3 540 33526 9

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
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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 Dr. Christine J. C. Boyle
Technical University of Braunschweig Augustastraße 32
Institute of Microbiology 02826 Görlitz
Spielmannstraße 7 Germany
38106 Braunschweig e-mail: c.boyle@tu-bs.de
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
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
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Preface
Healthy plant roots are not only colonized by mycorrhizal fungi and rhi-
zobial 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 fac-
tors. Recently it has become clear that some of these interactions with
endophytic bacteria and fungi can be latently pathogenic and/or mutualis-
tic. 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 agricul-
ture 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 chap-
ters. 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
a book in this series.
Braunschweig, Barbara Schulz

Görlitz and Zürich, Christine Boyle
June 2006 and Thomas Sieber
Contents
1 What are Endophytes? 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
References ............................................................................. 50
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
References ............................................................................. 66
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
References ............................................................................. 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
References ............................................................................. 103
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
References ............................................................................. 126
8 Endophytic Root Colonization by Fusarium Species:

Histology, Plant Interactions, and Toxicity 133
Charles W. Bacon, Ida E. 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
References ............................................................................. 147
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
References ............................................................................. 173
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
References ............................................................................. 188
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
References ............................................................................. 203
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
References ............................................................................. 220
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
References ............................................................................. 242
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
References ............................................................................. 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
References ............................................................................. 276
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
References ............................................................................. 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
References ............................................................................. 314
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
References ............................................................................. 333
19 Application of Molecular Fingerprinting Techniques

to Explore the Diversity of Bacterial Endophytic Communities 337
Leo S. van Overbeek, Jim van Vuurde, Jan D. van Elsas
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
References ............................................................................. 350
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 Agri-
cultural 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
63B York Street, Department of Biology, Mount Allison University, 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 Biotechnol-
ogy, 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, Switzer-
land
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,
The Netherlands
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.
land
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 Sci-
ences, Institute of Integrative Biology, Forest Pathology and Dendrology,
8092 Zürich, Switzerland
Hallmann, Johannes
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Ne-
matologie und Wirbeltierkunde, Toppheideweg 88, 48161 Münster, Ger-
many
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, Au-
burn, 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, Wagenin-
gen, The Netherlands
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,
10125 Torino, Italy
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.
land
Salinas, Jesús
Schulz, Barbara
Institute of Microbiology, Technical University of Braunschweig, Spiel-
mannstraße 7, 38106 Braunschweig, Germany
Sieber, Thomas N.
Swiss Federal Institute of Technology, Department of Environmental Sci-
ences, 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, Wagenin-
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 vari-
able is the usage of the term “endophyte” for variable life history strate-
gies 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, Spiel-
mannstraße 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.)
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. 13 by Rice and Currah) and Phialocephala 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 symptom-
less, 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 ap-
parent harm” (Petrini 1991), which presumably refers to an absence of
macroscopically visible symptoms. Aware of the determinative discrep-
ancies, 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 (Ta-
ble 1.1). They do, however, differ somewhat in their modes of colonisation.
Bacteria primarily colonise intercellularly (Hinton and Bacon 1995; Hall-
mann 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 advanta-
geous 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).
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,
1 What are Endophytes?
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
3
The assemblages of fungi that colonise plant roots are diverse (Van-
denkoornhuyse et al. 2002). In contrast to endophytic growth in the above-
ground 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 ba-
sidiomycete 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 bacte-
ria 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 molecu-
lar 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 trans-
mission 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 ubiqui-
tous, 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 (Hall-
mann 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
Organisms detected at any one moment in asymptomatic plant tissue and
arbitrarily named “endophytes” include microorganisms with different
life history strategies. Endophytes represent, both as individuals and col-
lectively, a continuum of mostly variable associations: mutualism, com-
mensalism, 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, into an endophyte. 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 sapro-
phytic 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 syn-
thesis 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 atmo-
spheric nitrogen, which has been demonstrated not only for the nodule-
forming 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 ob-
servations 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; Lumy-
ong 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 de-
fence 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 re-
sults 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 abi-
otic environmental factors and on the tolerance of each of the partners to
these factors. In particular, many endophytes seem to be masters of pheno-
typic 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 interac-
tions are creative, having the potential for evolutionary development – the
symbioses can evolve both in the direction of more highly specialised mu-
tualisms 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 endosymbi-
otic 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 colonisa-
tion as a balance of antagonisms between host and endophyte (Fig. 1.1;
Schulz and Boyle 2005). This often fragile balance of antagonism is a mo-
mentary 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 part-

ners.
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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 publica-
tions have described the spectrum and population dynamics of indigenous
endophytic root endophytes for various plant species (Bell et al. 1995;
Gardner et al. 1982; Hallmann et al. 1997a, 1999; Hallmann 2003; Mahaf-
fee 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 fu-
ture commercialisation. This chapter reviews our current knowledge of
(1) population density, bacterial spectrum and bacterial diversity of en-
dophytic root bacteria, (2) factors influencing the population dynamics
of indigenous and introduced bacterial endophytes, (3) bacterial interac-
tions 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 105 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 104 cfu g−1 fresh weight in
Johannes Hallmann: Federal Biological Research Centre for Agriculture and Forestry, Insti-
tute 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,
Petersgasse 12, 8010 Graz, Austria

16 J. Hallmann, G. Berg
the stem and 103 cfu g−1 fresh weight in leaves. Generative organs such
as flowers, fruits and seeds are colonised by even lower numbers and, 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 104 to 106 cfu g−1 for cotton and sweetcorn
(McInroy and Kloepper 1994), 103 to 106 cfu g−1 for sugar beet (Jacobs et al.
1985), 4.0×102 to 1.3×104 cfu g−1 for cotton (Hallmann et al. 1997a, Misaghi
and Donndelinger 1990), 105 cfu g−1 for potato (Krechel et al. 2002) and
105 cfu g−1 for pine seedlings (Shishido et al. 1995). Some authors have even
reported population densities up to 1010 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
107 cfu g−1 are known from bacterial 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 105 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 physi-
ological characters often supported by ready-made tests such as API iden-
tification 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 con-
siderably 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 identi-
fication (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 endo-
phytic bacteria is affected by similar biotic and abiotic factors. However, it
also seems to be influenced by niche specialisation, differences in coloni-
sation pathway (seed-borne, rhizosphere, above-ground plant organs), or
some form of mutual exclusion operating between different bacterial pop-
ulations (Hallmann 2001; Sturz et al. 1997). Table 2.1 gives an overview
of endophytic bacteria commonly isolated from the roots of some culti-
vated 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, Sturz et al. 1997
Pseudomonas, Rhizobium
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, and Kloepper 1995
Phyllobacterium, Pseudomonas, Serratia,
Stenotrophomonas
Grapevine Enterobacter, Pseudomonas, Rahnella, Rhodococcus, Bell et al. 1995
Staphylococcus
Maize Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Lalande et al. 1989;
Corynebacterium, Curtobacterium, Enterobacter, McInroy and Kloepper
Micrococcus, Phyllobacterium, Pseudomonas, 1995
Serratia
Potato Agrobacterium, Arthrobacter, Bacillus, Krechel et al. 2002;
Chryseobacterium, Enterobacter, Micrococcus, Sturz 1995
Pantoea, Pseudomonas, Stenotrophomonas,
Streptomyces
Table 2.1. (continued)
Canola Acidovorax, Agrobacterium, Aureobacterium, Germida et al. 1998;

Bacillus, Cytophaga, Chryseobacterium, Graner et al. 2003;
Flavobacterium, Micrococcus, Rathayibacter, Misko and Germida
Pseudomonas 2002
Red clover Agrobacterium, Bacillus, Methylobacterium, Pantoea, Sturz et al. 1997
Pseudomonas, Rhizobium, Xanthomonas
Rice Serratia, Azoarcus Gyaneshwar et al. 2001;
Hurek et al. 1994
Rough Bacillus, Corynebacterium, Enterobacter, Gardner et al. 1982
lemon Pseudomonas, Serratia
Soybean Bacillus Bai et al. 2002
Sugar beet Bacillus, Corynebacterium, Erwinia, Lactobacillus, Jacobs et al. 1985
Peudomonas, Xanthomonas
Sugar cane Acetobacter Cavalcante and
Döbereiner 1988
Tomato Bacillus, Burkholderia, Chryseobacterium, Kluyvera, Munif 2001
Micrococcus, Pseudomonas, Serratia
Wheat Bacillus, Flavobacterium, Microbispora, Micrococcus, Coombs and Franco
Micromonospora, Nacardiodes, Rathayibacter, 2003;
Streptomyces Germida et al. 1998
Diverse Streptomyces Sardi et al. 1992
species
Table 2.2. Endophytic bacteria isolated from roots of various plants
Acetobacter diazotrophicus13 , A. pasteurianus9

Acidovorax delafieldii7,9,10 , A. facilis10
Acinetobacter baumannii10 , A. calcoaceticus10 , A. wolffi5 , A. radioresistens10
Aeromonas salmonicida9
Agrobacterium radiobacter7,8,9,10,12 , A. rhizogenes12 , A. rubi7,9 , A. tumefaciens12,15
Alcaligenes piechaudii9,12 , A. xylosoxydans9,12
Arthrobacter atrocyaneus10,11 , A. aurescens11 , A. crystallopodietes12 , A. ilicis15 ,
A. mysorens10,12 , A. nicotianae12 , A. pascens10,11
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 avium14 , B. bronchiseptica8

Brevibacterium acetylicum11
Brevundimonas diminuta9 , B. vesicularis7,9
Burkholderia cepacia7,8,9,12 , B. gladioli8,10,12 , B. pickettii7,8,9,12 , B. solanacearum12
Cellulomonas cellulans12 , C. flavigena10 , C. turbata12,15
Chryseobacterium balustinum9,11 , C. indologenes7,11 , C. meningosepticum7,11
Citrobacter freundii5 , C. koseri12
Clavibacter michiganensis2,7,11,12,15
Comamonas acidovorans7,8,9,10,11 , C. terrigena2 , C. testosteroni9,12,14
Curtobacterium allidum15 , C. citreum14 , C. flaccumfaciens2,10,12,14,15 , C. luteum14,15 ,
C. pusillum2,12,15
Cytophaga aquatilis10 , C. johnsonae9,10,11
Enterobacter aerogenes5 , E. asburiae8,10,12 , E. cancerogenus12 , E. cloacae2,5,8,12 ,
E. intermedius11 , E. taylorae7,8,12 , E. sakazakii5
Erwinia carotovora9,12 , E. chrysanthemi9
Escherichia coli12,14 , E. vulneris12
Flavimonas oryzihabitans12
Flavobacterium aquatile11 , F. indologenes12 , F. meningosepticum12 , F. resinovorum9,10,11
Gluconobacter oxidans9
Gordonia polyisoprenivorans3
Herbaspirillum spp.13
Hydrogenophaga flava12 , H. pseudoflava10,12
Kingella kingae15
Klebsiella ozaenae2 , K. planticola12 , K. pneumoniae2,12 , K. terrigena12
Kluyvera ascorbata12 , K. cryocrescens8,9,10,12
Kocuria kristinae11
Lactobacillus kefir10
Leuconostoc pseudomesenteroides10
Methylobacterium extorquens14 , M. fuijsawaense12 , M. mesophilicum12 , M. radiotolerans12 ,
M. rhodinum14
Microbacterium imperiale12 , M. laevaniformans12
Micrococcus agilis12 , M. halobius9,10,11 , M. kristinae12 , M. luteus8,9,10,11,12 , M. lylae10,12 ,
M. roseus12 , M. varians10,12,15
Micromonospora endolithica3 , M. peucetica3
Moraxella bovis2 , M. phenylpyruvica10
Mycobacterium aichiense3 , M. bohemicum3 , M. cookii3 , M. flavescens3 , M. heidelbergense3 ,
M. palustre3
Neisseria flavescens, N. mucosa9
Nocardia pseudobrasiliensis3
Nocardiodes albus4
Ochrabactrum anthropi12
Paenibacillus alvei10 , P. pabuli12 , P. polymyxa12
Pantoea agglomerans2,5,11,12,14,15 , P. ananas12 , P. stewartii9
Paracoccus denitrificans7
Pedicoccus pentosaceus9
Photobacterium angustum9
Phyllobacterium myrsinacearum7,8,9,12,14 , P. rubiacearum7,8,9,10,12
Plesiomonas shigelloides10
Providencia spp.5
Pseudomonas aeruginosa5,9 , P. cepacia5 , P. chlororaphis6,7,10,11,12 , P. cichorii2,6,12,15 ,
P. coronofaciens12 , P. corrugata2,6,10,14,15 , P. flectens10 , P. fluorescens5,6,7,9,11,12 , P. fragi14 ,
P. fulva14 , P. marginalis2,6,9,12 , P. mendocina6,9,12 , P. pseudoalcaligenes6 ,
P. putida2,5,6,7,9,11,12 , P. rubrisubalbicans9,12 , P. saccharophila8,10,12 , P. savastanoi6,10,12 ,
P. stutzeri9 , P. syringae2,6,9,11,12 , P. tolaasii14,15 , P. vesicularis12,14 , P. viridiflava6,11
Rahnella aquatilis2
Rhizobium etli10 , R. japonicum12 , R. leguminosarum14 , R. loti14 , R. meliloti15
Rhodococcus coprophilus3 , R. luteus2
Salmonella cholerasuis7
Serratia liquefaciens5,12 , S. marcescens12 , S. plymuthica12
Shigella spp.5
Shingobacterium heparinum11
Sphingomonas capsulata9 , S. paucimobilis9,12,15 , S. thalpophilum15
Staphylococcus capitis12 , S. epidermidis12 , S. haemolyticus11
Stenotrophomonas maltophilia9,11,12
Streptomces argenteolus4 , S. bikiniensis4 , S. caviscabies4 , S. cyaneus11 , S. galilaeus4 ,
S. halstedii11 , S. maritimus4 , S. pseudovenezuelae4 , S. scabies4 , S. setonii4 , S. tendae4 ,
S. thermolineatus3 , S. violaceusniger11
Variovorax campestris, V. paradoxus7,9,12
Vibrio cholerae9
Xanthobacter agilis9,10
Xanthomonas agilis7 , X. campestris2,8,9,12,14,15 , X. oryzae15
Yersinia frederiksenii12 , Y. pseudotuberculosis10
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 bacte-
rial density into a single number to facilitate comparisons between plant
species, habitats, etc., as well as elucidation of changes in community re-
lationships (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 mod-
ified 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, Ma-
haffee 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 cultur-
able bacteria, as different methods will give different results. Key factors
affecting the bacterial spectrum recovered are (1) length of surface sterili-
sation, (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 tem-
perate conditions in Bonn, Germany, with that of tomato roots grown under
tropical conditions in Bogor, Indonesia. In Germany, 38 species compris-
ing 21 genera were isolated, whereas in Indonesia, 50 species comprising
32 genera were isolated. Twenty-four bacterial species were exclusively iso-
lated 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).
How is it possible that a bacterial species colonises such different regions
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 oc-
curred (Cho and Tiedje 2000). But adaptations also seem to occur within
a given environment. For example, Pseudomonas fluorescens strains iso-
lated 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 bac-
terial 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, Coma-
monas, Erwinia, Ochrobactrum, and Yersinia, were recovered only from
cotton roots, while other genera, such as Arthrobacter, Citrobacter, Flavi-
monas, Microbacterium and Stenotrophomonas, were recovered exclusively
from sweetcorn roots. Plant-species-specific factors such as root architec-
ture, 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 apoplas-
tic 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 tume-
faciens, 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). Differ-
ences in the endophytic spectrum were also reported to occur in potato
tubers of four potato cultivars (Sturz et al. 1999). Although the two bacte-
rial species Curtobacterium flaccumfaciens and Pseudomonas cichorii oc-
curred 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 cul-
tivars. 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 bacte-
rial 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: mod-
ern cultivars supported a more diverse endophytic community than ancient
land races (Germida and Siciliano 2001). Furthermore, endophytic bacte-
ria 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 con-
tinuously challenged by soil-borne pathogens, mutualistic symbionts and
various soil conditions. As a result, plant defence mechanisms might be in-
duced. How do these parameters affect the endophytic bacterial spectrum?
2.6.1
Plant Pathogens
While the beneficial effects of endophytic bacteria to control plant patho-
gens 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 fun-
gal pathogens, Mahaffee et al. (1997a, 1997b) reported that root infection
by Rhizoctonia solani promoted colonisation of the two introduced bacte-
rial 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 Methy-
lobacterium spp. (Araújo et al. 2002). A third group of plant pathogens,
plant parasitic nematodes, significantly increased the total number of bac-
terial 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 ex-
clusively 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 exclu-
sively 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 sym-
bionts 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 non-
treated plants. Total bacterial density was significantly higher in treated
(1.6×104 cfu g−1 ) than in non-treated (3.2×103 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
spectrum of bacterial root endophytes is increased.
2.6.4
Agricultural Practices
Besides the plant itself and plant-associated microorganisms, agricultural
practices can also influence the spectrum and population dynamics of bac-
terial 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 rhizo-
sphere, 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 else-
where in this book [see Chaps. 3 (Kloepper and Ryu), 4 (Berg and Hall-
mann), 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 secre-
tion systems, known for their role in bacterial pathogenicity, are present in
many plant-associated Pseudomonas strains and may confer induced resis-
tance, 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, which not
only have excellent antagonistic properties against plant pathogens, but are
also known to cause human diseases, especially of debilitated or immuno-
suppressed 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 en-
dorhiza (Krechel et al. 2002; Reiter et al. 2002). In some cases, human-
pathogenic 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, and O. 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 contam-
ination 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 ap-
plications. 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 coloni-
sation have been discussed and their potential to manage endophytic com-
munities 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 surface-
sterilized 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 disease-
producing 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 metabo-
lites 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 necrosis-
producing pathogens, and this process is termed systemic acquired resis-
tance (SAR). Pieterse et al. (1998) proposed that ISR and SAR can be differ-
entiated not only by the elicitor but also by the signal transduction pathways
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 path-
way 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 sys-
temic 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 cu-
cumber plants in the field or from cucumber seeds. Bacillus pumilus strain
INR7 was isolated from a surface-sterilized stem of a surviving cucum-
ber 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 cumula-
tive 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 treat-
ment with strain INR7 (Zehnder et al. 1997b). In both years of the study,
the season-long average number of cucumber beetles per plant was signif-
icantly lower on plants treated with INR7 than on nonbacterized plants.
Reduced beetle feeding on plants treated with strain INR7 was confirmed in
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 con-
sume cucurbitacins without toxicity, apparently as an evolutionary adap-
tation 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 explana-
tion 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 bee-
tles 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 split-
root 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 an-
thracnose 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 cotyle-
dons. On tomato, the effect of 90-166 was to delay symptom development
over time. The area under the disease progress curve (AUDPC) was signif-
icantly 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 treat-
ments and as drenches upon transplanting 2 weeks after seeding. CMV
was rub-inoculated with carborundum onto leaves 1 week after trans-
planting. 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 sig-
nificantly 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 test-
ing.
In another study with CMV in tomato, Murphy et al. (2003) used vari-
ous 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 dis-
ease severity based on symptoms, decreased disease incidence based on
enzyme-linked immunosorbent assay (ELISA), and decreased virus accu-

mulation, compared to controls.
Three field trials were conducted with various formulations of the endo-
phytic 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 in-
oculated 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 re-
duce 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, ap-
plication of all three strains as a soil drench to 4-week-old plants of three
tobacco cultivars resulted in significant reductions in the mean percent-
age 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

was inoculated onto leaves 5 weeks after planting.
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) and Colletotrichum gloeosporioides, green kuang futsoi
(Brassica chinensis var. parachinensis) and Rhizoctonia solani, and cucum-
ber 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.
Further evidence for the concept of using strain mixtures of Bacillus 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 bacte-
ria 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 pro-
tected 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) by strains B. 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 sus-
pensions 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 signif-
icant 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
of a foliar pathogen. However, based on the tenant of physical separation,
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 char-
acterized 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. fluo-
rescens, some publications before 1997 refer to 89B-61 as P. putida. Later
publications designate the strain as P. fluorescens based on repeated fatty
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 at a point 1.5 cm above the point
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 le-
sion numbers and size of anthracnose following challenge inoculation of
leaves with C. orbiculare. This induced resistance was associated with colo-
nization 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 in-
vestigate the pattern of internal colonization of cotton by the ISR-eliciting
strain 89B-61. Results from isolation studies indicated that, following treat-
ment of seeds, 89B-61 colonized roots internally at a mean population of
1.1×103 cfu/g, while the bacterium was not recovered from stems, cotyle-
dons, 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 intercel-
lular 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 con-
sidered 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 demon-
strated 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 sta-
tistically 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
to the epidermis and outer cortex of roots treated with SE34, while in
nonbacterized roots, the pathogen colonized the cortex, endodermis, and
the paratracheal parenchyma cells. This reduction in fungal colonization by
SE34 was associated with strengthening of the epidermal and cortical cell
walls. In addition, roots treated with SE34 exhibited newly formed barriers
beyond the site of fungal infection. These barriers were cell wall appositions
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. Treat-
ment 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 coloniza-
tion 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. In-
terestingly, 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 obser-
vations, 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 phy-
toalexins, and second the synthesis and accumulation of other molecules
including chitinases and other hydrolytic enzymes such as β-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 re-
vealed a higher frequency of autofluorescent epidermal cells, which are
related to accumulation of phenolic compounds, at the sites of fungal pene-
tration 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.
A few studies on signal pathways have been reported with endophytic
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 Ara-
bidopsis against Peronospora parasitica were investigated by Iavicoli et al.
(2003) using various transgenic and mutant plant lines: NahG (for degra-
dation 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, and npr1-1.
In another study of signaling pathways, Ryu et al. (2003b) used endo-
phytic 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),
ISR elicited by both strains was dependent on NPR1 and ISR elicited by
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, and fad3-2 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 en-
dophytic 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 bac-

teria was recently opened by the finding that volatile organic compounds
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 pre-
venting movement of soluble compounds. When IN937a was placed on
one side of an I-plate, Arabidopsis plants growing on the other side ex-
hibited 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 sep-
arate 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 promo-
tion has been extended to use in production agriculture and horticulture
through the development of two products. These two products are dis-
cussed, 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 us-
ing 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 prepa-
ration 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 Effects reported and systems useda Reference

identification
IN937a Reduced incidence or severity of vegetable diseases Jetiyanon et al.
Bacillus amy- (caused by Cucumber mosaic virus (CMV), Sclerotium 2002;
loliquefaciens rolfsii, Ralstonia solanacearum, Colletotrichum Jetiyanon and
gloeosporioides, and Rhizoctonia solani) in greenhouse Kloepper 2003
and field trials in Thailand
Reduced incidence of CMV on tomato in the greenhouse Zehnder et al.
2000
When applied to tomato with the non-endophyte Bacillus Murphy et al.
subtilis GB03, reduced severity of CMV in the greenhouse 2003
Reduced incidence and severity of tomato mottle virus in Murphy et al.
the field. Also reduced numbers of the white fly vector 2000
feeding on plants
Volatile organic compounds of the strain elicit growth Ryu et al.
promotion of Arabidopsis and ISR against Erwinia 2003a, 2004
carotovora subsp. carotovora
Component of the product, BioYield (Gustafson LLC, Kloepper et al.
http://www.gustafson.com) 2004
IN937b Reduced incidence of CMV on tomato in the greenhouse Zehnder et al.
Bacillus 2000
subtilis When applied to tomato with the non-endophyte Murphy et al.
B. subtilis GB03, reduced severity of CMV in the 2003
greenhouse
the field. Also reduced numbers of the white fly vector 2000
feeding on plants
90-166 Reduced incidence and delayed development of Liu et al. 1995a
Serratia symptoms of Fusarium wilt of cucumber, caused by
marcescens Fusarium oxysporum f. sp. cucumerinum
Reduced severity of bacterial angular leaf spot of Liu et al. 1995b
cucumber, caused by Pseudomonas syringae pv.
lachrymans
Reduced severity of anthracnose, caused by Liu et al. 1995c
Colletotrichum orbiculare, in two cucumber cultivars
over a 5-week period after bacterial treatment
Reduced incidence of CMV on cucumber and tomato Raupach et al.
and reduced the area under the disease progress curve 1996
(AUDPC)
Decreased severity of tobacco wildfire, caused by Press et al.
Pseudomonas syringae pv. tabaci 1997

identification
Wild-type strain reduced severity of cucumber Press et al.
anthracnose. A siderophore-negative mutant did not elicit 2001
ISR and colonized roots internally at lower populations
than the wild-type
Decreased severity of tobacco blue mold, caused by Zhang et al.
Peronospora tabacina, in NahG transgenic tobacco lines 2002a
that degrade salicylic acid
Decreased severity of tobacco blue mold in microtiter Zhang et al.
plate assays and detached leaf assays. Reduced pathogen 2002b
sporulation
Reduced symptoms of Pseudomonas syringae pvs. tomato Ryu et al.
and maculicola on Arabidopsis 2003b
Peronospora tabacina 2004
SE34 Bacillus Reduced incidence or severity of vegetable diseases Jetiyanon et.
pumilus (caused by cucumber mosaic virus, Sclerotium rolfsii, al. 2002;
Ralstonia solanacearum, Colletotrichum gloeosporioides, Jetiyanon and
and Rhizoctonia solani) in greenhouse and field trials in Kloepper 2003
Thailand
sporulation
Decreased severity of tobacco blue mold when applied as Zhang et al.
both seed treatment and drench in the greenhouse. 2004
Elicitation of ISR was associated with growth promotion
Decreased severity of tomato late blight, caused by Yan et al. 2002
Phytophthora infestans, and decreased germination of
sporangia and zoospores of the pathogen
Reduced incidence of CMV on tomato in the greenhouse Zehnder et al.
2000
When applied to tomato with the non-endophyte Murphy et al.
greenhouse
the field 2000
Reduced incidence of Fusiform rust, caused by Enebak and
Cronartium quercuum f. sp. fusiforme, on loblolly pine Carey 2000

identification
Restricted colonization of pea roots by F. oxysporum f. sp. Benhamou et
pisi and induced formation of structural barriers in the al. 1996
plant
Reduced damage of tomato roots to F. oxysporum f. sp. Benhamou et
radicis-lycopersici and induced structural and al. 1998
cytochemical barriers in the plant
INR7 Reduced incidence or severity of vegetable diseases Jetiyanon et.
Bacillus (caused by CMV, Sclerotium rolfsii, Ralstonia al. 2002;
pumilus solanacearum, Colletotrichum gloeosporioides, and Jetiyanon and
(available as Rhizoctonia solani) in greenhouse and field trials in Kloepper 2003
the product Thailand
Yield Shield;
Decreased severity of anthracnose (caused by Wei et al. 1996
Gustafson
Colletotrichum orbiculare) and angular leaf spot (caused
LLC)
by Pseudomonas syringae pv. lachrymans) on cucumber
in field trials
Decreased the incidence of cucurbit wilt disease, caused Zehnder et al.
by Erwinia tracheiphila, in field trials 2001
Decreased numbers of cucumber beetles on plants in the Zehnder et al.
field 1997b
Decreased beetle feeding activity and transmission of Zehnder et al.
E. tracheiphila on cucumber in cages where beetles had 1997a
a choice between bacterial-treated and nontreated plants
When applied to tomato with the non-endophyte Murphy et al.
greenhouse
Reduced incidence of Fusiform rust, caused by Enebak and
Cronartium quercuum f. sp. fusiforme, on loblolly pine Carey 2000
in field trials
89B-61 Decreased severity of tobacco blue mold, caused by Zhang et al.
Pseudomonas Peronospora tabacina 2004
fluorescens
Decreased severity of Fusarium wilt of cotton, caused by Chen et al.
(earlier
Fusarium oxysporum f. sp. vasinfectum 1995
referred to
as G8-4) Following seed treatment of cotton, 89B-61 reached an Quadt-
internal root population of 1.1×103 cfu/g. Bacteria were Hallmann et
not detected inside cotyledons, stems, or leaves al. 1997

identification
Decreased severity of tomato late blight, caused by Yan et al. 2002
Phytophthora infestans, and decreased germination of
sporangia and zoospores of the pathogen when applied to
seeds
sporulation
Decreased severity of tobacco wildfire, caused by Park and
Pseudomonas syringae pv. tabaci. Activated the promoter Kloepper 2000
for PR1a [a pathogenesis-related (PR) protein]
Reduced number of lesions of Colletotrichum orbiculare Jeun et al. 2004
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 Wei et al. 1991
on cucumber
Colonized roots internally at log 4.0 cfu/g at 2 weeks after Kloepper et al.
emergence when applied as seed treatments. Bacteria 1992
were not detected in leaves or stems
CHA0 Reduced severity of Tobacco necrosis virus (TNV) on Maurhofer et
Pseudomonas tobacco. al. 1994
fluorescens Severity of TNV on tobacco was reduced equivalently by Maurhofer et
the wild-type strain and a transgenic strain carrying al. 1998
pchAB genes for synthesis of salicylic acid.
Reduced sporulation of Peronospora parasitica on Iavicoli et al.
Arabidopsis 2003
aIn 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 ex-
ert 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) ex-
hibited 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 delin-
eating 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 endo-
phytic bacilli is associated with increased plant growth, and the relationship
between ISR and growth promotion should be further investigated. Elu-
cidation 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 ac-
tivity due to the development of resistant pathogen populations – and
to public pressure to develop production systems favourable to the envi-
ronment (Whipps 2001). In this respect antagonistic bacteria provide an
environmentally sound alternative to protect plants against attack by fun-
gal 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 Han-
delsman 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. Fur-
thermore, endophytic bacteria are known to promote plant growth by the
production of plant hormones, enhanced nutrient availability and nitro-
gen 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 cultivation-
dependent 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 culti-

vation-independent methods (reviewed by Smalla 2004) analyse endo-
phytic communities directly from root tissue. Techniques using bacterial
DNA provide information on the structural diversity of the entire endo-
phytic community, while techniques using bacterial RNA identify metabol-
ically active bacteria, e.g. those which are of interest after pathogen infec-
tion. 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 non-
antagonistic 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 microar-
rays (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 in-
sights 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 in-
teractions 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 an-
swered 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 in-
terfere 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 qual-
itative perspective. Commonly used methods for bacterial identification
include morphological and physiological characterisation, fatty methyl-
ester 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 en-
dophytic bacteria, e.g. in a clone library. Antagonistic activity of endophytic
bacteria is generally tested by in vitro inhibition of fungal pathogens in dual
cultures and then confirmed in bioassays on host plants. Quantitative anal-
ysis to detect antagonistic bacteria is time-consuming as all the endophytic
bacteria have to be screened for their antagonistic potential. Primers de-
veloped 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 Phytoph-
thora 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 por-
tion 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 Reference

antagonists (%)
Rice Achlya klebsiana 25 Adhikari et al. (2001)
Pythium spinosum 75
Potato Verticillium dahliae 9 Krechel et al. (2002)
Rhizoctonia solani
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 endo-
phytic bacteria is found in general, some genera harbour more antago-
nistic 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 num-
ber of different plant species, but most studies have been done on potato.
Table 4.2 lists antagonistic species found in potato in five studies. Surpris-
ingly, 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 fungia
Pseudomonas putida
Species with antagonistic properties
Pseudomonas rhodesiae
Agrobacterium tumefaciens Pseudomonas reactans
Amycolatopsis meditarranei Pseudomonas straminea
Arthrobacter ilicis Pseudomonas synthaxa
Bacillus aquamarinus Pseudomonas syringae
Bacillus cereus Pseudomonas tolaasii
Bacillus megaterium Pseudomonas veronii
Clavibacter michigenensis Psychrobacter immobilis
Curtobacterium albidum Ralstonia paucula
Curtobacterium flaccumfaciens Rhizobium meliloti
Curtobacterium luteum Rhizomonas suberifaciens
Erwinia persicinus Sphigobacterium thalophilum
Flavobacterium sp. Sphingomonas adhaesiva
Frateuria aurantia Stenotrophomonas maltophilia
Frigoribacterium sp. Streptomyces turgidiscabies
Kingella kingae Streptomyces bottropensis
Kitasatosporia cystargenia Streptomyces diastatochromogenes
Methylobacterium sp. Streptomyces galilaeus
Micrococcus varians Streptomyces griseus
Paenibacillus sp. Streptomyces lavendulae
Pantoaea agglomerans Streptomyces setonii
Pantoaea anantis Streptomyces turgidiscabies
Pseudomonas cichorii Xanthomonas campestris
Pseudomonas corrugata Xanthomonas oryzae
Pseudomonas fluorescens a According to Sturz et al. (1999), Krechel
Pseudomonas graminis
et al. (2002), Reiter et al. (2002), Sessitsch
Pseudomonas migulae
et al. (2004), and A. Krechel et al., unpub-
Pseudomonas orientalis
lished 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, contained a higher propor-
tion of bacteria with proteolytic, cellulolytic and phosphatase activity than
the susceptible cultivar ‘Libraska’ (Graner et al. 2003). Modern wheat cul-
tivars 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 se-
lection 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 commu-
nities 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 com-
munities 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 en-
dophytic 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 in-
tensively 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 antibio-
sis), competition and lysis, and indirect mechanisms such as induced re-
sistance 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 mech-
anisms 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 noth-
ing 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 simi-
lar 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 bac-
teria 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 Bacil-
lus 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 mis-
souriensis and its culture filtrates were shown to suppress Plectosporium
tabacinum on lupins (El-Tarabily 2003). The importance of hydrolytic en-
zymes 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 ma-
jor 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 pathogenic-
ity of fungal pathogens. Nejad and Johnson (2000) described isolates of en-
dophytic bacteria that significantly improved seed germination and plant
growth of oilseed rape and tomato. Plant growth promotion mediated by en-
dophytic bacteria may be exerted by several mechanisms, e.g. production of
plant growth hormones, synthesis of siderophores, nitrogen fixation, solu-
bilisation 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, En-
terobacter, Staphylococcus, Azotobacter and Azospirillum produce plant
growth regulators such as ethylene, auxins or cytokinins, which are as-
sumed 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 fac-
tors. 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 (Bloem-
berg and Lugtenberg 2001; Whipps 2001). Fluorescent pseudomonads are
common members of the endorhiza, making them ideal candidates for bi-
ological control measures. Antagonistic isolates have been reported to oc-
cur 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). Be-
sides 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 com-
monly found within the genus Bacillus (Emmert and Handelsman 1999).
Bacillus spp. occur mainly in the soil/rhizosphere but have also been re-
ported 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, have been shown to significantly
control many fungal diseases. Gram-positive biocontrol bacteria other than
bacilli include actinomycetes such as Streptomyces, Microbispora or Nocar-
dioides (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. How-
ever, a few studies have achieved similar biocontrol effects under field
conditions (Tjamos et al. 2004; Faltin et al. 2004). For example, the endo-
phytic isolate Pseudomonas trivialis 3Re2-7 significantly reduced disease
incidence of Rhizoctonia solani on lettuce and sugar beet by 40% and 86%,
respectively (Faltin et al. 2004).
These examples illustrate the high potential of endophytic bacteria in
fungal pathogen control. However, further fieldwork is required to con-
firm their control efficacy in different climatic regions and under differ-
ent 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 condi-
tions is often inconsistent (Weller 1988). Therefore, enhancing the efficacy
62
Table 4.3. Examples of antagonistic
Pathosystem BCA Trial Application Results Reference

Cotton, Bean Bacillus cereus Greenhouse Root incubation in Reduction of disease incidence Pleban et al.
(Rhizoctonia solani) Bacillus subtilis bacterial suspension approx. 50% 1995, 1997
Bacillus pumilus
Bean Bacillus subtilis Greenhouse Root incubation in Reduction of disease incidence Pleban et al.
(Sclerotium rolfsii) Bacillus cereus bacterial suspension of 70–80% 1995
Cotton Aureobacterium saperdae Pot experiment Pierced with a needle Reduction of disease severity Chen et al.
(Fusarium oxysporum Bacillus pumilus and plant growth promotion 1995
f. sp. vasinfectum) Phyllobacterium rubiacearum
Pseudomonas putida
Burkholderia solanacearum
Tomato Pseudomonas Growth Bacterization of tissue Reduction of disease severity Sharma and
(Verticillium dahliae) chamber plantlets or seedlings and plant growth promotion Nowak 1998
Bean Pseudomonas fluorescens Plant growth Soil drenching Reduction of disease incidence Downing and
(Rhizoctonia solani) Rif1::tc4 (with chiA gene) chamber 107 cfu ml−1 up to 48%, only if introduced as Thomson
Introduction into the endophytes 2000
plant
Rice Pseudomonas fluorescens Pot experiments Rice seeds were Reduction of disease incidence Adhikari et al.
(Achlya klebsiana, Pseudomonas tolaasii soaked for 1 h in by 50–73%; plant growth 2001
Pythium spinosum) Pseudomonas veronii a bacterial suspension promotion (plant height and
Sphingomonas trueperi 108 cfu ml−1 dry weight)
Potato Streptomyces turgidiscabies Growth Seed treatment Reduction of fungal growth in Krechel et al.
[Rhizoctonia solani, Kitasatosporia cystargenia chamber vitro, of nematode infestation 2002
Verticillium dahliae, Streptomyces galilaeus ad planta; plant growth
Meloidogyne incognita Streptomyces griseus promotion up to 78%
(nematode)] Pseudomonas graminis
G. Berg, J. Hallmann
Pathosystem BCA Trial Application Results Reference

Balloon flower Bacillus sp. Pot experiments Soil inoculation Reduction of disease incidence Cho et al. 2003
(Rhizoctonia solani)
Pepper Bacillus subtilis Greenhouse Seed treatments Reduction of disease incidence Ahmed et al.
(Rhizoctonia solani, Bacillus licheniformis Root drenching of R. solani up to 55% and of 2003
Phytophthora capsici) P. capsici up to 55%
Eggplant, potato Bacillus spp. Greenhouse Root dipping Reduction of disease severity by Tjamos et al.
(Verticillium dahliae) Paenibacillus alvei Field Soil drenching 40–70%; yield increase 25% 2004
Bacillus amiloliquefaciens 107 cfu ml−1 talc-gum
xanthan formulation
Potato Serratia plymuthica Greenhouse Soil drenching Reduction of disease severity up Faltin et al.
(Rhizoctonia solani) Pseudomonas reactans Field Seed bacterization to 76% 2004
108 cfu ml−1
Wheat Streptomyces spp. Greenhouse Seed treatments 109 Reduction of black lesions Coombs et al.
(Gaeumannomyces Microbispora spp. to 1010 cfu ml−1 up to 71% 2004
graminis var.tritici) Nocardioides
4 Control of Plant Pathogenic Fungi with Bacterial Endophytes
63
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 bac-
teria. Basically, two types of formulation seem to be preferable for endo-
phytic 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 formula-
tions 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 bac-
teria, while also providing nutrient sources, are an interesting alternative
(Burghes 1998).
The delivery of BCAs into the host plant is a prerequisite for success-
ful biocontrol. Musson et al. (1995) studied eight methods for delivering
endophytic bacteria into cotton stems and roots: stab-inoculation of bac-
teria 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 bio-
control 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 endo-
phytes 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 geno-
types 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 commu-
nication, 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-of-
action, colonisation potential, survival, fitness, and adaptation to environ-
mental 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. The gene chiA was
cloned under the control of the tac promoter in the broad-host-range plas-
mid pKT240 and the integration vector pJFF350. P. fluorescens carrying
tac-chiA either on the plasmid or integrated into the chromosome sig-
nificantly 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 hope-
fully 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 for-
mation 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 cor-
tex. 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 en-
zymes help the passage of infection threads from cell to cell (van Spronsen
et al. 1994). Rhizobia are released from the infection threads into the cyto-
plasm of host cells by a process resembling endocytosis (Stacey et al. 1991).
Extensive cell division in the primordia leads to functional nodules contain-
ing 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
Supérieures, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
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
Supérieures, Sciences III, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland

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. Indeter-
minate 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 rhi-
zobial 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 reg-
ulators, 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, and nol), which are responsible for the synthesis of host-specific lipo-
chito-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 at-
tached 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 concen-
trate 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 sec-
independent; proteins are secreted across the bacterial inner- and outer-
membranes 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 carboxy-
terminal 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 Ca2+ -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 cation-
selective 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 Ca2+ , which could act as a second messenger thereby stimulating
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 Nod-
factor 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 ap-
pear to abort in the nodO/nodE double mutant, it is possible that NodE (an
76 M.M. Saad et al.
α-keto-acyl-synthase) is also involved in acylation of some components of

the infection thread. Furthermore, mobilising nodO into different rhizo-
bia 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 Nod-
factor 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 trans-
ferase 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 nodu-
lation by the multiple mutant nodFEMNTLO (of R. leguminosarum bv.
viciae), Walker and Downie (2000) proposed a role for NodO in the com-
plementation of Nod-factor defects. This mutant produces Nod-factors that
are devoid of decorations and results in abortion of infection-thread de-
velopment 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 nodu-
lation. 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 symbio-
sis, perhaps by amplifying or modifying other signal molecules. In this
way they could increase ion flux following pore formation in plasma mem-
branes, 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 multi-
protein 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
use type IV pili during the symbiotic interaction with legumes.
5.2.3
Type III Secretion Systems
Type III secretion systems (T3SSs) are characteristic of pathogenic Gram-
negative bacteria, where their function is to inject proteins into the cy-
toplasm 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 poly-
merise into extra-cellular components of the secretion apparatus forming
78 M.M. Saad et al.
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 appara-
tus 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 com-
ponents of T3SSs were found and named rhc (Rhizobium conserved) but
the same final letter as used to describe pathogenic T3SS genes was main-
tained (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 ab-
solutely 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). Knock-
out mutants in the T3SS machine of NGR234 cause three host-dependent
effects. As compared to the wild-type rhizobia, there can be a dramatic im-
pairment 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 unaf-
fected 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 pres-
ence 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 sensor-
regulator 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. Tran-
scriptional 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 bac-
terium to the plant cell and allow proteins to pass through the hollow pili.
As protein secretion is abolished in mutants of pili-genes, their pheno-
type 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 struc-
tures on its surface in a flavonoid and T3SS-dependent manner. These ap-
pendages were purified and shown to be composed predominantly of NopA
80 M.M. Saad et al.
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 identi-
fied in all T3SS-possessing rhizobia, suggesting that NopA is an essential
component of the secretion machinery. Mutation of nopA also blocks the se-
cretion 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 ef-
fectors 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 rhi-
zobial 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 iden-
tified (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 pro-
teins, 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
to NGR234 on the majority of legumes tested. This is another character-
istic of effector proteins of phytopathogens, for there are many of them
and they are thought to be redundant in function. NopL is important for
the efficient nodulation of Flemingia congesta (Marie et al. 2003), sug-
gesting 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 Nico-
tiana tabacum inhibited this plant’s ability to accumulate pathogenesis-
related (PR)-proteins in response to pathogen attack. We thus suggest that
NopL could suppress root-cell defence responses by disrupting the intra-
cellular signalling cascades required for activation of PR-genes (Bartsev et
al. 2004a).
Sequence analyses of NGR234 and USDA110 revealed the presence of
genes homologous to secreted effector proteins from other T3SS-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
82 M.M. Saad et al.
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 infec-
tion threads into cortical cells. Similarities between attempts by rhizobia
to colonise roots and the attack by pathogens of other plant cells are ob-
vious. 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 sophisti-
cated 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 respond-
ing 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 con-
jugal 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 trans-
port across the cell envelope (Christie 2001). Some symbiotic nitrogen-
fixing 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 loca-
tion 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 “mas-
ter keys”. Once the legume “doors” have been opened to allow rhizobia in,
different morphological and cytological changes in the roots occur. Nod-
factors play only secondary roles in the later steps of invasion, at which
time other signal molecules occupy centre stage. Bacterial SPSs and se-
creted proteins contribute to the infection process, where they assist in
infection thread development within the root hair, and help modify host-
defence mechanisms.
Acknowledgements. We thank D. Gerber for general support and encour-

agement. 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
Plants can be considered as complex microecosystems that provide different
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 “endo-
phyte”, 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 Balan-
siaceae, for which the most compelling evidence of plant–microbe mu-
tualism 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, Univer-
sity 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 Agricul-
tural 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 counter-
parts. The primary mechanisms thought to lead to beneficial effects for
the plant are nitrogen fixation (Boddey and Döbereiner 1995) and bio-
control 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 ben-
eficial 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 Hall-
mann)].
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) in dry interior regions
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 102 –104
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

from trees is the same as their isolation from agronomic crops, and suffers
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 knowl-
edge 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) in Texas, 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 in-
hibition 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 deni-
trificans reduced the number of diseased trees by 50% and decreased the
percentage of crown loss by 17%. In a subsequent trial, no reduction in
numbers of diseased trees was observed but preinoculation with the same
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 gen-
era Bacillus, Curtobacterium, Pseudomonas, Stenotrophomonas, Sphingo-
monas, Enterobacter, and Staphylococcus, have also been isolated from
phloem tissue of roots and branches of elm trees (Ulmus spp.: Mocali
et al. 2003). An attempt was also made to determine the correlation be-
tween the seasonal fluctuations in the structure of the endophytic bacterial
92 R. Anand et al.
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 pro-
motion 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 environmen-
tal 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 clas-
sification), 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
comprised of 550 species spread over seven families, each dating back to the
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 bac-
terial 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 Paenibacil-
lus, 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 acti-
nomycetes (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
may have involved one of the direct plant growth promotion mechanisms,
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.
94 R. Anand et al.
We have also looked for bacterial endophytes in lodgepole pine. After iso-
lation 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 inter-
nal 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 develop-
ment, and results in endophytic bacterial population sizes approaching
106 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 inoc-
ulation (Chanway et al. 2000). Five months after root inoculation, internal
stem bacterial populations reached 105 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 bacte-
rial 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 in-
creases 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 inhibi-
tion.
Population sizes of Pw2-R and Sm3-RN were generally below the assay
detection limit of ca. 102 cfu g−1 plant tissue, which led us to question how
effectively internal plant tissues were colonised at the onset of the exper-
iment. Therefore, we also evaluated seedlings that were inoculated with
strains Pw2-R and Sm3-RN and grown in the greenhouse for 4 months
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. 103 –104 cfu g−1 tissue in the greenhouse. As
expected, mean seedling biomass also increased due to bacterial inocu-

lation in the greenhouse. Because seedling growth responses in the field
would be inseparable from those that occurred in the greenhouse, sim-
ple 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 ex-
pressed 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 inocu-
lated plants were double the control value. This was particularly interesting
in view of results with seedlings that we inoculated and planted immediately
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
and root RGRs of seedlings pretreated in the greenhouse before planting at
the same sites were 23–132% greater than controls, and endophytic popu-
lations in root tissues of between 102 and 4×104 cfu g−1 plant tissue were
detected in seedlings at three of the four sites. Similar effects on establish-
ment 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 inoc-
ulation 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
responses will be required before reliable recommendations can be made.
In addition, much more research is warranted to answer the many ques-
tions regarding the entry and operation of endophytic bacteria in conifers,
some of which are being actively pursued by our group.
6.4
Modes and Sites of Entry
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
96 R. Anand et al.
strain of Paenibacillus polymyxa in Arabidopsis thaliana; they observed

a slight degradation of the root tips within 5 h of inoculation. It was found
that P. polymyxa had two preferred zones of infection. The first is located
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 ca-
pabilities 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 rhi-
zosphere 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 demon-
strated 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 d-
galacturonic 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 mono-

meric 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 de-
fence 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), produc-
tion of plant growth regulators and antibiotics, or increased availability of
plant growth-limiting nutrients. Indirect mechanisms may involve suppres-
sion 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 de-
rive benefits from bacteria only indirectly through their mycorrhizal sym-
bionts (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 promo-
tion of these conifers by PGPR is independent of the mycorrhizal status of
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
property was advanced as a likely explanation of pine growth promotion
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,
98 R. Anand et al.
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 di-
azotrophic properties. This led to the intriguing possibility that lodgepole
pine harbours a systemically endophytic nitrogen-fixing bacterial popula-
tion, similar to that found in sugar cane (Boddey et al. 1995), which would
explain its ability to grow, and even thrive, under nitrogen-deficient condi-
tions 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 mecha-
nism of growth promotion by P. polymyxa strain Pw2-R, since seedlings in-
oculated 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 fixation in vivo should be drawn from a number of lines
of evidence, including a positive nitrogenase activity test (acetylene re-
duction 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
described as a unique and enigmatic type of mycorrhizae on lodgepole pine,
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
5 mm
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 2 mm
Recently, we tested the hypothesis that diazotrophic endophytes iso-

lated from internal tissues of immature and mature, naturally regener-
ated, lodgepole pine produce biologically significant amounts of N through
100 R. Anand et al.
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, west-
ern 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 proce-
dure and 16S rDNA sequencing identified them as belonging to the genus
Paenibacillus (strains P2b-2R, P18b-2R and C3b) as well as to the Flexibac-
ter group (strain P19a-2R). The three strains with names beginning with
“P” were originally isolated from pine tissues from the Williams Lake site.
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 ni-
trogen 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,
a loopful of each strain was separately inoculated into its own 1 l flask
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 (10,000 g for 30 min), and re-
suspended in sterile phosphate buffer (SPB). Strains P2b-2R and C3b were
resuspended to a density of ca. 107 cfu/ml and strains P18b-2R and P19a-2R
were resuspended to a density of ca. 106 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). Photosyntheti-

cally active radiation (PAR) at canopy level was ca. 300 µmol s−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
between endophytic strains can result in profoundly different effects on
plant growth and foliar nitrogen derived from the atmosphere (NDFA).
While large, statistically significant amounts of foliar NDFA were de-
tected 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 sup-
porting nitrogen-fixing bacteria in the plant. Seedling growth reduction
during the establishment of symbiosis is not uncommon, due to the en-
ergy 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 nitro-
gen (38%) compared to noninoculated controls, and derived 27% of foliar
N from the atmosphere. In trials 1 and 2, control seedlings were appar-
ently scavenging the small amount of soil N that was initially provided,
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) %NDFAa

1 8 30
2 9 66
3 9 27
a Calculated according to Rennie et al. (1978), where:

atom % 15 N excess(inoculated plant)
%NDFA = 1 − × 100
atom % 15 N excess (uninoculated plant)
102 R. Anand et al.
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 bac-
teria 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 exper-
iments 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-R and P2b-2R could also be used as vectors to deliver specific gene
products to plants. Such an approach may be more feasible than attempt-
ing to genetically alter the plant host directly. In addition, diazotrophic
endophytic bacteria hold great potential for reducing application of fer-
tilisers, especially of mineral N. Inoculation of forest seedlings with ef-
fective diazotrophic or plant growth promoting endophytic bacteria could
enhance growth and yield of trees significantly, especially at nutrient-poor
sites. However, there is much more work to be done if we are to under-
stand these plant/microbe associations to the degree that we can manage
them effectively for more efficient and sustainable nursery and field treat-
ments.
More knowledge of the population dynamics and activity of endophytic

bacteria in their host plants is required. A considerable research effort is also
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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Wilson D (1995) Endophyte: the evolution of a term, and clarification of its use and defini-
tion. Oikos 73:274–276
Zak B (1971) Characterization and classification of mycorrhizae of Douglas fir. II. Pseudot-
suga menziesii + Rhizopogon vinicolor. Can J Bot 49:1079–1084
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 chemi-
cally complex microcosm that provides different habitats for diverse com-
munities 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 con-
stant 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 mod-
ification 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 epi-
dermal and outer cortical cells of healthy roots inter- and intra-cellularly.
A comparatively small number of organisms, e.g. mycorrhizal fungi, en-
dophytic 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 envi-
ronmental 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 com-
munities 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 decision-
making 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 non-
mycorrhizal fungal root endophytes at all levels. The first part of this review
will be dedicated to biodiversity at the community level in relation to envi-
ronmental 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
obscuring the effects of different factors is that of site history, 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
owing in part to the great diversity of hosts, but also to the optimal growth
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 tem-
perate or the tropical zones, species diversity is distinctly reduced in arc-
tic-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)]. Cor-
respondingly, 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 precipita-
tion, as well as enhanced CO2 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
110
endophytes
Hosts and factors Observed Adjusted Number Even- Total Sample Pairwise Reference
species species of very ness number sizee similarities of
richnessa richnessb abundant indexd of communitiesf
speciesc isolates (2) (3) (4) (5)
(i) Dryas octopetala R Fisher et al.
(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 Kattner and
(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 Fisher et al.
(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 – –
T.N. Sieber, C.R. Grünig
(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 – – –
7 Biodiversity of Fungal Root-Endophyte Communities and Populations
111
112
(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
(3) Natural regeneration 18 10.9 ± 1.5 7.4 0.60 48 100 – – 0.57
in cleared windthrow
(4) Natural regeneration 17 9.3 ± 1.5 6.3 0.70 58 100 – – –
in low density forestg
(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)
(2) Natural regeneration 8 5.5 ± 1.0 4.7 0.79 20 75 – 0.55 0.53
in untouched windthrow
(3) Natural regeneration 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 – – –
T.N. Sieber, C.R. Grünig
(xi) Erica carnea R Cevnik et
(1) Control (Cd 1.4; Pb 171; Zn 61.8)h 9 8.2 ± 0.7 3.9 0.78 104 240 0.50 0.60 0.63 al. 2000
(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 Biodiversity of Fungal Root-Endophyte Communities and Populations
113
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 signifi-
cantly 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 differen-
tially 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

Soil pH had an effect on community composition but not on species di-
versity of endophytic fungi in Norway spruce roots (Picea abies) (Kattner
and Schönhar 1990) [Table 7.1(iii)]. The similarity of only 57% of the en-
dophyte communities in roots from neutral and acidic soils reflects either
the selectivity of soil pH or the historical presence/absence of certain en-
dophyte 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 af-
fected 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 carbo-
hydrates, 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, dif-
ferential 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 di-
versity 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 non-
submerged 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 ap-
proximately 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 Srid-
har 2002) [Table 7.1(v)]. Flooding and site conditions affected endophyte
species spectra but not species richness in roots of common reed (Phrag-
mites 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

species diversity and spectrum will then depend on the range of available
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 ce-
real roots, where it functions as an effective antagonist of various root
pathogens. For example, its presence in wheat roots was negatively cor-
related with the presence of Septoria nodorum, the causal agent of glume
blotch disease of wheat (Riesen and Sieber 1985; Sieber et al. 1988). Simi-
larly, M. bolleyi inhibited various Fusarium species and Gaeumannomyces
graminis var. tritici (Kirk and Deacon 1987; Reinecke 1978). Whether M. bol-
leyi interacts with these pathogens indirectly by inducing systemic resis-
tance in the host plant, or directly by either parasitising pathogens or
producing inhibitory metabolites, remains to be examined.
The phenological state of the roots and/or the season may influence en-
dophyte 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 estab-
lished 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 tem-
peratures 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 com-
munity in which the host species grows may, therefore, influence root-
endophyte 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 Cylin-
drocarpon didymum, were rare or absent in roots of Scots pine (Pinus
sylvestris) growing in monoculture. Likewise, P. fortinii, the dominant root
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 di-
versity of the current crop. For example, species richness and the number
of dominant species were significantly higher when wheat (Triticum aes-
tivum) 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 in-
different 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, bio-
cides, 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 endophyte-
host associations occur more frequently than others, suggesting host pref-
erence or adaptation. The similarity of the spectra of endophyte species
among host species was as low as 44% in an experiment performed by Nar-
isawa 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. Sec-
ondary 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). Correspond-
ingly, 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 regener-
ated 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 sig-
nificantly 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 es-
tablished seedlings of bishop pine (Pinus muricata) were colonised by DSE
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 pol-
lution 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 re-
duced (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. In this section, we will briefly present the history of the term
“DSE”, outline the diversity of DSE and give an overview of current knowl-
edge 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 mycor-
rhizae and roots of Picea abies and Pinus sylvestris. The tree-fungus sym-
biosis 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 relation-
ship 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 dif-
ferentiate 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 re-
lated to the take-all fungi (Gaeumannomyces graminis var. tritici and var.
avenae) of cereals and grasses in temperate areas and to G. 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
only under specific conditions. Cold treatment for up to 1 year was shown
to induce sporulation in some DSE isolates, e.g. in isolates of Chloridium
paucisporum, Phialophora finlandica, and Phialocephala fortinii (Wang and
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 may function as a mycorrhizal fungus and mediate nutrient
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
abundance it is very unlikely that P. 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 spa-
tial 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. Har-
ney 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 substi-
tutions) 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 iden-
tical 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 com-
parisons of the rDNA ITS regions. Two genets dominated and were isolated
from all sampling points within contiguous areas of at least 6.8 m2 and
5.3 m2 that overlapped by 3.6 m2 . Other genets were rare and were isolated
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 can be high even within single root pieces. For example, 8-
to 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
find the sources of this high diversity.
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 analy-
ses (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 was isolated as an endophyte
from each other were studied using 11 single-locus RFLP probes. The aver-
age 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 reproduc-
tively 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 de-
serves 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 specia-
tion through genetic drift (Carter et al. 2001). On the other hand, niche or
habitat specialisation may lead to sympatric speciation when local popula-
tions 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. Pos-
sibly, the cryptic species were the products of allopatric speciation in the
Fig. 7.2. Distribution of the four most frequently observed cryptic species (csp) of Phialo-
cephala fortinii s. l. within healthy fine roots of Norway spruce (Picea abies) collected at
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 succes-
sion of several glaciations and warmer inter-glacial periods had profound
effects on animals, plants, and, consequently, on fungi. During the Qua-
ternary, each species experienced many contractions/expansions of range,
leading to extinctions and foundations of populations, decreases and in-
creases 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 hetero-
geneities 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 ap-
pear 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.
MLH with identical ISSR fingerprinting patterns were common to at least
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 pop-
ulation 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 ge-
netic 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 an-
thropogenic 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 mi-
crosclerotia within the roots of the host.
Phialocephala fortinii is the most prominent DSE, especially in woody

plant species. P. fortinii s. l. is genotypically very diverse and forms a com-
plex 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 ecologi-
cal 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 geno-
typic diversity and presented several plausible explanations for these pat-
terns, 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, cli-
matic 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 geo-
graphic 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 ad-
dition to colonization of many plant species. The diversity of plant species
colonized by members of the genus Fusarium is amazing. A recent liter-
ature 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 pri-
mary incitants of root, stem, and ear rots in many agriculturally important
crops. For example, F. verticillioides (= F. moniliforme) is capable of col-
onizing 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 mycotox-
ins 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 Agricul-
ture, 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
production of characteristic toxic secondary metabolites (Bacon et al. 1986).
For example, fungi of the genus Neotyphodium (teleomorph = Epichloë)
are found only in the stems, leaves and seed of grasses but are not found
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 in-
clude 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 expres-
sion 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, teleomorph Gibberella
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 local-
ized 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, Fusar-
ium species are not necessarily obligate endophytes. Indeed, as presented
8 Root Endophytic Fusarium 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, pre-

dicts a transitory nature of the endophytic phase of Fusarium associations.
Understanding to what extent species or isolates of this genus are endo-
phytic 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 bene-
fits derived from such association that are indicated and characteristic of
those derived from the Neotyphodium grass endophytes (for review, 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).
Reports about the interactions of F. verticillioides with maize have been

contradictory since the initial description of this fungus as both a patho-
genic and a symptomless infection (Sheldon 1902; Voorhees 1934). Anatom-
ical features of pathogenic infections by Fusarium species, including F. verti-
cillioides, 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. verticil-
lioides into roots that occurred from the soil. Infection took place via the
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 pen-
etration (Voorhees 1934), spread of infection into the stele is prevented.
However, infection of the stele can occur from soil via the wounds pro-
duced by adventitious and lateral roots, suggesting that the degree and rate
of suberization in young seedlings can serve as the key to the nature of
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. ver-
ticillioides 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 fluoromet-
ric 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 A hypha of F. 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 in-
fected 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 horizon-
tally (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 distinc-
tion 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 asso-
ciation of parasitic fungi with vascular plants, we now can apply this to
endophytic Fusarium species.
Following the definition above, Fusarium species should be considered
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, are typical hemibiotrophs.
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 agricul-
tural 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. Com-
plete 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 light-
and 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 contam-
ination 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. oxys-
porum (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 compart-
mentalization 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 mem-
branes (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 struc-
tures characteristic of the usual pathogenic infections in rust and powdery
mildews such as haustoria. Virulent strains of F. verticillioides do have in-
tracellular hyphae that are not distinct haustoria (Fig. 8.4b–f) (Bacon and
Hinton 1996). However, haustoria represent only one type of nutrient ab-
sorption structure. Fungal cell-wall-to-plant-wall appositions, as observed
in most strains of F. verticillioides and other Fusarium species as well as
other fungal endophytes, are one of three fungus-plant cell nutrient absorb-
ing 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 ac-
complished, 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 ) in an intercellular space (i) of maize cells (h); bar 1 µm. b Another
view of the intercellular nature of hyphae in roots of maize plants approximately 8 weeks
old; bar 1 µm. c Two groups of hyphae in intercellular spaces of the root cortex; bar 1 µm
(from Bacon and Hinton 1996, with permission)
al. 2001). The accumulation of secondary compounds must require, a pri-

ori, 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 verticil-
lioides, 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 1 µm. 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 penetra-
tion peg (arrowhead); bar 1 µm. 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 1 µm. d Intercellular hyphae invading
another cell in stem tissue of a 3-week-old plant; bar 1 µm. e An intracellular hypha growing
between two cells along and between the middle lamellae; bar 1 µm. 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 his-
tological studies of these types of infections tend to be restricted to the spe-
cific 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; Ba-
con and Hinton 1996; Baayen and Rykenbuerg 1999; Kang and Buchenauer
2000), reinforcing our concept that most Fusarium species are hemibio-
trophic.
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 character-
istic of interactions that occur in different symbiotic systems (Honegger
1986). We do not know if the lack of specialized nutrient absorption struc-
tures 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 mutu-
alistic symbioses commonly found in lichens. However, the nature of the
association with a particular host may vary, as some endophytic associ-
ations 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 compat-
ibility, 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 va-
riety 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 accumu-
lation 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 fumon-
isins 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 culti-
var 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 out-
growths. 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 physi-
ology 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 my-
cotoxin 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 per-
formance, 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 evolu-
tionary and physiological implications since it has been established that
all maize isolates of this fungus can detoxify the host’s native antimicro-
bial 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 endo-
phytic 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 mi-
croorganisms. Fusaric acid is an antibiotic, showing activity against both
Gram-negative and -positive bacteria, especially those used for biocon-
trol. 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 produc-
tion 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 sec-
ondary metabolites is incomplete. Moniliformin is a weak mycotoxin and
an even better phytotoxin for specific hosts (Cole et al. 1973), but any over-
all 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 chemi-
cal 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, vi-
soltricin, chrysogine, culmorin, aurofusarin, equisetin, fusoproliferatum,
fusarochromanone, acuminatopyrone, fusamarin, chlamydosporol, addi-
tional derivatives of T-2 toxins, and several unidentified antibiotics. The
identities of the Fusarium species producing these metabolites and a more
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 produc-
tion and food safety. Fusarium species exist as intercellular root endophytes
in both cultivated and wild plants and their role during the symptom-
less 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 prob-
lems associated with this genus are global as their distribution is world-
wide, 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 sum-
marize, a major concern is that many Fusarium species produce mycotoxins
that are harmful to humans and animals ingesting food or feed products col-
onized 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 appropri-
ate 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 symptom-
less 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 adapta-
tions 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 en-
dophytes. 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 world-
wide; 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
in both cases the roots are exposed to light and air. Roots of epiphytic
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 dif-
ferences 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 accu-
rately, 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 coloniza-
tion; 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
important for the health of wild plants as well as crop plants (Hallmann et
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, which varied from 23% to 73%
of isolates from each orchid species. Most isolates from Pterostylis spp. were
also Gram-negative, while most isolates from all other orchids were Gram-
positive. 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 Nos-
toc were isolated from the surfaces of roots of the terrestrial orchid Calan-
the vestita, and Bacillus, Curtobacterium, Flavobacterium, Nocardia, Pseu-
domonas, 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 fix-
ation; 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 green-
house (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 inter-
esting 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 myc-
orrhizae 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 en-
dophytes 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 con-
cept of endophytes principally describes where an organism lives, without
assuming or excluding the possibility of benefit for either party. This dis-
tinction is complicated by the fact that mutualisms are part of a continuum
of symbiotic relationships, and a relationship that is mutualistic may be-
come 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 germi-
nation and seedling growth tests in vitro may not be entirely accurate in
distinguishing mycorrhizal fungi from non-mycorrhizal endophytes (Ras-
mussen 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 litera-
ture. 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 (Al-
conero 1969; Masuhara and Katsuya 1994; Carling et al. 1999; Rasmussen
2002). A more precise (but less convenient) description would be ‘presum-
ably 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,
the existing body of work on orchid endophytes is surprisingly small
(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, and Sebacina, are mycorrhizal in associations
with orchids, that other basidiomycete fungi may be mycorrhizal in myco-

heterotrophic 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 cul-
ture, and efforts to induce sporulation in vitro are often unsuccessful. Since
traditionally fungi are classified by their spores and spore-bearing struc-
tures, 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; La-
cap 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 microor-
ganisms 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 en-
dophytes. 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 ampli-
fication conditions may help reveal additional taxa.
9.6
Host Specificity of Orchid Endophytes
Host specificity of endophytes is important for estimates of global fun-
gal 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 fun-
gal 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 signif-
icant 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 mycor-
rhizal fungi, (see Arditti et al.1990; Rasmussen 1995, 2002; Taylor et al. 2002
for reviews), which applies to non-mycorrhizal endophytes as well. How-
ever, few attempts have been made to compare non-mycorrhizal endophytes
among orchid species using quantitative methods. Given the diversity of
endophytic fungi in orchid roots and the variation in methods among stud-
ies, 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 fungi reported from orchid rootsa . 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., Chaetomium sp. 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
159
160

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
P. Bayman, J.T. Otero

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
161
162

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., Costa Rica Richardson 1993
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
163
Maxillaria sp. Colletrotrichum crassipes, Hadrotrichum sp., Xylaria sp. Costa Rica Richardson 1993
164

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, Costa Rica Richardson 1993
Pithomyces maydicus, Xylaria sp.
Pleurothallis pantasmi Hadrotrichum sp., Humicola sp., Nectria peziza Costa Rica Richardson 1993
Pleurothallis periodica Chaetomium subspirale, Cladosporium cladosporioides, Costa Rica Richardson 1993
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, Costa Rica Richardson 1993
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, Costa Rica Richardson 1993
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, Costa Rica Richardson 1993
Lasiodiplodia theobromae, Nectria haematococca
Stelis endresii Curvularia cymbopogonis, Nectria haematococca, Costa Rica Richardson 1993
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
165
Nectria haematococca, Nodulisporium sp., Periconiella sp.

166

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
a The 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)
b Also 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)
was isolated from leaves and roots of Cypripedium 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 relation-
ships 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 indi-
rectly 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 myco-
heterotrophic orchids using fungal-specific (or in some cases, basidiomy-
cete-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 ectomycor-

rhizal. 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 identify-
ing 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 be-
cause 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-hetero-
trophic orchid, Wullschlaegelia. Forty-one morphospecies of endophytes
were isolated from plants of W. calcarata in Puerto Rico (J.T. Otero, unpub-
lished; Table 9.1). Common endophytes included Xylaria, Trichoderma, Col-
letotrichum and various dematiaceous hyphomycetes. These genera were
not randomly distributed among sites (χ2 = 31.84, df = 17, P = 0.004).
A morphospecies accumulation curve suggested that most of the cultur-
able endophytic fungi in the roots were isolated (Fig. 9.1). It is likely that
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 mor-
phology 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 litho-
phytic 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 en-
dophytes was done in La Selva, Costa Rica (Richardson et al. 1993; Richard-
son 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). Rel-
ative 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 epi-
phytic orchids in Puerto Rico (Table 9.1). The most common endophytic
fungi have been fairly consistent from study to study. A variety of endo-
phytic 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 To-
lumnia, 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 mor-
phospecies) 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
the sample did not include Fusarium (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, unpub-
lished). There was no apparent difference between the fungal communities
of the two orchids. The dominant species were Xylaria spp.; Rhizoctonia-
like fungi were much less common than in the orchids cited above, and few
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 were isolated, up to four species occurring in a single
plant. Frequency of Xylaria species and Rhizoctonia did not differ signifi-
cantly between roots and leaves, which is perhaps not surprising given that
roots of epiphytes are exposed to light and air. Frequency of both Rhizocto-
nia 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, endophytic fungi were
isolated from roots of lithophytic plants (Bayman et al. 2002). The most
common genera were Guignardia (isolated from 22% of root pieces), Col-
letotrichum (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? Trem-
blay et al. (1998) isolated fungi from roots of Lepanthes caritensis, an orchid
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, particu-
larly 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 endo-
phytes (Table 9.1) are fungi that are ubiquitous in soil and as endophytes
of other plants. There are marked differences between terrestrial and epi-
phytic 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 differ-
ences in temperate vs. tropical mycofloras and are not particular to orchid-
associated 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 phy-
toalexins 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). Accord-
ing to Rasmussen (1995), “...all underground parts of terrestrial orchids
must either accommodate the endophyte (i.e., mycorrhizal fungus) or ac-
tively 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; Bidar-
tondo 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 signif-
icantly lower rate of infection with Xylaria than would be expected from
the frequency of each genus alone (Bayman et al. 2002). Presence of Col-
letotrichum also showed a significant, negative correlation with Guignar-
dia. This suggests there may be competition or antagonism between these
genera; alternatively, their colonization or growth could be favored by dif-
ferent 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). Propicona-
zole 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 de-
creased 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 harm-
ing 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 microorgan-
isms, which are more common than pathogens. Their importance becomes
obvious when an antibiotic designed to kill a pathogen affects the commu-
nity of commensals as well, causing a secondary condition – for example,
vaginal yeast infections in women who are taking antibiotics for bladder in-
fections. 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 Fungal Endophytes in Submerged Roots
Felix Bärlocher
10.1
Introduction
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 con-
sider 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. Bern-
stein 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; Saikkon-
nen 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 defini-
tion 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 bal-
anced antagonism: pathogens overcome the host’s defences to the extent
that they cause visible damage, whereas endophytic virulence is only suf-
ficient 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
unique secondary metabolites and enzymes affecting plants, herbivores and
microbes, with potential applications in medicine and agriculture (Petrini
et al. 1992). The continuum of endophyte interactions with plants also pro-
vides 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 colonisa-
tion 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 hy-
phomycetes, a heterogeneous group of aquatic fungi, are an indispens-
able 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 pro-
ducing conidia, whose shapes are predominantly tetraradiate (four arms)
or sigmoid. Both types have been shown to increase the conidium’s likeli-
hood 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 de-
clines 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 per-
sist 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 (Bär-
locher 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 (Har-
tig) Deighton. It is a remarkably widespread and versatile species: it is
a well-known plant pathogen, has been implicated in human infections,
and is a common stream fungus. Morphologically, there is no difference
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 Tetra-
cladium 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 Al-
nus 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, respec-
tively, compared to the 19% that were colonised by the most common
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 typi-
cal aquatic hyphomycetes, they also found species of the genera Fusarium
and Cylindrocarpon. Members of these two taxa are often found on leaves
182 F. Bärlocher
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 fusir-
amos, 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, suggesting that roots are colonised by fungi settling on surfaces and
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 produc-
tion 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 sec-
ondary resource for aquatic hyphomycetes. Their existence as endophytes
may provide them with a head start in the use of root detritus, a possi-
ble 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 hyphomy-
cetes 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 esculen-
tum (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
roots or pneumatophores (which cycle between exposure to air and immer-
sion in salt or brackish water) of Avicennia officinalis L., Rhizophora mu-
cronata 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, includ-
ing 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 unidirec-
tional 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)
Root
Fungus Substrate section References
Anguillospora filiformis Acer spicatum B, X Raviraja et al. 1996;
Greath. Sridhar and Bärlocher 1992b
Betula papyrifera B, X Sridhar and Bärlocher 1992a
Picea glauca B Sridhar and Bärlocher 1992a
A. longissima Mangifera indica B, X Iqbal et al. 1995
(de Wild.) Ingold Populus hybrida B, X Iqbal et al. 1995
Salix babylonica B, X Iqbal et al. 1995
Articulospora antipodea Picea glauca B, X Sridhar and Bärlocher 1992a
Roldán
A. atra Alnus glutinosa B Fisher et al. 1991
Descals Picea glauca B, X Sridhar and Bärlocher 1992a
A. tetracladia Acer spicatum B, X Fisher et al. 1991
Ingold Alnus glutinosa B, X Fisher et al. 1991; Sridhar
and Bärlocher 1992a, 1992b
Picea glauca B, X Sridhar and Bärlocher 1992a
A. proliferata Mangifera indica B, X Iqbal et al. 1995
Jooste, Radon & Merwe Populus hybrida B, X Iqbal et al. 1995
Bacillispora inflata Mangifera indica B Iqbal et al. 1995
Iqbal & Bhatty Populus hybrida B Iqbal et al. 1995
Salix babylonica B Iqbal et al. 1995
184 F. Bärlocher
Root
Campylospora Salix babylonica B Iqbal et al. 1995
chaetocladia
Ranzoni
Clavariopsis aquatica Alnus glutinosa B, X Fisher et al. 1991
de Wild. Picea glauca X Sridhar and Bärlocher 1992a
Populus hybrida B Iqbal et al. 1995
C. azlanii Nawawi Mangifera indica B Iqbal et al. 1995
Cylindrocarpon aquaticum Acer spicatum B, X Sridhar and Bärlocher 1992a
(Nils.) Marvanová & Mangifera indica B, X Iqbal et al. 1995
Descals Picea glauca B Sridhar and Bärlocher 1992a
Populus hybrida B, X Iqbal et al. 1995
Filosporella sp. Alnus glutinosa B Fisher et al. 1991
F. fistucella Alnus glutinosa B Marvanová and Fisher 1991
Marvanová & Fisher
F. versimorpha Alnus glutinosa B Marvanová et al. 1992
Marvanová et al.
Flagellospora curvula Mangifera indica B Iqbal et al. 1995
Ingold Populus hybrida B Iqbal et al. 1995
F. fusarioides Mangifera indica B, X Iqbal et al. 1995
Iqbal Populus hybrida B, X Iqbal et al. 1995
F. penicillioides Mangifera indica B, X Iqbal et al. 1995
Ingold Populus hybrida B, X Iqbal et al. 1995
Fontanospora fusiramosa Alnus glutinosa R Marvanová et al. 1997
Marvanova et al.
Geniculospora sp. Picea glauca B, X Sridhar and Bärlocher 1992b
Heliscus lugdunensis Acer spicatum B, X Sridhar and Bärlocher 1992a
Sacc. & Therry 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
Lunulospora curvula Alnus glutinosa B Fisher et al. 1991
Ingold 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
Mangifera indica B Iqbal et al. 1995
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 R Raviraja et al. 1996
torresiana
M. acerina *Avicennia officinalis R Ananda and Sridhar 2002
(Hartig) Deighton
M. clavata Iqbal Betula papyrifera B, X Sridhar and Bärlocher 1992a
M. iqbalii sp. ind. F. Bareen Mangifera indica B, X Iqbal et al. 1995
Phalangispora constricta Picea glauca B, X Sridhar and Bärlocher 1992b
Nawawi & Webster
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
Picea glauca B 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. Populus hybrida B Iqbal et al. 1995
T. setigerum Picea glauca B Sridhar and Bärlocher 1992b
(Grove) Ingold
Tricladium chaetocladium Alnus glutinosa B Fisher et al. 1991
Ingold Alnus glutinosa B Fisher et al. 1991
Tricellula aquatica Mangifera indica B Iqbal et al. 1995
Webster
Triscelophorus acuminatus Angiopteris evecta R Raviraja et al. 1996
Nawawi Christela dentata R Raviraja et al. 1996
Coffea arabica B Raviraja et al. 1996
Diplazium esculatum R Raviraja et al. 1996
Hevea brasiliensis B, X Raviraja et al. 1996
186 F. Bärlocher
Root
torresiana
*Rhizophora R Ananda and Sridhar 2002
mucronata
*Sonneratia caseolaris R Ananda and Sridhar 2002
T. konajensis Antipteris evecta R Raviraja et al. 1996
Sridhar & Kaveriappa Christela dentata R Raviraja et al. 1996
Coffea arabica B Raviraja et al. 1996
Macrothylpteris R Raviraja et al. 1996
torresiana
T. monosporus Angiopteris evecta R Raviraja et al. 1996
Ingold Christela dentata R Raviraja et al. 1996
Coffea arabica B, X Raviraja et al. 1996
Diplazium esculatum R Raviraja et al. 1996
torresiana
Mangifera indica B Iqbal et al. 1995
Tumularia aquatica Alnus glutinosa B Fisher et al. 1991
(Ingold)
Marvanová & Descals
Varicosporium elodeae Alnus glutinosa B Fisher et al. 1991
Kegel 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 sug-
gest that aquatic fungi can produce diffusible antibiotics. For example,
Massarina aquatica, the teleomorph of Tumularia aquatica, releases anti-
fungal 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 an-
tibacterial 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, such as wood, favour colonisation by species capable of defending
captured resources (Shearer 1992). It is currently unknown how such com-
pounds 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 fun-
gal pathogens, endophytes and saprobes. Alternatively, plant detritus, pre-
colonised by fungal biotrophs or saprotrophs may have fallen into streams.
Some of these fungi may subsequently have adapted to dispersal and re-
production in water. The most comprehensive analysis of fungal gene se-
quences 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
other types of imported terrestrial detritus, such as leaves. Over time, this
may have favoured adaptation to life in running water. Or, terrestrial leaves
infected with endophytes were shed and landed in streams. Even water-
films on soil or between layers of terrestrial leaf layer may have selected
for tetraradiate spore shapes (Bandoni 1975) and predisposed some fungi
for their eventual evolution into aquatic hyphomycetes. There are several
reports of aquatic hyphomycete conidia in rainwater dripping from trees
188 F. Bärlocher
(Ando and Tubaki 1984; Bärlocher 1992; Czeczuga and Orlowska 1999), and
Widler and Müller (1984) isolated two undescribed species of Gyoerffyella
and Varicosporium from green twigs. Iqbal et al. (1995) reported 14 species
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 Nematophagous Fungi
as Root Endophytes
Luis V. Lopez-Llorca, Hans-Börje Jansson,
José Gaspar Maciá Vicente, Jesús Salinas
11.1
Introduction
Nematophagous fungi constitute a group of fungal antagonists to nema-
todes. 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 em-
ployed (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, ju-
veniles 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, Univer-
sidad de Alicante, Apdo. 99, 03080 Alicante, Spain, E-mail: jgmv@ua.es
Jesús Salinas: Departamento de Ciencias del Mar y Biología Aplicada, Universidad de Ali-
cante, 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 saprophyti-

cally 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, have the capacity to
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 vermi-
form nematodes (juveniles and adults), whereas the fourth group infects
nematode females and eggs (Jansson and Lopez-Llorca 2001). Nematode-
trapping 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 nema-
todes 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 ne-
matode cuticle or are ingested. These fungi are generally obligate parasites
of nematodes (Fig. 11.1b). The toxin-producing fungi, comprising for in-
stance 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) and Hohenbuehelia 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 endopar-
asitic 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 ne-
matode egg infected by the egg-parasitic fungus Pochonia rubescens. (From Lopez-Llorca
and Claugher 1990, courtesy of Elsevier). Bars a, b 5 µm; c 4 µm
to be Ascomycetes with their teleomorph in Orbilia spp. (Pfister 1997).

Other nematode-trapping fungi, e.g. Stylopage and Cystopage spp. are Zy-
gomycetes, 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 nematode-
trapping 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 oligospora forms 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 be-
fore traps are fully developed, nematodes can be captured in the adhesive
covering the traps. The adhesive has been partially characterised and ap-
pears 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 fun-
gus Drechmeria coniospora, where the fibrils appear directed whether ne-
matodes are present or not (Jansson and Nordbring-Hertz 1988). After
adhesion, the fungus penetrates the nematode cuticle from the trap, prob-
ably using both mechanical and enzymatic means. Since the nematode
cuticle contains mainly proteinaceous material (Bird and Bird 1991), ex-
tracellular proteolytic enzymes involved in cuticle penetration have been
studied. The major protease appears to be subtilisin PII. This serine pro-
tease 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
are then formed outside the nematode corpus to start a new infection
cycle.
Pochonia spp. adhere to nematode egg shells by means of an appresso-
rium 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 there-
fore 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). Fol-
lowing 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 myco-
parasitic 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 with-
out 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
external root colonisation (ectorhizosphere). Lately, colonisation in the root
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 nema-
tode-trapping fungi in field soils planted with barley, pea or white mustard.
The pea rhizosphere harboured by far the highest frequency of nematode-
trapping fungi: 19 times higher than in the root free soil. The number
of species of nematode-trapping fungi was also higher in the pea rhizo-
sphere, 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 ne-
matophagous fungi was followed for 3 months. Monacrosporium ellipsospo-
rum 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 exu-
dates (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. This effect is possibly due to increased leakage of
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)
when penetrating plant cell walls of epidermis and cortex cells, but never
Fig. 11.2. a Formation of appressoria (arrow) by Arthrobotrys 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 abun-
dant 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. chlamy-
dosporia 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 labora-
tory.
The growth of the two nematophagous fungi in plant roots appears to re-
semble 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 investi-
gation. 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, which infects nematodes by means of adhesive
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, does not seem
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 (teleomorph Hohenbuehelia) is a peculiar genus of basidio-
mycetous nematophagous fungi. It comprises about 15 species, some of
Table 11.1. Endophytic root colonisation of barley by the four ecological groups of ne-
matophagous 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 vermi-
culite 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
surface as did H. 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. It forms basid-
iocarps and its standard means of living is saprophytic growth on decaying
wood. The most common species, P. ostreatus, is a commercially culti-
vated 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 immo-
bilises 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 Hir-
sutella rhossiliensis 3 weeks after inoculation, forming conidiophore with phialide (arrow).
b Colonisation of cortex of barley roots by the nematode-trapping basidiomycete Nematoc-
tonus robustus 2 weeks after inoculation. c Colonisation of cortex and epidermis of barley
roots by the nematode-trapping basidiomycete Nematoctonus robustus 1 week after inocu-
lation. 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 nematode-
trapping fungus Arthrobotrys dactyloides 2 weeks after inoculation. f Colonisation of cortex
of barley roots by the nematode-trapping fungus Arthrobotrys dactyloides 2 weeks after
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 sur-
face 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 ne-
matode. With trapping and endoparasitic nematophagous fungi, such ex-
periments 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 ne-
matophagous 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 nema-
todes. These phytopathogenic nematodes have an endoparasitic behaviour
and a life span of at least a month – too long for our axenic system. Al-
though 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 speci-
ficity 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
described yet). Since the nematode lays eggs in root tissue, one could expect
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., and the final goal of our studies is to develop bio-
logical 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., may give the fungi an opportunity to infect nematode eggs in egg sacks
of root-knot nematodes inside the roots and reduce subsequent spread and
infection of roots by the second stage juveniles. Structures resembling trap-
ping 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 sup-
pressiveness 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. chlamydospo-
ria 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, partic-
ularly for biocontrol purposes.
11.3
Concluding Remarks
The role of the host plant in the tritrophic relationship between nema-
tophagous 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 agroe-
cosystem 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. An-
other 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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mycorrhiza fungi (AMF) on pyrethrum in Kenya. Int J Pest Manage 47:135–140
12 Molecular Diversity and Ecological
Roles of Mycorrhiza-Associated Sterile
Fungal Endophytes in Mediterranean
Ecosystems
Mariangela Girlanda, Silvia Perotto, Anna Maria Luppi
12.1
Introduction
Under natural conditions, plant roots sustain considerable fungal diversity
(Vandenkoornhuyse et al. 2002). In healthy plants, colonisation of root
tissues is not restricted to mycorrhizal symbionts; other fungi can also grow
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 ubiqui-
tous 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, repre-
senting 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,
10125 Torino, Italy
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, sporula-
tion 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 coni-
dial 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 indi-
cated 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 rep-
resentatives 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 at-
tribute 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. In-
deed, 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 sub-
antarctic 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 op-
portunity 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 gen-
erally 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 “gar-
rigue” 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 di-
versity of plants and their associated mycorrhizal types: sclerophyllous
and evergreen shrubs, and small trees bearing arbuscular, ericoid, arbu-
toid 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 ecolog-
ical 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; Mediter-
ranean heather) were selected for isolation of DSE fungi. Using both molec-
ular 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 my-
corrhizal roots of the two different host pairs (Pinus halepensis / Rosmari-
nus officinalis, Quercus ilex / Erica arborea), morphotypes were recognised
based on macro- and micro-scopic somatic features and assessed for con-
sistency 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 pub-
lic DNA databases as well as for phylogenetic reconstructions carried out
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, car-

ried 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 (<80% sequence identity), being limited mostly to the 5.8S gene re-
gion. These morphotypes would therefore represent fungi having no close
relatives in GenBank or hitherto undescribed fungi. They could therefore
be given taxonomic placement only at a high level based on 18S rDNA se-
quence data, and were assigned to the Dothideomycetidae and Chaetothyri-
omycetidae. Interestingly, one such morphotype displayed significant ITS
identity with a DSE morphotype obtained from E arborea roots and mature
Q ilex woodland and garrigue soil in the Q. ilex / E. arborea investigations
(Bergero et al. 2000, 2003; see below). Better sequence matches referred to
the entire ITS region, (94–95% sequence identity), and were obtained for
a different morphotype with Diaporthe/Phomopsis sequences. Diaporthe is
a genus belonging to the Valsaceae (Diaporthales, Sordariomycetidae) that
has anamorphs primarily in Phomopsis (Kirk et al. 2001). Results from phy-
logenetic analyses based on ITS, 18S and elongation factor-1 alpha (EF1-α)
gene sequences indicate that this morphotype, which is likely identifiable
as a Diaporthe/Phomopsis species, would, however, be distinct from those
currently represented in GenBank.
A single morphotype could be identified with confidence to the species
level as Rhizopycnis vagum D.F. Farr, based on a nearly identical ITS se-
quence (approx. 99% identity over 500 bp, i.e. the entire ITS region). Such
an in silico identification is consistent with colony features and morphology
of somatic structures, including characteristic, tuberculate dark chlamy-
dospores. R. vagum is a recently described coelomycete (gen. et sp. nov.;
Farr et al. 1998), which was originally known to contribute to ‘vine de-
cline’ of cucurbit crops in several parts of the world. It was first reported
from cantaloupe in the Rio Grande Valley of Texas and was subsequently
isolated from melon, watermelon and cucumber in Guatemala, Honduras,
California, Spain and Central Italy (Mertely et al. 1991; Miller et al. 1996;
Bruton and Miller 1997a, 1997b, 1997c; Gwinne et al. 1997; Aegerter et al.
2000; Armengol et al. 2000, 2003; Montuschi 2001). In Central and South-
ern Italy R. vagum has also been isolated from tomato roots exhibiting
typical corky root symptoms (Porta-Puglia et al. 2001). Teleomorphic con-
nections are unknown for R. vagum, but phylogenetic ITS-based analyses
placed this fungus in a sister-group to Massarina walkeri Shoemaker, C.E.
Babc. & J.A.G. Irwin, a teleomorphic species belonging to Lophiostomat-
aceae, Pleosporales (Dothideomycetidae; Girlanda et al. 2002; Armengol
et al. 2003). Massarina walkeri is the teleomorph of Acrocalymma med-
icaginis, a coelomycete exhibiting morphological resemblance to R. vagum
(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 Phialo-
cephala spp., Phialophora spp., Cadophora finlandia (Wang & Wilcox) Har-
rington & 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; Har-
rington 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, a typ-
ical ericoid Mediterranean shrub. In the climax woodland, disturbances
such as human activities and fire, which play a key role in shaping Mediter-
ranean 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 disap-
pearance 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 re-
gion 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 Col-
orado (Monreal et al. 1999; McLean et al. 1999; Vrålstad et al. 2002a; Bergero
et al. 2003; and unpublished sequences). In particular, Sd9 shared high se-
quence 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, re-
spectively, 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-related group or of separate, unspecified groups often receiving poor
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 Hy-
menoscyphus ericae and the fungal symbiont forming Piceirhiza bicolorata
ectomycorrhizae, belonging to a monophyletic aggregate possibly compa-
rable 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
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) pro-
files 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, inde-
pendently 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 speci-
ficity unresolved, there is evidence from RAPD data on Mediterranean DSE
that some DSE genotypes may actually associate with both ecto- and endo-
mycorrhizal 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 E arborea as a bait plant, we have shown that soil
from mature Q ilex forest 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 temper-
ate 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 decom-
posing organic debris in the complex soil environment, or whether they
exist primarily as root associates of different plants, including ectomyc-
orrhizal 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 as-
sociation with host roots DSE isolates have been shown to increase host
foliar P and N concentrations and plant biomass under some experimen-
tal 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 dissimi-
lar from that of conventional mycorrhizal symbionts. It should also be con-
sidered 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
may be even more prevalent than conventional mycorrhizal fungi, colonis-
ing 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 peri-
ods when the latter are inhibited by environmental conditions (Jumpponen
and Trappe 1998a).
Inoculation experiments in E arborea with DSE isolates obtained from
either this plant or Q ilex have shown that, irrespective of the host of ori-
gin, some strains are capable of forming typical intracellular hyphal coils,
suggesting ericoid mycorrhizal behaviour (Bergero et al. 2000, 2003). Mor-
photype 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 < 0.05
(ANOVA, Tukey post-hoc test; different values have no common letter)
phenotypic plasticity in the production of distinct interfaces in different

hosts, e.g. intracortical microsclerotia and hyphal coils, is not unusual
and is reminiscent of the behaviour of some conventional mycorrhizal
symbionts, such as fungi capable of forming ectomycorrhiza on tree hosts
and endomycorrhiza on non-photosynthetic, “mycoheterotrophic” orchids
(Taylor et al. 2002; see Chap. 9 by Bayman and Otero). Recently, the ability
of a fungus grouping with Cadophora finlandia in the “Hymenoscyphus
ericae aggregate” to simultaneously form both ectomycorrhizas in asep-
tic synthesis with Pinus sylvestris seedlings and ericoid mycorrhizas in
Vaccinium myrtillus was demonstrated (Villarreal-Ruiz et al. 2004). This
suggests that ericoid and ectomycorrhizal fungi may be part of a com-
mon guild in boreal and temperate woodland ecosystems (Vrålstad 2004).
Of other fungal isolates from the same aggregate, only isolates of ericoid
mycorrhizal origin formed classic ericoid mycorrhizal associations, while
ectomycorrhiza formation was restricted to isolates of ectomycorrhizal ori-
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 morpho-
types isolated from the Q. ilex/ E. arborea host pair in Mediterranean plant
communities, were only able to form typical ericoid mycorrhiza in the lat-
ter host under the conditions tested, despite the fact that RAPD data for
the Sd9 morphotype demonstrate the presence of the same genetic individ-
ual 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 in-
teractions, which can in turn affect the diversity and dynamics of plant
communities. Mycelia of DSE fungi might interlink roots of different hosts,
which could translocate nutrients via the hyphae of such shared fungal as-
sociates, 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 envi-
ronments. 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 in-
vestigation (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 dif-
fer from situations in which crop pathogens are endophytic in weeds in
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 poly-
morphisms 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 was confirmed in
greenhouses and growth chambers under different temperature regimes.
Melon seedling inoculation demonstrated virulence on this host, establish-
ing inherent pathogenic potential on a cucurbit plant, with at least some
endophytic Italian isolates being more aggressive, under certain experi-
mental 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 num-
bers 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. En-
dophytic fungi colonising asymptomatically plant parts may also include
pathogens that have extended latency periods before development of dis-
ease, 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 bar-
riers, 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 con-
ventional 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 vir-
ulence 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 spec-
trum of DSE taxa known to associate with ecto- and endo-mycorrhizal
hosts, pointing to broad taxonomic boundaries of these endophytes. Se-
quencing 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. ITS-
based 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 in-
cluded, 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 prob-
lem 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 pos-
sibility that taxonomically diverse fungi have evolved not only different
patterns of biome and host specificity, but also diverse functional signifi-
cance 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 cre-
dence 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 informa-
tion derived from axenic culture work with isolated DSE and the influences
of plant and fungal genotypes, as well as local abiotic and biotic environ-
ments, 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%,
CEBIOVEM.
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13 Oidiodendron maius:
Saprobe in Sphagnum Peat,
Mutualist in Ericaceous Roots?
Adrianne V. Rice, Randolph S. Currah
13.1
Introduction
Oidiodendron maius Barron is a hyphomycete species isolated from peat,
soil, decaying organic matter, and plant roots throughout temperate ecosys-
tems, including peatlands, forests, and heathlands (e.g. Barron 1962; Nord-
gren 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 conidio-
phores 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 can be induced when the species is
grown on autoclaved lichen (Rice and Currah 2002) (Fig. 13.1c). Molecular
evidence confirms the position of O. maius among other species of Oidio-
dendron and their teleomorphic counterpart, Myxotrichum (Hambleton et
al. 1998).
The distribution of O. maius seems to parallel that of members of the Er-
icaceae (blueberries, cranberries, rhododendrons, etc.), a family that often
dominates the vegetation in arctic and alpine meadows, temperate heath-
lands, 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 5 µm, c 80 µm
based on a predilection in both plants and fungus for acidic, nutrient-

poor, 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 ben-
efits 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 fastid-
ious 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, con-
text 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 possibil-
ities 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, pos-
sibly 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.
One type of ecosystem that may be supporting O. maius both as a saprobe
and a mycorrhizal associate is acidic Sphagnum peatlands, found through-
out 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 peat-
lands 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 mycor-
rhizal 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
of conifers and ericaceous shrubs in peatlands, and degrade the Sphagnum
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; Nord-
gren 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). Al-
though 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.
The first report of O. maius from the roots of ericaceous shrubs appeared
in 1989, when it was isolated from ericoid mycorrhizal roots of Rhododen-
dron (Douglas et al. 1989). Since then most records of O. maius have been
based on isolates from presumably healthy ericaceous roots (e.g. Hamble-
ton 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 rela-
tively 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 mycor-
rhizal fungi than saprobes, with all arbuscular and most ectomycorrhizal
fungi unable to reproduce in the absence of their hosts (Read 2002). En-
dorhizal fungi allied to the Helotiales are notable exceptions (Addy et
al. 2005). For example, Rhizoscyphus ericae, an ericoid mycorrhizal fun-
gus that also forms ectomycorrhizas (Vrålstad et al. 2000, 2002), produces
chains of arthroconidia in culture, and in rare instances has formed dis-
cocarps (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 appear-
ing directly on Sphagnum peat, but it was only rarely encountered when the
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 addi-
tion 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% (Thor-
mann 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 Sphag-
num 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 in-
termediate 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) and hyphae (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 3 µm, b 20 µm, c 5 µm, 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 fre-
quent 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 ecten-
domycorrhizas 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 includ-
ing 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 metal-
contaminated 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
layer of root cells (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 inter-
twined, 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 associ-
ation (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 cul-

ture 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 Rhizoscy-
phus, as R. ericae (Zhang and Zhuang 2004). Scytalidium vaccinii has been
confirmed as the anamorph of R. ericae (Egger and Sigler 1993; Hamble-
ton 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 cul-
ture. 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 us-
ing 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 endo-
phytes.
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
were based on misidentified strains of O. 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 in-
dicator 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 ap-
pears 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 Gym-
nascella 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 asso-
ciation 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. Inocula-
tion of salal (Gaultheria shallon) with four isolates of O. 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; Berg-

ero 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 in-
oculation 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, but not the Oidiodendron 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 sim-
ilar to the dynamics in orchid mycorrhizas in which the orchid “ex-
ploits” 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. Rhododen-
dron, 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 evolution-
ary 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 move-
ment 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
The occupation of multiple niches within a given environment could confer
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 my-
corrhizal 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 nutri-
ents 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 eri-
coid 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). Decomposi-
tion of Sphagnum by O. maius may release a significant amount of carbon
and nutrients from the peat and, instead of releasing carbon into the at-
mosphere 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 phos-
phorus 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 as-
sociations 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, includ-
ing 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. peri-
conioides from Rhododendron brachycarpum grown in pot cultures con-
taining 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. Usually considered a root endophyte,
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

in birch snags and birch and pine stumps, where it is presumably occupying
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
species is abundant.
13.6
Conclusions
Oidiodendron maius forms associations with the roots of ericaceous shrubs,
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 environ-
ment, although the benefits to O. maius are unclear. It remains necessary
to investigate the benefits to both partners and demonstrate what envi-
ronmental 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 to-
wards 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 eri-
coid 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 Engi-
neering Research Council of Canada are gratefully acknowledged.
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14 Mycorrhizal and Endophytic Fungi
of Epacrids (Ericaceae)
John W.G. Cairney
14.1
Introduction
Epacrids are a group of over 450 species of woody plants that were conven-
tionally 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 classifica-
tion regarding epacrids as Styphelioideae, one of eight Ericaceae subfami-
lies (Kron et al. 2002). Although epacrids occur in several southern hemi-
sphere 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 ba-
sic soils (Keighery 1996). Despite being geographically and hydrologically
disparate, these habitats characteristically encompass soils in which avail-
ability of mineral nutrients is relatively poor (Read 1996).
In common with many other Ericaceae taxa, epacrids produce extremely
fine lateral roots known as hair roots. Up to three orders of hair roots may be
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 gen-
erally reveals longitudinally oriented surface hyphae from which perpen-
dicular 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 Ash-
ford 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 gener-
ally taken to indicate mycorrhizal infection. Aside from putatively ericoid
mycorrhizal ascomycetes, arbuscular mycorrhiza-like infections, suggest-
ing 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 associa-
tion. In the case of apparent arbuscular mycorrhizal infection at least, it
seems most plausible that this reflects opportunistic infection by arbuscu-
lar 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. Nonethe-
less, 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 con-
centrated largely on the rDNA internal transcribed spacer (ITS) region.
Assemblages of isolated endophytes have thus been grouped on the ba-
sis 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. Compar-
ison 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 pub-
lished 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 er-
icae 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 mycor-
rhiza with northern hemisphere Ericaceae (Vrålstad et al. 2002). These iso-
lates have been obtained from epacrids at alpine, dry sclerophyll forest, 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 simi-
larity to Capronia (Chaetothyriales) and Thielavia (Sordariales) (Midgley
et al. 2002, 2004a), and, while some of these are also endophytes of non-
ericaceae 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
250 J.W.G. Cairney
(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, un-
published data). Isolates with close sequence similarity to Phialocephala
fortinii and other DSE taxa, although occasionally obtained from lower el-
evation 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 differ-
ent 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 en-
dophyte 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
Where multiple isolations have been made from root systems of individ-
ual 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 east-
ern Australia. Between 99 and 227 isolates were obtained from each plant
and ITS-RFLP analysis suggested that the assemblage from each plant com-
prised 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, demon-
strated that the isolates that dominated the assemblages were widespread
throughout the hair root systems (Midgley et al. 2002, 2004a). These dom-
inant RFLP-types were shown to form typical ericoid mycorrhiza-like coil
structures in gnotobiotic mycorrhizal infection experiments with W. pun-
gens as host (Midgley 2003; Midgley et al. 2004a), suggesting that each
root system was dominated by a single putative ericoid mycorrhizal fun-
gal 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 mycor-
rhizal 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 myc-
orrhizal 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
252 J.W.G. Cairney
fungi, particularly where comparisons of relative abundance of taxa be-

tween 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 competi-
tiveness 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 mycor-
rhizal fungi from epacrids vary in their responses to water stress (Hutton et
al. 1996b; Chen et al. 2003). Furthermore, the extent of ericoid mycorrhiza-
like hair root colonisation varies seasonally for W. pungens in eastern Aus-
tralian 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. pun-
gens 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 re-
garded 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 hemi-
sphere. 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 ecolog-
ical 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 er-
icae 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 1 cm, b 50 µm
254 J.W.G. Cairney
mycorrhizal fungi (Berch et al. 2002), none of the isolates from epacrids that
have close sequence identity to these fungi formed mycorrhiza-like struc-
tures 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
abilities of isolates in these groups. The only basidiomycete so far isolated
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 epi-
dermal 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 eri-
coid mycorrhizal associations evolved in a common ancestral host plant
group (Cullings 1996). Fossil evidence and extant biogeographical pat-
terns 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 sclero-
phyll 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 veg-
etation (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 sapro-
troph (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 pu-
tative 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 acquisi-
tion.
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 in-
ositol 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 sub-
strates 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
256 J.W.G. Cairney
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 geno-
types 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 puta-
tive mycorrhizal fungus from W. pungens and six isolates of an unknown
Helotiales putative mycorrhizal fungal taxon from W. pungens and L. parv-
iflorus 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 in-
traspecific variation was observed on all substrates, one taxon produced
significantly more biomass on most substrates than the other. For all sub-
strates, 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 pu-
tative ericoid mycorrhizal fungal taxa from W. pungens suggests that all
are broadly similar in their abilities to utilise organic nitrogen and phos-
phorus 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 Mutualistic Interactions
with Fungal Root Endophytes
Barbara Schulz
15.1
Introduction
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 interac-
tions 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 (Jump-
ponen 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 endo-
phytes may lead to induced disease resistance (Picard et al. 2000; Benhamou
and Garand 2001), improved growth of the host (Kimura et al. 1992; Jump-
ponen 2001; Mucciarelli et al. 2002; Ernst et al. 2003), abiotic stress tolerance
(Barrow and Aaltonen 2001; Redman et al. 2002; Barrow 2003) or protec-
tion 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 deal-
ing with mutualistic interactions between non-mycorrhizal root-colonising
endophytic fungi with their plant hosts. When reading this chapter it is im-
portant 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, Spiel-
mannstraße 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 Fusar-
ium and dark septate endophytes (DSE; Jumpponen and Trappe 1998; Sieber
2002).
Histological studies of colonisation by DSE have focussed on Phialo-
cephala 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 hya-
line 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 cylin-
der (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 un-
published) 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 de-
fence 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 myc-
orrhizal structures. For example, in the roots of at least 19 plant species,
the dematiaceous hyphomycete H. chaetospira grew intra- or intercellu-
larly 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 Pop-
ulus tremuloides grown in an experimental field plot (Kaldorf et al. 2004).
This is also the case for the DSE Phialophora finlandia, which formed ecten-
domycorrhizal 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 nodo-
rum 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. The endophyte formed an external enveloping mycelial
sheet around the root with only little penetration of the root cortex (Muc-
ciarelli 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 phe-
notypic plasticity. Colonisation can be inter- and/or intra-cellular, limited
to the roots or apparently systemic within the entire plant. The morpholo-
gies 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 biolog-
ically 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
which organ the endophytes were isolated.
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 re-
quired for the synthesis of secondary metabolites without some beneficial
effect for survival. As virulence factors, we have hypothesised that sec-
ondary 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 in-
cluding 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 nema-
tode 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 endo-
phytic 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 biolog-
ically 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, Cryptosporiop-
sis, 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 Neoty-
phodium, 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
to the roots, e.g. that by Phoma in Vulpia ciliata spp. ambigua, which in-
creased shoot biomass, root lengths, and tiller numbers of the host (New-
sham 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 ob-
served 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 phospho-
rous 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 acquisi-

tion 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 phospho-
rous, 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 Oidio-
dendron 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 or-
ganic 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 mycor-
rhiza (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 ef-
fects 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 inter-
action (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 en-
hancement 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 ax-
enic 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 < 0.05, significant
in comparison to the control according to Kruskal-Wallis ANOVA on ranks (Schulz et al.
2002)
Mucciarelli et al. (2003) found that colonisation by a non-sporulating en-

dophyte increased the biomass of the roots of peppermint. Application of
a methanolic culture extract of P. fortinii to the seedlings also increased
host biomass (Table 15.1; Römmert et al. 2002), as Varma et al. (2000)
had reported for shoots of maize not only when colonised by Piriformo-
spora indica, but also following treatment with methanolic mycelial culture
extracts.
It is possible that the enhancement of growth of L. decidua was, perhaps
in part, due to synthesis of indole acetic acid (IAA) by the endophytes
studied, since both the endophytes synthesised IAA in vitro (Römmert
et al. 2002). This hypothesis is supported by the fact that colonisation by
the pathogen Heterobasidion annosum, which also produced IAA in vitro,
initially improved growth of shoots of L. decidua (Fig. 15.1; Römmert et al.
2002). The synthesis of phytohormones is not unusual for fungal pathogens
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
+ Culture extract (n = 8) 1.0* 44.0*
Control (n = 10) 0.08 16.9
T. aestivum L.
+ Culture extract (n = 20) 0.84 1.6
Control (n = 20) 0.82 2.4
∗P < 0.05
(Tudzynski 1997; Tudzynski and Sharon 2002). For example, the yield loss
caused by infections with Pythium group F, a minor pathogen that induces
symptomless infections in tomato cultures, is ascribed to IAA (Rey et al.
2001).
However, small molecular weight metabolites may also be responsible
for growth promotion, as was found by Kimura et al. (1992), who identified
the novel altechromones A and B in the culture broth of an Alternaria sp.
When these substances were applied to the seedlings of lettuce, root growth
was improved by ∼ 50%. Similarly, fumonisin, which on the one hand is
a potent mycotoxin of Fusarium, also improves development of the roots
of maize (Yates et al. 1997).
Host adaptation may be involved in the growth-enhancing effects of
some root endophytes. In contrast to colonisation of the roots of seedlings
of L. decidua by P. fortinii, colonisation of Triticum aestivum L. (wheat)
led to no significant enhancement of growth (data not shown). The culture
extract was also ineffective when applied to this non-host (Table 15.1).
Similarly, inoculation of the roots of aseptically grown seedlings of Carex
firma and C. curvula with a DSE led to a significant increase in production
of dry matter in C. firma, but not when the fungus was inoculated into
C. curvula (Haselwandter and Read 1982).
In this context, Holland (1997) provided evidence for a very intriguing
hypothesis, giving a clue as to one reason why plants may need microbial
interactions. He suggested that the microbial synthesis of so-called phyto-
hormones might well be microbial signals to the plant that active growth
can be accelerated, that endophytic microbes are present to degrade and
recycle plant waste products resulting from growing tissue. For example,
Methylobacterium spp. that synthesise cytokinin in vitro, are found en-

dophytically 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 sig-
nals 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 multi-
ple 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 car-
bon transport. These results suggest that fungal endophytes are not only
involved in a balanced antagonism with their hosts, but also may be re-
sponsible for a balanced regulation of growth. Thus, in at least some inter-
actions, 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
The idea of endophyte mediated induced resistance is an extension of
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 Schön-
beck 1992; Hallmann and Sikora 1994; Sieber 2002). For example, root
270 B. Schulz
inoculations of cabbage plants with soilborne endophytic Acremonium al-

ternatum decreased damage due to larvae of the diamondback moth on the
leaves (Raps and Vidal 1998). However, the authors suggested that compe-
tition 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 disjunc-
tion 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 mechan-
ical defence response also limits colonisation by subsequent pathogens.
Only a few of the endophytic isolates from a given host are effective pro-
tectants when reinoculated into the host. Of 322 endophytic fungi isolated
from Chinese cabbage (Brassica campestris), only 16 isolates, including Het-
eroconium 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 ex-
tensively 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 ax-
enically 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 me-
chanical 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 oligan-
drum 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 abi-
otic 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 mycorrhiza-
tion, 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 energy-
rich 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 ac-
cumulated 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 con-
clusion 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 mutu-
alistically 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 (Ta-
ble 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, phy-
tohormones, nutrients, elicitins and colonisation (Fig. 15.2). These factors
may have more than one benefit for the host. Potential advantages of the in-
teractions 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 mir-
ror 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
Phoma fimeti Vulpia ciliate Newsham 1994
Stagonospora spp. Phragmites Ernst et al. 2003
australis
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 Redman et al. 2002
lanuginosum
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 mutu-

alistic 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 with-
out 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 ad-
equately 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 un-
known 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 Sci-
ences, 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 (Brun-
drett 2004). VAM Vesicular arbuscular mycorrhizas, ECM ectomycorrhizas
Criteria Mycorrhizal Parasitic Endophytic

Morphology Specialised hyphae in Specialised hyphae Relatively
specialised plant organ unspecialised hyphae
Development Synchronised Often synchronised Not synchronised
Impact on fungus Obligate requirement Fungus obligately or Fungus moderately or
for plant supplied facultatively weakly dependent
nutrients (VAM and dependent on plant
ECM)
Impact on plant Strong or weak benefit Strong or weak harm Weak harm or benefit
Nutrient transfer Synchronised transfer, Active or passive Passive transfer,
fungus a strong sink transfer, fungus fungus not a strong
a strong or weak sink 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 plant-
fungus relationships, as mycorrhizas are defined by morphological criteria
(Brundrett 2004). Differences between endophytic associations and myc-
orrhizal associations are summarised in Table 16.1. The most important
of these differences are the absence of substantial fungus-to-plant nutri-
ent transfer in endophytic associations and the lack of synchronised de-
velopment 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 associa-
tions 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 associ-
ations. 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 de-
fined above), it seems likely that mycorrhizal fungi have endophytic phases
involving long-term coexistence with plants without active growth or nu-
trient 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
the fungus with first access to nutrients that can be transferred to hyphae
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 dif-
fers 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 po-
tentials 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 maintain-
ing 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
284 M.C. Brundrett
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) and hyphae (arrow) in a rhizome
scale A and nonmycorrhizal root B. C, D Dense growth by unknown fungi (arrows) on the
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 coloni-
sation 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 myc-
orrhizal fungi from their healthy young roots (Brundrett 1991). As implied
by this definition, endophytic colonisation of nonmycorrhizal plant roots
by VAM fungi is common in older roots, but is considered to be of limited
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 faculta-
tively mycorrhizal species in which VAM associations are present or absent
as a result of soil conditions (see Brundrett 1991). These issues are best il-
lustrated by the debate about the role of mycorrhizal associations in sedges
(Cyperaceae and allied families) that has been ongoing for decades (Pow-
ell 1975; Tester et al. 1987; Brundrett 1991; Muthukumar et al. 2004). The
statement by Muthukumar et al. (2004) that the Cyperaceae is a mycor-
rhizal 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 dis-
tinguish 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 Glom-
eromycotan fungi is difficult to interpret is their colonisation of roots of
ectomycorrhizal (ECM) plants in species with dual mycorrhizal associa-
tions. 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-
286 M.C. Brundrett
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 well-
defined Hartig net is not always clear cut, as “superficial” ECM roots with
a thin or patchy Hartig net occur in synthesis experiments using host-
fungus 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, Tulas-
nellales, 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 (Rhizan-
thella 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 cul-
tures of bacteria, actinomycetes and common endophytes such as Fusarium
and Verticillium, as well as ECM fungi and ericoid fungi (Richardson and
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 sub-
stantially 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 (Tulas-
nella and Sebacina) closely related to orchid fungi that formed mycorrhiza-
like associations with hepatics. These bryophytes also contained ascomy-
cetes, 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
288
Fungus Endophyte Pathogen Ectomycorrhizal Ericoid my- Orchid mycorrhizas Other role
corrhizas
Tomentella spp. Primary role Myco-heterotrophic Decomposition of
(Thelephoraceae) (Köljalg et al. 2000) orchids wood?
(McCormick et al. 2004;
Taylor et al. 2002)
Rhizoctonia complex S.L. Common: e.g. Major role: See below Many orchid associates Mycorrhizas in hepatics
Pinus sylvestris (see text) (see text) (Kottke et al. 2003)a .
(Sen et al. 1999) Saprophytic
Sebacina spp. Ericaceae? (Glen et al. 2002; Suspected Some orchids (Warcup Myco-heterotrophic
(in Rhizoctonia complex) (Allen et al. 2003) Selosse et al. 2002a; role? (Allen 1981; Y. Bonnardeaux, orchids (Selosse et al.
Urban et al. 2003)a et al. 2003) personal 2002b; Taylor et al.
communication) 2003)a . Saprophytic?
Thanatephorus gardneri Some endophytic Melaleuca uncinata Myco-heterotrophic Saprophytic?
(in Rhizoctonia complex) activity and other plants orchid Rhizanthella
(Mursidawati (Warcup 1985; gardneri (Warcup 1985;
2003) Mursidawati 2003) Mursidawati 2003)
Phialocephala spp. Widespread ECM of trees
(DSE fungi) (see text) (Harney et al. 1997)
Fusarium spp. Common (Kuldau e.g. Tropical orchids Saprophytic phases
and Yates 2000; Pamphile (Bayman et al. 2002;
Redman et al. and Azevedo Abdul Karim 2005)
2001) 2002
M.C. Brundrett
Fungus Endophyte Pathogen Ectomycorrhizal Ericoid Orchid Other role

mycorrhizas mycorrhizas
Hymenoscyphus ericae Picea mariana roots Six tree spp. Primary role Mycorrhizas of
(Piercey et al. 2002), (Vrålstad et al. 2000)a (see text) bryophytes (Duckett
Bryophyte and Read 1995)a
(Chambers et al. 1999)
Oidiodendron spp. ECM roots of Quercus ilex Primary role? Some isolates are
(Bergero et al. 2000) (see text) strong saprophytes
(Piercey et al. 2002)
Mycelium radicis In ECM of Betula platyphylla Trees
atrovirens (Hashimoto and (Sakakibara et
Hyakumachi 2001) al. 2002)a
a Role not fully-established
16 Roles of Endophytic and Mycorrhizal Fungi
289
290 M.C. Brundrett
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, par-
asites, 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 en-
dophytically 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 ben-
efits 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, an ECM fungus that may not
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.
Despite the common occurrence of endophytic activity by mycorrhizal
fungi, we have very little knowledge of its ecological significance. Dam-
age 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 antago-
nistic 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 confu-
sion 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 rela-
tively new and more research is required to test the relative efficiency with
which they detect different categories of fungi. In contrast, identification of
mycorrhizal associations by morphology allows a much higher degree of
replication and can be more accurate than DNA-based studies, which some-
times fail to detect the most important fungi in roots. The interpretation
of fungal presence data provided by molecular tools that allow miniscule
292 M.C. Brundrett
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 micro-
scopic techniques. One such study by Sakakibara et al. (2002) found that
microscopic identification of fungi by morphotypes and molecular meth-
ods (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 my-
corrhizal and non-mycorrhizal fungi in roots. However, due to the increas-
ing recognition of the importance of “endophytes” as a specific category of
specialised plant-inhabiting fungi, it is no longer appropriate to call mycor-
rhizal 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 des-
ignate 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 endophyt-
ically 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 syn-
chronised 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, the number of roles fungi have does seem to be limited, presum-
ably because they cannot be proficient at all of these roles. 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 as-
sociations. 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 Isolation Procedures
for Endophytic Microorganisms
Johannes Hallmann, Gabriele Berg, Barbara Schulz
17.1
Introduction
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,
but at the same time be strong enough to eliminate epiphytes from the root
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.
The most commonly used isolation procedures combine surface sterilisa-
tion of the root tissue with either maceration of the plant tissue and streak-
ing 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 primar-
ily on culturable microorganisms. However, increasing interest in non-
culturable endophytic microorganisms has recently led to the application
Johannes Hallmann: Federal Biological Research Centre for Agriculture and Forestry, Insti-
tute 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,
Petersgasse 12, 8010 Graz, Austria
Barbara Schulz: Institute of Microbiology, Technical University of Braunschweig, Spiel-
mannstraße 7, 38106 Braunschweig, Germany

300 J. Hallmann et al.
of molecular methods for their identification. This chapter summarises iso-

lation 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 in-
formation 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 microorgan-
isms. 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 inciden-
tals, (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 guar-
anteed 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 ster-
ilised 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, sonifica-
tion 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 sterilisa-
tion. 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 hypochlo-
rite, 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 be-
cause 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 sam-
ples with complete sterilisation by 40%, but at the same time decreases
the population densities of endophytic bacteria from 4.36×103 cfu/ml to
Table 17.1. Protocols for surface sterilisation of root tissue
302
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 hypochloritea 1–2 min hirsutum, Pinus contorta McInroy and Kloepper 1995;
c) Two to four rinses in sterile water var. latifolia, Zea mays Shishido et al. 1995
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
d) Rinse in sterile water
a) Wash under running tap water Roots 1- to 5 mm in Various herbaceous Sardi et al. 1992
b) Exposure to propylene oxide vapor 1h diameter and woody plants
Physical sterilisation
a) Shake vigorously in 0.9% NaCl solution 20 min 13- to 14-week-old roots Solanum tuberosum Sessitsch et al. 2002
containing 0.3 g acid-washed glass beads
b) Five Rinses in sterile water
Fungi
36% Formaldehyde Musa acuminata Cao et al. 2002
J. Hallmann et al.
time diameter
a) Wash under running tap water Nodule roots, fine hair Triticum aestivum, Oberholzer-Tschütscher 1982;
b) 96% Ethanol 0.5–1.0 min roots, nonlignified Erica carnea Sieber et al. 1988;
c) 2–2.5% Sodium hypochlorite 1–4 min roots, lignified roots Crous et al. 1995b
d) 96% Ethanol 0.5 min
a) Wash under running tap water Adventitious roots, fine Lolium perenne, Picia abies Skipp and Christensen 1989;
b) 0.3–2% Sodium hypochlorite 1–3 min hair roots various species of Ericaceae, Kattner and Schönhar 1990;
c) Rinse in sterile water various alpine plant species Hambleton and Currah 1997;
Lycopersicon esculentum Stoyke and Currah 1991b .
Hallmann and Sikora 1994
a) Wash under running tap water Roots Hordeum vulgare, Boyle et al. 2001
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; Petrini et al. 1992;
17 Isolation Procedures for Endophytic Microorganisms
b) 75% Ethanol 3 min 6- to 9-month-old roots Aphelandra tetragona Werner et al. 1997b
c) 3–5% Sodium hypochloritea 0.5 min
d) 75% Ethanol
0.01% Mercuric chloride 5–15 min Fine roots Lupinus spp., O’Dell and Trappe 1992b
Oxytropis campestris
5% Hydrogen peroxide 5–15 min Fine roots Lupinus spp., O’Dell and Trappe 1992b
Oxytropis campestris
303
304
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 1996b
b) 99% Ethanol 1 min roots, 0–5.3 mm in
c) 35% Hydrogen peroxide 5 min diameter
Physical sterilisation
a) Cut core into pieces Boring cores from which Picea abies Maifeld 1998b
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 Fagus sylvatica, Picea abies, Görke 1998b
b) 70% Ethanol 1 min undergoing secondary Pinus sylvestris, Betula
c) 10% Sodium hypochlorite 3 min growth pendula
e) Rinse twice in sterile water
f) Discard bark
g) Move pieces quickly through
flame of Bunsen burner
a Concentrations of sodium hypochlorite represent percent active chlorine
b Adapted from Sieber 2002
J. Hallmann et al.
5.75 × 10 cfu/ml (A. Munif, personal communication). The effect of the

sterilising agent penetrating the plant tissue on the bacterial cells was visu-
ally demonstrated by treating the root tissue with a tetrazolium-phosphate
buffer solution (Patriquin and Döbereiner 1978). While metabolically ac-
tive 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) bi-
phenyl can be used to monitor the relative progression of the applied agent
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 surface-
sterilised 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
the medium, surface sterilisation is considered complete. A further check is
to test the effect of the sterilising agent directly on fungal or bacterial cells.
Roots are dipped into a fungal or bacterial suspension of known density,
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 microorgan-
isms that emerge from the root fragments are subsequently transferred to
fresh media. This method is commonly used for endophytic fungi (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 endo-
phytic bacteria (Araújo et al. 2001). This method selects for fast-growing
microorganisms, therefore representing more a qualitative than a quan-
titative approach. When studying microbial diversity, slow growing mi-
croorganisms 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 endo-
phytic 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 (Zin-
niel 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 de-
vices are advisable. For the latter, optimal time and intensity of maceration
need to be empirically determined. Following maceration of the surface-
sterilised 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, necessitating cooling; the sample should be immediately diluted to
decrease the concentrations of toxic compounds to ineffective levels. Al-
ternatively, 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 3,000 g (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 cryo-
scanning 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
at the same time and the overall time requirement may in effect be no more
than for other methods.
Whereas surface sterilisation, followed by subsequent plating of macer-
ated 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)
microorganisms can hide in niches that are not reached by the sterilising
agent or “stick” to the host tissue, resulting in false identifications of endo-
phytes; and (3) the sterilising agent may penetrate the root tissue and kill
endophytes, so that microbial densities will be underestimated. These dis-
advantages 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 centrifuga-
tion, 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 pres-
sure bomb commonly used for measuring plant water status is also effective
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
the root into the pressure cylinder, collection of plant sap from the root with a sterile Pasteur
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 microor-
ganisms. 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 imme-
diately 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
sterilised roots resulted in slightly higher bacterial numbers for the latter
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 vas-
cular 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 sur-
face 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). Compar-
ing 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 dex-
trose 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 in-
cluded in the isolation media. Sublethal doses of fungicides restrict radial
growth of fungal colonies, preventing overgrowth (Bills 1996). For exam-
ple, 1−2 mg/l Cyclosporin A (active component of Sandimmune; Novartis,
Basel, Switzerland) may be added to the growth medium to suppress fast-
growing 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 micro-
bial numbers as they do not record viable but non-culturable cells, e.g.
obligate biotrophs, and microorganisms with unknown growth require-
ments. 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 iden-
tify the active genes. In most cases target sequences are amplified using
the polymerase chain reaction (PCR). The 16S rRNA gene (rDNA) has be-
come a frequently employed phylogenetic marker with which to describe
bacterial diversity in natural environments (Vossbrinck et al. 1987). Sim-
ilarly, 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 (bac-

teria) or 18S and 28S rDNA (fungi) genes allow for analysis of the struc-
ture 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 electrophore-
sis (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-conformation-
polymorphism (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 endo-
phytes 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
represent about 1% of the total community to be detectable in a finger-
print (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 activ-
ity to certain ribotypes, such as the incorporation of bromide oxyuridine
(BrdU) or 13 C into the nucleic acids of growing cells (Borneman 1999). Fur-
thermore, 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 en-
dophytic 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 ergos-
terol (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 endo-
phytic taxa. Real-time PCR (Vaitilingom et al. 1998; Schena et al. 2004) is
presumably the most accurate method for quantifying microbial colonisa-
tion 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 sterilisa-
tion, vacuum and pressure extraction, cultivation independent (molecular)
methods and cultivation dependent (isolation and morphological) identifi-
cation, 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 Microbial Interactions with Plants:
a Hidden World?
Guido V. Bloemberg, Margarita M. Camacho Carvajal
18.1
Introduction
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 essen-
tial 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 (includ-
ing 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 dis-
cover 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 en-
dophytes 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, Insti-

tute 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 microorgan-
isms in the plant environment. We will briefly discuss some of the available
techniques and give examples of how these techniques have been valu-
able 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 microbiol-
ogy 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 colonisa-
tion by single bacterial cells and bacterial microcolonies of the rhizosphere
of both tomato and Arabidopsis thaliana, and to monitor gene expres-
sion (Fig. 18.1). No background β-galactosidase activity from tomato or
Arabidopsis cells was observed. In particular we focused on the differ-
ences 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 envi-
ronment. 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 Light microscopy analysis of tomato and Arabidopsis thaliana root colonisa-
tion 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 ac-
tivity, 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 environ-
ment 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 bacteria-
root 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 phase-
contrast microscopy and differential interference microscopy (DIC) to visu-
alise 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 bac-
terial 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 coloni-
sation 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 com-
pounds, including proteins, which, after excitation with light of a certain
wavelength, will emit light of a longer wavelength due to energy loss dur-
ing the process of absorption and excitation. Confocal laser scanning mi-
croscopy (CLSM) is a highly sophisticated form of fluorescence microscope.
The use of CLSM for visualising fluorescent molecules results in higher res-
olution 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 decon-
volution 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 (Invitro-
gen 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 pub-
lished 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, antibodies or probes targeted to 16S ribosomal RNA genes, the use of
autofluorescent proteins as markers does not require any preparatory steps
such as fixing with formaldehyde or ethanol, which might affect the biolog-
ical 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) species-
independent application (pro- and eukaryotes), (3) non-invasive analysis
without the need for exogenous substrates or energy, and (4) in vivo mon-
itoring while preserving the integrity of the living cells. Colour- and opti-
mised variants of GFP, such as enhanced GFP (EGFP), enhanced cyan flu-
orescent 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 rhizo-
sphere (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 legu-
minous plants (Stuurman et al. 2000), the infection process of Fusarium
oxysporum f.sp. radicis lycopersici (Lagopodi et al. 2002) and the interac-
tions 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 sur-
face preferentially at the junctions between cells, which are presumed sites
of root exudation, where they proliferate and divide, resulting in the for-
mation 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 bac-
terial exopolysaccharides, the production of which is increased in bacterial
biofilms (O’Toole et al. 2000; Sutherland 2001) and which could form a bar-
rier 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, al-
though 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 gno-
tobiotic 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 be-
tween 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 biore-
mediation 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 sur-
face 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, car-
rot roots, and mycorrhizal mycelium showed that mucus-producing mutant
strains of CHA0 can better adhere to the root, indicating that acidic extracel-
lular polysaccharides contribute to root colonisation (Biancotto et al. 2001).
Invasion of plant tissue has been extensively studied for rhizobial infec-
tion 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 fol-
low 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 bac-
terial 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 mi-
croscopes, 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 rela-
tively 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. flu-
orescens at the root tip were metabolically most active and that bacteria
from the surrounding soil population entered the rhizosphere 2 days after
seed inoculation (Lübeck et al. 2000). Another dual marker system was de-
veloped 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 deriva-
tives 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 P putida cells (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 sys-
tem 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 GFP-
based 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 sys-
tems 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 rfp on 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, sys-
tems 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 rhizo-
sphere, such as the identification of a putative ABC transporter of putrescine
(Allaway et al. 2001). Interestingly, uptake of putrescine from the rhizo-
sphere 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 fur-
ther 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 vi-
sualised by light- and electron-microscopy with the advantages and disad-
vantages of these techniques as discussed above. The use of reporters such
as lacZ or gfp requires genetic transformation, which is much more com-
plicated 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 F oxysporum f. 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 suc-
cessfully 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 chlamy-
dospores 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 coloni-
sation 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 at-
tachment 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 non-
invasive, 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 inter-
actions. Visualisation of interactions between phytopathogenic fungi and
biocontrol agents will help to understand these interactions and facili-
tate 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 fol-
lowing similarities: (1) the sites of colonisation of the tomato root surface
by Fusarium are strikingly similar to those of the Pseudomonas biocon-
trol 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 differ-
ential 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 ob-
served 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 apply-
ing the purified antifungal metabolite phenazine-1-carboxamide produced
by P. chlororaphis PCL1391.
Studying the molecular basis of the interactions between fungi and bac-
teria 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 optimisa-
tion of autofluorescent proteins as reporters are making substantial con-
tributions 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 prop-
erties 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 contribu-
tions in valuable discussions for optimising and developing microscopic
techniques, interpretation of results and technical assistance.
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19 Application of Molecular Fingerprinting
Techniques to Explore the Diversity
of Bacterial Endophytic Communities
Leo S. van Overbeek, Jim van Vuurde, Jan D. van Elsas
19.1
Introduction
Many bacteria associated with plants are able to penetrate and live as
endophytes in roots and other tissues. Like most bacteria in natural envi-
ronments, 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, us-
age of molecular techniques reveals higher species diversity than classi-
cal isolation methods. Molecular fingerprinting of bacterial endophytic
populations can expand our knowledge of populations that were previ-
ously 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 ecosys-
tems 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 bacte-
rial 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 espe-
cially 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, Wagenin-
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 phyllo-
sphere). 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 coloni-
sation, 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
bacterial species are able to become established. The plant xylem was
demonstrated to be a selective environment for endophytic bacteria such
as Acetobacter diazotrophicus (Dong et al. 1994), Bacillus pumilus (Ben-
hamou 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 condi-
tions (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 Hall-
mann)], 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 micro-
bial 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 com-
Example Reference
munity compo-
sition in plants
Other micro- Colonisation and nodulation by Rhizobium Sturz and
organisms leguminosarum in red clover was promoted by Christie 1996
endophytic isolates of Bacillus insolitus, Bacillus brevis
and Agrobacterium rhizogenes
Genotype Higher diversity in root-endophyte composition was Germida and
observed in recent versus ancient cultivars of wheat Siciliano 2001
Differences in endophyte composition and density Adams and
were observed in different cotton cultivars Kloepper 2002
Propagating Enterobacter cloacae applied to sterilised corn seeds Hinton and
material resulted in endophytic colonisation of emerging plants Bacon 1995
Optimal colonisation was obtained by inoculation Sharma and
of in vitro explants with endophytic strain Nowak 1998
Pseudomonas sp. PsJN resulting in increased resistance
against Verticillium dahliae in tomato
Gradient in disease-suppressing endophytes observed Sturz et al.
from peel to the centre of potato tubers 1999
Agricultural Crop rotation and tillage management of agricultural Peters et al.
practices fields resulted in differences in number of disease 2003
suppressive endophytes in potato
Endophytic communities in corn were influenced Seghers et al.
by herbicide, feritiliser and compost treatment of soil 2004
Localisation Non-nodular nitrogen fixing strain Gluconacetobacter Dong et al.
within plant diazotrophicus was isolated from apoplastic 1994
fluid of sugarcane
Endophytic community structure in potato differed Garbeva et al.
between the epidermis and internal stem and between 2001
lower and upper stem parts
Temperature Endophytic colonisation of tomato by strain Pillay and
Pseudomonas sp. PsJN was highest in roots and shoots Nowak 1997
at 10◦ C and lowest at 30◦ C
influencing endophytic populations. These studies make it clear that fac-

tors 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 popula-
tions in plants can shift depending on the intricate interplay of different
factors. Eventually, climax populations of endophytes will become estab-
lished.
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 struc-
ture of endophyte communities and for determining the influence of var-
ious endophyte communities on plant health and crop yield. Genes re-
sponsible 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 suppres-
sion 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 environ-
mental 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 re-

stricted 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). Multi-
ple sequence detection is crucial for molecular community fingerprinting
of environmental samples, e.g. using PCR coupled with denaturing gradi-
ent 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. com-
petitive PCR amplification of a pool of 16S rRNA gene fragments, is the
same, whereas final analyses of the fragments from environmental sam-
ples differs. The first step in all methods is extraction and purification
of total community nucleic acids. This step is critical, as high molecular
weight DNA pure enough for PCR amplification is required. Secondly, total
microbial community DNA is PCR-amplified using primers spanning frag-
ments 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 dena-

turing 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 steril-
isation 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 con-
taminants 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
method is effective in determining the absence of cultivable cells and spores
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 frag-
ile 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 ap-
plication 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 respec-
tive 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 sam-
ples (Garbeva et al. 2001; Araújo et al. 2002). Suspended cells are lysed
in SDS solution and occasionally CTAB (cetyl trimethyl ammonium bro-
mide) treatment is included to remove plant-derived exopolysaccharides
(Garbeva et al. 2001; Reiter et al. 2003b). DNA is recovered using stan-
dard 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 nu-
cleic acids for subsequent molecular fingerprinting. Commercially avail-
able 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 lim-
ited to the recovery of plant DNA. Garbeva et al. (2001) also applied an
alternative protocol, in which DNA was extracted from bacterial cells dis-
lodged 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 ap-
plied to DNA extraction from different plants (tomato, leek, chrysanthe-
mum, lettuce) followed by endophytic fingerprinting with PCR-DGGE in
our laboratories. It is advisable to optimise the nucleic acid extraction pro-
tocols for each newly studied plant species or plant part, although we found
both methods applicable to stems and roots of different plants without fur-
ther 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 sam-
ples 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 (Schmalen-
berger 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 in-
dividual 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 pos-
sess 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 mito-
chondrial 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 fix-
ation, for several important crops. These cultivable endophytic beneficials,
which will to a great extent control plant health, belong to a limited num-
ber 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), Acti-
nomyces 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 (Chain-
treuil 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 prop-
erties, 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 posi-
tive 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 finger-
print 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 impor-
tant known endophytes. For pathogen suppression, taxonomically related
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, includ-
ing Ralstonia sp., appeared to be suitable for studying R. solanacearum
and its taxonomically nearest relatives (Gomes et al. 2001). Primers cover-
ing 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 First step: Pseudomonas spp. specific Garbeva
spp. PsR: 5 -GGTCTGAGAGGATGATCAGT-3 et al. 2004
and pSf: 5 -TTAGCTCCACCTCGCGGC-3
Second step: Pseudomonas spp. specific
f968: 5 -AACGCGAAGAACCTTAC- 3 with GC clamp and PsR
First step: eubacterial Reiter et
8f: 5 -AGAGTTTGATCCTGGCTCCAG-3 and 926r: al. 2003b
5 -CCGTCAATTCCTTT(AG)AGTTT-3
Second step: Pseudomonas spp. specific
8f with CG clamp and PSMGx: 5 -ĆCTTCCTCCCAACTT-3
Burkholderia First step: Burkholderia spp. specific Salles et
spp. Burk3: 5 -CTGCGAAAGCCGGAT-3 and BurkR: al. 2002
5 -TGCCATACTCTAGCYYGC-3
Second step: Burkholderia spp. specific
Burk3 with GC clamp and r1378:
5 -CGGTGTGTACAAGGCCCGGGAACG-3
Actinomyces First step: Actinomyces spp. specific Heuer et
spp. f243: 5 -GGATGAGCCCGCGGCCTA-3 and r1378 al. 1997
Second step: eubacterial
f984: 5 -AACGCGAAGAACCTTAC-3
with GC clamp and r1378
Bacillus spp. First step: Bacillus spp. specific Garbeva
Bacf: 5 -GGGAAACCGGGGCTAATACCGGAT-3 and r1378 et al. 2003
f968 with GC clamp and r1378
α-Proteo- First step: α-proteobacteria specific Gomes et
bacteria f203 α: 5 -CCGCATACGCCCTACGGGGGAAAGATTTAT-3 al. 2001
and r1494: 5 -CTACGG(T/C)TACCTTGTTACGAC-3
β-Proteo- First step: β-proteobacteria specific Gomes et
bacteria f203 β: 5 -CGCACAAGCGGTGGATGA-3 and r1494 al. 2001
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. Subse-
quent sequencing and phylogenetic analysis theoretically enables identifi-
cation, provided that the sequences found correspond to known sequences
in the databanks. From the sequence information, new probes can be con-
structed 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 chro-
mosome (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 inci-
dence 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 agro-
chemical 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 popula-
tions. 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 ac-
tivity staining, cultivation and PCR-DGGE to isolate and identify cultivable
and non-cultivable bacteria associated with storage and vascular tissue of
potato tubers. Sliced potato tubers were incubated in water agar contain-
ing 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 dom-
inant 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 de-
tection techniques. The advantage of molecular fingerprinting techniques
over other techniques is the possibility to detect entire bacterial commu-
nities 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 bac-
teria and traditional culture-based endophyte research as complementary
routines.
19.7
Conclusions
Molecular fingerprinting methods are required as tools to supplement con-
ventional methods in endophyte research to study cultivable and non-
cultivable populations in plants. The intricacies of plants make these tech-
niques 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 sub-
sequent 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 sub-
stances from plant-associated microorganisms, which may be important
for the pharmaceutical and crop protection industries.
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Subject Index
Abies alba 304 Anguillospora

Abiotic stress 271 – filiformis 183
Acephala applanata 121 – longissima 181, 183
Acer spicatum 182–185 Angular leaf spot 35
Acetobacter 7 Antagonism 54, 56–59, 67, 68
– diazotrophicus 26, 338 – balanced 172
Achlya klebsiana 55 Antagonistic 53–58, 61, 64–67
Acremonium 163 Antagonistic bacteria 58
– alternatum 270, 274 Antagonistic endophytic bacteria used to
– cf. curvulum 232 control fungal pathogens. BCA
– cucurbitacearum 216 Biocontrol agent 62
– killense 159 Anthostomella aracearum 166
– pteridii 166 Antibiosis 53, 58, 59, 67
– strictum 164, 237 Antibiotic 180, 186
Acrocalymma medicaginis 210 Antifungal 57–60, 66
Actinoplanes missouriensis 59, 61 Antipteris evecta 186
Acuminatopyrone 146 Aphelandra tetragona 303
Adaptations 4 Apoplastic fluid 307
Adhesive 194 Appressorium 192, 194, 197
Aequorea victoria 326 Arabidopsis 43
Agricultural practices 25 – thaliana 96, 322
Agrobacterium 321 Arbuscular mycorrhiza 119, 209, 248, 266
– tumefaciens 23, 56, 82, 346 Arbutoid mycorrhiza 209
Agrobacterium-based Armillaria jezoensis 161
transformation 331 – mellea 160, 161, 168
Agrochemicals 117 Armillariella mellea 161
Air pollutants 118 Arthrinium sp. 163, 165
Allozyme 122 Arthrobacter 155
Alnus glutinosa 110, 181, 183–186, – ilicis 56
188, 189 Arthrobotrys 192
α-proteobacteria 346 – dactyloides 196, 198–200
Altechromones 268, 274 – ferox 192
Alternaria alternata 162, 165 – oligospora 193, 197, 198
Altitude 109 Articulospora
AMF 109, 117, 119 – antipodea 183
Amycolatopsis meditarranei 56 – atra 183
Anamorph 187 – proliferata 183
Angiopteris evecta 182, – tetracladia 183
184–186 Artificial inoculation 16
356 Subject Index
Ascochyta sp. 163, 166 – mutualistic 107

Ascomycetes 193, 208 – neutral 107, 110, 114
Ascomycota 219 – pathogenic 107, 120
Aspergillus 163 Beneficial 340
Assemblages 4 Benomyl 118
Astroloma pinifolium 212 Benzoxazinoids 145
Asymptomatic 6 β-glucoronidase 136
Aureobasidium caulivorum 166 β-proteobacteria 346
Auricularia polytricha 161 Betula
Aurofusarin 146 – papyrifera 117, 182–185
Avicennia – pendula 112, 304
– officinalis 183, 185 – platyphylla 118
Azoarcus 2, 7, 11, 96 – pubescens 212
Azospirillum 39 Bidirectional carbon transport 269
– brasilense 330 Biocides 117
Biodiversity 107, 157
B trichothecene 144 Biofilms 327
BAC 348 Biological control 6, 33, 53, 54, 57–61,
Bacillispora 64–67, 69, 191
– inflata 183 Biomass 115
Bacillus 56, 61, 66, 68, 91, 155, 321 Biotrophic 147
– amyloliquefaciens 36 BioYield 49
– aquamarinus 56 Bishop pine 118
– cereus 26, 56, 59, 68 Blue mold 37
– licheniformis 65 Bog 229, 232
– megaterium 22, 56 Bordetella pertussis toxin exporter 82
– polymyxa 94 Bracken 115
– pumilus 34, 36, 65, 338, 349 Bradyrhizobium japonicum 77
– subtilis 36, 58, 59, 65 Brassica chinensis var.
Bacteria 154 parachinensis 38
– nitrogen-fixing 155 Bremisia argentifolii 37
Bacterial 338 Burkholderia 54
Bacterial artificial chromosome 348 – cepacia 26, 65
Bacterial diversity 21 – solanacearum 61
Bacterial endophytes 90 Butenolide 146
Bacterial identification 16
Bacterial speck 39 Cadophora finlandia 211, 216
Bacterial spectrum 16 Cajanus cajan 79
Bacteroids 71 Calanthe 155
Balance of antagonisms 264 Callose 197
Balanced antagonism 7, 219, 269 Calluna vulgaris 236
Balansia 134 Calonectria kyotensis 162
Basidiomycetes 192 Campylospora
BCA 355 – chaetocladia 184
Bead beating 343 – purvula 181
Beauvericins 264 Capronia 212, 249, 253
Behaviour 107 Capsicum annuum 38
– antagonistic 107, 119 Capsular polysaccharides 73
Subject Index 357
Carbohydrates 114 Colletotrichum 163–165, 168

Carbon sink 272 – acutatum 165
Carbon transport 266 – crassipes 162–165
Catenaria anguillulae 193 – gloeosporioides 38, 163, 166
Cell organelles 345 – magna 6
Cell wall appositions 197, 270, 271 – orbiculare 36
Centrifugation 307 Colonisation 262, 263, 273
Centrospora acerina 181 Commensalism 6
Cephalanthera austinae 167 Common reed 115
Ceratobasidium 156, 166 Communities 107, 108, 110, 113–115,
Ceratocystis fagacearum 91 117, 118, 127, 329, 337
Ceratorhiza, Epulorhiza 166 Community composition 108
Cereal 116, 120 Competition 59, 115, 120
Chaetomium aureum 165 – interspecific 115, 125
– funicola 164, 265, 274 Competitive advantage 107
– homopilatum 162 Competivity 109
– subspirale 163, 164 Confocal laser scanning microscopy 325
Chaetosphaeria endophytica, Conidia 197
Colletotrichum gloeosporioides 166 Conidial traps 196
Chaetosticta cf. perforata 165 Coniothyrium sp. 166
Chaetothyriomycetidae 210, 212 Continuum 6
Chemical messengers 117 Corallorhiza 167
Chinese cabbage 112 Corn (Zea mays) 90
Chitinase 194 Cortinarius 286
Chlamydospores 118, 125, 197 Cotton 40
Chlamydosporol 146 Cronartium quercuum f. sp. fusiforme 38
Chloridium paucisporum 120, 211, 262 Crop protection 118
– virescens 165 Crotalaria juncea 78
Chloroplasts 345 Cryptocline 163, 164, 166
Christela Cryptosporiopsis 4, 163–166, 262, 264,
– dentata 182, 184–186 266, 271, 274
Chrysogine 146 – radicicola 116
Chytridiomycetes 193 Cucumber anthracnose 36
Citrus jambhiri 302 Cucumber beetles 35
Cladorrhinum foecumdissimum 274 Cucumber mosaic virus 36
Cladosporium cladosporioides 164 Cucumis sativus 302
Clavariopsis Cucurbit 210, 217
– aquatica 184 Culmorin 146
– azlanii 184 Cultivation-independent methods 311
Clavibacter michigenensis 56 Culture extracts 266
Climate 108, 109 Culture morphology 120
Climate change 108 Curtobacterium 91, 155
Clone libraries 345 – albidum 56
Clover 111, 117 – flaccumfaciens 23, 56
CO2 109 – luteum 56
Codinaea parva 162 Curvularia 164, 165, 271, 274
Coelomycete 210 – cymbopogonis 165
Coffea arabica 182, 184, 185, 186 – pallescens 166
358 Subject Index
Cyanobacteria 155 Diversity 5, 337

Cyclic β-glucans 73 – fungal 180
Cylindrocarpon 181 Diversity indices 21
– aquaticum 184 DNA 344
– destructans 159, 181 DON 144
– didymum 116, 123 Dothideomycetidae 210
Cypripedium calceolus 167 Douglas-fir (Pseudotsuga menziesii) 98
Cystopage 193 Drechmeria coniospora 193, 194
Cytogloeum sp. 166 Drechslera australensis 163
Cytokinin 269 – ellisii 163
Dryas octopetala 109, 110
Dactylaria sp. 164 DSE 1, 108, 109, 114, 117–122, 125, 126,
Dactylorhiza majalis 167 207, 209, 254, 262, 265, 266, 268, 270, 272,
Dark septate endophytes 1, 108, 109, 114, 274, 290
117–122, 125, 126, 207, 209, 254, 262, 265, DSM 207
266, 268, 270, 272, 274, 290 Dual ECM/VAM associations 286
Dark septate fungi 290
Decision-making 108 Ear rots 133
Defence metabolites 145, 271 ECM 109
Defence responses 8 Ecosystem 108, 109
Definition of fungal endophytes 134 – forest 109, 112, 113, 117, 121, 123, 124
Degenerate primers 341 – pristine 108
Deleterious 346 Ectendomycorrhiza 4, 214, 215, 263, 286
Denaturing gradient gel Ectomycorrhiza 209, 215–217, 263, 266
electrophoresis 341 – defining 286
Dendrobium moschatum 155 – intermediate associations 286
Deoxynivalenol 144 Ectorhizosphere 195
Detection 5 Egg- and female parasitic fungi 192
Detrimental 340 Eggplant 112
Deuteromycetes 192 Elm (Ulmus) 91
Diacetoxyscripenol 146 Endoparasitic fungi 192
Diaporthales 210 Endophyte 1
Diaporthe 210, 218 – dark septate 158
Differential interference – roles 282
microscopy 324 Endophytic
Differential labelling 329 – definition 281
Dikaryomycota 180 Endophytic phase 283
Diplazium Enniatins 146
– esculentum 182, 185, 186 Enterobacter 91
Discosoma 326 – amnigenus 27
Disease 179 – asburiae 24, 40
Disease incidence 61–63 – cloacae 27
Disease severity 61–63 Environment 109, 339
Distinguish endophytic from mycorrhizal – alpine 109, 119–121
associations 285 – arctic 109
Disturbances 108, 117 – temperate 109, 118, 120, 121
– anthropogenic 108, 117, 125 – tropical 109
– natural 108, 117, 121 Environmental factors 107
Subject Index 359
Epacrid mycorrhiza 212 Flavobacterium 155

Epacridaceae 247 Flavonoids 72
Epacris pulchella 250 Flemingia congesta 81
Epichloë 125, 134 Flexibacter 100
Epicoccum 169 Flooding 115
– andropogonis 162–165 Fluorescence microscopy 325
– nigrum 163, 165 Fluorescent in situ hybridisation 340
Epiphytes 154 Fomes 168
Epulorhiza, Moniliopsis 166 Fomitopsis pinicola 231
Equisetin 146 Fontanospora fusiramosa 182, 184
Ergot alkaloids 134 Food safety 27
Erica arborea 209, 254 Forest-management 117
– carnea 109, 110 Foundations 124
Ericaceae 212, 227 Founder genome 125
Ericales 234 Fumonisins 144
Ericoid mycorrhiza 4, 209, 212, Fungal plant pathogen 58
262, 290 Fungi
Erwinia carotovora 24 – ancestral 180, 187
– herbicola 330 – Ascomycetes 169
– persicinus 56 – Basidiomycetes 169
– tracheiphila 34 – biomass 182
Erythromyces crocicreas 161 – clavicipitaceous 179
Ethanol 301 – Coelomycetes 169
European beech 116 – Deuteromycetes 169
Exoenzymes 7 – foliicolous 187
Exploitation 6 – Hyphomycetes 169
Exploitive 1 – lignicolous 187
Extinctions 124 – mycorrhizal 153
Extracellular enzymes 255 – phyllosphere 179
Extracellular polysaccharides 73 Fungi in roots
Extraction 341 – identification 291
Fungicide 118, 172
Fagus sylvatica 116, 304 Fusamarin 146
Favolaschia thwaitesii 159 Fusaric acid 145
Fen 229 Fusarium 116, 133, 163, 167, 181,
Ferns 183 262–264, 270, 271, 287
Fertilisation 25 – culmorum 140
Fertilisers 108, 117, 118 – graminearum 133, 139, 140, 144,
Filosporella 182, 184 145, 147
– fistucella 184 – moniliforme 4, 133, 268, 274, 276
– versimorpha 184 – nivale 140
Fingerprinting 337 – oxysporum 140, 160, 162, 163, 166
Fire 211 – oxysporum f. sp. melonis 140
Flagella assembly 77 – oxysporum f. sp. pisi 41
Flagellospora – oxysporum f. sp. radicis lycopersici
– curvula 184 41, 324
– fusarioides 184 – oxysporum f. sp. vasinfectum 40
– penicillioides 184 – sambucinum 164
360 Subject Index
– verticillioides 4, 7, 133, 136, 139, 140, Heliscus lugdunensis 184, 187

144, 145, 147 Helotiales 212, 253
Fusarium wilt 40 Hemibiotrophic 138, 147
Fusarochromanone 146 Herbaspirillum 321
Fusiform rust 38 – seropedicae 338
Fusoproliferatum 146 Herbicide 180
Herbivores 107, 179
Gaeumannomyces graminis 116, 120, 198 Heterobasidion annosum 230, 267
Ganoderma 168 Heteroconium chaetospira 4, 13, 262,
– australe 161 270, 274
Gap formation 118 Hevea
Gaultheria shallon 212, 237, 249, 250, 260 – brasiliensis 182, 185
GC clamp 342 Hexalectris spicata 167
Gelatinosporium spp. 166 Hirsutella rhossiliensis 198–200
Genetic diversity 121, 122, 125 Histology 262
Geniculospora 184 Hohenbuehelia 192, 198
Genotype 339 Holm oak 209
Geographical isolation 124 Hordeum vulgare 303
Geography 22, 108 Horizontal transmission 5
Geotrichopsis sp. 164 Host adaptation 268
Germination Host defence 116, 270
– seed 156 Host preference 5, 116, 117
Gibberella fujikuroi 134
Host specificity 116
Glaciations 124
HrpF 81
Gliocladium roseum 166
Human pathogens 26, 346
Globodera pallida 194
Humicola 163, 164
Glomerella cingulata 162, 163, 165, 166
– fuscoatra 159
Glomeromycota 180
Hyaline hyphae 263
– endophytic phases 283
Hydrogen peroxide 301
Gluconacetobacter diazotrophicus 338
Hymenochaete crocicreas 161
Glume blotch 116
Hymenoscyphus ericae 212, 216, 249,
Gossypium hirsutum 302
253, 258, 290
Grass 120, 125
Hyphal coils 192
Green fluorescent protein 326
Hyphomycetes 115
Green kuang futsoi 38
– aquatic 115, 180
Gremmeniella abietina 121
Hypoxylon cf. unitum 163–165
Group-specific PCR 346
Growth enhancement 266, 274
Growth stage 339 IAA 267
Guignardia 162, 163, 170 Immunogold labeling 40
Gymnascella dankalienses 236 Index of association 123
Gyoerffyella 188 Indole acetic acid 267
Induced resistance 6, 25, 33, 269
Hadrotrichum 162–165, 169 Infection asymptomatic 179
Hair roots 247 Infection threads 71
Halep pine 209 Inhibition 117
Hartig net 119 Insect pests 107
Heavy metals 113, 118 Interaction 24, 108, 115, 116, 126
Helicobacter pylori 172 – among microorganisms 108
Subject Index 361
– host-endophyte 180 Lotus japonicus 78

– hyphal 116 Loweporus tephroporus 161
– multitrophic 108, 115 Lucerne 211
Inter-glacial periods 124 Lunulospora curvula 184
ITS (Internal transcribed spacer) Lycoperdon perlatum 161
120–122, 208, 220 Lycopersicon esculentum 303
ISSR (Inter-simple sequence repeat) PCR Lyophyllum shimeji 161
122, 123, 125, 251 Lysis 53, 58, 59
Interspecific hybrids 125
Isolation procedures 299 Maceration 306
Isozyme 121 Macrothelypteris torresiana 182, 185, 186
ITS-RFLP 209 Maize 111
ITS-RFLP analysis 251 Maize take-all fungi 120
Malbranchea sp. 163
Kaskaskia sp. 166
Mangifera indica 182–186
Kingella kingae 56
Mangrove 115
Kitasatosporia cystargenia 56
Marasmius coniatus 161
Klebsiella pneumoniae 338
Massarina aquatica 186
Kobresia myosuroides 212
– walkeri 210
Lactic acid bacteria 346 Mechanical defence reactions 2
Larix decidua 262 Media 309
Lasiodiplodia 169 Medicago sativa 302
– theobromae 162–166 Mediterranean ecosystems 208
Lasmeniella sp. 163 Mediterranean heather 209
Late blight 37 Melanconium sp. 166
Latent pathogen 2, 218 Melanotus alpiniae 164
Leafage killers 117 Meloidogyne incognita 24, 196, 264
Lecanicillium lecanii 192 Melon 112
Lentinula edodes 161 Mercuric chloride 301
Lenzites betulinus 161 Mesorhizobium loti 77
Leotiales 208 Metabolic activity 330
Lepanthes 170 Metabolite 109, 114, 116, 117, 121, 264
Leptodontidium 4 – novel 187
– orchidicola 11, 159, 160, 171, 211, 262 – inhibitory 116
Leptosphaerulina australis 164, 165 – secondary 109, 117, 121, 125, 180
Leucaena leucocephala 76 Metagenome 348
Life history strategies 1 Methodology 21
Lignin 270 Microascus cinereus 166
Lipo-polysaccharides 73 Micrococcus varians 56
Live oak (Quercus fusiformis) 91 Microcyclus sp. 166
Loam 114 Microdochium bolleyi 114, 116, 120
Loblolly pine 38 Microporus affinus 161
Lodgepole pine (Pinus contorta Dougl. var. Microsclerotia 118, 119, 125, 266
latifolia) 90 Microspermy 241
Logging 108 Minerals 114
Lolium perenne 118, 120, 303 Mitochondria 345
Long cayenne pepper 38 MLH 123–125
Lophiostomataceae 210 Molecular techniques 337
362 Subject Index
Monacrosporium ellipsosporum 196 Nectria alata 162, 165

– haptotylum 193 – haematococca 162–165
Moniliopsis 166 – ochroleuca 162, 163, 165
Monosporascus cannonballus 216 – peziza 164
Monotropes 238 – radicicola 165
Morchella 286 Nematoctonus 192, 198
Morphospecies 157 – pachysporus 198, 199
Mortierella elongata 270, 274 – robustus 198–200
MRA 119 Nematode 117
Mucor 164, 165 – entomopathogenic 117
Multilocus haplotypes 123, 124 Nematode-trapping fungi 192
Musa acuminata 302 Nematophagous fungi 6, 191
Mutation 125 Neoplaconema napelli 165
– rate 109, 116, 125 Neottia nidus-avis 167
– somatic 125 Neotyphodium 134
Mutualism 6, 155 Nicotiana tabacum 81
Mutualistic root symbioses 261 Nigrospora sphaerica 164
Mutualistic symbionts 338 Nitrogen 118
Mycelium radicis atrovirens 119, 166, 290 – fixation 90, 155
Mycena orchidicola 159, 171 – uptake 265
– osmundicola 161 Nitrogen-fixing bacteria 25
– thuja 160 Nivalenol 146
Mycobacterium 155 NOx 118
Mycobiont 235 Nocardia 155
Mycocentrospora 185 nod-box 72
– acerina 181 NodD 72
– clavata 185 Nod-factors 72
– iqbalii 185 NodO 75
Mycoheterotroph 4, 238 Nodulisporium 162, 163, 165, 166
Mycoparasitism 195 – gregarium 166
Mycorrhiza 116, 117, 153 Non-cultivable 311, 341
– arbuscular 109, 119 Non-invasive 326
– definition 281 NopL 81
– ecto- 109 NopX 80
– ericoid 229 Norway spruce 114, 121–124
– evolution 292 Nostoc 155
– orchid 238 Nucleic acids 344
Mycorrhizal fungi 1, 25
– designation 292 Ochrobactrum anthropi 27
– endophytic phase 283 Oidiodendron 249
– inoculum 283 – cerealis 236
– roles 282 – chlamydosporicum 236
– structures 262 – citrinum 236
Mycorrhizin 264 – flavum 236
Mycotoxin 7, 143, 144, 268, 275 – griseum 236
Myriogenospora 134 – maius 2, 4, 227, 262, 266,
Myxotrichaceae 227 274, 290
Myxotrichum setosum 236 – periconioides 236
Subject Index 363
– rhodogenum 236 – cf. heterocornis 163, 164

– scytaloides 232, 236 – poppola 163
Oliveonia 166 Pestalotiopsis 171
Optical trapping microscopy 331 – aquatica 163, 165
Optimum carrying capacity 16 – gracilis 163
Orbilia 193 – papposa 162–164
Orchid 4, 153, 216 Pesticides 108
Orchid mycorrhizas 287 Pezicula 8, 11, 13
– designation 287 Pezizales 208
Orchidaceae 153 Pezizella ericae 236
– evolution 293 pH 110, 114
Ordination 179 Phaeocryptopus gaeumannii 313
Organic amendment 26 Phaeoseptoria cf. vermiformis 163
Organicization 231 Phalangispora constricta 185
Oryza sativa 304 Phase-contrast microscopy 324
Oscillatoria 155 Phaseolus vulgaris 303
Oxytropis campestris 303 Phellinus 168
Phenolic metabolites 271
Phenotypic plasticity 263
Pachyrhizus tuberosus 78 Phenylpropanoids 271
Paecilomyces 164, 165 Pheromones 117
Paenibacillus 93 Phialospora sp. 166
– polymyxa 96 Phialocephala 158, 211, 262
Pantoea agglomerans 56, 349 – compacta 121
– anantis 56 – dimorphospora 121
Papillae 197 – fortinii 2, 11, 108, 109, 114–126, 159,
Parasitic 1 211, 214, 217, 240, 250, 252, 254, 262, 266,
Parasponia andersonii 71 270, 274, 290
Pathogen 53–55, 57–60, 67, 107, 116, – scopiformis 121
121, 179 Phialophora 120, 211, 262
PCR 121–123, 157 – finlandica 120, 249
PCR-DGGE 341 – graminicola 120
PCR-single strand conformational – radicicola 120
polymorphism (SSCP) 341 – zeicola 120
PCR-TGGE 341 Phloem 338
PCR-T-RFLP 341 Phoma 163, 164, 166, 265
Pea 41 Phomatospora berkeleyi 166
Peat 227 Phomopsis 163, 169, 210, 218
Peatlands 227 – cf. orchidophila 162–166
Pelotons 154 Phosphorus 114, 265, 266, 269
Penicillium 163, 170 Photosynthates 109
– thomii 232 Phragmites australis 111, 115
Periconia macrospinosa 114 Phyllobacterium 93
Periconiella sp. 162, 165 – rubiacearum 61
Peronospora parasitica 43 Phyllosticta capitalensis 166
– tabacina 37 – colocasiicola 166
Pestalotia 163, 164, 169 Physiological relationships 143
– adusta 166 Phytoalexin 171
364 Subject Index
Phytohormones 7, 267, 268, 273 Population densities 15

Phytophthora cactorum 55 Population genetics 119, 122
– cinnamomi 230 Populations 108, 121, 123, 338
– infestans 37 Populus hybrida 182–186
Phytoplasms 338 Potato (Solanum tuberosum) 90, 111, 117
Phytosphere 348 Pratylenchus 201
Phytosterol 270 Preceding crop 117
Picea abies 110, 114, 119, 121–124, Pressure bomb method 21, 309
303, 304 Pressure extraction 308
– glauca 118, 182–186 Primers 341
Piceirhiza bicolorata 212 Proteolytic enzymes 194
Pili 77 PR-proteins 81
Pinus contorta 302 Pseudallescheria boydii 165
– halepensis 209 Pseudoanguillospora 185
– muricata 118 Pseudogymnoascus roseus 236
– sylvestris 112, 116, 119, 212, 216, 304 Pseudomonas 54, 56, 60, 66–69, 91,
– taeda 38 155, 321
Piriformospora indica 4, 13, 201, 262, – chlororaphis 61, 94
267, 272 – chlororaphis PCL1391 327
Pisum sativum 75 – cichorii 23, 56
Pithomyces maydicus 162–165 – corrugata 56
Plant and Fungus Interactions 134 – denitrificans 91
Plant defence mechanisms 25 – fluorescens 22, 34, 56, 61, 65, 66,
Plant disease 65, 67 68, 146
Plant genotype 23 – fluorescens CHA0 329
Plant growth 53, 58, 60, 62, 66, 68 – fluorescens SBW25 330
Plant health 53, 345 – fluorescens WCS365 322
Plant pathogens 24 – graminis 56, 61
Plant species 22 – migulae 56
Plant symbionts 25 – orientalis 56
Plant-associated 338 – putida 22, 56, 61, 91, 330
Plant-fungus coevolution 282 – reactans 56
Plant-metagenomic approach 348 – rhodesiae 56
Plants – straminea 56
– myco-heterotrophic 154 – synthaxa 56
Plasmodiophora brassicae 270 – syringae 56, 90
Plasticity 9, 269, 273 – syringae pv. lachrymans 34
Plectosporium tabacinum 59, 61 – tolaasii 56, 61
Pleosporales 208, 210 – trivialis 61
Pleurotus djamor 198, 199 – veronii 56, 61
– ostreatus 192 Pseudomycorrhiza 119
Pochonia 193 Pseudotsuga menziesii 117
– bulbillosa 232 Psychilis kraenzlinii 170
– chlamydosporia 193, 197, 198 Psychrobacter immobilis 56
– rubescens 193 Pteridium aquilinum 115, 303
Pollution 108, 113, 118 Pterostylis 155
Polymerase chain reaction 121, 123, 340 Purification 341
Polyphasic 348 Pyrenochaeta cf. rubi-idaei 164
Subject Index 365
Pythium 268 Root-colonising 321

– oligandrum 271, 274 Roots, adventitious 155
– spinosum 55 Rosemary 209
Rosmarinus officinalis 209
Quantification 5, 313 Rot 133
Quercus ilex 209, 255 Rough lemon (Citrus jambhiri) 90
Quorum sensing 324
16S rDNA 341
Ralstonia paucula 56
Salicylic acid 34
– picketti 346
Salix babylonica 182–186
– solanacearum 38, 340
Salmonella enterica 27
Ramichloridium apiculatum 166
Saprobe 2, 228
– cf. subulatum 164
Saprophytic 266
RAPD (Random amplified polymorphic
Saprotrophic symbiont 219
DNA) 122, 214
Scanning electron microscopy 324
rDNA 208, 219
Sclerroderis canker 121
Real-time PCR 340
Recombination 122, 125 Scots pine 116
Red fluorescent protein 326 Scytalidium vaccinii 236
Reporter genes 322 Seawater 115
Resistance 116 Sebacina 156, 166, 238, 250
Restriction fragment length sec pathway 78
polymorphism 121 Secondary metabolites 264, 273
Restriction patterns 120 Sedges 120
RFLP 121, 122, 125 Seed borne 137
Rhizobacteria 34, 201 Seed treatment 118
Rhizobiaceae 321 Selective media 311
Rhizobium etli 25 Septoria nodorum 116, 118
– leguminosarum 25, 75 Sequences, ribosomal 180
– meliloti 56, 71, 76 Serendipita 166
– sp. NGR234 71 Serine protease 194
Rhizoctonia 4, 156, 166, 287 Serratia 54, 56
– solani 24, 38, 55, 59, 61, 195 – marcescens 26, 35, 65, 66, 338, 346
Rhizomonas suberifaciens 56 – plymuthica 58, 61, 66, 67
Rhizophora mucronata 110, 115, 183, 186 Sink 269, 270, 272
Rhizoplane 107 Sinorhizobium meliloti 329
Rhizopycnis vagum 210 Ski slopes 118
Rhizoscyphus ericae 231 SO2 118
Rhizosphere 107, 114, 117, 125, 322 Sodium hypochlorite 301
Rhodococcus 155 Soil suppressiveness 202
Ribosomal 340 Soil texture 114
Ribosomal RNA 120 Soil-rhizosphere-root continuum 107
Rice (Oryza sativa) 90, 120 Solanum tuberosum 90, 111, 117, 302
– crown sheath rot 120 Sonification 300
RNA 343 Sonneratia caseolaris 183, 186
rRNA 120, 121 Sordaria fimicola 159
Root endophytes, multiple roles 290 Sordariomycetidae 210
Root nodules 71 Spanish oak (Quercus texana) 91
Root turnover 109 Spatiotemporal patterns 108
366 Subject Index
Speciation 123, 125, 126 Surfactants 305

– allopatric 123, 125 Symbiont 235
– sympatric 123 Symbiosis 1
Species 110–113, 345 Sympatric occurrence 123
– abundance 108, 115, 121 Symptomless root infections 139
– competitive 107, 116, 118 Systemic acquired resistance 33
– cryptic 123–126 Systemic fungal root infections 269
– diversity 108–110, 114–119, 121–126 Systemic resistance 271
– evenness 108
– identification 120 T-2 toxins 146
– richness 108, 109, 114, 115, 117, 118 Take-all fungus 198
– spectrum 108, 109, 115–117 Taxon 344
– typing 120 Tecomella undulata (Bignoniaceae) 90
Specificity 5, 158 Teleomorph 181, 186, 187
Sphagnum 229 Temperature gradient gel
Sphigobacterium thalophilum 56 electrophoresis 341
Sphingomonas 91 Tephrosia vogelii 78
– adhaesiva 56 Terminal restriction fragment length
– trueperi 61 polymorphism 341
Spore production 182 Tetrabrachium
Sporormia minima 160 – elegans 185
Spruce 229 Tetracladium 185
Stagonospora 263, 265, 274 – furcatum 185
Staphylococcus xylosus 27 – marchalianum 181, 185
Stem rot 133 – setigerum 185
Stenotrophomonas 91 Tetrazolium 348
– maltophilia 26, 56 Thailand 38
Sterilising agents 301 Thanatephorus 156, 166
Sterility check 300, 305 – gardneri 287
Stimulation 117 Thermomyces verrucosus 159
Strain mixtures 38 Thielavia 249, 253
Strawberry 112 – basicola 160
Streptomyces 93, 321 Thuja occidentalis 117
– bottropensis 56 Tidal level 115
– diastatochromogenes 56 Tillandsia 155
– galilaeus 56 Tissue type 339
– griseus 56 Tobacco 36
– lavendulae 56 Tolumnia variegata 169
– scabies 55 Tomato 36, 37, 112, 210
– setonii 56 Tomato mottle virus 37
– turgidiscabies 56 Total community nucleic acids 341
Stress tolerance 274 Total microflora 324
Stylopage 193 Toxicity 179
Succession 122, 124 Toxin-producing fungi 192
Sugar beet 111 Toxins 264
Sugarcane Saccharum officinarum 90 Trametes hirsuta 161
Suppression 346 Trapping organs 192
Surface sterilisation 5, 300 Tricellula aquatica 185
Subject Index 367
Trichocladium opacum 160 Vitis vinifera 329

Trichoderma 163, 168, 195 Volatile organic compounds 44
Tricholoma 286 Vomitoxin 144
Trichosporiella multisporum 159
Tricladium chaetocladium 185 Water regime 114, 115
– splendens 181 Weather 109
Triscelophorus Weevil larvae 117
– acuminatus 185 Westerdykella 270, 274
– konajensis 186 Western red cedar (Thuja plicata) 100
– monosporus 186 Wetland 115
Triticum aestivum 111, 114, 117, 302 Wheat (Triticum aestivum) 96, 114,
Troposporella sp. 162–164 116–118
Tsuga 212 White x Engelmann hybrid spruce (Picea
Tubercularia 164 glauca x P. enelmannii 92
Tuberculate mycorrhizae 98 Whitefly 37
Tulasnella 156, 166 Wildfire 36, 118
Tumularia aquatica 186 Windthrow 112, 117, 118
Type 1 121 Woodland 115
Type I secretion systems 73 Woollsia pungens 212, 249, 251, 253,
Type II secretion systems 77 258–260
Wortmannin 146
Urban development 108 Wullschlaegelia calcarata 168
Xanthomonas 155
Vaccinium corymbosum 253 – campestris 55, 56
– macrocarpon 253 – campestris pv. vesicatoria 81
– myrtillus 216 – oryzae 56
Vacuum 308 Xylaria 162, 163, 164, 168
Valsaceae 210 – arbuscula 163
Vanilla 171 – cf. cubensis 163
Varicosporium 188 – cf. curta 163
– elodeae 186 – corniformis 163
– giganteum 186 – enteroleuca 163
Vascular cylinder 2 – hypoxylon 163
Vascular tissue 309 – mellisii 163
Velamen 154 – multiplex 163
Verticillium 193, 287 – obovata 163
– chlamydosporium 264, 275 – polymorpha 163
– dahliae 55, 59 Xylella fastidiosa 313, 329
– lecanii 166 Xylem 338
– longisporum 24, 55, 270
Vicia sativa 75 Yield shield 44
Vigna unguiculata 78 Ypsilonidium 166
Vine decline 210, 217
Virulence factors 7, 264 Zea mays 302
Virulent pathogens 2 Zearalenone 144
Visoltricin 146 Zygomycetes 193

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Soil Biology

Series Editor: Ajit Varma 9

Dr. Thomas N. Sieber

Library of Congress Control Number: 2005938057

Braunschweig, Barbara Schulz

1 What are Endophytes? 1

Part I Endophytic Bacteria

3 Bacterial Endophytes as Elicitors of Induced Systemic Resistance 33

4 Control of Plant Pathogenic Fungi with Bacterial Endophytes 53

5 Role of Proteins Secreted by Rhizobia in Symbiotic

6 Research on Endophytic Bacteria: Recent Advances

Part II Endophytic Fungi

8 Endophytic Root Colonization by Fusarium Species:

9 Microbial Endophytes of Orchid Roots 153

10 Fungal Endophytes in Submerged Roots 179

11 Nematophagous Fungi as Root Endophytes 191

12 Molecular Diversity and Ecological Roles of Mycorrhiza-Associated

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

13 Oidiodendron maius: Saprobe in Sphagnum Peat,

14 Mycorrhizal and Endophytic Fungi of Epacrids (Ericaceae) 247

15 Mutualistic Interactions with Fungal Root Endophytes 261

16 Understanding the Roles of Multifunctional Mycorrhizal

Part III Methods

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

18 Microbial Interactions with Plants: a Hidden World? 321

19 Application of Molecular Fingerprinting Techniques

Subject Index 355

Cairney, John W.G.

Carvajal, Margarita M. Camacho

Elsas, Jan D. van

Luppi, Anna Maria

Overbeek, Leo S. van

Vicente, José Gaspar Maciá

Vuurde, Jim van

IAA indole acetic acid

Soil Biology, Volume 9

Criteria Bacteria Fungi

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

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

latent pathogens. The majority can produce phytotoxic metabolites (Schulz

tors, nutritional status, as well as the developmental stages of the part-

Marler M, Pedersen D, Mitchell OT, Callaway RM (1999) A polymerase chain reaction

Tudzynski B (1997) Fungal phytohormones in pathogenic and mutualistic associations In:

Soil Biology, Volume 9

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

Plant Bacterial taxa Reference

Table 2.1. (continued)

Canola Acidovorax, Agrobacterium, Aureobacterium, Germida et al. 1998;

Table 2.2. Endophytic bacteria isolated from roots of various plants

Acetobacter diazotrophicus13 , A. pasteurianus9

Table 2.2. (continued)

Bordetella avium14 , B. bronchiseptica8

Table 2.2. (continued)

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

As endophytic bacteria represent a subset of the rhizosphere colonisers,

their susceptibility to the fungal wilt pathogen Verticillium longisporum.

Eberl L (1999) N-acyl homoserinelactone-mediated gene regulation in Gram-negative bac-

Lalande R, Bissonnette N, Coutlée D, Antoun H (1989) Identiﬁcation of rhizobacteria from

Soil Biology, Volume 9

teria or other nonpathogenic microorganisms, while SAR is elicited by

enzyme-linked immunosorbent assay (ELISA), and decreased virus accu-

planting elicited signiﬁcant reductions in disease severity when P. infestans

reduced the severity of Fusarium wilt of cotton. In this system, 89B-61