costs of purchasing an electron microscope and its maintenance are 

relatively high.


Fig.18.2. A–D Scanning electron microscopy (SEM) analyses of tomato seed and root colonisation 

by Pseudomonas putida WCS358. A Seed coat colonisation 1 day after inoculation. 

B Seed coat colonisation monitored 3 days after inoculation. C Attachment on and early 

colonisation of the root surface. D Microcolony formed at an interjunction of root cells. 

Bars A, B 10 µm; C, D 1 µm. Images provided by T.F.C. Chin-A-Woeng, Leiden University, 

Leiden, The Netherlands 

18.2.3 

Epifluorescence Microscopy and the Application 

of Auto-Fluorescent Proteins 

Fluorescence microscopy is based on the presence of fluorescent compounds, 

including proteins, which, after excitation with light of a certain 

wavelength, will emit light of a longer wavelength due to energy loss during 

the process of absorption and excitation. Confocal laser scanning microscopy 

(CLSM) is a highly sophisticated form of fluorescence microscope. 

The use of CLSM for visualising fluorescent molecules results in higher resolution 

and lower autofluorescence background compared to traditional 

fluorescence microscopy In addition, the resolution and sharpness of the 

digital images produced by CLSM can be improved by the use of deconvolution 

software that corrects for small defects in the optical lenses. In


many applications fluorescent tags are coupled to compounds specifically 

binding to certain molecules, such as DNA or RNA, in the living cell. 

Such compounds are able to diffuse or to be transported into the cell. 

Some will diffuse or penetrate differentially through the cell membranes 

of Gram positive and Gram negative bacteria. Various fluorescent staining 

kits are commercially available. For example, the ViaGram Red kit (Invitrogen 

Molecular Probes, Breda, The Netherlands) contains three fluorescent 

compounds that offer the possibility to distinguish between Gram positive 

and negative bacteria and to test their viability. 

Green fluorescent protein (GFP) has become the most frequently used 

reporter in the biological sciences since its application as a marker was published 

by Chalfie et al. (1994). GFP was isolated from the jellyfish Aequorea 

victoria and is extensively applied to mark cells, to visualise proteins within 

cells and to monitor gene expression. In contrast to the use of fluorescent 

stains,antibodiesorprobestargetedto16SribosomalRNAgenes,theuseof 

autofluorescent proteins as markers does not require any preparatory steps 

such as fixing with formaldehyde or ethanol, which might affect the biological 

material and the biological processes studied. In many cases, fixation 

results in death of the living cells. GFP as a reporter has many advantages, 

which include (1) stability (due to its barrel protein structure), (2) speciesindependent 

application (pro- and eukaryotes), (3) non-invasive analysis 

without the need for exogenous substrates or energy, and (4) in vivo monitoring 

while preserving the integrity of the living cells. Colour- and optimised 

variants of GFP, such as enhanced GFP (EGFP), enhanced cyan fluorescent 

protein (ECFP) and enhanced yellow fluorescent protein (EYFP), 

with shifted excitation and emission maxima, and increased brightness and 

stability have been developed and used for multiple colour imaging (Yang 

et al. 1998; Tsien 1998; Matus 1999; Ellenberg 1999). Useful information on 

the properties and commercial availability of autofluorescent proteins can 

be found on the following website: www.clontech.com/clontech/. 

The development of GFP variants with altered excitation and emission 

wavelengths and the red fluorescent protein (RFP or DsRed), which was 

isolated from the coral Discosoma (Matz et al. 1999), allows for differential 

staining of bacterial strains for simultaneous visualisation using CLSM in 

one system. We made use of broad host-range cloning vectors for Gram 

negative bacteria, which are stably maintained without antibiotic pressure 

(Heeb et al. 2000) to tag Pseudomonas and Rhizobium with autofluorescent 

proteins encoded by egfp (green), ecfp (cyan), eyfp (yellow), ebfp (blue) 

and rfp (red)(Stuurman et al. 2000; Bloemberg et al. 2000). Subsequently, 

we were able to visualise simultaneously and clearly differentiate up to 

three different bacterial populations at the single cell level in the rhizosphere 

(Bloemberg et al. 2000). GFP was shown to be an excellent marker 

for monitoring root colonisation at the single cell level by Pseudomonas


spp. (Bloemberg et al. 1997, 2000), the interactions of Rhizobium with leguminous 

plants (Stuurman et al. 2000), the infection process of Fusarium 

oxysporum f.sp. radicis lycopersici (Lagopodi et al. 2002) and the interactions 

between biocontrol strains of Pseudomonas sp. and F oxysporum f.sp. 

radicis lycopersici. (Bolwerk et al. 2003). Besides whole plasmid systems, 

valuable transposons have been constructed for integration of gfp into 

bacterial chromosomes under constitutive expression (Burlage et al. 1995; 

Tombolini et al. 1997; Unge et al. 1997). 

18.3 

Visualisation of Bacterium-Plant Interactions 

Studying microbial communities and their interactions with plants has 

been highly facilitated by using combinations of GFP, its colour variants 

cyan and yellow fluorescent protein and DsRed as markers. Tomato root 

colonisation studies showed that Pseudomonas cells adhere to the root surface 

preferentially at the junctions between cells, which are presumed sites 

of root exudation, where they proliferate and divide, resulting in the formation 

of microcolonies (Bloemberg et al. 1997, 2000; Fig. 18.3A). SEM and 

fluorescence microscopy studies revealed that microcolonies are covered 

with a mucoid-like layer of unknown origin (Chin-A-Woeng et al. 1997; 

Bloemberg et al. 1997). It can be hypothesised that this layer contains bacterial 

exopolysaccharides, the production of which is increased in bacterial 

biofilms (O’Toole et al. 2000; Sutherland 2001) and which could form a barrier 

for signal molecules such as acyl homoserine lactones. Interestingly, we 

observed that some plant cells were colonised intracellularly (Fig. 18.3B). 

Bacterial cells were also observed close to the openings of the stomata 

(Fig. 18.3C), which could form another site of endophytic colonisation, although 

we have not observed endophytic colonisation by the Pseudomonas 

strains we have used. 

Microcolonies are most frequently initiated by one cell, but cells from 

external sources can attach to the colony as revealed by a study performed 

with a mixture of P. fluorescens WCS365 cells expressing ecfp, egfp and 

rfp (Bloemberg et al 2000). It is also expected that cells will detach from 

the microcolony to colonise other parts of the growing root. This shows 

that bacterial microcolonies are not static, but dynamic entities that can 

be invaded by external bacterial cells after the formation of the initial 

microcolony. It is of great fundamental interest to find out which genes, 

molecules and traits are involved in the attachment and departure of cells. 

Studies of an inoculant containing a mixture of P. chlororaphis PCL1391 and 

P. fluorescens WCS365 differentially labelled with auto-fluorescent proteins 

has shown that (1) predominantly mixed colonies are present on and in


Fig.18.3. A–F Confocal laser scanning microscopy (CLSM) analysis of tomato surfaces 

colonised by Pseudomonas spp. and Fusarium oxysporum f. sp. radicis lycopersici labelled 

with autofluorescent proteins. Tomato seedlings were grown upon inoculation in a gnotobiotic 

sand system. Plants were taken out after 7 days of growth and examined for the 

presence of green fluorescent protein (gfp)- or red fluorescent protein (rfp)-expressing 

organisms. A Colonisation of the root surface by P. fluorescens expressing enhanced GFP 

(egfp). B Colonisation of the lumen of a root cell. C Colonisation of a stoma. D Simultaneous 

imaging of root surface colonisation by a mixture of P. putida PCL1444 expressing egfp 

and P. putida PCL1446 expressing rfp. E, F Interactions between P. chlororaphis PCL1391 

expressing rfp and the phytopathogenic fungus Fusarium oxysporum f. sp. radicis lycopersici 

expressing gfp during colonisation. Bars 10 µm. D courtesy of E. Lagendijk; E, F courtesy of 

T. Lagopodi and A. Bolwerk


older parts of the root, and (2) in a mixture of PCL1391 and WCS365, 

PCL1391 predominantly epiphytically colonised the root hairs (Dekkers et 

al. 2000), indicating differences in attachment and colonising abilities between 

these two closely related Pseudomonas spp. Differential labelling of 

strains has also been used to study the colonisation pattern of polyaromatic 

hydrocarbon (PAH)-degrading bacteria, which had been isolated for bioremediation 

studies. Interestingly, it was shown that these bacterial strains 

can form communities on the root as a response to the presence of PAHs 

in the soil (Fig. 18.3D). 

Results of SEM and CLSM studies show that rhizobacteria, such as 

Pseudomonas spp. used for biocontrol, colonise the seed and root surface 

at the same positions as phytopathogenic fungi (Chin-A-Woeng et al. 

1997; Bloemberg et al. 1997; Tombolini et al. 1999; Lugtenberg et al. 2001; 

Lagopodi et al. 2002; Bolwerk et al. 2003; Fig. 18.3). Visualisation is required 

to explore and fully understand the interactions between organisms. For 

example, visualisation of the relationship among P. fluorescens CHA0, carrot 

roots, and mycorrhizal mycelium showed that mucus-producing mutant 

strains of CHA0 can better adhere to the root, indicating that acidic extracellular 

polysaccharides contribute to root colonisation (Biancotto et al. 2001). 

Invasion of plant tissue has been extensively studied for rhizobial infection 

that results in the nitrogen-fixing symbiosis with leguminous plants. 

Tagging with GFP made it possible to follow early nodulation events, for 

example of Sinorhizobium meliloti on alfalfa (Gage et al. 1996), and to follow 

rhizobial cells in the infection thread during its growth into the root 

cortex. Visualisation of rhizobia in the infection thread made it possible to 

calculate the rhizobial growth rate (Gage et al. 1996). GFP tagging was also 

successfully applied to study the movement of Rhizobium bacterothe root 

nodules (Stuurman et al. 2000). To achieve optimal fluorescence of the bacterial 

cells, sectioning of the plant material was required to prevent loss of 

light intensity during transmission through the plant tissue. Sectioning of 

the plant tissue was also successful in studying the endophytic colonisation 

of (1) rice roots and shoots by Herbaspirillum sp. (Elbeltagy et al. 2001) and 

(2) Vitis vinifera by the bacterial pathogen Xylella fastidiosa (Newman et 

al. 2003). If preservation is preferred or necessary, plant tissue sections can 

be fixed with paraformaldehyde, which does not affect the folding and the 

fluorescence of GFP (Stuurman et al. 2000; Elbeltagy et al. 2001). 

Due to different emission and excitation spectra, a combination of GFP 

and DsRed is very suitable for visualisation of two populations of cells 

and for providing the possibility of studying competition events. Infection 

threads can contain mixed S. meliloti populations that can give rise to mixed 

populations of bacteroids in the root nodules (Gage 2002). Since not every 

laboratory is equipped with state of the art confocal laser scanning microscopes, 

combinations of different reporter genes should be considered.


Such combinations have been shown to be powerful tools for studying root 

colonisation and gene expression in the rhizosphere. Useful constructs 

have been made to deliver gfp and gusA in mini-Tn5 transposons or in 

plasmids (Ramos et al. 2002) for chromosomal insertion (Xi et al. 1999). 

Applying these constructs to the study of Azospirillum brasilense on and in 

wheat roots showed that A. brasilense preferentially colonises intercellular 

spaces and points of lateral root emergence where it is expected that relatively 

large quantities of nutrients will be released from the root cells (Xi 

et al. 1999; Ramos et al. 2002). Others have combined immunofluorescence 

and an rRNA-targeting probe to monitor the presence of organisms and 

metabolic activity in the rhizosphere. For example, P. fluorescens DR54 cells 

were analysed in the sugar beet rhizosphere, showing that cells of P. fluorescens 

attheroottipweremetabolicallymostactiveandthatbacteria 

from the surrounding soil population entered the rhizosphere 2 days after 

seed inoculation (Lübeck et al. 2000). Another dual marker system was developed 

with gfp and the luxAB genes encoding bacterial luciferase, which 

as a biomarker is dependent on cellular energy status. This construct was 

used to show that metabolic activity of P. fluorescens SBW25 was detectable 

on all parts of wheat and that this strain colonises specific sites of the seed 

(Unge et al. 1999; Unge and Jansson 2001). 

18.4 

Most Recent Developments in Visualising 

Plant-Microorganism Interactions 

The stability of GFP can be regarded as one of its advantages. However, this 

makes GFP unsuitable for studying transient gene expression. GFP derivatives 

carrying at their C-terminus amino acid tags for the recognition of 

specific proteases have reduced half-life times of GFP to 1–1.5 h (Andersen 

et al. 1999). The use of such unstable GFP variants has made it possible 

to analyse transient gene expression in the rhizosphere, for example, the 

monitoring of ribosomal activity in Pputidacells (Ramos et al. 2000). 

These variants have recently been applied to the study of several aspects 

of interactions between plants and microorganisms. For example, a system 

was constructed for the detection of acyl homoserine lactones (AHL), 

showing that quorum sensing and cross talk occur in microcolonies in the 

rhizosphere (Andersen et al. 2001; Steidle et al. 2001). Examples of GFPbased 

expression systems for studying the interaction of the bacterium 

with the plant are given by (1) Leaveau and Lindow (2001), who showed 

that foliar growth of Erwinia herbicola on bean is driven by the utilisation 

of sugars, e.g. fructose and/or sucrose; and (2) Aldon et al. (2000), who 

showed that the strong induction of hrp genes in the presence of the host


plant cell depends specifically on physicalcontact between the bacterium 

and its target cell. The development and application of such reporter systems 

contribute significantly to increasing our fundamental knowledge of 

bacterial physiology and behaviour on the plant (Leveau and Lindow 2002). 

The addition of a constitutively expressed rfpon the reporter construct or 

delivered in trans on a second plasmid should make it possible to follow the 

presence of bacterial cells and their gene expression. DsRed is specifically 

suited to this since it has been isolated from a different organism and 

its homology with GFP is extremely low, which excludes the possibility 

of recombination when gfp and rfp are present in one cell. DsRed has 

a longer folding (maturation) time than GFP, which might reduce or delay 

its detection after it is produced. However, an enhanced RFP that matures 

rapidly has recently been developed (Sörensen et al. 2003) and will improve 

the usefulness of rfp. 

In order to identify genes expressed in the plant environment, systems 

based on differential fluorescence induction and optical trapping 

microscopy have been developed (Allaway et al. 2001). Several genes have 

been isolated that have led to new insights into bacterial life in the rhizosphere, 

such as the identification of a putative ABC transporter of putrescine 

(Allaway et al. 2001). Interestingly, uptake of putrescine from the rhizosphere 

environment was identified as an important trait for competitive 

root colonisation (Kuiper et al. 2001). New systems for promoter trapping 

based on the combination of an antibiotic resistance marker and GFP further 

extend the possibilities for researchers to identify genes specifically 

expressed in the plant environment (Izallalen et al. 2002). 

18.5 

Visualisation of Plant-Fungus Interactions 

Fungi frequently colonise the internal and external plant environment as 

pathogens, mutualists or organisms without apparent effect on the plant. 

Fungal structures, including hyphae, fruiting bodies and spores can be visualised 

by light- and electron-microscopy with the advantages and disadvantages 

of these techniques as discussed above. The use of reporters such 

as lacZ or gfp requires genetic transformation, which is much more complicated 

for fungi than for bacteria. However, there is a growing number of 

fungal species for which transformation methods, including conventional 

protoplast transformation, ballistic bombardment and the more recently 

developed highly successful Agrobacterium-based transformation method 

for filamentous fungi (de Groot et al. 1998), are available Two examples will 

be discussed to illustrate the use of GFP in visualisation of fungi interacting 

with plants and microorganisms in the plant environment.


In order to visualise tomato root colonisation and infection processes in 

vivo we marked Foxysporumf. sp. radicis-lycopersici, which causes tomato 

foot and root rot, with GFP. A protocol to produce Fusarium protoplasts 

with the use of a mixture of hydrolytic enzymes was optimised and successfully 

applied for cotransformation of the protoplasts with two plasmids, 

one of which harboured sgfp, which is an optimised gfp variant (Lagopodi 

et al. 2002). This resulted in an efficient tagging and the constitutive sgfp 

expression was stable for at least nine subcultures. Homogeneity of the 

fluorescent signal was clearly visible in the hyphae as well as in chlamydospores 

and conidia. Since, after transformation, the sgfp-harbouring 

plasmid is integrated randomly into the chromosome, hyphal morphology, 

growth rate and pathogenicity were tested and found to be unaffected in 

the transformants tested CLSM was used to analyse colonisation, infection 

and disease processes of the isolate in detail on/in tomato roots, including 

the following interesting aspects: (1) an overview of the complete colonisation 

pattern of the tomato rhizosphere; (2) the very first steps of contact 

with the root, which took place in the root hair zone by mingling and attachment 

of hyphae to the root hairs, suggesting a chemotactic response 

towards the root hairs; (3) the preferential colonisation of the grooves along 

the junctions of the epidermal cells, which is similar to the colonisation 

patterns of rhizobacteria; (4) the absence of specific infection sites, such as 

sites of emergence of secondary roots, root tips or wounded tissue and the 

absence of specific infection structures, e.g. appressoria (Lagopodi et al. 

2002). This study illustrates the powerful use of GFP as a marker as a noninvasive, 

convenient, fast and effective approach for studying plant-fungus 

interactions. 

Fungi interact with other microorganisms in the plant environment. The 

use of different autofluorescent proteins is an excellent tool with which to 

distinguish microorganisms from each other and to visualise their interactions. 

Visualisation of interactions between phytopathogenic fungi and 

biocontrol agents will help to understand these interactions and facilitate 

the development of efficient biocontrol applications. We have studied 

the interaction between F. oxysporum f. sp. radicis-lycopersici tagged with 

GFP and the biocontrol strain P. chlororaphis PCL1391 tagged with DsRed 

(Bolwerk et al. 2003). CLSM studies of the single strains showed the following 

similarities: (1) the sites of colonisation of the tomato root surface 

by Fusarium are strikingly similar to those of the Pseudomonas biocontrol 

strains PCL1391 and WCS365; (2) both preferentially colonise sites 

between the junctions of two root cells. Studying the interactions between 

Pseudomonas biocontrol strains and Fusarium in the rhizosphere by differential 

labelling showed that (1) penetration of the fungal hyphae was not 

observed where bacterial microcolonies were present, and (2) Pseudomonas 

bacteria attached to and colonised Fusarium hyphae (Fig. 18.3F, G). The


latter could be a novel biocontrol trait and preliminary studies showed that 

Pseudomonas strains show a chemotactic response towards Fusarium (De 

Weert et al. 2004). (3) Many stress responses in the fungal hyphae were observed 

in the presence of the biocontrol agent including increased vacuole 

formation, loss of growth directionality, curly growth, “swollen bodies” and 

increased hyphal branching. Similar responses could be induced by applying 

the purified antifungal metabolite phenazine-1-carboxamide produced 

by P. chlororaphis PCL1391. 

Studying the molecular basis of the interactions between fungi and bacteria 

is an emerging field with great relevance for plant microbiology and 

plant pathology. Future studies will benefit greatly from tools developed 

for visualisation of these organisms. 

18.6 

Future Perspectives 

The development of highly sophisticated microscopy tools and optimisation 

of autofluorescent proteins as reporters are making substantial contributions 

to a better fundamental understanding of how microorganisms 

interact with plants and the endemic microflora. More reasonably priced 

tools for microscopy will facilitate and stimulate such studies in the future. 

Visualisation is, however, dependent on whether the microorganims can 

be cultured and transformed with genetic constructs that harbour reporter 

gene(s). Some important endophytes cannot be cultured outside the plant, 

which is severely hampering their study. Discovery and understanding of 

plant growth requirements, including chemical signals and physical properties 

for providing suitable conditions for culturing, as well as genetic 

accessibility of these microorganisms, represents a major challenge. 

Acknowledgements. We thank all the members of the Microbiology section 

and Gerda Lamers of the Institute of Biology Leiden for their contributions 

in valuable discussions for optimising and developing microscopic 

techniques, interpretation of results and technical assistance. 


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19 

19.1 

Introduction 

Application of Molecular Fingerprinting 

Techniques to Explore the Diversity 

of Bacterial Endophytic Communities 

Leo S. van Overbeek, Jim van Vuurde, Jan D. van Elsas 

Many bacteria associated with plants are able to penetrate and live as 

endophytes in roots and other tissues. Like most bacteria in natural environments, 

endophytic bacteria may be non-cultivable, and detectable only 

by microscopical and molecular techniques (Garbeva et al. 2001; Araújo 

et al. 2002; Sessitsch et al. 2002; Reiter et al. 2003b). Consequently, usage 

of molecular techniques reveals higher species diversity than classical 

isolation methods. Molecular fingerprinting of bacterial endophytic 

populations can expand our knowledge of populations that were previously 

inaccessible. So far, little information is available on the possibilities 

for employing molecular methods to detect bacterial populations inside 

plants. There is, however, overwhelming information from related ecosystems 

such as the soil and rhizosphere so that adaptation of methods to 

studies on bacterial endophyte communities should be feasible. 

In this chapter we will briefly summarise the factors affecting bacterial 

endophyte populations in plants and provide an extended account of 

the available molecular detection and fingerprinting techniques, including 

their integration with other methods, to study such bacterial endophyte 

communities. Exploitation of molecular fingerprinting techniques is especially 

relevant for studying those endophytic populations that show clear 

effects on plant growth and development. Special emphasis will be placed 

on agricultural production systems. 

Leo S. van Overbeek: Plant Research International B.V, Droevendaalsesteeg 1, 6708 PB, 

Wageningen, The Netherlands, E-mail: leo.vanoverbeek@wur.nl 

Jim van Vuurde: Plant Research International B.V, Droevendaalsesteeg 1, 6708 PB, Wageningen, 


Jan D. van Elsas: Groningen University, Department of Microbial Ecology, Biological Center, 

Kerklaan 30, 9751 NN, Haren, The Netherlands 





338 L.S.v. Overbeek et al. 

19.2 

Colonisation by Bacterial Endophytes 

Bacterial populations residing on the surface or inside plants are commonly 

referred to as plant-associated bacteria. Most of these populations can 

colonise both internal and external tissue of plants and therefore a strict 

separation between ‘genuine’ endophytes and plant-associated bacteria 

cannot always be made. Endophytes thus comprise populations strictly 

bound to, and occasionally occupying, internal plant organs. 

The initial step in the route to the internal plant system for soil and aerial 

bacteria is colonisation of the phytosphere (vis rhizosphere and phyllosphere). 

Endophytes occur in the rhizosphere (Sturz 1995; Hallman et al. 

1997; Sturz and Nowak 2000) or the phyllosphere (Pillay and Nowak 1997). 

Colonisation of internal plant tissue requires adaptation of the bacterial cell 

to a highly specific and restrictive environment. Therefore, active colonisation, 

systemic spread and reproduction inside plants must be considered 

as important features of ‘genuine’ endophytic associations. 

Certain plant organs may be environments in which only highly adapted 

bacterialspeciesareabletobecomeestablished.Theplantxylemwas 

demonstrated to be a selective environment for endophytic bacteria such 

as Acetobacter diazotrophicus (Dong et al. 1994), Bacillus pumilus (Benhamou 

et al. 1996, 1998), Gluconacetobacter diazotrophicus (Cocking 2003), 

Herbaspirillum seropedicae (James et al. 2002), Klebsiella pneumoniae 

(Dong et al. 2003) and Serratia marcescens (Gyaneshwar et al. 2001). Sieve 

tubes (phloem) are a habitat for a restricted group of pathogens, so called 

phytoplasms (Bové and Garnier 2003). 

Endophytes can exert diverse effects on the performance of their host 

at different stages of growth and under different environmental conditions 

(Hallmann et al. 1997; Sturz and Nowak 2000). The effects described 

for endophytes inoculated into different plant species may be beneficial 

or detrimental [see Chaps. 3 (Kloepper and Ryu) and 4 (Berg and Hallmann)], 

although, most often, clear effects cannot be observed. The effect 

of bacterial populations associated with plants has been well described for 

pathogens and mutualistic symbionts; however, the role of species showing 

no obvious effects on plant health during the association may, in general, 

be underestimated. 

19.3 

Shifts of Bacterial Endophyte Communities 

Being growing entities, plants have a dynamic interaction with their microbial 

inhabitants. Table 19.1 summarises studies on biotic and abiotic factors

19 Molecular fingerprinting of endophytic communities 339 

Table 19.1. Factors influencing endophyte colonisation and community structure 

Factors affecting 

endophytic communitycomposition 

in plants 

Other microorganisms 

Example Reference 

Colonisation and nodulation by Rhizobium 

leguminosarum in red clover was promoted by 

endophytic isolates of Bacillus insolitus, Bacillus brevis 

and Agrobacterium rhizogenes 

Genotype Higher diversity in root-endophyte composition was 

observed in recent versus ancient cultivars of wheat 

Differences in endophyte composition and density 

were observed in different cotton cultivars 

Propagating 

material 

Agricultural 

practices 

Localisation 

within plant 

Enterobacter cloacae applied to sterilised corn seeds 

resulted in endophytic colonisation of emerging plants 

Optimal colonisation was obtained by inoculation 

of in vitro explants with endophytic strain 

Pseudomonas sp. PsJN resulting in increased resistance 

against Verticillium dahliae in tomato 

Gradient in disease-suppressing endophytes observed 

from peel to the centre of potato tubers 

Crop rotation and tillage management of agricultural 

fields resulted in differences in number of disease 

suppressive endophytes in potato 

Endophytic communities in corn were influenced 

by herbicide, feritiliser and compost treatment of soil 

Non-nodular nitrogen fixing strain Gluconacetobacter 

diazotrophicus was isolated from apoplastic 

fluid of sugarcane 

Endophytic community structure in potato differed 

between the epidermis and internal stem and between 

lower and upper stem parts 

Temperature Endophytic colonisation of tomato by strain 

Pseudomonas sp. PsJN was highest in roots and shoots 

at 10 ◦ C and lowest at 30 ◦ C 

Sturz and 

Christie 1996 

Germida and 

Siciliano 2001 

Adams and 

Kloepper 2002 

Hinton and 

Bacon 1995 

Sharma and 

Nowak 1998 

Sturz et al. 

1999 

Peters et al. 

2003 

Seghers et al. 

2004 

Dong et al. 

1994 

Garbeva et al. 

2001 

Pillay and 

Nowak 1997 

influencing endophytic populations. These studies make it clear that factors 

influencing plant growth also influence endophytic populations. The 

most important factors governing endophytic community structures are 

(1) the environment, (2) characteristics of the host plant species (genotype, 

growth stage, tissue type), and (3) endophyte populations already present 

in the plant.


Inoculation of in vitro plants with endophyte biocontrol strains aimed 

to suppress diseases in potato has been proposed by Nowak (1998). The 

rational for inoculation of in vitro transplants with endophytes was that 

already established populations will have a competitive advantage over 

organisms invading from the rhizosphere. The composition of populations 

in plants can shift depending on the intricate interplay of different 

factors. Eventually, climax populations of endophytes will become established. 

19.4 

Molecular methods to Study Bacterial Endophytes 

Methods to detect beneficial and detrimental endophytic populations are 

important in studying the effects of agricultural management on the structure 

of endophyte communities and for determining the influence of various 

endophyte communities on plant health and crop yield. Genes responsible 

for beneficial or detrimental interactions may be the targets for 

detection. The structural nitrogen fixation (nif H) gene present in many 

nitrogen-fixing species has been detected in bacterial communities inside 

and near plants (Reiter et al. 2003a; Tan et al. 2003). Polymerase chain 

reaction (PCR)-based detection of genes involved in pathogen suppression 

has been developed for pyrrolnitrin and pyoluteorin (De Souza and 

Raaijmakers 2003) and ketosynthase (Metsä-Ketelä et al. 2002; Moffitt and 

Neilan 2003) genes. The flagellar subunit protein gene, fliC, which codes 

for an important protein responsible for host colonisation by the pathogen 

Ralstonia solanacearum (Tans-Kersten et al. 2001), was used to establish 

a sensitive PCR-based method to detect this pathogen in soil (Schönfeld et 

al. 2003). Although most of these detection systems were developed with 

the intention of studying bacterial communities in other habitats, they can 

easily be applied to study bacterial communities in plants. 

The most commonly applied targets for molecular detection in environmental 

samples are genes that can be used for taxonomical differentiation, 

such as the small and large subunit genes of the ribosomal operon (16S and 

23S, respectively), ribosomal RNA (rRNA) genes, and the RNA polymerase 

β subunit gene rpoB (Dahllöf et al. 2000). A specific 16S rDNA-based PCR 

system aimed at detecting R. solanacearum was, for instance, developed 

by Boudazin et al. (1999), whereas a 23S rDNA gene-directed probe was 

developed by Wullings et al. (1998), and applied for detection of the same 

pathogen using fluorescent in situ hybridisation (FISH) in tomato (Van 

Overbeek et al. 2002). A real-time PCR system based on the rpoB gene 

has been applied to detect Bacillus anthracis in different environmental 

samples (Qi et al. 2001).


Molecular techniques aimed at detecting specific genes are often restricted 

in their application to single targets. Degenerate primers suitable 

for PCR detection can overcome this restriction by targeting multiple genes 

as, for example, applied to the genes encoding ketosynthase (Metsä-Ketelä 

et al. 2002; Moffitt and Neilan 2003) and nif H (Steward et al. 2004). Multiple 

sequence detection is crucial for molecular community fingerprinting 

of environmental samples, e.g. using PCR coupled with denaturing gradient 

gel electrophoresis (PCR-DGGE). Molecular detection techniques are 

suitable tools for detecting both cultivable and non-cultivable endophytic 

populations in plants. 

19.5 

Molecular Fingerprinting of Endophyte Communities 

19.5.1 

Basic Concept of Molecular Fingerprinting 

Shifts in endophytic populations and the effect of introduction of selected 

endophytes on bacterial communities have recently been studied using 

molecular fingerprinting techniques (Garbeva et al. 2001; Sessitsch et al. 

2002; Araújo et al. 2002; Reiter et al. 2003b; Conn and Franco 2004; Seghers 

et al. 2004). Different methods have been applied, such as PCR-DGGE 

(Garbeva et al. 2001; Araújo et al. 2002; Reiter et al. 2003b) and PCR 

followed by terminal restriction fragment length polymorphism (PCR- 

T-RFLP; Sessitsch et al. 2002). Other molecular fingerprinting techniques, 

such as PCR followed by temperature gradient gel electrophoresis (PCR- 

TGGE; e.g., Felske et al. 1998b) and PCR-single strand conformational 

polymorphism (PCR-SSCP; Schwieger and Tebbe 1998; Schmalenberger 

and Tebbe 2003), have not yet been applied to microbial populations in 

plants. However, these methods have been successfully applied in studies 

of other environments, e.g. bulk and rhizosphere soils. 

The principle of all these community fingerprinting techniques, i.e. competitive 

PCR amplification of a pool of 16S rRNA gene fragments, is the 

same, whereas final analyses of the fragments from environmental samples 

differs. The first step in all methods is extraction and purification 

of total community nucleic acids. This step is critical, as high molecular 

weightDNApureenoughforPCRamplificationisrequired.Secondly,total 

microbial community DNA is PCR-amplified using primers spanning fragments 

of the 16S rDNA gene. Most commonly, primers that target regions 

in the 16S rDNA, which is conserved for all eubacterial species, are used. 

However, depending on the focus of the study, primers targeting specific 

groups of microorganisms, e.g. eubacteria, fungi or archeabacteria, should


be applied. Separation of PCR products is performed in gels with denaturing 

gradients (DGGE) or temperature gradients (TGGE), or otherwise, 

e.g. SSCP and T-RFLP. A further requirement for PCR-DGGE and PCR- 

TGGE is the presence of a GC clamp: a stretch of around 40 nucleotides 

of mostly G and C residues. The GC clamp is required for immobilising 

denatured DNA fragments within the gel matrix. T-RFLP is recommended 

for studying habitats with low species richness (Engebretson and Moyer 

2003), which is often the case for internal plant environments (Garbeva et 

al. 2001; Sessitsch et al. 2002). T-RFLP has appeared to be more sensitive 

for detection of weak bands than PCR-DGGE (Sessitsch et al. 2002; Conn 

and Franco 2004). 

It is not our intention to describe the details of all methods in full. To 

obtain more details about the techniques described in this chapter, we 

refer readers interested in this topic to the Molecular microbial ecology 

manual (Akkermans et al. 2001). We will focus on critical steps in these 

fingerprinting techniques when studying bacterial endophyte populations 

as well as the potential offered by these methods. Further, the intricate tasks 

and challenges posed by the need to characterise non-cultivable endophytes 

will be considered. 

19.5.2 

Sample Preparation 

To discriminate bacteria inside plants (‘genuine’ endophytes) from those 

attached or living adjacent to plants, a critical evaluation of methods used 

for sample preparation is required. Contamination of samples by bacterial 

cells from the outside of plants should be avoided. However, surface sterilisation 

of plant parts prior to nucleic acid extraction may not be sufficient 

to clean the material of surface nucleic acids. Even minor contamination 

may lead to false positive bands in fingerprints due to the sensitivity of 

the PCR amplification steps. Moreover, endospores from spore-forming 

bacterial species may survive these treatments – these are notorious contaminants 

in endophyte studies (Bent and Chanway 2002). Commonly, 

surface-sterilised stem parts are incubated on agar medium to check for 

the absence of colony growth from the outside (Araújo et al. 2002; Reiter 

et al. 2003b; see Chap. 17 by Hallman et al.). Colonies formed from the 

plant surface will develop adjacent to the plant part and not within it. This 

methodiseffectiveindeterminingtheabsenceofcultivablecellsandspores 

originating from the surface that may have survived surface sterilisation. 

However, non-cultivable contaminating bacteria will not be detected using 

this method.


For molecular detection, aseptic handling during sample preparation 

is extremely important. Surface sterilisation of plant parts followed by 

aseptic removal of the outer layer (epidermis) is an effective approach to 

remove any traces of external microbial life and thus avoid contamination 

of the sample from the outside (Garbeva et al. 2001; Sessitsch et al. 2002). 

However, Garbeva et al. (2001) and Sessitsch et al. (2002) concluded that 

this approach is feasible only with robust plant parts such as stems and 

(potato) tubers but not with fragile structures such as leaves and roots. 

A clear distinction between bacteria residing inside and outside these fragile 

structures thus cannot easily be made using molecular fingerprinting. 

This may be too restrictive when studying ‘genuine’ endophytes. A possible 

remedy to selectively isolate DNA and/or RNA from endophytes in finer 

structured organs may be a pretreatment with DNase and/or RNase prior 

to nucleic acid extraction. 

19.5.3 

Nucleic Acid Extraction 

In principle, both DNA and RNA can be extracted from plant samples 

to evaluate endophytic populations. Comparison of fingerprints generated 

from DNA and RNA samples may reveal the activity of particular endophyte 

populations due to proposed higher ribosomal numbers in metabolically 

active cells. Felske et al. (1998b) and Duarte et al. (1998) used this approach 

in soil samples. For plant samples, this comparative approach has so far 

been applied only once, by Reiter et al. (2003b). The quality and purity 

of nucleic acid extracts from plants are the technical constraints for application 

of total plant microbial community DNA and RNA in molecular 

fingerprinting analyses. 

Contamination by polymerase-inhibiting compounds from plants, such 

as (poly) phenolics, cannot always be avoided. These vary with the respective 

plant species. Community DNA extracts should be diluted in Tris-EDTA 

(pH 8) buffer prior to PCR amplification to avoid inhibition of polymerase 

activity during PCR. 

Although DNA extraction procedures applied in different laboratories 

vary, they do not differ fundamentally. Most common procedures include 

pulverisation in liquid nitrogen followed by bead beating (Sessitsch et al. 

2002; Reiter et al. 2003b) or bead beating of fresh and sliced plant samples 

(Garbeva et al. 2001; Araújo et al. 2002). Suspended cells are lysed 

in SDS solution and occasionally CTAB (cetyl trimethyl ammonium bromide) 

treatment is included to remove plant-derived exopolysaccharides 

(Garbeva et al. 2001; Reiter et al. 2003b). DNA is recovered using standard 

procedures; i.e. extraction with phenol and chloroform, precipitation


in isopropanol and final wash steps in 70% ethanol. Crude extracts may 

be further purified using Wizard DNA clean up (Promega, Leiden, The 

Netherlands) prior to PCR amplification (Garbeva et al. 2001). 

The procedures described above have proven suitable for obtaining nucleic 

acids for subsequent molecular fingerprinting. Commercially available 

extraction kits, such as the Mo Bio UltraClean soil DNA isolation kit 

(Mo Bio Laboratories, BIOzym TC, Landgraaf, The Netherlands), appeared 

to be less efficient in the recovery of high quality DNA than both methods 

described by Garbeva et al. (2001), and was also our experience in our 

laboratories. Although the Mo Bio soil DNA isolation system proved its 

validity for recovery of DNA from different soils, it appeared to be limited 

to the recovery of plant DNA. Garbeva et al. (2001) also applied an 

alternative protocol, in which DNA was extracted from bacterial cells dislodged 

by incubating sliced plant parts in buffer. Cells were collected by 

centrifugation of the incubation buffer and DNA was extracted from the 

cell pellet. The two protocols – DNA extraction from macerated plants and 

dislodged cells – were compared by PCR-DGGE analysis (Garbeva et al. 

2001). The latter method yielded a clearer pattern with more distinctive 

bands in the DGGE gel. A modification of the standard protocol in which 

endophytic cells were collected by centrifugation resulted in higher quality 

DNA, presumably because there was less contamination with plant-derived 

phenolics. 

Both methods described by Garbeva et al. (2001) were successfully applied 

to DNA extraction from different plants (tomato, leek, chrysanthemum, 

lettuce) followed by endophytic fingerprinting with PCR-DGGE in 

our laboratories. It is advisable to optimise the nucleic acid extraction protocols 

for each newly studied plant species or plant part, although we found 

both methods applicable to stems and roots of different plants without further 

adaptation. Only optimised sample preparation and DNA extraction 

procedures can guarantee successful molecular fingerprinting analysis. 

19.5.4 

PCR and Molecular Community Fingerprinting 

The target sequences present in the total plant-extracted nucleic acid samples 

serve as templates for PCR. To assess microbial community structures 

in environmental samples, primers that target conserved regions of 16S 

rDNA genes are most commonly used. Molecular community fingerprints 

will, in principle, reveal all bands from 16S rDNA genes present in total plant 

nucleic acid extracts. Each band should represent one taxon. In practice, 

bands representing more than one taxon have been reported (Schmalenberger 

and Tebbe 2003). Also, single isolates represented by more than one


band in fingerprints have been observed, as shown for Bacillus sp. strain 

Sal1 (Garbeva et al. 2001). The number of individual bands in fingerprints 

cannot thus simply be translated to the number of species present in the 

plant. 

Estimation of the relative population size in molecular fingerprints is 

possible by measuring band intensities. However, linear amplification of individual 

target sequences in complex DNA extracts will probably not occur. 

Therefore, individual bands cannot be used in a straightforward manner 

for direct quantification. For additional information about cell numbers of 

individual populations, other methods are required. Therefore, molecular 

fingerprint analysis is most valuable to demonstrate microbial community 

shifts and to compare microbial community structures in different samples. 

Plant cell organelles, such as chloroplasts and mitochondria, also possess 

16S rDNA genes, and primers targeted to bacteria will also amplify 

these genes. Therefore, extra bands may be expected upon fingerprinting 

of the amplified products. Identification of individual PCR fragments in 

random clone libraries may fail as a result of the high abundance of cell 

organelles in total plant DNA extracts. A strategy to exclude 16S rDNA 

amplicons of eukaryotic origin is based on pre-amplification with a primer 

(799F, Escherichia coli numbering) that does not anneal to chloroplast DNA 

(Chelius and Triplett 2001). Clone libraries made in our laboratories by PCR 

amplification with primers 799F and 1401R with potato community DNA 

extract as template revealed that chloroplast amplicon numbers were lower 

in comparison with clone libraries made with eubacterial primers 968F 

and 1401R. PCR-DGGE analysis from the same amplicons revealed lower 

band intensity at the position where chloroplast bands were expected when 

primers 799F and 1401R were applied. Application of primers that target 

specific microbial groups also exclude amplicons of chloroplast and mitochondrial 

origin (Sessitsch et al. 2002; Reiter et al. 2003b). Group-specific 

primers may therefore be best suited for endophyte community structure 

analyses. 

19.5.5 

Group-Specific Molecular Community Fingerprinting 

Candidate bacterial endophytes have been characterised for improvement 

of plant resistance and increased nutrient acquisition, e.g. by nitrogen fixation, 

for several important crops. These cultivable endophytic beneficials, 

which will to a great extent control plant health, belong to a limited number 

of taxonomic groups. Therefore, molecular tools for detection of these 

beneficial populations in plants will certainly gain importance in the near 

future. Taxonomic groups representing beneficial endophytes are Aceto-


bacter and Gluconacetobacter spp. (Dong et al. 1994; Cocking 2003), Actinomyces 

spp. (Zinniel et al. 2002; Castillo et al. 2003; Coombs et al. 2004), 

Bacillus spp. (Benhamou et al. 1998; Bacon and Hinton 2002; Reva et al. 

2002), Burkholderia spp. (Balandreau et al. 2001), Bradyrhizobium (Chaintreuil 

et al. 2000), Enterobacter spp. (Hinton and Bacon 1995), Pseudomonas 

spp. (Duijff et al. 1997; Rediers et al. 2003), Rhizobium and Sinorhizobium 

spp. (Reiter et al. 2003a) and Serratia spp. [Press et al. 1997; Benhamou 

et al. 2000; Tan et al. 2001; Kamensky et al. 2003; see Chaps. 2 (Hallmann 

and Berg) and 3 (Kloepper and Ryu)]. However, some of these taxonomic 

groups also contain members with non-beneficial or even deleterious properties, 

as was the case for certain Serratia marcescens isolates (Gyaneshwar 

et al. 2001; Bruton et al. 2003). Endophytic taxa with potentially negative 

impacts on human health may belong to the group of Enterobacteriaceae. 

Association and endophytic colonisation of plants with human bacterial 

pathogens such as Salmonella spp. has recently been reported (Guo et al. 

2001, 2002; Cooley et al. 2003; Dong et al. 2003). The lactic acid bacteria, on 

the contrary, represent a group of bacteria that are presumed to have positive 

effects on human health. A group-specific primer system for lactic acid 

bacteria was developed by Heilig et al. (2002). Nevertheless, endophytic 

colonisation by lactic acid bacteria has not yet been reported, although it 

is known that representatives of this group live in close association with 

plants (Ennahar et al. 2003). 

Group-specific PCR systems that may be applied for molecular fingerprint 

analysis of taxonomically important groups of bacterial endophytes 

are presented in Table 19.2. The most important taxons amplified by primers 

described in literature are Pseudomonas spp., Bacillus spp, Burkholderia 

spp. and Actinomyces spp. Fingerprints from group-specific PCR will give 

an impression of all species with close taxonomic relationships to importantknownendophytes.Forpathogensuppression,taxonomicallyrelated 

species may be the best competitors for water, nutrients and available 

space. For instance, the plant pathogen R. solanacearum was suppressed by 

a closely related species, R. picketti, on the rhizoplane of tomato (Shiomi 

et al. 1999). Group-specific primers covering all β-proteobacteria, including 

Ralstonia sp., appeared to be suitable for studying R. solanacearum 

and its taxonomically nearest relatives (Gomes et al. 2001). Primers covering 

α-proteobacteria (Gomes et al. 2001) are important because the group 

of α-proteobacteria harbours pathogenic species, such as Agrobacterium 

tumefaciens, as well as beneficial nitrogen-fixing species such as Rhizobium 

and Bradyrhizobium spp.


Table 19.2. Group-specific primer systems for detection of some endophytic bacterial taxa 

(groups) 

Bacterial group Nested primer system Reference 

Pseudomonas 

spp. 

Burkholderia 

spp. 

Actinomyces 

spp. 

First step: Pseudomonas spp. specific Garbeva 

et al. 2004 

PsR: 5 ′ -GGTCTGAGAGGATGATCAGT-3 ′ 

and pSf: 5 ′ -TTAGCTCCACCTCGCGGC-3 ′ 

Second step: Pseudomonas spp. specific 

f968: 5 ′ -AACGCGAAGAACCTTAC- 3 ′ with GC clamp and PsR 

First step: eubacterial Reiter et 

al. 2003b 

8f: 5 ′ -AGAGTTTGATCCTGGCTCCAG-3 ′ and 926r: 

5 ′ -CCGTCAATTCCTTT(AG)AGTTT-3 ′ 

Second step: Pseudomonas spp. specific 

8f with CG clamp and PSMGx: 5 ′ -ĆCTTCCTCCCAACTT-3 ′ 

First step: Burkholderia spp. specific Salles et 

al. 2002 

Burk3: 5 ′ -CTGCGAAAGCCGGAT-3 ′ and BurkR: 

5 ′ -TGCCATACTCTAGCYYGC-3 ′ 

Second step: Burkholderia spp. specific 

Burk3 with GC clamp and r1378: 

5 ′ -CGGTGTGTACAAGGCCCGGGAACG-3 ′ 

First step: Actinomyces spp. specific Heuer et 

al. 1997 

f243: 5 ′ -GGATGAGCCCGCGGCCTA-3 ′ and r1378 

Second step: eubacterial 

f984: 5 ′ -AACGCGAAGAACCTTAC-3 ′ 

with GC clamp and r1378 

Bacillus spp. First step: Bacillus spp. specific Garbeva 

et al. 2003 

Bacf: 5 ′ -GGGAAACCGGGGCTAATACCGGAT-3 ′ and r1378 


f968 with GC clamp and r1378 

α-Proteobacteria 

β-Proteobacteria 

First step: α-proteobacteria specific Gomes et 

al. 2001 

f203 α:5 ′ -CCGCATACGCCCTACGGGGGAAAGATTTAT-3 ′ 

and r1494: 5 ′ -CTACGG(T/C)TACCTTGTTACGAC-3 ′ 


f984 with GC clamp and r1378 

First step: β-proteobacteria specific Gomes et 

al. 2001 

f203 β:5 ′ -CGCACAAGCGGTGGATGA-3 ′ and r1494 


f984 with GC clamp and r1378


19.5.6 

Molecular Identification of Species and Genes 

Bands in molecular fingerprints reveal neither the identity nor the function 

of individual species. For identification of bands of noncultivable species, 

individual bands are isolated and, if necessary, the DNA is cloned. Subsequent 

sequencing and phylogenetic analysis theoretically enables identification, 

provided that the sequences found correspond to known sequences 

in the databanks. From the sequence information, new probes can be constructed 

for follow-up studies using FISH (e.g. Felske et al. 1998a; Amann 

and Ludwig 2000). Noncultivable species may be identified, though their 

function and ecological roles will remain unresolved. 

Metagenome analysis may represent a new and promising approach in 

endophyte research (Rondon et al. 2000). Using this approach the function 

of genes from the noncultivable fraction present in different habitats can be 

unravelled. Large fragments (>50 kb) are required, prepared from soil or 

other environmental DNA samples and cloned into bacterial artificial chromosome 

(BAC) vectors. Expression studies and sequence data of genes and 

operons of cloned fragments should reveal some of the genetic properties 

of noncultivable species in the ecosystem under study. Due to the high incidence 

of beneficial bacteria among endophytes and other plant-associated 

bacteria commonly observed in many different studies, we expect that the 

phytosphere will become an interesting object for metagenome analysis 

studies. In the near future, use of the plant-metagenomic approach may 

result in new compounds for the development of pharmaceutical and agrochemical 

products. 

19.6 

Integration of Detection Techniques 

19.6.1 

Polyphasic Approach 

Molecular analysis of plant ecosystems has limitations with respect to the 

detection, function, activity and ecological behavior of individual populations. 

Complementary data are often required, e.g. the use of conventional 

techniques of cultivation and microscopy (Van Elsas et al. 1998). 

In an unpublished study we used a combination of in situ microbial activity 

staining, cultivation and PCR-DGGE to isolate and identify cultivable 

and non-cultivable bacteria associated with storage and vascular tissue of 

potatotubers.Slicedpotatotuberswereincubatedinwateragarcontaining 

triphenyl tetrazolium chloride, a colourless dye that changes into red


formazan in the presence of microbial activity. Vascular bundles in tubers 

appeared to be hot spots for microbial activity and small samples extracted 

from the bundles were used for isolation and PCR-DGGE analysis. Two 

dominant isolates from the vascular bundle were identified by partial 16S 

rDNA gene sequence analysis as Pantoea agglomerans and Bacillus pumilis 

Comparison of the bands of these isolates with those of the PCR-DGGE 

fingerprints from total vascular community DNA revealed that three dominant 

bands did not match any of the isolates. A combination of plating 

and PCR-DGGE made it clear that dominant noncultivable species must be 

present in the vascular bundles of potato tubers. Additionally, the in situ 

metabolic activity staining facilitated sampling of potentially interesting 

areas by demonstrating hot spots of microbial activity in tuber tissue. 

The application of multiple detection methods will aid in increasing our 

knowledge of plant ecosystems. Molecular fingerprinting methods must 

be considered as supplementary tools to the already existing panel of detection 

techniques. The advantage of molecular fingerprinting techniques 

over other techniques is the possibility to detect entire bacterial communities 

instead of only cultivable populations. For some ecosystems, such as 

soil and water, molecular fingerprinting is nowadays considered a routine 

method. A challenge for future endophyte research will be the integration 

of molecular fingerprinting methods for cultivable and non-cultivable bacteria 

and traditional culture-based endophyte research as complementary 

routines. 

19.7 

Conclusions 

Molecular fingerprinting methods are required as tools to supplement conventional 

methods in endophyte research to study cultivable and noncultivable 

populations in plants. The intricacies of plants make these techniques 

difficult to perform. The presence of chloroplast and mitochondrial 

DNA in total plant DNA extracts, the abundance of plant DNA versus 

bacterial DNA, and the presence of (poly) phenolics may hamper subsequent 

analyses. Group (taxon)-specific primer systems can circumvent 

co-amplification of 16S rDNA sequences from plant cell organelles. The 

plant metagenome concept offers great perspectives for isolating new substances 

from plant-associated microorganisms, which may be important 

for the pharmaceutical and crop protection industries.



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Subject Index 

Abies alba 304 

Abiotic stress 271 

Acephala applanata 121 

Acer spicatum 182–185 

Acetobacter 7 

– diazotrophicus 26, 338 

Achlya klebsiana 55 

Acremonium 163 

– alternatum 270, 274 

–cf.curvulum 232 

– cucurbitacearum 216 

– killense 159 

– pteridii 166 

– strictum 164, 237 

Acrocalymma medicaginis 210 

Actinoplanes missouriensis 59, 61 

Acuminatopyrone 146 

Adaptations 4 

Adhesive 194 

Aequorea victoria 326 

Agricultural practices 25 

Agrobacterium 321 

– tumefaciens 23, 56, 82, 346 

Agrobacterium-based 

transformation 331 

Agrochemicals 117 

Air pollutants 118 

Allozyme 122 

Alnus glutinosa 110, 181, 183–186, 

188, 189 

α-proteobacteria 346 

Altechromones 268, 274 

Alternaria alternata 162, 165 

Altitude 109 

AMF 109, 117, 119 

Amycolatopsis meditarranei 56 

Anamorph 187 

Angiopteris evecta 182, 

184–186 

Anguillospora 

– filiformis 183 

– longissima 181, 183 

Angular leaf spot 35 

Antagonism 54, 56–59, 67, 68 

– balanced 172 

Antagonistic 53–58, 61, 64–67 

Antagonistic bacteria 58 

Antagonistic endophytic bacteria used to 

control fungal pathogens. BCA 

Biocontrol agent 62 

Anthostomella aracearum 166 

Antibiosis 53, 58, 59, 67 

Antibiotic 180, 186 

Antifungal 57–60, 66 

Antipteris evecta 186 

Aphelandra tetragona 303 

Apoplastic fluid 307 

Appressorium 192, 194, 197 

Arabidopsis 43 

– thaliana 96, 322 

Arbuscular mycorrhiza 119, 209, 248, 266 

Arbutoid mycorrhiza 209 

Armillaria jezoensis 161 

– mellea 160, 161, 168 

Armillariella mellea 161 

Arthrinium sp. 163, 165 

Arthrobacter 155 

– ilicis 56 

Arthrobotrys 192 

– dactyloides 196, 198–200 

– ferox 192 

– oligospora 193, 197, 198 

Articulospora 

– antipodea 183 

– atra 183 

– proliferata 183 

– tetracladia 183 

Artificial inoculation 16

356 Subject Index 

Ascochyta sp. 163, 166 

Ascomycetes 193, 208 

Ascomycota 219 

Aspergillus 163 

Assemblages 4 

Astroloma pinifolium 212 

Asymptomatic 6 

Aureobasidium caulivorum 166 

Auricularia polytricha 161 

Aurofusarin 146 

Avicennia 

– officinalis 183, 185 

Azoarcus 2, 7, 11, 96 

Azospirillum 39 

– brasilense 330 

B trichothecene 144 

BAC 348 

Bacillispora 

– inflata 183 

Bacillus 56, 61, 66, 68, 91, 155, 321 

– amyloliquefaciens 36 

– aquamarinus 56 

– cereus 26, 56, 59, 68 

– licheniformis 65 

– megaterium 22, 56 

– polymyxa 94 

– pumilus 34, 36, 65, 338, 349 

– subtilis 36, 58, 59, 65 

Bacteria 154 

– nitrogen-fixing 155 

Bacterial 338 

Bacterial artificial chromosome 348 

Bacterial diversity 21 

Bacterial endophytes 90 

Bacterial identification 16 

Bacterial speck 39 

Bacterial spectrum 16 

Bacteroids 71 

Balance of antagonisms 264 

Balanced antagonism 7, 219, 269 

Balansia 134 

Basidiomycetes 192 

BCA 355 

Bead beating 343 

Beauvericins 264 

Behaviour 107 

– antagonistic 107, 119 

– mutualistic 107 

– neutral 107, 110, 114 

– pathogenic 107, 120 

Beneficial 340 

Benomyl 118 

Benzoxazinoids 145 

β-glucoronidase 136 

β-proteobacteria 346 

Betula 

– papyrifera 117, 182–185 

– pendula 112, 304 

– platyphylla 118 

– pubescens 212 

Bidirectional carbon transport 269 

Biocides 117 

Biodiversity 107, 157 

Biofilms 327 

Biological control 6, 33, 53, 54, 57–61, 

64–67, 69, 191 

Biomass 115 

Biotrophic 147 

BioYield 49 

Bishop pine 118 

Blue mold 37 

Bog 229, 232 

Bordetella pertussis toxin exporter 82 

Bracken 115 

Bradyrhizobium japonicum 77 

Brassica chinensis var. 

parachinensis 38 

Bremisia argentifolii 37 

Burkholderia 54 

– cepacia 26, 65 

– solanacearum 61 

Butenolide 146 

Cadophora finlandia 211, 216 

Cajanus cajan 79 

Calanthe 155 

Callose 197 

Calluna vulgaris 236 

Calonectria kyotensis 162 

Campylospora 

– chaetocladia 184 

– purvula 181 

Capronia 212, 249, 253 

Capsicum annuum 38 

Capsular polysaccharides 73

Subject Index 357 

Carbohydrates 114 

Carbon sink 272 

Carbon transport 266 

Catenaria anguillulae 193 

Cell organelles 345 

Cell wall appositions 197, 270, 271 

Centrifugation 307 

Centrospora acerina 181 

Cephalanthera austinae 167 

Ceratobasidium 156, 166 

Ceratocystis fagacearum 91 

Ceratorhiza, Epulorhiza 166 

Cereal 116, 120 

Chaetomium aureum 165 

– funicola 164, 265, 274 

– homopilatum 162 

– subspirale 163, 164 

Chaetosphaeria endophytica, 

Colletotrichum gloeosporioides 166 

Chaetosticta cf. perforata 165 

Chaetothyriomycetidae 210, 212 

Chemical messengers 117 

Chinese cabbage 112 

Chitinase 194 

Chlamydospores 118, 125, 197 

Chlamydosporol 146 

Chloridium paucisporum 120, 211, 262 

– virescens 165 

Chloroplasts 345 

Christela 

– dentata 182, 184–186 

Chrysogine 146 

Chytridiomycetes 193 

Citrus jambhiri 302 

Cladorrhinum foecumdissimum 274 

Cladosporium cladosporioides 164 

Clavariopsis 

– aquatica 184 

– azlanii 184 

Clavibacter michigenensis 56 

Climate 108, 109 

Climate change 108 

Clone libraries 345 

Clover 111, 117 

CO2 109 

Codinaea parva 162 

Coelomycete 210 

Coffea arabica 182, 184, 185, 186 

Colletotrichum 163–165, 168 

– acutatum 165 

– crassipes 162–165 

– gloeosporioides 38, 163, 166 

– magna 6 

– orbiculare 36 

Colonisation 262, 263, 273 

Commensalism 6 

Common reed 115 

Communities 107, 108, 110, 113–115, 

117, 118, 127, 329, 337 

Community composition 108 

Competition 59, 115, 120 

– interspecific 115, 125 

Competitive advantage 107 

Competivity 109 

Confocal laser scanning microscopy 325 

Conidia 197 

Conidial traps 196 

Coniothyrium sp. 166 

Continuum 6 

Corallorhiza 167 

Corn (Zea mays) 90 

Cortinarius 286 

Cotton 40 

Cronartium quercuum f. sp. fusiforme 38 

Crop protection 118 

Crotalaria juncea 78 

Cryptocline 163, 164, 166 

Cryptosporiopsis 4, 163–166, 262, 264, 

266, 271, 274 

– radicicola 116 

Cucumber anthracnose 36 

Cucumber beetles 35 

Cucumber mosaic virus 36 

Cucumis sativus 302 

Cucurbit 210, 217 

Culmorin 146 

Cultivation-independent methods 311 

Culture extracts 266 

Culture morphology 120 

Curtobacterium 91, 155 

– albidum 56 

– flaccumfaciens 23, 56 

– luteum 56 

Curvularia 164, 165, 271, 274 

– cymbopogonis 165 

– pallescens 166


Cyanobacteria 155 

Cyclic β-glucans 73 

Cylindrocarpon 181 

– aquaticum 184 

– destructans 159, 181 

– didymum 116, 123 

Cypripedium calceolus 167 

Cystopage 193 

Cytogloeum sp. 166 

Cytokinin 269 

Dactylaria sp. 164 

Dactylorhiza majalis 167 

Dark septate endophytes 1, 108, 109, 114, 

117–122, 125, 126, 207, 209, 254, 262, 265, 

266, 268, 270, 272, 274, 290 

Dark septate fungi 290 

Decision-making 108 

Defence metabolites 145, 271 

Defence responses 8 

Definition of fungal endophytes 134 

Degenerate primers 341 

Deleterious 346 

Denaturing gradient gel 

electrophoresis 341 

Dendrobium moschatum 155 

Deoxynivalenol 144 

Detection 5 

Detrimental 340 

Deuteromycetes 192 

Diacetoxyscripenol 146 

Diaporthales 210 

Diaporthe 210, 218 

Differential interference 

microscopy 324 

Differential labelling 329 

Dikaryomycota 180 

Diplazium 

– esculentum 182, 185, 186 

Discosoma 326 

Disease 179 

Disease incidence 61–63 

Disease severity 61–63 

Distinguish endophytic from mycorrhizal 

associations 285 

Disturbances 108, 117 

– anthropogenic 108, 117, 125 

– natural 108, 117, 121 

Diversity 5, 337 

– fungal 180 

Diversity indices 21 

DNA 344 

DON 144 

Dothideomycetidae 210 

Douglas-fir (Pseudotsuga menziesii) 98 

Drechmeria coniospora 193, 194 

Drechslera australensis 163 

– ellisii 163 

Dryas octopetala 109, 110 

DSE 1, 108, 109, 114, 117–122, 125, 126, 

207, 209, 254, 262, 265, 266, 268, 270, 272, 

274, 290 

DSM 207 

Dual ECM/VAM associations 286 

Ear rots 133 

ECM 109 

Ecosystem 108, 109 

– forest 109, 112, 113, 117, 121, 123, 124 

– pristine 108 

Ectendomycorrhiza 4, 214, 215, 263, 286 

Ectomycorrhiza 209, 215–217, 263, 266 

– defining 286 

– intermediate associations 286 

Ectorhizosphere 195 

Egg- and female parasitic fungi 192 

Eggplant 112 

Elm (Ulmus) 91 

Endoparasitic fungi 192 

Endophyte 1 

– dark septate 158 

– roles 282 

Endophytic 

– definition 281 

Endophytic phase 283 

Enniatins 146 

Enterobacter 91 

– amnigenus 27 

– asburiae 24, 40 

– cloacae 27 

Environment 109, 339 

– alpine 109, 119–121 

– arctic 109 

– temperate 109, 118, 120, 121 

– tropical 109 

Environmental factors 107


Epacrid mycorrhiza 212 

Epacridaceae 247 

Epacris pulchella 250 

Epichloë 125, 134 

Epicoccum 169 

– andropogonis 162–165 

– nigrum 163, 165 

Epiphytes 154 

Epulorhiza, Moniliopsis 166 

Equisetin 146 

Ergot alkaloids 134 

Erica arborea 209, 254 

– carnea 109, 110 

Ericaceae 212, 227 

Ericales 234 

Ericoid mycorrhiza 4, 209, 212, 

262, 290 

Erwinia carotovora 24 

– herbicola 330 

– persicinus 56 

– tracheiphila 34 

Erythromyces crocicreas 161 

Ethanol 301 

European beech 116 

Exoenzymes 7 

Exploitation 6 

Exploitive 1 

Extinctions 124 

Extracellular enzymes 255 

Extracellular polysaccharides 73 

Extraction 341 

Fagus sylvatica 116, 304 

Favolaschia thwaitesii 159 

Fen 229 

Ferns 183 

Fertilisation 25 

Fertilisers 108, 117, 118 

Filosporella 182, 184 

– fistucella 184 

– versimorpha 184 

Fingerprinting 337 

Fire 211 

Flagella assembly 77 

Flagellospora 

– curvula 184 

– fusarioides 184 

– penicillioides 184 

Flavobacterium 155 

Flavonoids 72 

Flemingia congesta 81 

Flexibacter 100 

Flooding 115 

Fluorescence microscopy 325 

Fluorescent in situ hybridisation 340 

Fomes 168 

Fomitopsis pinicola 231 

Fontanospora fusiramosa 182, 184 

Food safety 27 

Forest-management 117 

Foundations 124 

Founder genome 125 

Fumonisins 144 

Fungal plant pathogen 58 

Fungi 

– ancestral 180, 187 

– Ascomycetes 169 

– Basidiomycetes 169 

– biomass 182 

– clavicipitaceous 179 

– Coelomycetes 169 

– Deuteromycetes 169 

– foliicolous 187 

– Hyphomycetes 169 

– lignicolous 187 

– mycorrhizal 153 

– phyllosphere 179 

Fungi in roots 

– identification 291 

Fungicide 118, 172 

Fusamarin 146 

Fusaric acid 145 

Fusarium 116, 133, 163, 167, 181, 

262–264, 270, 271, 287 

– culmorum 140 

– graminearum 133, 139, 140, 144, 

145, 147 

– moniliforme 4, 133, 268, 274, 276 

– nivale 140 

– oxysporum 140, 160, 162, 163, 166 

– oxysporum f. sp. melonis 140 

– oxysporum f. sp. pisi 41 

– oxysporum f. sp. radicis lycopersici 

41, 324 

– oxysporum f. sp. vasinfectum 40 

– sambucinum 164


– verticillioides 4, 7, 133, 136, 139, 140, 

144, 145, 147 

Fusarium wilt 40 

Fusarochromanone 146 

Fusiform rust 38 

Fusoproliferatum 146 

Gaeumannomyces graminis 116, 120, 198 

Ganoderma 168 

– australe 161 

Gap formation 118 

Gaultheria shallon 212, 237, 249, 250, 260 

GC clamp 342 

Gelatinosporium spp. 166 

Genetic diversity 121, 122, 125 

Geniculospora 184 

Genotype 339 

Geographical isolation 124 

Geography 22, 108 

Geotrichopsis sp. 164 

Germination 

– seed 156 

Gibberella fujikuroi 134 

Glaciations 124 

Gliocladium roseum 166 

Globodera pallida 194 

Glomerella cingulata 162, 163, 165, 166 

Glomeromycota 180 

– endophytic phases 283 

Gluconacetobacter diazotrophicus 338 

Glume blotch 116 

Gossypium hirsutum 302 

Grass 120, 125 

Green fluorescent protein 326 

Green kuang futsoi 38 

Gremmeniella abietina 121 

Group-specific PCR 346 

Growth enhancement 266, 274 

Growth stage 339 

Guignardia 162, 163, 170 

Gymnascella dankalienses 236 

Gyoerffyella 188 

Hadrotrichum 162–165, 169 

Hair roots 247 

Halep pine 209 

Hartig net 119 

Heavy metals 113, 118 

Helicobacter pylori 172 

Heliscus lugdunensis 184, 187 

Helotiales 212, 253 

Hemibiotrophic 138, 147 

Herbaspirillum 321 

– seropedicae 338 

Herbicide 180 

Herbivores 107, 179 

Heterobasidion annosum 230, 267 

Heteroconium chaetospira 4, 13, 262, 

270, 274 

Hevea 

– brasiliensis 182, 185 

Hexalectris spicata 167 

Hirsutella rhossiliensis 198–200 

Histology 262 

Hohenbuehelia 192, 198 

Holm oak 209 

Hordeum vulgare 303 

Horizontal transmission 5 

Host adaptation 268 

Host defence 116, 270 

Host preference 5, 116, 117 

Host specificity 116 

HrpF 81 

Human pathogens 26, 346 

Humicola 163, 164 

– fuscoatra 159 

Hyaline hyphae 263 

Hydrogen peroxide 301 

Hymenochaete crocicreas 161 

Hymenoscyphus ericae 212, 216, 249, 

253, 258, 290 

Hyphal coils 192 

Hyphomycetes 115 

– aquatic 115, 180 

Hypoxylon cf. unitum 163–165 

IAA 267 

Immunogold labeling 40 

Index of association 123 

Indole acetic acid 267 

Induced resistance 6, 25, 33, 269 

Infection asymptomatic 179 

Infection threads 71 

Inhibition 117 

Insect pests 107 

Interaction 24, 108, 115, 116, 126 

– among microorganisms 108


– host-endophyte 180 

– hyphal 116 

– multitrophic 108, 115 

Inter-glacial periods 124 

ITS (Internal transcribed spacer) 

120–122, 208, 220 

ISSR (Inter-simple sequence repeat) PCR 

122, 123, 125, 251 

Interspecific hybrids 125 

Isolation procedures 299 

Isozyme 121 

ITS-RFLP 209 

ITS-RFLP analysis 251 

Kaskaskia sp. 166 

Kingella kingae 56 

Kitasatosporia cystargenia 56 

Klebsiella pneumoniae 338 

Kobresia myosuroides 212 

Lactic acid bacteria 346 

Larix decidua 262 

Lasiodiplodia 169 

– theobromae 162–166 

Lasmeniella sp. 163 

Late blight 37 

Latent pathogen 2, 218 

Leafage killers 117 

Lecanicillium lecanii 192 

Lentinula edodes 161 

Lenzites betulinus 161 

Leotiales 208 

Lepanthes 170 

Leptodontidium 4 

– orchidicola 11, 159, 160, 171, 211, 262 

Leptosphaerulina australis 164, 165 

Leucaena leucocephala 76 

Life history strategies 1 

Lignin 270 

Lipo-polysaccharides 73 

Live oak (Quercus fusiformis) 91 

Loam 114 

Loblolly pine 38 

Lodgepole pine (Pinus contorta Dougl. var. 

latifolia) 90 

Logging 108 

Lolium perenne 118, 120, 303 

Long cayenne pepper 38 

Lophiostomataceae 210 

Lotus japonicus 78 

Loweporus tephroporus 161 

Lucerne 211 

Lunulospora curvula 184 

Lycoperdon perlatum 161 

Lycopersicon esculentum 303 

Lyophyllum shimeji 161 

Lysis 53, 58, 59 

Maceration 306 

Macrothelypteris torresiana 182, 185, 186 

Maize 111 

Maize take-all fungi 120 

Malbranchea sp. 163 

Mangifera indica 182–186 

Mangrove 115 

Marasmius coniatus 161 

Massarina aquatica 186 

– walkeri 210 

Mechanical defence reactions 2 

Media 309 

Medicago sativa 302 

Mediterranean ecosystems 208 

Mediterranean heather 209 

Melanconium sp. 166 

Melanotus alpiniae 164 

Meloidogyne incognita 24, 196, 264 

Melon 112 

Mercuric chloride 301 

Mesorhizobium loti 77 

Metabolic activity 330 

Metabolite 109, 114, 116, 117, 121, 264 

– novel 187 

– inhibitory 116 

– secondary 109, 117, 121, 125, 180 

Metagenome 348 

Methodology 21 

Microascus cinereus 166 

Micrococcus varians 56 

Microcyclus sp. 166 

Microdochium bolleyi 114, 116, 120 

Microporus affinus 161 

Microsclerotia 118, 119, 125, 266 

Microspermy 241 

Minerals 114 

Mitochondria 345 

MLH 123–125 

Molecular techniques 337


Monacrosporium ellipsosporum 196 

– haptotylum 193 

Moniliopsis 166 

Monosporascus cannonballus 216 

Monotropes 238 

Morchella 286 

Morphospecies 157 

Mortierella elongata 270, 274 

MRA 119 

Mucor 164, 165 

Multilocus haplotypes 123, 124 

Musa acuminata 302 

Mutation 125 

– rate 109, 116, 125 

– somatic 125 

Mutualism 6, 155 

Mutualistic root symbioses 261 

Mutualistic symbionts 338 

Mycelium radicis atrovirens 119, 166, 290 

Mycena orchidicola 159, 171 

– osmundicola 161 

– thuja 160 

Mycobacterium 155 

Mycobiont 235 

Mycocentrospora 185 

– acerina 181 

– clavata 185 

– iqbalii 185 

Mycoheterotroph 4, 238 

Mycoparasitism 195 

Mycorrhiza 116, 117, 153 

– arbuscular 109, 119 

– definition 281 

– ecto- 109 

– ericoid 229 

– evolution 292 

– orchid 238 

Mycorrhizal fungi 1, 25 

– designation 292 

– endophytic phase 283 

– inoculum 283 

– roles 282 

– structures 262 

Mycorrhizin 264 

Mycotoxin 7, 143, 144, 268, 275 

Myriogenospora 134 

Myxotrichaceae 227 

Myxotrichum setosum 236 

Nectria alata 162, 165 

– haematococca 162–165 

– ochroleuca 162, 163, 165 

– peziza 164 


Nematoctonus 192, 198 

– pachysporus 198, 199 

– robustus 198–200 

Nematode 117 

– entomopathogenic 117 

Nematode-trapping fungi 192 

Nematophagous fungi 6, 191 

Neoplaconema napelli 165 

Neottia nidus-avis 167 

Neotyphodium 134 

Nicotiana tabacum 81 

Nigrospora sphaerica 164 

Nitrogen 118 

– fixation 90, 155 

– uptake 265 

Nitrogen-fixing bacteria 25 

Nivalenol 146 

NOx 118 

Nocardia 155 

nod-box 72 

NodD 72 

Nod-factors 72 

NodO 75 

Nodulisporium 162, 163, 165, 166 

– gregarium 166 

Non-cultivable 311, 341 

Non-invasive 326 

NopL 81 

NopX 80 

Norway spruce 114, 121–124 

Nostoc 155 

Nucleic acids 344 

Ochrobactrum anthropi 27 

Oidiodendron 249 

– cerealis 236 

– chlamydosporicum 236 

– citrinum 236 

– flavum 236 

– griseum 236 

– maius 2, 4, 227, 262, 266, 

274, 290 

– periconioides 236


– rhodogenum 236 

– scytaloides 232, 236 

Oliveonia 166 

Optical trapping microscopy 331 

Optimum carrying capacity 16 

Orbilia 193 

Orchid 4, 153, 216 

Orchid mycorrhizas 287 

– designation 287 

Orchidaceae 153 

– evolution 293 

Ordination 179 

Organic amendment 26 

Organicization 231 

Oryza sativa 304 

Oscillatoria 155 

Oxytropis campestris 303 

Pachyrhizus tuberosus 78 

Paecilomyces 164, 165 

Paenibacillus 93 

– polymyxa 96 

Pantoea agglomerans 56, 349 

– anantis 56 

Papillae 197 

Parasitic 1 

Parasponia andersonii 71 

Pathogen 53–55, 57–60, 67, 107, 116, 

121, 179 

PCR 121–123, 157 

PCR-DGGE 341 

PCR-single strand conformational 

polymorphism (SSCP) 341 

PCR-TGGE 341 

PCR-T-RFLP 341 

Pea 41 

Peat 227 

Peatlands 227 

Pelotons 154 

Penicillium 163, 170 

– thomii 232 

Periconia macrospinosa 114 

Periconiella sp. 162, 165 

Peronospora parasitica 43 

– tabacina 37 

Pestalotia 163, 164, 169 

– adusta 166 

– cf. heterocornis 163, 164 

– poppola 163 

Pestalotiopsis 171 

– aquatica 163, 165 

– gracilis 163 

– papposa 162–164 

Pesticides 108 

Pezicula 8, 11, 13 

Pezizales 208 

Pezizella ericae 236 

pH 110, 114 

Phaeocryptopus gaeumannii 313 

Phaeoseptoria cf. vermiformis 163 

Phalangispora constricta 185 

Phase-contrast microscopy 324 

Phaseolus vulgaris 303 

Phellinus 168 

Phenolic metabolites 271 

Phenotypic plasticity 263 

Phenylpropanoids 271 

Pheromones 117 

Phialospora sp. 166 

Phialocephala 158, 211, 262 

– compacta 121 

– dimorphospora 121 

– fortinii 2, 11, 108, 109, 114–126, 159, 

211, 214, 217, 240, 250, 252, 254, 262, 266, 

270, 274, 290 

– scopiformis 121 

Phialophora 120, 211, 262 

– finlandica 120, 249 

– graminicola 120 


– zeicola 120 

Phloem 338 

Phoma 163, 164, 166, 265 

Phomatospora berkeleyi 166 

Phomopsis 163, 169, 210, 218 

– cf. orchidophila 162–166 

Phosphorus 114, 265, 266, 269 

Photosynthates 109 

Phragmites australis 111, 115 

Phyllobacterium 93 

– rubiacearum 61 

Phyllosticta capitalensis 166 

– colocasiicola 166 

Physiological relationships 143 

Phytoalexin 171


Phytohormones 7, 267, 268, 273 

Phytophthora cactorum 55 

– cinnamomi 230 

– infestans 37 

Phytoplasms 338 

Phytosphere 348 

Phytosterol 270 

Picea abies 110, 114, 119, 121–124, 

303, 304 

– glauca 118, 182–186 

Piceirhiza bicolorata 212 

Pili 77 

Pinus contorta 302 

– halepensis 209 

– muricata 118 

– sylvestris 112, 116, 119, 212, 216, 304 

– taeda 38 

Piriformospora indica 4, 13, 201, 262, 

267, 272 

Pisum sativum 75 

Pithomyces maydicus 162–165 

Plant and Fungus Interactions 134 

Plant defence mechanisms 25 

Plant disease 65, 67 

Plant genotype 23 

Plant growth 53, 58, 60, 62, 66, 68 

Plant health 53, 345 

Plant pathogens 24 

Plant species 22 

Plant symbionts 25 

Plant-associated 338 

Plant-fungus coevolution 282 

Plant-metagenomic approach 348 

Plants 

– myco-heterotrophic 154 

Plasmodiophora brassicae 270 

Plasticity 9, 269, 273 

Plectosporium tabacinum 59, 61 

Pleosporales 208, 210 

Pleurotus djamor 198, 199 

– ostreatus 192 

Pochonia 193 

– bulbillosa 232 

– chlamydosporia 193, 197, 198 

– rubescens 193 

Pollution 108, 113, 118 

Polymerase chain reaction 121, 123, 340 

Polyphasic 348 

Population densities 15 

Population genetics 119, 122 

Populations 108, 121, 123, 338 

Populus hybrida 182–186 

Potato (Solanum tuberosum) 90, 111, 117 

Pratylenchus 201 

Preceding crop 117 

Pressure bomb method 21, 309 

Pressure extraction 308 

Primers 341 

Proteolytic enzymes 194 

PR-proteins 81 

Pseudallescheria boydii 165 

Pseudoanguillospora 185 

Pseudogymnoascus roseus 236 

Pseudomonas 54, 56, 60, 66–69, 91, 

155, 321 

– chlororaphis 61, 94 

– chlororaphis PCL1391 327 

– cichorii 23, 56 

– corrugata 56 

– denitrificans 91 

– fluorescens 22, 34, 56, 61, 65, 66, 

68, 146 

– fluorescens CHA0 329 

– fluorescens SBW25 330 

– fluorescens WCS365 322 

– graminis 56, 61 

– migulae 56 

– orientalis 56 

– putida 22, 56, 61, 91, 330 

– reactans 56 

– rhodesiae 56 

– straminea 56 

– synthaxa 56 

– syringae 56, 90 

– syringae pv. lachrymans 34 

– tolaasii 56, 61 

– trivialis 61 

– veronii 56, 61 

Pseudomycorrhiza 119 

Pseudotsuga menziesii 117 

Psychilis kraenzlinii 170 

Psychrobacter immobilis 56 

Pteridium aquilinum 115, 303 

Pterostylis 155 

Purification 341 

Pyrenochaeta cf. rubi-idaei 164


Pythium 268 

– oligandrum 271, 274 

– spinosum 55 

Quantification 5, 313 

Quercus ilex 209, 255 

Quorum sensing 324 

Ralstonia paucula 56 

– picketti 346 

– solanacearum 38, 340 

Ramichloridium apiculatum 166 

– cf. subulatum 164 

RAPD (Random amplified polymorphic 

DNA) 122, 214 

rDNA 208, 219 

Real-time PCR 340 

Recombination 122, 125 

Red fluorescent protein 326 

Reporter genes 322 

Resistance 116 

Restriction fragment length 

polymorphism 121 

Restriction patterns 120 

RFLP 121, 122, 125 

Rhizobacteria 34, 201 

Rhizobiaceae 321 

Rhizobium etli 25 

– leguminosarum 25, 75 

– meliloti 56, 71, 76 

– sp. NGR234 71 

Rhizoctonia 4, 156, 166, 287 

– solani 24, 38, 55, 59, 61, 195 

Rhizomonas suberifaciens 56 

Rhizophora mucronata 110, 115, 183, 186 

Rhizoplane 107 

Rhizopycnis vagum 210 

Rhizoscyphus ericae 231 

Rhizosphere 107, 114, 117, 125, 322 

Rhodococcus 155 

Ribosomal 340 

Ribosomal RNA 120 

Rice (Oryza sativa) 90, 120 

– crown sheath rot 120 

RNA 343 

rRNA 120, 121 

Root endophytes, multiple roles 290 

Root nodules 71 

Root turnover 109 

Root-colonising 321 

Roots, adventitious 155 

Rosemary 209 

Rosmarinus officinalis 209 

Rot 133 

Rough lemon (Citrus jambhiri) 90 

16S rDNA 341 

Salicylic acid 34 

Salix babylonica 182–186 

Salmonella enterica 27 

Saprobe 2, 228 

Saprophytic 266 

Saprotrophic symbiont 219 

Scanning electron microscopy 324 

Sclerroderis canker 121 

Scots pine 116 

Scytalidium vaccinii 236 

Seawater 115 

Sebacina 156, 166, 238, 250 

sec pathway 78 

Secondary metabolites 264, 273 

Sedges 120 

Seed borne 137 

Seed treatment 118 

Selective media 311 

Septoria nodorum 116, 118 

Sequences, ribosomal 180 

Serendipita 166 

Serine protease 194 

Serratia 54, 56 

– marcescens 26, 35, 65, 66, 338, 346 

– plymuthica 58, 61, 66, 67 

Sink 269, 270, 272 

Sinorhizobium meliloti 329 

Ski slopes 118 

SO2 118 

Sodium hypochlorite 301 

Soil suppressiveness 202 

Soil texture 114 

Soil-rhizosphere-root continuum 107 

Solanum tuberosum 90, 111, 117, 302 

Sonification 300 

Sonneratia caseolaris 183, 186 

Sordaria fimicola 159 

Sordariomycetidae 210 

Spanish oak (Quercus texana) 91 

Spatiotemporal patterns 108


Speciation 123, 125, 126 

– allopatric 123, 125 

– sympatric 123 

Species 110–113, 345 

– abundance 108, 115, 121 

– competitive 107, 116, 118 

– cryptic 123–126 

– diversity 108–110, 114–119, 121–126 

– evenness 108 

– identification 120 

– richness 108, 109, 114, 115, 117, 118 

– spectrum 108, 109, 115–117 

– typing 120 

Specificity 5, 158 

Sphagnum 229 

Sphigobacterium thalophilum 56 

Sphingomonas 91 

– adhaesiva 56 

– trueperi 61 

Spore production 182 

Sporormia minima 160 

Spruce 229 

Stagonospora 263, 265, 274 

Staphylococcus xylosus 27 

Stem rot 133 

Stenotrophomonas 91 

– maltophilia 26, 56 

Sterilising agents 301 

Sterility check 300, 305 

Stimulation 117 

Strain mixtures 38 

Strawberry 112 

Streptomyces 93, 321 

– bottropensis 56 

– diastatochromogenes 56 

– galilaeus 56 

– griseus 56 

– lavendulae 56 

– scabies 55 

– setonii 56 

– turgidiscabies 56 

Stress tolerance 274 

Stylopage 193 

Succession 122, 124 

Sugar beet 111 

Sugarcane Saccharum officinarum 90 

Suppression 346 

Surface sterilisation 5, 300 

Surfactants 305 

Symbiont 235 

Symbiosis 1 

Sympatric occurrence 123 

Symptomless root infections 139 

Systemic acquired resistance 33 

Systemic fungal root infections 269 

Systemic resistance 271 

T-2 toxins 146 

Take-all fungus 198 

Taxon 344 

Tecomella undulata (Bignoniaceae) 90 

Teleomorph 181, 186, 187 

Temperature gradient gel 

electrophoresis 341 

Tephrosia vogelii 78 

Terminal restriction fragment length 

polymorphism 341 

Tetrabrachium 

– elegans 185 

Tetracladium 185 

– furcatum 185 

– marchalianum 181, 185 

– setigerum 185 

Tetrazolium 348 

Thailand 38 

Thanatephorus 156, 166 

– gardneri 287 

Thermomyces verrucosus 159 

Thielavia 249, 253 

– basicola 160 

Thuja occidentalis 117 

Tidal level 115 

Tillandsia 155 

Tissue type 339 

Tobacco 36 

Tolumnia variegata 169 

Tomato 36, 37, 112, 210 

Tomato mottle virus 37 

Total community nucleic acids 341 

Total microflora 324 

Toxicity 179 

Toxin-producing fungi 192 

Toxins 264 

Trametes hirsuta 161 

Trapping organs 192 

Tricellula aquatica 185


Trichocladium opacum 160 

Trichoderma 163, 168, 195 

Tricholoma 286 

Trichosporiella multisporum 159 

Tricladium chaetocladium 185 

– splendens 181 

Triscelophorus 

– acuminatus 185 

– konajensis 186 

– monosporus 186 

Triticum aestivum 111, 114, 117, 302 

Troposporella sp. 162–164 

Tsuga 212 

Tubercularia 164 

Tuberculate mycorrhizae 98 

Tulasnella 156, 166 

Tumularia aquatica 186 

Type 1 121 

Type I secretion systems 73 

Type II secretion systems 77 

Urban development 108 

Vaccinium corymbosum 253 

– macrocarpon 253 

– myrtillus 216 

Vacuum 308 

Valsaceae 210 

Vanilla 171 

Varicosporium 188 

– elodeae 186 

– giganteum 186 

Vascular cylinder 2 

Vascular tissue 309 

Velamen 154 

Verticillium 193, 287 

– chlamydosporium 264, 275 

– dahliae 55, 59 

– lecanii 166 

– longisporum 24, 55, 270 

Vicia sativa 75 

Vigna unguiculata 78 

Vine decline 210, 217 

Virulence factors 7, 264 

Virulent pathogens 2 

Visoltricin 146 

Vitis vinifera 329 

Volatile organic compounds 44 

Vomitoxin 144 

Water regime 114, 115 

Weather 109 

Weevil larvae 117 

Westerdykella 270, 274 

Western red cedar (Thuja plicata) 100 

Wetland 115 

Wheat (Triticum aestivum) 96, 114, 

116–118 

White x Engelmann hybrid spruce (Picea 

glauca x P. enelmannii 92 

Whitefly 37 

Wildfire 36, 118 

Windthrow 112, 117, 118 

Woodland 115 

Woollsia pungens 212, 249, 251, 253, 

258–260 

Wortmannin 146 

Wullschlaegelia calcarata 168 

Xanthomonas 155 

– campestris 55, 56 

– campestris pv. vesicatoria 81 

– oryzae 56 

Xylaria 162, 163, 164, 168 

– arbuscula 163 

– cf. cubensis 163 

– cf. curta 163 

– corniformis 163 

– enteroleuca 163 

– hypoxylon 163 

– mellisii 163 

– multiplex 163 

– obovata 163 

– polymorpha 163 

Xylella fastidiosa 313, 329 

Xylem 338 

Yield shield 44 

Ypsilonidium 166 

Zea mays 302 

Zearalenone 144 

Zygomycetes 193

References

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