Working with Mycorrhizas in Forestry and Agriculture SUB Gdttingen 206 384661 Mark Brundrett, Neale Bougher, Bernie Dell, Tim Grove and Nick Malajczuk
CONTENTS Chapter I. INTRODUCTION 1.1. MYCORRHIZAL ASSOCIATIONS I A. Association types I B. Host plants 2 C. Mycorrhizal fungi 3 D. Working with mycorrhizal associations 6 1.2. THE STRUCTURE AND DEVELOPMENT OF MYCORRHIZAL ROOTS 8 A. Root systems 8 B. Tissues 9 C. Cells 9 1.3. ECTOMYCORRHIZAL ASSOCIATIONS 13 A. Development 13 B. Root systems 16 C. Fungal structures 16 1.4. VESICULAR-ARBUSCULAR MYCORRHIZAL ASSOCIATIONS PRODUCED BY GLOMALEAN FUNGI 20 A. Development 20 B. Mycorrhizal roots 21 C. Fungal structures 24 1.5. THE INFLUENCE OF ROOT STRUCTURE ON MYCORRHIZAL FORMATION 26 1.6. IDENTIFYING AND DEFINING MYCORRHIZAL ASSOCIATIONS 32 A. VAM associations 32 B. ECM associations 33 C. Dual associations 34 D. Facultative associations and non-mycorrhizal plants 35 E. Recommendations 37 1.7. WORKING WITH MICROSCOPES 39 A. Dissecting microscopes 39 B. Compound microscopes 40 C. Photography 41 Chapter 2. WORKING WITH ECTOMYCORRHIZAL FUNGI 43 2.1. INTRODUCTION 43 A. Fungal taxonomy 43 B. The biodiversity of Australian fungi 46 2.2. COLLECTING, PROCESSING AND DESCRIBING ECTOMYCORRHIZAL FUNGI 49 A. Finding and collecting fungi 49 B. Recording habitat data 53 C. Describing fresh specimens 56 D. Photographing specimens 56 E. Making spore prints 57 F. Preserving fungal specimens by air drying 58 I
2.3. DESCRIBING THE MACROSCOPIC MORPHOLOGY OF FUNGI 59 A. Describing colours of fungi. 61 B. Recording details of the main morphological characters of fungi 62 2.4. MICROSCOPIC MORPHOLOGY OF FUNGAL FRUIT BODIES 71 A. Microscopic structures 71 B. Observing and measuring microscopic structures 76 C. Spore size and shape 79 D. Spore wall structure and ornamentation 80 E. Illustration of microscopic structures 84 F. Compiling detailed descriptions of fungi 85 G. Glossary of terminology 91 2.5. MANAGEMENT OF COLLECTIONS AS A GENETIC RESOURCE 92 A. Herbaria 92 B. Databases 97 2.6. AN INTRODUCTION TO THE AUSTRALIAN ECTOMYCORRHIZAL FUNGI 100 A. Ascomycetes 102 B. Basidiomycetes 107 C. Gasteromycetes 108 D. Aphyllophorales 114 E. Agaricales I 16 F. Mushrooms with gills 117 G. Fungi, with predominantly white spore prints 120 H. Fungi with predominantly brown spore prints 124 I. Fungi with predominantly black spore prints 131 J. Mushrooms with pores 131 K. Truffle-like Agaricales 135 Chapter 3. WORKING WITH GLOMALEAN FUNGI 141 3.1. INTRODUCTION 141 3.2 TAXONOMY OF GLOMALEAN FUNGI 144 A. Spore-based taxonomy 144 B. Identifying Glomalean fungi by root colonisation patterns 151 3.3. SEPARATING SPORES FROM SOIL 155 A. Sieving and centrifugation 155 B. Working with spores 158 C. Spore germination 158 D. Herbarium specimens and slide preparations 161 3.4. ISOLATING AND PROPAGATING GLOMALEAN FUNGI 162 A. Soils and nutrient supplements 164 B. Host plants 164 C. Pot culture initiation 165 D. Maintenance of pot cultures 170 E. Quality control sampling of pot cultures 170 F. Inoculum of Glomalean fungi 171
Chapter 4. EXAMINING MYCORRHIZAL ASSOCIATIONS 173 4.1. MYCORRHIZAL ROOT SAMPLES 174 A. Washing and sampling roots 174 B. Glasshouse investigations of mycorrhizal relationships 175 C. Field collections of roots 177 D. Other methods of studying roots 178 4.2. CLEARING AND STAINING MYCORRHIZAL ROOTS 179 A. Clearing roots with KOH 179 B. Staining roots with Chlorazol black E (CBE) or trypan blue 181 C. Working with darkly pigmented roots 181 D. Alternative methods 182 E. Sample storage and slide preparation 182 4.3. MEASURING ROOT COLONISATION BY MYCORRHIZAL FUNGI 184 A. Roots and VAM fungi 184 B. Quantifying ectomycorrhizal associations 187 C. Identifying ectomycorrhizal fungi 189 4.4. BIOASSAY MEASUREMENTS OF MYCORRHIZAL INOCULUM IN SOILS 193 4.5. SECTIONING FRESH MYCORRHIZAL ROOT MATERIAL 196 A. Sectioning roots 198 B. Clearing and staining sections 200 C. Some histochemical staining procedures 200 4.6. ADVANCED STAINING AND MICROSCOPY PROCEDURES 202 A. Comparison of methods - 202 B. Electron microscopy 204 C. Scanning electron microscopy 207 D. Plastic sections for light and transmission electron microscopy 208 4.7. THE VALUE OF HISTOLOGICAL INFORMATION 212 Chapter 5. PURE CULTURE ISOLATION OF FUNGI AND THE PRODUCTION OF INOCULUM 217 5.1. INTRODUCTION 217 A. Aseptic techniques 221 B. Culture media 221 C. Media additions 225 5.1. ISOLATING FUNGI INTO AXENIC CULTURE 228 A. Isolation from fruit bodies ^ \ 228 B. Isolation of fungi from within mycorrhizal roots 229 C. Spore germination. 231 D. Identification of mycorrhizal fungi and microbial contaminants in culture 232 5.3. MAINTENANCE OF FUNGAL ISOLATES 235 A. Transferring fungi to start new cultures 235 B. Preservation of fungal cultures 237 5.4. THE SYNTHESIS OF ECTOMYCORRHIZAL ASSOCIATIONS 239 A. Seed surface sterilisation 239 B. Mycorrhizal synthesis in sterile culture 241 5.5. LARGE-SCALE INOCULUM PRODUCTION 243 A. Types of inoculum 243 B. Spore-based inoculum forms 245 C. Mycelium-based inoculum forms 249 D. Storage of inoculum 253
Chapter 6. MANAGEMENT OF MYCORRHIZAL PLANTS 255 6.1. ASSESSING THE BENEFITS OF MYCORRHIZAL INOCULATION 256 A. Soil and site factors 256 B. Plant root systems 261 C. Characteristics of fungal isolates 265 6.2. MYCORRHIZAL PLANTS IN THE GLASSHOUSE 269 A. Glasshouse experimentation 269 B. Selecting superior fungal isolates 274 6.3. MYCORRHIZAL SEEDLINGS IN THE NURSERY 278 A. Potting mix components 278 B. Pasteurising potting mixes 281 C. Seedling containers 283 D. Disease and pest control 287 6.4. MINERAL NUTRITION OF MYCORRHIZAL PLANTS 288 A. Nutrient requirements 288 B. Fertilising eucalypts in nursery containers 291 C. Fertilising eucalypts in field trials 296 D. Identifying nutrient disorders in the nursery 297 E. Analysis of potting mix, soil, water and fertilizer 302 6.5. ASSESSMENT OF MYCORRHIZAL FORMATION 303 Chapter 7. FIELD EXPERIMENTS: PLANNING, DESIGN, MEASUREMENT AND DATA ANALYSIS 307 7.1. INTRODUCTION 307 A. Experimental process 308 B. Planning experiments 308 C. The basis for comparing treatments 308 7.2. EXPERIMENTAL DESIGN 314 A. Selecting treatments 314 B. Choice of design 316 C. Replication and randomisation 317 D. Examples of designs and plot layouts 319 E. Minimising effects of extraneous variation 321 7.3. EXPERIMENTAL PROCEDURES 323 A. Site selection 323 B. Site preparation and experiment establishment 325 C. Maintenance of the experiment 332 D. Measurements and observations 334 7.4. DATA PROCESSING AND STATISTICAL ANALYSIS 340 REFERENCES 347