Symbiotic Fungi (eBook)

Foreword 6
References 7
Preface 9
Contents 11
Contributors 14
Symbiosis: The Art of Living 21
1.1 Introduction 21
1.2 History of Symbiosis 22
1.3 Symbiosis of Bacteria 1.3.1 Symbiotic Association of Bacteria with Leguminous Plants 24
1.3.2 Symbiotic Association of Bacteria with Nonleguminous Plants 26
1.3.3 Establishment of the Mutualistic Relationship Between Rhizobia and Legumes 26
1.4 Symbiosis of Actinomycetes 32
1.5 Symbiosis Between Blue–Green Alga and Fungus: Geosiphon pyriforme 33
1.6 Symbiosis Between Algae and Fungi: Lichens 35
1.7 Symbiosis in Bryophytes 35
1.8 Symbiosis in Pteridophytes 36
1.9 Symbiosis in Gymnosperms 36
1.9.1 Coralloid Roots of Cycas 37
1.10 Symbiosis in Angiosperms 37
1.10.1 Mycorrhizal Symbiosis 37
1.11 Conclusions 45
References 45
Analysis of Rhizosphere Fungal Communities Using rRNA and rDNA 49
2.1 Introduction 49
2.2 Materials 2.2.1 Equipment 50
2.2.2 Materials 51
2.2.3 Procedure 51
2.3 Results 2.3.1 Nucleic Acid Extraction and Reverse Transcription from the Andropogon gerardii Rhizosphere 54
2.3.2 Community Assessment Using Reverse-Transcribed cDNAs and Environmental rDNAs 55
2.4 Conclusions 58
References 59
Use of Mycorrhiza Bioassays in Ecological Studies 61
3.1 Introduction 61
3.2 Assessment of Infectivity of AM Fungi in Soil 62
3.2.1 Direct Assessment of AM Fungi 62
3.2.2 Indirect Assessment: Mycorrhiza Bioassay 64
3.3 Purpose of Bioassays for Infectivity Assessment of AM Fungi 66
3.4 Conclusions 67
References 67
In Vivo Model Systems for Visualisation, Quantification and Experimental Studies of Intact Arbuscular Mycorrhizal Networks 71
4.1 Introduction 71
4.2 Structure of Pre-symbiotic Mycelium of AM Fungi: Visualisation and Quantification of Anastomosis 4.2.1 Occurrence and Frequency of Anastomosis 72
4.2.2 Dynamics of Anastomosis Formation in Living Hyphae 73
4.2.3 Cytochemical Analyses of Anastomosing Hyphae 75
4.2.4 Remarks 76
4.3 Visualisation and Quantification of Intact Mycelial Networks Spreading from Mycorrhizal Roots 4.3.1 Experimental System 76
4.3.2 Quantification of the Extent and Structure of the Mycorrhizal Network 78
4.3.3 Viability of the Mycorrhizal Network 79
4.3.4 Remarks 79
4.4 Visualisation of Belowground Links Between Plants of Different Species, Genera and Families 4.4.1 Experimental System 80
4.4.2 Occurrence and Frequency of Anastomoses Within and Between Mycorrhizal Networks 80
4.4.3 Remarks 82
4.5 Conclusions 82
References 83
Measurement of Net Ion Fluxes Using Ion- Selective Microelectrodes at the Surface of Ectomycorrhizal Roots 85
5.1 Introduction 85
5.2 Principle of Ion Activities Measurement with Ion- Selective Microelectrodes 5.2.1 What Is an Ion- Selective Microelectrode? 86
5.2.2 Expression of the Voltage Difference Across the Cocktail 87
5.3 Principle of Flux Measurement 5.3.1 Expression of Local Diffusion Flux in a Solution 88
5.3.2 Estimation of Ion Fluxes at the Root Surface 89
5.4 Equipment and Microelectrode Fabrication 5.4.1 Experimental Set- up 90
5.4.2 Making the Microelectrodes 93
5.4.3 Internal Silanization of the Pulled Borosilicate Tube or Microelectrode 93
5.4.4 Backfilling the Pipette 94
5.4.5 Calibration 95
5.4.6 Microelectrode Selectivity 96
5.5 Setting up the Electrophysiological Measurements 96
5.5.1 Determination of the Distances from the Root Surface for the Measurement Points 97
5.5.2 Factors Affecting the Calculation of the Concentration 99
5.5.3 Using a Complex Perfusing Solution 102
5.6 A Case Study: Measurement of NO3 103
Net Fluxes 103
into Ectomycorrhizal Short Roots 5.6.1 Validation of Flux Measurements into Coniferous Plants 103
5.6.2 Variation of NO3 105
Net Fluxes into Ectomycorrhizal Short 105
Roots: Effect of N Source Supplied to the Plants 105
5.7 Conclusions 106
References 107
Assessment of Phosphatase Activity Associated with Mycorrhizal Fungi by Epi- Fluorescent Microscopy 109
6.1 Introduction 109
6.2 Phosphatase Activity 110
6.3 Equipment and Laboratory Material 6.3.1 Equipment 110
6.3.2 Laboratory Material 110
6.3.3 Solutions 111
6.3.4 Protocol for Assessment of Phosphatase Activity with ELF- 97 Substrate 112
6.4 Various Applications of ELF-97 Substrate in Mycorrhizal Research 115
6.4.1 Phosphatase Activity Associated with Ectomycorrhizal Fungi 115
6.4.2 Phosphatase Activity Associated with Arbuscular Mycorrhizal Fungi 115
6.4.3 Other Applications of the ELF-97 Substrate 116
6.5 Advantages and Limitation of ELF-97 Substrate 117
6.6 Conclusions 118
References 118
In Vitro Compartmented Systems to Study Transport in Arbuscular Mycorrhizal Symbiosis 120
7.1 Introduction 120
7.2 Equipment and Laboratory Material 7.2.1 Equipment 121
7.2.2 Laboratory Material 121
7.3 Culture Media 7.3.1 Composition 122
7.3.2 Stock Solutions 123
7.3.3 Preparation of Culture Media 123
7.4 Transport Studies with Root-Organ Cultures (ROC) 124
7.4.1 Adding Medium to the Petri Plates 125
7.4.2 Selection of a Root-Organ 126
7.4.3 Inoculation of the Root with AM Fungal Propagules 127
7.5 Transport Studies with Autotrophic Plants 128
7.5.1 Surface-Sterilization/Scarification 129
7.5.2 Medium 130
7.5.3 The Half-Closed Arbuscular MycorrhizalÒPlant ( HAMÒ P) In Vitro Culture System ( Voets et al. 2005) 130
7.5.4 The Arbuscular MycorrhizalÒ Plant (AMÒP) In Vitro Culture System ( Dupre • de Boulois et al. 2006) 132
7.6 Labelling with Isotopic Tracers 135
7.6.1 Stable or Radio-Isotopic Tracer? 136
7.6.2 Adding the Isotopic Tracer 137
7.6.3 Exposure Time 137
7.6.4 Harvest 139
7.7 Conclusions 139
References 139
Use of the Autofluorescence Properties of AM Fungi for AM Assessment and Handling 142
8.1 Introduction 142
8.2 Equipment and Laboratory Material 8.2.1 Equipment 143
8.2.2 Laboratory Material 143
8.3 Sample Preparation 8.3.1 Whole Root Samples 144
8.3.2 Root Section Samples 144
8.3.3 Isolation of Intraradical Fungal Structures 145
8.3.4 Spores 146
8.4 Bright-Field Microscopy, Epifluorescence Microscopy and Lambda- Scan 146
8.5 Autofluorescence of AM Fungal Structures 149
8.6 Viability of Fungal Structures 153
8.7 Autofluorescence Localization 155
8.8 Spore Autofluorescence and Flow Cytometry 156
8.9 Conclusions 158
References 158
Role of Root Exudates and Rhizosphere Microflora in the Arbuscular Mycorrhizal Fungi- Mediated Biocontrol of Phytophthora nicotianae in Tomato 160
9.1 Introduction 160
9.2 Zoospore Chemotaxy in P. nicotianae 161
9.3 Biocontrol Mediated by AMF on P. nicotianae Infecting Tomato 162
9.4 Induction of Tomato Plant Defense Mechanisms Following Mycorrhizal Colonization 162
9.5 Effect of Mycorrhizal Root Exudates on P. nicotianae Zoospore Chemotaxy In Vitro 163
9.6 Effect of Mycorrhizal Root Exudates on Tomato Infection by P. nicotianae in Soil 165
9.7 Effect of Mycorrhizal Root Exudates on the Rhizosphere Bacterial Community Structure 166
9.7.1 Antagonistic Potential of Bacteria Associated with Spores of G. mosseae 168
9.7.2 Other Mechanisms Contributing to the Biocontrol Induced by AMF on P. nicotianae 169
9.8 Conclusions 170
References 170
Assessing the Mycorrhizal Diversity of Soils and Identification of Fungus Fruiting Bodies and Axenic Cultures 178
10.1 Introduction 178
10.2 General Characteristics of Mycorrhizae 179
10.3 Classical Fungal Processing and Identification 10.3.1 Field Notes, Processing, Fungal Identification 179
10.3.2 Herbarium Facilities 181
10.4 Isolation of Fungal Cultures from Soils, Mycorrhizosphere and Sporophores 10.4.1 Preparations 183
10.4.2 Isolation from Fruiting Bodies and Spores 183
10.4.3 Indirect Methods for Screening 184
10.4.4 Soil Dilution Technique for Enumeration of Fungi 184
10.4.5 Direct Plate Technique 186
10.4.6 WetÒSieving and Decanting Technique for the Extraction of Spores of AMF ( Fig. 10.4) 186
10.4.7 Techniques for Large Volumes of Soil 187
10.5 Preservation and Maintenance 10.5.1 Culture Collections 188
10.5.2 Cultures in Herbaria 189
10.6 Modern Molecular Methods Used in Fungal Identification 189
10.6.1 ÎÎExtraction-FreeÌÌ Preparation of PCR-Ready Material 192
10.6.2 PEX Extraction 192
10.6.3 Choice of PCR Target 197
10.6.4 PCR Troubleshoot 198
10.6.5 Sequencing and Editing Sequences 199
10.6.6 Cloning 200
10.6.7 Database Queries and Alignment 201
10.6.8 Phylogenetic Placement 202
10.7 Conclusions 203
References 203
Isolation of Metabolically Active Arbuscules and Intraradical Hyphae from Mycorrhizal Roots 208
11.1 Introduction 208
11.2 Isolation 11.2.1 Isolation of Arbuscules and Intraradical Hyphae with Enzymatic Digestion 209
11.2.2 Isolation of Arbuscules and Intraradical Hyphae without Enzymatic Digestion 211
11.2.3 Measurement of Metabolic Activity of Isolated Arbuscules and Hyphae 211
11.3 Conclusions 213
References 213
Interaction with Soil Microorganisms 215
12.1 Introduction 215
12.2 Fungal Growth Promotion in Bacterium–Fungus Co- Cultures: An Indication of Mycorrhiza Helper Function 216
12.3 Rapid Fungal Responses to Bacteria and Their Metabolites: Bacterium– Fungus Suspension Cultures 218
12.4 Materials 12.4.1 Reagents for Culture 219
12.4.2 Organisms 220
12.5 Procedures 12.5.1 Culture on Solid Media 221
12.5.2 Suspension Cultures 222
12.5.3 Co-Cultures 223
12.5.4 Simple Culture System for the Inoculation of Norway Spruce Roots with Actinomycetes 224
12.5.5 Impact of Pre-Inoculation with Mycorrhization Helper Bacteria on Heterobasidion Root Rot 224
12.5.6 Impact of Volatiles of Mycorrhization Helper Bacteria on Heterobasidion Root Rot 225
12.5.7 Physiological Screening of Host Plant Viability 225
References 226
Isolation, Cultivation and In Planta Visualization of Bacterial Endophytes in Hanging Roots of Banyan Tree ( Ficus bengalensis) 229
13.1 Introduction 229
13.2 Isolation and Cultivation of Endophytes from Plant Roots 230
13.2.1 Selection of Plant Material 230
13.2.2 Isolation of Endophytic Microorganisms 230
13.2.3 Cultivationof Endophytes 232
13.3 Protocol for Isolation and Cultivation of Bacterial Endophytes from Hanging Roots of Banyan Tree 13.3.1 Requirements 232
13.3.2 Media for Cultivation 233
13.3.3 Method 233
13.4 In Planta Visualization/Localization of Endophytes 234
13.4.1 Vital Staining Method 234
13.4.2 Electron Microscopy 235
13.5 Protocol for Detection of Endophytic Bacteria by Vital Staining 235
13.5.1 Equipment 235
13.5.2 Chemicals and Reagents 236
13.5.3 Procedure 236
13.6 Protocol for Detection of Bacterial Endophytes by Transmission Electron Microscopy 237
13.6.1 Requirements 237
13.6.2 Procedure 239
13.7 Conclusions 241
References 242
Micro-PIXE Analysis for Localization and Quantification of Elements in Roots of Mycorrhizal Metal- Tolerant Plants 244
14.1 Introduction 244
14.2 Materials and Procedures 14.2.1 Equipment for Sample Preparation 245
14.2.2 Laboratory Materials 245
14.2.3 Specimen Preparation 245
14.2.4 Micro-PIXE Analysis 249
14.3 Example of Micro-PIXE Analysis 252
14.3.1 Sample Preparation and Micro-PIXE Analysis 252
14.3.2 Results 257
14.4 Conclusions 258
References 258
Functional Genomic of Arbuscular Mycorrhizal Symbiosis: Why and How Using Proteomics 260
15.1 Introduction 261
15.2 General Considerations About the Biological Material 262
15.2.1 Root Handling for Protein Extraction Protocols of 262
15.3 Materials and Apparatus 15.3.1 Products and Buffers for Protein Extraction 263
15.3.2 Products and Buffers for 2DE 264
15.3.3 Products for Protein Staining 265
15.3.4 Equipment 265
15.4 Procedure 268
15.4.1 Extraction of Total and Soluble Proteins 268
15.4.2 Combined Extraction of RNA and Proteins 269
15.4.3 Protein Extraction 270
15.4.4 Measure of Protein Content in Samples 270
15.4.5 Two-Dimensional Electrophoresis 271
15.4.6 Protein Detection 275
15.5 A Case Study Applied to Dissect the Early Stages of Medicago Truncatula Mycorrhizal Symbiosis: When Transcriptomics and Proteomics are Working Together 281
15.5.1 In Silico Analysis 282
15.5.2 Proteomic Analysis 283
15.6 Conclusions 286
15.7 New Insights 287
References 288
Using Stable Carbon Isotope Labelling in Signature Fatty Acids to Track Carbon Allocation in Arbuscular Mycorrhiza 292
16.1 Introduction 292
16.2 Microbial Biomass Distribution 293
16.3 294
C Labelling 16.3.1 Timing of Measurements 294
16.3.2 Labelling in Monoxenic AM Cultures 294
16.3.3 Labelling in Pot Experiments 295
16.3.4 Labelling in the Field 295
16.4 Analytical Techniques 16.4.1 Homogenization of Samples 296
16.4.2 Lipid Analysis 296
16.4.3 Signature Fatty Acids 296
16.4.4 Determination of 297
C Enrichment in Crude Tissue 297
Samples and Fatty Acids 297
16.4.5 Quantification of Transferred Carbon 298
16.5 Sensitivity and Specificity of the Method 298
16.6 Conclusions 299
References 300
N Enrichment Methods to Quantify Two-Way Nitrogen Transfer Between Plants Linked by Mychorrhizal Networks 302
17.1 Introduction 302
17.2 Use of 303
N Isotopes for Investigating Nitrogen 303
Transfer Between Plants 303
17.3 Experimental Design for Investigating Two-Way Nitrogen Transfer Between Plants 304
17.3.1 Quantification of Nitrogen Transfer Between Plants 305
17.4 Conclusions 307
References 307
Analyses of Ecophysiological Traits of Tropical Rain Forest Seedlings Under Arbuscular Mycorrhization: Implications in Ecological Restoration 309
18.1 Introduction 309
18.1.1 Fragmentation 309
18.1.2 Restoration 310
18.1.3 What is the Role of Arbuscular Mycorrhizal Fungi in Habitat Recovery? 310
18.2 Objective 311
18.3 Case Study 18.3.1 Study Site 312
18.3.2 Methods 313
18.3.3 Results 314
18.3.4 Remarks 315
18.4 Restoration and Arbuscular Mycorrhizae Fungi 318
18.5 Conclusions 319
References 319
Techniques for Arbuscular Mycorrhiza Inoculum Reduction 322
19.1 Introduction 322
19.2 Solarization 323
19.3 Steam Sterilization 324
19.4 Pasteurization 325
19.5 Gamma (g)-Irradiation 326
19.6 Chemicals 326
19.7 Soil Disturbance 328
19.8 Crop Rotation 329
19.9 Other Methods 329
19.10 Conclusions 330
References 331
Best Production Practice of Arbuscular Mycorrhizal Inoculum 334
20.1 Introduction 334
20.2 Working Hypotheses (Following Feldmann 1998) 335
20.3 Basic Assumptions for the Inoculum Production ( Following Feldmann and Grotkass 2002) 335
20.3.1 The Planning Phase: Define what you Need 336
20.3.2 The Analytical Phase: Make your AMF Isolate a Strain and Describe its Abilities 338
20.3.3 The Adaption Phase: Direction Instead of Screenings 341
20.3.4 The Up-Scaling Phase: One Further Step Only 342
20.4 Conclusions 347
References 350
The Use of AMF and PGPR Inoculants Singly and Combined to Promote Microplant Establishment, Growth and Health 352
21.1 Introduction 352
21.2 Mode of Action and Safety of Inoculants 354
21.2.1 Arbuscular Mycorrhizal Fungi 354
21.2.2 Plant Growth Promoting Rhizobacteria (PGPR) 355
21.3 Selection, Production and Formulation of Inoculants 357
21.3.1 Arbuscular Mycorrhizal Fungi 357
21.3.2 Plant Growth-Promoting Rhizobacteria 358
21.4 Use of Inoculants in Micropropagation 21.4.1 Arbuscular Mycorrhizal Fungi 360
21.4.2 Plant Growth-Promoting Rhizobacteria 364
21.5 Holistic Strategies for the Use of Inoculants 365
21.6 Conclusions 367
References 367
Co-Culture of Linum album Cells and Piriformospora indica for Improved Production of Phytopharmaceuticals 376
22.1 Introduction 376
22.2 Development of Plant Cell Cultures 22.2.1 Germination of Seeds 378
22.2.2 Initiation of Callus Cultures 378
22.2.3 Initiation of Suspension Cultures 379
22.3 Development of Fungal Culture 22.3.1 Maintenance of Piriformospora indica 379
22.3.2 Initiation of Fungal Culture 380
22.4 Establishment of Co-Culture of L. album with P. indica 22.4.1 Development of Co- Culture ( Fig. 22.1) 382
22.5 Analysis 22.5.1 Growth in Terms of Dry Cell Weight 383
22.5.2 Determination of Fungal and Plant Biomass 383
22.5.3 Extraction and Estimation of Podophyllotoxin 384
22.5.4 Phenylalanine Ammonia Lyase (PAL) Enzyme Extraction and Assay 385
22.6 Conclusions 385
References 385
Fungal Elicitors for Enhanced Production of Secondary Metabolites in Plant Cell Suspension Cultures 388
23.1 Introduction 388
23.2 Development of Plant Cell Cultures 23.2.1 Germination of Seeds 390
23.2.2 Initiation of Callus Cultures 390
23.2.3 Initiation of Suspension Cultures 391
23.3 Development of Elicitors 23.3.1 Preparation of Elicitors 391
23.3.2 Addition of Elicitors 392
23.4 Analysis 23.4.1 Growth in Terms of Dry Cell Weight 393
23.4.2 Extraction and Estimation of Podophyllotoxin 394
23.5 Conclusions 394
References 394
Auxin Production by Symbiotic Fungi: Bioassay and HPLC- MS Analysis 396
24.1 Introduction 396
24.2 Equipment and Laboratory Material 24.2.1 Equipment 397
24.2.2 Laboratory Material 398
24.3 Interaction on the Same Medium: Piriformospora/ Arabidopsis 24.3.1 Preparation of Slope Agar Plates 398
24.3.2 Inoculation of the Fungus 400
24.3.3 Adding Arabidopsis Seeds to the Petri Dish 400
24.3.4 Growth Conditions 402
24.3.5 Evaluation of Roots 402
24.4 Interaction on Two Different Media: Truffles/ Arabidopsis 402
24.4.1 Preparation of Dual Bioassay Plates 402
24.4.2 Inoculation of the Fungus 402
24.4.3 Adding Arabidopsis Seeds to the Petri Dish 403
24.4.4 Growth Conditions and Evaluation 403
24.4.5 Estimation of the IAA Amount Produced by the Fungus 404
24.4.6 IAA Quantification in Agar Plates 404
24.4.7 Extraction of the Agar Plates for IAA Determination 405
24.4.8 HPLC-ESI-MS/MS Determination of IAA 405
24.5 Examples of IAA Production by Fungi 24.5.1 Piriformospora indica 406
24.5.2 Truffles 407
References 407
Siderophores of Mycorrhizal Fungi: Detection, Isolation and Identification 408
25.1 Introduction 408
25.2 Isolation of Hydroxamate Siderophores from Fungal Culture Filtrates 409
25.3 Chemical Assays 412
25.3.1 Chrome Azurol S (CAS) Assay ( Modified After Schwyn and Neilands 1987) 412
25.4 Separation of Ferric Hydroxamates by HPLC 413
25.5 Mass Spectrometry 414
25.5.1 Gas Chromatography: Mass Spectrometry 414
25.6 NMR Spectroscopy 414
25.7 Conclusions 415
References 416
Biology and Molecular Approaches in Genetic Improvement of Cultivated Button Mushroom ( Agaricus Bisporus) 418
26.1 General Biology 26.1.1 Morphology and Life Cycle 418
26.1.2 Taxonomy and Nomenclature 420
26.1.3 Nutritional Requirements 423
26.1.4 Environmental Requirements 424
26.1.5 Sexuality and Breeding 426
26.2 Trouble Shooting 433
References 434
Index 437