SPRINGER LAB MANUAL

Springer Berlin Heidelberg New York Barcelona Budapest Hong Kong London Milan Paris Santa Clara Singapore Tokyo

Ajit Varma (Ed.)

Mycorrhiza Manual

With 114 Figures

Springer

Professor Dr. AJIT VARMA

Jawaharlal Nehru University School of Life Sciences 110067 New Delhi India

ISBN-13: 978-3-540-62437-0 DOl: 10.1007/978-3-642-60268-9

e-ISBN-13: 978-3-642-60268-9

Library of Congress Cataloging-in-Publication Data.

Mycorrhiza mauual! Ajit Varma (ed.). p. cm. - (Springer lab manual)

Includes biliographical references and index.

I. Mycorrhizas - Laboratory manuals. I. Varma, A. (Ajit), 1940-II. Series. QK604.2.M92M945 1998 579.5'17852 - dc21

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©Springer-Verlag Berlin Heidelberg 1998

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Foreword

The survival of mankind requires that we modify deeply our relationship to natural resources, and especially those with living organisms. In par­ticular, new management roles have to be defined to exploit what for us is an unavoidable resource: the plant. We can postulate from present-day knowledge that plant survival and fitness in a given ecosystem are not conceivable without the establishment of beneficial relationships between plants and microorganisms, in particular those living in the soil. However, we are still a long way from understanding the contribution of each type of microorganism to such processes, although those developing pre­ferentially or exclusively in roots, like mycorrhizal fungi, must playa key role.

The intimate symbiotic relationships developed between mycorrhizal fungi and plants, since land colonization by the latter, have led to an interdependence between these organisms for many basic processes. The fungi require plants to accomplish their life cycle; this is the case, for example, in fructification of many edible fungi, such as truffles or bolets. Plants depend heavily on mycorrhizal fungi for many important func­tions, such as mineral nutrition and abiotic or biotic stress resistance. Substantial evidence has accumulated in recent years about how rational use of these microsymbionts could significantly contribute to decreasing fertilizer and pesticide use in agriculture, especially if combined with other beneficial soil microorganisms (Nz fixers, pathogen antagonists, PGPR, etc.).

The commercial importance of mycorrhizal fungi lies in their poten­tial to maintain a reasonable level and quality of production, whilst minimising the negative impacts on the environment resulting from agri­cultural practices developed during this century, which entirely ignore the existence of beneficial soil microorganisms. Mycorrhizal fungi act as a major link between plants and soil, and should therefore be considered a central pivot for new strategies in the development of biologically orien­tated agricultural practices.

However, although the science of mycorrhizology is expanding rapidly, it is still generally considered of secondary importance (see, for example,

VI Foreword

research carried out on root development and physiology without consid­ering mycorrhizal fungi!).

The great merit of this book is to bring together worldwide specialists in the science of mycorrhizology, in order to present up-to-date techniques for research aimed at understanding and exploiting mycorrhizal systems, and so meet future challenges of using them in sustainable agricultural practices.

Dijon, September 1997 Dr. S. Gianinazzi

Preface

Nearly all terrestrial plants in this universe have or could have my­corrhizae of one type or another. The major types of mycorrhizae of importance in agriculture, forestry, florihorticulture, viticulture and arboriculture are arbuscular mycorrhizae (AM) and ectomycorrhizae. The evidence gathered so far is very strong that plants evolved with mycor­rhizae, and, in natural ecosystems are highly dependent on them for their contributions to growth, health and sustainability. Mycorrhizae are in­volved in many fundamental plant processes because they link plants and soil and induce changes in the host plant physiology. If our crop plants, other than those that are nonmycotrophic, lack mycorrhizae, it is prob­ably because of the detrimental effects of agricultural and forestry prac­tices on these fungi. Plant growth and "health" are supported in many ways by the rhizosphere microbes, and the key among these microbes is mycorrhizal fungi.

Rhizosphere or, more appropriately, "mycorrhizosphere" microorgan­isms influence many chemical reactions by means of their metabolites, and mycorrhizae play a crucial role in facilitating both microbial and plant functions as mediators of exchanges between them. Mycorrhizae improve the "health" and development of plants by enhancing nutrition, modifying physiological functions of plants, reducing plant response to environmental stresses, and modifying the chemistry and biology of the rhizosphere in ways that alter nutrient cycling and suppress activity of root pathogens. The extraradical phase of mycorrhizae extends into the soil, and generates significant changes in soil aggregation, organic matter accumulation, and microbial activity in the "hyphasphere" soil; all these changes improve the structure and "health" of the soil (sustainability of soil). If any type of microorganism could induce and orchestrate interac­tions and functions of the soil in relation to the growth and health of plants, it is the friendly mycorrhizal fungi.

The benefits of mycorrhiza in suppressing plant diseases are: aiding in the reclamation of mine spoils; maintenance of nutrient recycling sys­tems, structure and stability of soils; reducing the need for excessive fertilizers and pesticides; stabilizing ecosystems exposed to air pollutants;

VIII Preface

and maintaining the biodiversity of all ecosystems, making them a key element in maintaining healthy plants on the planet Earth.

The science of mycorrhizology is expanding rapidly and will soon make mycorrhiza connections with other sciences of plant systematics, ecology, and physiology; horticulture; agronomy; soil science; climatology; mo­lecular biology, biochemistry, genetics, and certainly plant pathology.

Mycorrhizal research is entering a challenging and exciting period when molecular and genetic tools can be used synergistically. The devel­opment of techniques permitting studies of the mycorrhizal fungi, which are at best difficult to culture, will expand our understanding of the value and functioning of below-ground root-fungal symbiosis. In this manual, every possible effort has been made to describe the protocols in a simple and illustrative version for a wide audience, including specialists, stu­dents, and beginners, to pick up the thread and conduct comprehensive research on these complex but eco-friendly symbiotic fungi.

In planning this treatise I extended invitations to prepare chapter(s) to distinguished scientists throughout the world. I wish to sincerely thank these eminent authors for their scholarly contributions, their enthusiasm for this joint venture, and their patient cooperation in responding gener­ously and submitting the manuscripts promptly. I have learned much during the editorial process and hope that this volume will provide a similar stimulus to its users. I offer my sincere appreciation to Dr. Dieter Czeschlik, Executive Editor, Biology, Springer and his coworkers for timely publication.

This book is dedicated to my friends and well-wishers, especially Dr. Hannes Schuepp (Wadenswil, Switzerland), Professor David J Read (Sheffield, UK), Professor Bertold Hock (Miinchen, Germany), Professor Paola Bonfante (Torino, Italy), Drs. Vivienne Gianinazzi-Pearson, Silvio Gianinazzi (Dijon, France), my students especially Sudha and my wife Gita, our children Amit with Neeti, Anshul with Sravana and Ruchita with Avijit.

TUM, Freising-Weihenstephan, Germany September 1997

Ajit Varma

Contents

Chapter 1 Mycorrhizae, the Friendly Fungi: What We Know, What Should We Know, and How Do We Know? .......................... 1 A.VARMA

Chapter 2 Approaches to the Study of Interactions Between Arbuscular Mycorrhizal Fungi ...................... 25 S. JUNIPER, L. K. ABBOTT, and F. JAYASUNDARA

Chapter 3 Application of Molecular Techniques to Ecological Studies of Symbioses in Actinorhizal Plants .......................... 41 C. T. WHEELER, N. R. McEWAN, A. SELLSTEDT, and G. SANDSTROM

Chapter 4 Characterizing Effects of Mycorrhizal Fungi on Plant Population Structure .............................. 65 R. T. KOIDE and D. L. SHUMWAY

Chapter 5 Evaluation of V esicular-Arbuscudar Mycorrhizal Colonisation by Staining .............................................. 77 S. DICKSON, MANDEEP, and S. E. SMITH

Chapter 6 Mycorrhizosphere Microorganisms: Screening and Evaluation. . . . 85 K. G. MUKER]I, MANDEEP, and A. VARMA

Chapter 7 Methods for Cultivating Edible Ectomycorrhizal Mushrooms 99 I. R. HALL and Y. WANG

Chapter 8 Isolation and Regeneration of Protoplasts from Ectomycorrhizal Fungi ................................ 115 R. HAMPP, C. STULTEN, and U. NEHLS

X Contents

Chapter 9 Electrophoretic Analytical Technique for the Genetic Identification of Mycorrhizal Fungi of the Genus Tuber and Their Mycelium and Mycorrhiza ........................ 127 S. URBANELLI, P. SALLICANDRO, and E. DE VITO

Chapter 10 The Use of Polytetrafluoroethylene (PTFE) Hydrophobic Membranes to Study Transport ofNls by Mycorrhizal Hyphae .............................. 151 B. FREY, I. BRUNNER, P. CHRISTIE, A. WIEMKEN, and P. MADER

Chapter 11 Application of Methods of Quantitative Histochemistry on Mycorrhizal Roots ..................................... 159 R. HAMPP, C. SCHAEFFER, and M. GUTTENBERGER

Chapter 12 Measuring Production and Activity of Plant Cell Wall-Degrading Enzymes in Ectomycorrhizal Fungi M. BURKE and J. W. G. CAIRNEY

Chapter 13 Biochemical Analysis of Polygalacturonases

173

by Ericoid Mycorrhizal Fungi ............................... 187 S. PEROTTO, V. COMETTI, J. D. COISSON, I. PERUGINI, and P. BONFANTE

Chapter 14 Methods for Determining Extracellular and Surface-Bound Phosphatase Activities in Ectomycorrhizal Fungi .............. 217 M. TIBBETT, S. M. CHAMBERS, and J. W. G. CAIRNEY

Chapter 15 Determination of Glutathione, Glutathione Disulphide and Two Related Enzymes - Glutathione Reductase and Glucose-6-Phosphate Dehydrogenase, in Fungal and Plant Cells ........... 227 I. KRANNER

Chapter 16 Identification and Characterzation of Siderophores of Mycorrhizal Fungi ....................................... 243 K. HASELWANDTER and G. WINKELMANN

Chapter 17 Polydonal Antibodies for the Detection of Arbuscular Mycorrhizal Fungi .............................. 255 A. HAHN, C. GOBEL, and B. HOCK

Contents XI

Chapter 18 Monoclonal Antibodies for the Identification of Arbuscular Mycorrhizal Fungi ............................ 271 C. GOBEL, A. HAHN, T. GIERSCH, and B. HOCK

Chapter 19 Targeted Approaches for Detecting Changes in Protein Expression with Mycorrhiza Development: Hydrolytic Isoenzyme Analyses and Immunological Detection of Known Proteins in Root Extracts .......................................... 289 E. DUMAS-GAuDOT, B. DASSI, S. SLEZACK, V. GIANINAZZI-PEARSON, and S. GIANINAZZI

Chapter 20 Untargeted Approaches to the Analysis of Gene Expression in Arbuscular Mycorrhiza: Analyses of Polypeptides (in Vivo and in Vitro) and of Translated mRNA Products E. DUMAS-GAUDOT, A. SAMRA, V. GIANINAZZI-PEARSON, and S. GIANINAZZI

Chapter 21 The Molecular Route to Understanding V AM Symbiosis G. DELP, G. M. RosEwARNE, and S. J. BARKER

Chapter 22 Molecular Approaches to Investigate Biodiversity

311

327

in Mycorrhizal Fungi ...................................... 353 L.LANFRANCO,S.PEROTTO,S.LoNGATO,A.MELLo, V. COMETTI, and P. BONFANTE

Chapter 23 DNA Isolation and Development of Molecular Tools for Identification and Detection of V A Mycorrhizal Fungi A. ZEZE, K. OPHEL-KELLER, and S. E. SMITH

Chapter 24 PCR in Studies of AM Fungi: from Primers to Application D. VAN TUINEN, B. ZHAO, and V. GIANINAZZI-PEARSON

Chapter 25

373

387

PCR Cloning of Genes from Arbuscular Mycorrhizal Fungi 401 P. FRANKEN, B. RESSIN, L. LAPOPIN, V. GIANINAZZI-PEARSON, and S. GIANINAZZI

Chapter 26 RAPD and ITS Analysis of Orchid Mycorrhizal Fungi .......... 413 G. C. SAUNDERS and S. J. OWENS

XII Contents

Chapter 27 Isolation and Partial Characterisation of Double-Stranded RNA-Containing Viruses of Orchid Mycorrhizal Fungi. . . . . . . . .. 425 J. D. JAMES, G. C. SAUNDERS, and S. J. OWENS

Chapter 28 Identification and Characterization of Genes by Mutant Complementation ........................................ 437 U. NEHLS and R. HAMPP

Chapter 29 Spatial Regulation of Genes in Ectomycorrhiza: mRNA in Situ Localization and ~-Glucuronidase Detection. . . . . . . . . . . . . . . . . .. 449 D. TAGU and S. HAWKINS

Chapter 30 Genomic Fingerprinting of Ectomycorrhizal Fungi by Microsatellite-primed PCR .... . . . . . . . . . . . . . . . . . . . . . . . . . .. 463 F. MARTIN, G. COSTA, C. DELARUELLE, and J. DIEZ

Chapter 31 Large-Scale Sequencing of Anonymous Ectomycorrhiza cDNA Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 475 F. MARTIN and C. VOIBLET

Chapter 32 Production of Biomass of Arbuscular Mycorrhizal Fungi in the Glass Bead Compartment System ...................... 495 D. REDECKER, H. THIERFELDER, and D. WERNER

Chapter 33 DNA Amplification Fingerprinting of Mycorrhizal Fungi and Associated Plant Materials Using Arbitrary Primers ........ 499 P. M. GRESSHOFF, F. GHASSEMI, R. A. BREWER, and E. G. O'NEILL

Chapter 34 Quantification of AMF-Induced Modifications to Root System Architecture and Longevity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 515 J. E. HOOKER, G. BERTA, G. LINGUA, A. FUSCONI, and S. SGORBA TI

Subject Index ............................................ 533