Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology
VOLUME 24
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BOTANICAL RESEARCH ...
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Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology
VOLUME 24
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J. A. CALLOW
School of Biological Sciences, University of Birmingham, Birmingham, UK
Editorial Board J. H. ANDREWS
H. G. DICKINSON M. KREIS R. M. LEECH R. A. LEIGH E. LORD D. J. READ I. C. TOMMERUP
University of Wisconsin-Madison, Madison, USA University of Oxford, Oxford, UK Universitb de Park-Sud, Orsay, France University of York, York, UK Rothamsted Experimental Station, Harpenden, U K University of California, Riverside, USA University of Shefield, Shefield, UK CSIRO, Perth, Australia
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology edited by
J. H. Andrews
I. C. Tommerup
and
Department of Plant Pathology, The University of Wisconsin-Madison Madison, USA
CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Perth, PO Wembley 6014, Australia
Series editor
J. A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK
VOLUME 24
1997
ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto
This book is printed on acid-free paper Copyright
0 1997 by ACADEMIC PRESS
All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www. apnet .com Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co.uk/ap/ A catalogue record for this book is available from the British Library ISBN 0-12-005924-X Typeset by Keyset Composition, Colchester, Essex Printed in Great Britain by Hartnolls Limited, Bodmin, Cornwall 97 98 99 00 01 02 EB 9 8 7 6 5 4 3 2 1
CONTENTS
CONTRIBUTORS TO VOLUME 24 ..................................... CONTENTS OF VOLUMES 13-23
xv
....................... ,............ .... xvii
SERIES PREFACE ................................. .......................... ....... xxiii PREFACE .................................................................................
xxv
Contributions of Population Genetics to Plant Disease Epidemiology and Management M. G. MILGROOM and W. E. FRY I.
Introduction _........._................................_...................,...... ..... ..
1
11.
Population Genetics and Plant Pathology ..... ................... .............. A. What is (and what isn’t) Population Genetics? ........................ B. Brief history of Population Genetics in Plant Pathology ............
3 3 4
111. What can Population Genetics Contribute to Epidemiology and Plant Disease Management? ....... .................................. . . . ......... .......... A. The Concept of a Population ............................................... B. Genetic Variation in Populations .....................,............... ...,. C. The Use of Neutral Genetic Markers to Estimate Ecologically Important Variation ........................... ...................,........,.. .. D. The Interface between Population Genetics and Epidemiology ... E. Population Characteristics vary among Populations ....... ..,. ... . ...
10 13 18
IV. Examples of the Integration of Population Genetics and Epidemiology A. Fungicide Resistance in Pyrenophora teres ........................... ... B. Epidemiology and Population Genetics of Phytophthora infestans
19 21
... ....
24
V.
Future Contributions of Population Genetics to Plant Pathology
5 5 8
19
Acknowledgements ....................................................................
24
...............................................................................
25
References
vi
CONTENTS
A Molecular View Through the Looking Glass: the Pyrenopeziza brassicae-Brassica Interaction A . M . ASHBY I . Introduction I1. The A. B. C. D.
...........................................................................
P . brassicae-Brassica Interaction ......................................... The Fungus ..................................................................... Pathogenesis .................................................................... Sexual Morphogenesis ....................................................... Disease Epidemiology .......................................................
32 32 32 33 33 34
111. Molecular Techniques in the Analysis of the P. brassicae-Brassica Interaction .............................................................................
38 .
IV. Molecular Analysis of Pathogenesis ........................................... A . Surface Growth and Penetration: the Role of Cutinase ........... B . Subcuticular Growth: the Role of Protease ........................... C . Using Reporter Genes to Measure Fungal Biomass In Planta . . D . Proposed Role of Extracellular Protease in Pathogenicity ........ E . Implications for Disease Control .........................................
40 41 42 45 47 47
V. Analysis of the Hemibiotrophic Phase: the Role of Cytokinins ....... A . Biochemical Analysis of Cytokinin Production by P. brassicae . B . Molecular Analysis of P. brassicae Cytokinins ....................... C . The Role of Cytokinins in Pathogenicity .............................. D . Implications for Disease Control .........................................
47 49 49 51 51
VI . Analysis of Sexual Morphogenesis ............................................. A . Biochemical Analysis: Identification of a Post-Mating Factor ... B . Molecular Analysis ........................................................... C . Sexual Morphogenesis in P . brassicae: a Speculative Summary . D. Implications .....................................................................
51 52 53 58 60
VII . A Molecular View through the Looking Glass: the P. brassicaeBrassica Interaction ....................................................................
60
...............................................................
65
.................................................................
65
............................................................................
65
VIII . Concluding Remarks Acknowledgements References
vii
CONTENTS
The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi M . CHAMBERLAIN and D . S. INGRAM
.............................................................................
I . Introduction
71
..............
72
I11. Comparisons of Genetic Variation. Physiological Costs and Fitness between Asexual and Sexual Systems ........................................... A . Does the Sexual System Generate more Variants? ................... B . Is the Sexual System Physiologically more Costly? ................... C . Are Sexual Progeny more “Fit”? .............................................
73 73 74 74
I1. Initiation of Asexual Sporulation and Sexual Reproduction
IV. Maintaining and Changing the Balance between Reproductive Processes .................................................................................... A . Species-determined Equilibria ............................................... B . Genotype-determined Equilibria ........................................... C. Seasonally Maintained Equilibria .......................................... D . Physical and Nutritional Factors ........................................... E . Density and Competition ..................................................... F. The Effect of Mycelial Extracts and Specific Morphogens .........
80
...............................................................................
81
....................................................................
82
Acknowledgements References
76 77 77 77 78
......................
V. Trade-off between Asexual and Sexual Reproduction VI . Conclusion
74 75
...............................................................................
82
The Role of Leucine-Rich Repeat Proteins in Plant Defences D . A . JONES and J . D . G . JONES I . Introduction
...........................................................................
I1. Resistance Genes Encoding Proteins with Extracytoplasmic LRRs .. A . Resistance Genes Encoding Membrane-anchored Proteins with Extracytoplasmic LRRs and no Kinase Domain .................... B . A Resistance Gene Encoding a Membrane-anchored Protein with Extracytoplasmic LRRs and a Cytoplasmic Kinase Domain ..... C . Avirulence Determinants that Interact with Resistance Proteins Containing Extracytoplasmic LRRs ..................................... D . Activation of Plant Defences by Resistance Genes Encoding Proteins Containing Extracytoplasmic LRRs ............................
90 91 91 95 97 99
I11. Resistance Genes Encoding Proteins with Cytoplasmic LRRs ......... 101 A . Resistance Genes Encoding Proteins with Cytoplasmic LRRs and 102 Potential Leucine Zippers ..................................................
...
CONTENTS
Vlll
B . Resistance Genes Encoding Proteins with Cytoplasmic LRRs and Homology to the Cytoplasmic Domains of Toll and the Interleukin-1 Receptor ........................................................... C . A Gene Encoding a Protein with Cytoplasmic LRRs that is Required for a Resistance Gene to Function ........................ D . Avirulence Determinants that Interact with Resistance Proteins Containing Cytoplasmic LRRs ............................................ E . Activation of Plant Defences by Resistance Proteins Containing Cytoplasmic LRRs ........................................................... IV . Defence-related Genes Encoding Proteins with Extracytoplasmic LRRs ................................................................................... A . Polygalacturonase-inhibiting Proteins ................................... B . LRR Extensins ................................................................ C . A Viroid-induced LRR Protein ..........................................
108 109 113 114 119 120 127 131
V. Genes Encoding Proteins of Unknown Function with Extra131 cytoplasmic LRRs ................................................................... 131 A . The AWJL Proteins of Wheat ............................................ 135 B . Receptor-like Protein Kinases ............................................ VI . A Gene Encoding a Protein of Unknown Function with Cytoplasmic 137 LRRs ...................................................................................
VII . The A. B. C.
Evolution of Plant LRR Proteins ........................................ The Evolution of LRR Proteins in the Eukaryotes ................ Evolutionary Clues Provided by Intron Arrangements ............ Evolution of Different Specificities in LRR Proteins ..............
138 138 141 143
VIII . The Structure and Molecular Specificity of Plant LRR Proteins ...... A . Inferences about the Structure of Plant LRRs by Comparison with the Known Structure of Porcine Ribonuclease Inhibitor ... B . Inferences about the Structure and Interactions of Extracellular Plant LRR Proteins Based on their Potential Patterns of Glycosylation .................................................................. C . Inferences about the Interactions between Plant LRR Proteins and their Ligands Based on Comparisons with the Interactions between Ribonuclease Inhibitors and Ribonucleases .............. XI . Concluding Remarks ...............................................................
144 144 147 150 153
.................................................................
156
References ............................................................................
156
Acknowledgements
ix
CONTENTS
Fungal Life-styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens. Plant Endophytes and Saprophytes R . J . RODRIGUEZ and R . S . REDMAN I . Introduction
...........................................................................
I1. Plant Pathogens
........
.......................................................
171
...................................................................
174
...........................................................................
176
...............................................................
179
..........................................
182
I11. Plant Endophytes IV. Saprophytes
V . Life-style Crossroads
VI . Life-styles and Ecosystem Dynamics
VII . Fungal Biology in Agricultural Versus Natural Ecosystems VIII . The Evolution of Agriculture IX . Conclusion
............
...................................................
............................................................................
.........................
183 184 186
....................................
187
............................................................................
187
Acknowledgements References
169
Cellular Interactions oetween Plants and Biotrophic Fungal Parasites M . C . HEATH and D . SKALAMERA I . Introduction
.............................................................................
I1 . Why do Fungal and Oomycetous Parasites form Intracellular Structures? A . General Characteristics of Plant-Haustorium Interfaces ............. B . Maintenance of a Differentiated Extrahaustorial Membrane ...... C . Solute Transport Across the Plant-Parasite Interface ................ D . Other Roles of the Haustorium ............................................ E . Concluding Remarks ........................................................... 111. Of what Significance are the Plant Cellular Rearrangements that Accompany Parasite Invasion? .................................................... A . Defensive Responses to Parasite Invasion ............................... B . Parasite-Induced Changes in the Plant’s Endomembrane System C . Associations of Intracellular Fungal Structures with the Plant Nucleus ............................................................................ D . Changes in the Plant Cytoskeleton ........................................ E . Concluding Remarks ........................ ..............................
196 198 198 199 202 204 205 206 206 207 209 210 210
CONTENTS
X
IV. Why do Biotroph-Invaded Cells Die in Resistant Plants? ................ A . Is Cell Death the “Default State” following Cell Penetration? ... B . Do Invaded Cells Die in Host and Non-host Plants for the Same Reason? ........................................................................... C. Arguments for Cell Death in Resistant Plants being a Form of Programmed Cell Death ...................................................... D . Arguments against Cell Death in Resistant Plants being a Form of Programmed Cell Death ...................................................... E . Cellular Mechanisms of Cell Death ....................................... F. Concluding Remarks ........................................................... V . Conclusions References
210 211 212 214 215 216 218
..............................................................................
219
...............................................................................
219
Symbiology of Mouse-Ear Cress (Arubidopsis thalhna) and Oomycetes E . B . HOLUB and J . L . BEYNON I . Introduction
............................................................................
I1. Defining a New Research Arena of Plant Biology ......................... A . The Dawn of Arabidopsis ................................................... B . Relevant Trends in Modern Biology ..................................... C . A Rebirth in Plant Pathology .............................................. D . A Copernican Perspective ...................................................
228 229 229 230 231 232
I11. Symbionts from the Wild .......................................................... A . The Phytobiont: Arabidopsis thaliana ................................... B . The Biotrophs: Peronospora parasitica and Albugo candida ...... C . Three Rs of Symbiosis ....................................................... D . The Phenotypes of Interactions: Consequences of Recognition ..
233 233 235 238 240
IV. Molecular Genetics of Natural Variation ...................................... A . Building Models: Predicting the Host Genotype ..................... B . Nonallelism: Juggling with Apples and Oranges ..................... C . Major Complexes of Recognition Genes: How Big is a Cluster? D . A Natural Anomaly of Susceptible Origin .............................
243 246 248 254 258
V . Mutations: Revealing Complexity from Black and White ................ 259 260 A . A Myriad of Columbia Mutants ........................................... 261 B . Surprising Extremes in Wassilewskija ................................... VI . Avenues of Future Research
......................................................
262
................................................................ ...................................................................
268 269
..............................................................................
269
VII . Concluding Remarks Acknowledgements References
xi
CONTENTS
Use of Monoclonal Antibodies to Detect. Quantify and Visualize Fungi in Soils F. M . DEWEY. C . R . THORNTON and C . A . GILLIGAN I . Introduction
...........................................................................
I1. Production of Species-specific and Genus-specific Monoclonal Antibodies ................................................................................... A . Selection and Preparation of Immunogens ............................ B . Selection of Hybridoma Cell Lines Secreting Specific Antibodies C . Choice of Antibody Subclass .............................................
276 278 278 279 279
I11. Assay Formats ....................................................................... 280 A . Enzyme-linked lmmunosorbent Assays (ELISAs) .................. 281 B . Membrane Assays ............................................................ 282 IV. Sample Preparation ................................................................. 283 A . Extraction of Antigens from Soil ........................................ 283 B. Elimination and Reduction of Interference from Soil Components .......................................................................... 286 V. Detection
..............................................................................
VI . Quantification ........................................................................ A . Estimation of Biomass ...................................................... B . Immunological Estimation of Colony-forming Units ............... C. Setting of Thresholds for Detection and Quanta1 Assay Systems D . False-negative Results ....................................................... VII . Visualization
..........................................................................
VIII . Concluding Remarks
289 289 293 296 298 300 302
.................................................................
304
............................................................................
304
Acknowledgements References
...............................................................
287
Function of Fungal Haustoria in Epiphytic and Endophytic Infections P. T. N . SPENCER-PHILLIPS I . Introduction
.............................................................................
I1. Strategies for Nutrition and Biotrophy: an Overview
309
.......................
311
I11. The Challenge of Biotrophic Nutrient Accumulation ....................... A . The Apoplastic Environment ................................................
313 314
xii
CONTENTS
IV. The Role of Haustoria and Intercellular Hyphae in Transfer Intercept A . Vascular Association: General Considerations ......................... B . Minor Vein Type. Mechanisms of Phloem Loading and Interception Strategy ................................................................. C . Evolutionary Aspects ..........................................................
316 317 318 324
..............................................
325
................................................
327
....................................................................
328
References ...............................................................................
328
V . Alternative Functions of Haustoria VI . Summary and Research Priorities Acknowledgements
Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria B . HAUBOLD and P. B . RAINEY I . Introduction
.............................................................................
335
I1. Quantifying Genetic Variation: the Indirect Approach ....................
337
I11. The Neutral Theory: Historical Background to the Study of Bacterial 338 Population Genetics .................................................................. IV. Population Structure .................................................................. A . Genetic Structure ........................................... B . Phylogenetic Structure ........................................................ C. Spatial and Temporal Structure ............................................
340 340 342 343
......................................................
345
VI . Conclusions ..............................................................................
347
V . The Metapopulation Concept
Acknowledgements
....................................................................
References ...............................................................................
347 347
Asexual Sporulation in the Oomycetes A . R . HARDHAM and G . J . HYDE I . Introduction
.............................................................................
I1. Sporangiogenesis ....................................................................... A . Induction .......................................................................... B . Morphological Development ..................................................
353 357 357 359
...
CONTENTS
Xlll
C. Synthesis of Zoospore-specific Components during Sporangio361 genesis ............................................................................. 111. Zoosporogenesis
....................................................................... 377 A . Induction .......................................................................... 377 B . The Process of Cleavage ...................................................... 378 C. Synthesis of Zoospore-specific Components during Zoosporo384 genesis ............................................................................. D . Polarization of Zoosporic Organelles ..................................... 386 E . Zoospore Discharge ............................................................ 386 F. Conclusions ....................................................................... 389
....................................................................
390
...............................................................................
390
Acknowledgements References
Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution? J . WOSTEMEYER. A . WOSTEMEYER and K . VOIGT I . Horizontal or Lateral Gene Transfer: where does it Occur?
.............
399
I1. Bacteria as Recipients of Foreign DNA ........................................
401
I11. Fungi as Recipients of Foreign DNA ...........................................
410
IV . Plants as Recipients of Foreign DNA ........................................... A . Agrobacteriurn turnefaciens ................................................... B . Rhizobiurn Interactions with Legumes ...................................
416 416 418
.......................................................
420
V. VI .
Interkingdom Gene Transfer
.........................
422
...............................................................................
424
Refevance of Lateral Gene Transfer for Evolution References
The Origins of Phytophthora Species Attacking Legumes in Australia J . A . G . IRWIN. A . R . CRAWFORD and A . DRENTH I . Introduction
...........................................................................
432
I1. Legumes in Australia ..............................................................
432
111. Phytophthora Species Attacking Legumes in Australia
A . Taxonomy
.................. 433 ....................................................................... 433
xiv
CONTENTS
B . Asexual Life Cycle ........................................................... C . Sexual Life Cycle ............................................................. IV. Genetic Variation and Possible Origins of Phytophthora Species Attacking Legumes ................................................................. A . Phytophthora sojae .......................................................... B . Phytophthora medicaginis ................................................... C . Phytophthora vignae .......................................................... D . Phytophthora macrochlamydospora ......................................
436 437 437 438 439 439 440
V . Evolutionary Relationships between Phytophthora Species Attacking 440 Legumes ............................................................................... VI . Evolution of Species and Host Specificity in Phytophthora Species attacking Legumes .................................................................. A . Biological Species and Speciation ........................................ B . Evolution of Host Specificity in Phytophthora .......................
442 442 443
VII . Evolution of Cultivar Specificity in Phytophthora sojae ....................
448
..............................................................
450
.................................................................
451
VIII . Concluding Remarks Acknowledgements
............................................................................
451
INDEX ...............................................................................
457
References
CONTRIBUTORS TO VOLUME 24
A. M. ASHBY, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK J . L. BEYNON, Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK M. CHAMBERLAIN, (previously known as M. Jurand) Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK A. R. CRAWFORD, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia F. M. DEWEY, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U K A. DRENTH, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia W. E. FRY, Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA C. A. GILLIGAN, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK A. R. HARDHAM, Plant Cell Biology Group, The Research School of Biological Sciences, The Australian National University, P. 0. Box 475, Canberra 2601, Australia B. HAUBOLD, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK M. C . HEATH, Department of Botany, University of Toronto, Toronto, Ontario M5S lA1, Canada E. B. HOLUB, Plant Pathology and Weed Science Department, Horticulture Research International- Wellesbou me, Warwickshire CV35 9EF, UK G. J . HYDE, School of Biological Science, University of New South Wales, Kensington, NSW 2033, Australia D. S . INGRAM, Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK J . A. G. IRWIN, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia D. A. JONES, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK J . D. G. JONES, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, U K M. G. MILGROOM, Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA
xvi
CONTRIBUTORS TO VOLUME 24
P. B. RAINEY, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U K R. S . REDMAN, National Biological Service, NW Biological Science Center, Seattle, W A 98115, U S A R. J. RODRIGUEZ, National Biological Service, NW Biological Science Center, Seattle, W A 98115, U S A D. SKALAMERA, Department of Botany, University of Toronto, Toronto, Ontario M5S lA1, Canada P. T. N. SPENCER-PHILLIPS, Department of Biological Sciences, University of the West of England, Bristol BS16 l Q Y , UK C. R. THORNTON, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U K K . VOIGT, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie and Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany A. WOSTEMEYER, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany J. WOSTEMEYER, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany
xvii
CONTENTS TO VOLUMES 1 S 2 3
Contents of Volume 13 Interactions Between Photosystems N. R. BAKER and A. N. WEBBER
Cyanobacterial Water-Blooms C. S. REYNOLDS
Determinants of Yield of Secondary Products in Plant Tissue Cultures H. A. COLLIN
Contents to Volume 14 Protein Targeting R. J. ELLIS and C. ROBINSON
Control of Isoprenoid Biosynthesis in Higher Plants J. C. GRAY
Dunaliella: A Green Alga Adapter to Salt M. GINZBURG
Contents to Volume 15 Perception of Gravity by Plants T. BJORKMAN
Crassulacean Acid Metabolism: a Re-Appraisal of Physiological Plasticity in Form and Function H. GRIFFITHS
xviii
CONTENTS TO VOLUMES 13-23
Potassium Transport in Roots L. V. KOCHIAN and W. J. LUCAS
Sporogenesis in Conifers R. I. PENNELL
Contents of Volume 16 Lipid Metabolism in Algae J. L. HARWOOD and A . L. JONES
The Alteration of Generations P. R. BELL
The Formation and Interpretation of Plant Fossil Assemblages R. A . SPICER
Primary Productivity in the Shelf of North-West Europe P. M. HOLLIGAN
Contents of Volume 17 Plant Evolution and Ecology During the Early Cainozoic Diversification M. E. COLLINSON
Origin and Evolution of Angiosperm Flowers E. M. FRIIS and P. K . ENDRESS
Bacterial Leaf Nodule Symbiosis I. M. MILLER
Fracture Properties of Plants J. F. V. VINCENT
CONTENTS TO VOLUMES 13-23
xix
Contents of Volume 18 Photosynthesis and Stomatal Responses to Polluted Air, and the Use of Physiological and Bacterial Responses for Early Detection and Diagnostic Tools H. SAXE
Transport and Metabolism of Carbon and Nitrogen in Legume Nodu1es J. G. STREETER
Plants and Wind P. VAN GARDINGEN and J. GRACE
Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues T. C. VOGELMANN, G. MARTIN, G. CHEN and D. BUTI'RY
Contents of Volume 19 Oligosaccharins S. ALDINGTON and S. C. FRY
Are Plant Hormones Involved in Root to Shoot Communication? M. B. JACKSON
Second-Hand Chloroplasts: Evolution of Cryptomonad Algae G. I. McFADDEN
The Gametophytdporophyte Junction in Land Plants R. LIGRONE, J. G. DUCKETT and K. S. RENZAGLIA
Contents of Volume 20 Global Photosynthesis and Stomatal Conductance: Modelling the Controls by Soil and Climate F. I. WOODWARD and T. M. SMITH
In vivo NMR Studies of Higher Plants and Algae R. G. RATCLIFFE
xx
CONTENTS TO VOLUMES 13-23
Vegetative and Gametic Development in the Green Alga Chlamydomonas H. VAN DEN ENDE
Salicylic Acid and its Derivatives in Plants: Medianes, Metabolites and Messenger Molecules W. S. PIERPOINT
Contents of Volume 21 Defense Responses of Plants to Pathogens E. KOMBRINK and I. E. SOMSSICH
On the Nature and Genetic Basis for Resistance and Tolerance to Fungal Wilt Diseases of Plants C. H. BECKMAN and E. M. ROBERTS
Implication of Population Pressure on Agriculture and Ecosystems A. H. EHRLICH
Plant Virus infection: Another Point of View G. A. DE ZOETEN
The Pathogens and Pests of Chestnuts S. L. ANAGNOSTAKIS
Fungal Avirulence Genes and Plant Resistance Genes: Unraveling the Molecular Basis of Gene-for-Gene Interactions P. J. G. M. DE WIT
Phytoplasmas: Can Phylogeny Provide the Means to Understand Pathogenicity B. C. KIRKPATRICK and C. D. SMART
Use of Categorical Information and Correspondence Analysis in Plant Disease Epidemiology S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEIN-GEBBINCK
CONTENTS TO VOLUMES 13-23
xxi
Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the “Arbuscular” (VA) Mycorrhizal Symbiosis F. A. SMITH and S. E. SMITH
Calcium Ions as Intracellular Second Messengers in Higher Plants A. A. R. WEBB, M. R. McAINSH, J. E. TAYLOR and I. M. HETHERINGTON
The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective B. R. JORDAN
Rapid, Long-Distance Signal Transmission in Higher Plants M. MALONE
Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A. J. S. McDONALD and W. J. DAVIES
Contents of Volume 23 The Value of Indexing for Disease Control Strategies D. E. STEAD, D. L. EBBELS and A. W. PEMBERTON
Detecting Latent Bacterial Infections S. H. DE BOER, D. A. CUPPELS and R. GITAITIS
Sensitivity of Indexing Procedures for Viruses and Viroides H. HUTTINGA
Detecting Propagules of Plant Pathogenic Fungi S. A. MILLER
Assessing Plant-Nematode Infestations and Infections K. K. BARKER and E. L. DAVIS
xxii
CONTENTS TO VOLUMES 13-23
Potential of Pathogen Detection Technology for Management of Diseases in Glasshouse Ornamental Crops I. G. DINESEN and A. VAN ZAAYEN
Indexing Seeds for Pathogens J. LANGERAK, R. W. VAN DEN BULK and A. A. J. M. FRANKEN
A Role for Pathogen Indexing Procedures in Potato Certification S. H. DE BOER, S. A. SLACK, G. VAN DEN BOVENKAMP and I. MASTENBROEK
A Decision Modelling Approach for Quantifying Risk in Pathogen Indexing C. A. LBVESQUE and D. M. EAVES
Quality Control and Cost Effectiveness of Indexing Procedures C. SUTULAR
SERIES PREFACE
Advances in Botanical Research is one of Academic Press’ longest standing serials, and has established an excellent reputation over more than 30 years. Advances in Plant Pathology, although somewhat younger, has also succeeded in attracting a highly respected name for itself over a period of more than a decade. The decision has now been made to bring the two serials together under the title of Advances in Botanical Research incorporating Advances in Plant Pathology. The resulting synergy of the merging of these two serials is intended to greatly benefit the plant science community by providing a more comprehensive resource under one “roof”. John Andrews and Inez Tommerup, the previous editors of Advances in Plant Pathology, are now on the editorial board of the new series. Our joint aim is to continue to include the very best articles, thereby maintaining the status of a high impact factor review series.
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PREFACE
All plants, whether in the wild or cultivation, co-exist and interact with numerous phylogenetically and ecologically diverse microbes. Some are potential plant pathogens. Plant pathology is a complex field of study; the future is an exciting one with rapidly evolving scientific approaches to research, many of them at the cusp of development. Articles in this volume analyse developments in plant defence mechanisms, pathogen reproduction, growth, survival and population genetics and dynamics. The recognition of disease resistance in plants is ancient. Plant resistance genes were recognized a century ago, pathogen virulence and avirulence genes 50 years ago, the first pathogen avirulence genes were isolated in the last decade and the first plant resistance genes in the past 4 years. Jones and Jones focus on key resistance genes that encode proteins containing leucine-rich repeats or that require a protein with leucine-rich repeats to function. They discuss evolution, molecular specificity and the role in plant defence mechanisms of leucine-rich proteins, a protein class involved in specific protein-protein interactions. Precision and efficiency in analysing disease phenotype variation and bridging the conceptual gap between genetical and functional aspects of symbiosis is underpinned by basic principles and modern research tools. Holub and Beynon examine the concepts and approaches involved in associating host resistance specificity with a single locus, determining whether the phenotype is actually due to single o r multiple genes and unravelling the interacting network of genes involved in disease expression. Ashby shows how, by employing a multidisciplinary approach using biochemical, molecular biological and classical plant pathology to dissect a host-pathogen interaction into its component parts, new leads towards developing novel control strategies are emerging. Heath and Skalamera examine the hypotheses and supporting evidence about cellular interactions between plants and biotrophic fungal parasites. They question why fungal biotrophic parasites form intracellular structures, the significance of cellular rearrangements that accompany invasion and why invaded resistant cells die. Their synthesis throws new interpretative light on the complex associations of haustoria and cells in compatible and incompatible interactions. Spencer-Phillips has taken on the challenge of exploring the roles of intercellular hyphae and haustoria in intercepting and transferring organic and inorganic nutrients from host tissue in epiphytic and endophytic infections. To persist, pathogens must produce propagules. Chamberlain and Ingram consider asexual and sexual reproduction of fungal pathogens for their ability
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to generate genetic variation, the physiological and fitness costs of the processes, how the balance is maintained and what are the trade-offs between the two reproductive processes. Hardham and Hyde synthesize the current state of knowledge of sporangiogenesis and zoospore production in oomycetes. Adoption of function-oriented approaches and new techniques reveal similarities in the process of sporulation across the taxon where previously diversity was thought to be the case. The importance of capitalizing on recent technical advances to develop accurate and reliable methods for detecting pathogens in soil, a microbially , chemically and physically complex environment, is the theme of Dewey, Thornton and Gilligan’s chapter. They emphasize the need to couple this with new approaches to sampling techniques and methods of statistical analysis to quantify soil pathogens. Evolution of plants is influenced by their pathogens, and vice versa. Evolution of pathogens may also be influenced by other microbes. That plant rhizosphere and soil communities provide opportunities for horizontal gene transfer amongst microbes and plants is the thesis of Wostemeyer, Wostemeyer and Voigt. They highlight the significance of foreign DNA transfers for recipient organism evolution. Legumes, like most agricultural crop species, have been relocated amongst continents, in some cases with their pathogens. Irwin, Crawford and Drenth discuss approaches to unravelling the possible evolutionary origins and genetic variation of Phytophthoru species associated with the pasture legumes in Australia. That information is used in searches for sources of host resistance. Can a pathogen life-style be defined? Rodrigues and Redman point out deficiencies in current understanding of fungal biology related to cross-overs in behaviour by organisms amongst the categories plant pathogen, endophyte or saprophyte. Prominent pathogens move between categories and they may influence natural and agricultural ecosystem community structure and dynamics. Haubold and Rainey challenge microbial pathologists and phytopathologists to consider questions relating to the extent and significance of genetic variation within plant-colonizing bacterial pathogens. If effective biological control strategies are to be developed, then temporal and spatial variation must be understood. They also stress that understanding the mechanisms, rates and extent of recombination in natural populations are essential if genetically engineered bacteria are to be safely exploited in agriculture. Milgroom and Fry illustrate how the practical need to understand pathogen variation and evolution is the most significant application of population genetics to epidemiology and disease management. Sufficient knowledge exists for this critical step in integration to be taken in a few diseases, and they highlight the experimental structures needed for others to follow that route. John H Andrews Inez C Tommerup
Contributions of Population Genetics to Plant Disease Epidemiology and Management
M. G. MILGROOM and W. E. FRY
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA
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11. Population Genetics and Plant Pathology .....,,..............,................ A. What is (and what isn’t) Population Genetics? ........................ B. Brief history of Population Genetics in Plant Pathology .............
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I. Introduction
111. What can Population Genetics Contribute to Epidemiology and Plant Disease Management? ........................................................ A. The Concept of a Population ................................................ B. Genetic Variation in Populations ........................................... C. The Use of Neutral Genetic Markers to Estimate Ecologically Important Variation ................................................,............ D. The Interface between Population Genetics and Epidemiology . . . E. Population Characteristics vary among Populations ............... .... IV. Examples of the Integration of Population Genetics and Epidemiology ......................................................... ........ .... A. Fungicide Resistance in Pyrenophora teres .............................. B. Epidemiology and Population Genetics of Phytophthora infestans ............................................................................ V. Future Contributions of Population Genetics to Plant Pathology . ... ... Acknowledgements .................................................................... References ...............................................................................
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I. INTRODUCTION Population genetics and genetic variation in plant pathogens are subjects that have generated much interest since the late 1980s. Almost every recent issue Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology
ISBN 0-12-005924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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of major plant pathological and mycological journals has at least one article on genetic variation of a plant pathogen species. The visibility of this area in plant pathology is also evident from the number of recent reviews of concepts and methods appropriate to population genetic studies with plant pathogens (Burdon, 1992; Leung et al., 1993; McDermott and McDonald, 1993; McDonald and McDermott, 1993; Anderson and Kohn, 1995; Brasier, 1995; Milgroom, 1995a, 1996), as well as recent reviews of population genetic studies of specific organisms (Fry et al., 1992; Wolfe and McDermott, 1994; Kohn, 1995; Leslie, 1995; McDonald et al., 1995; Milgroom, 1995b). Although there is an exciting potential for population genetics to contribute to plant pathology, it is difficult to predict whether it will become an indispensable part of plant pathology, or fade away as fashions change. The study of population genetics of plant pathogens is still in its infancy, with some of the requisite growing pains associated with development into a mature discipline within plant pathology. Its rapid emergence has been made possible, in part, by technological advances that have increased the ease with which one can find polymorphic genetic markers; further fueling this rise is the apparent fashion of using molecular biological techniques. As a result, researchers are undertaking numerous studies in which genetic variation is described and quantified on various scales. The future of this field, from the perspective of population genetics, will be to progress beyond the description of genetic variation, towards an emphasis on evolutionary processes. Its future with respect to epidemiology is an open question, and the subject of this chapter. Whether population genetics becomes an integral discipline within plant pathology depends, in part, on whether it can be integrated with epidemiology and disease management. Evolutionary biology and population genetics have the potential to deliver much basic information about plant pathogens. Two books in the 1980s (Wolfe and Caten, 1987; Leonard and Fry, 1989) initiated the integration, but much has happened in this field since they were published. Useful integration of these fields will require mutual understanding and better communication between geneticists and epidemiologists. The value of applying population genetics in plant pathology is not always obvious, and is sometimes impeded by biological and conceptual obstacles. We will try to highlight some of the ways population genetic studies relate to epidemiology and disease management, and identify the various obstacles. We begin with an overview of population genetics and a brief account of its history in plant pathology. This is followed by some of the general principles that unite population genetics and epidemiology. Finally, we finish by describing research on two pathosystems in which links between population genetics and epidemiology are relatively strong.
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11. POPULATION GENETICS IN PLANT PATHOLOGY A . WHAT IS (AND WHAT ISN’T) POPULATION GENETICS?
Population genetics is a field concerned with determining the extent and pattern of genetic variation in populations with the goal of understanding the evolutionary processes affecting the origin and maintenance of genetic variation. The conceptual framework is based on evolutionary biology and on the processes affecting the genetic composition of populations: selection, mutation, gene flow, genetic drift and mating systems. With an understanding of the relative importance of different evolutionary processes, predictions can be made about changes in the genetic composition of populations. Sometimes predictions can be made for responses of pathogen populations to selection by various management practices. The term “population genetics” has sometimes been interpreted broadly among plant pathologists to include any study on genetic variation. This interpretation is perhaps one reason why population genetics is not fully understood in plant pathology as a discipline distinct from systematics and diagnostics. Although knowledge of genetic variation is key to population genetic studies, it is but the raw material from which inferences are made. In population genetics, evolutionary inferences are made based on the dynamics of gene and genotype frequencies within and between populations. Systematics is fundamentally different: phylogenetic inferences are made at the species level (or higher) from morphological and genetic differences among taxa. Although there is overlap of concepts and techniques between population genetics and systematics, the scale is different, in both time and space. Systematics focuses on differences between species or higher taxa, which have evolved over relatively long time scales, and often in different locations. Most emphasis in population genetics is on microevolutionary events within populations of single species, and on time scales that are generally shorter than those required for speciation. However, this distinction is not always clear, especially when taxonomic complexity is revealed by closer inspection of genetic variation within species complexes (e.g. Vilgalys and Cubeta, 1994; Leslie, 1995). Furthermore, the distinction between microevolution and macroevolution is somewhat arbitrary because microevolutionary events may lead eventually to speciation (Brasier, 1995). Population genetics is sometimes confused with those studies of genetic variation in which the goal is to find diagnostic genetic markers among species or among different phenotypes within species. The search for diagnostic markers and studies of population genetics share the need for genetic variation as the basic empirical observation, but the objectives are very different. Genetic differences exploited for diagnostic purposes are more akin to systematics than to population genetics. Neither the population concept nor that of evolution, however, is considered rigorously in the search for
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diagnostic tools. Similarly, the use of molecular genetic markers in population genetics must be distinguished from molecular biology, where questions are addressed at a subcellular as opposed to a population level of organization. Clearly, a discipline is defined by the questions asked, not the tools used. B. BRIEF HISTORY OF POPULATION GENETICS IN PLANT PATHOLOGY
Plant pathologists began their studies of population genetics when they discovered variation in physiological races (now often referred to as “pathotypes”) within populations of plant pathogenic fungi. Pathotypes are defined solely by their abilities to cause disease on host plants with different resistance genes. From a management perspective, deployment of resistance genes in host plant populations has become a fundamental concept in plant pathology (Vanderplank, 1963). Successful deployment of resistance genes depends on knowledge of the genetic composition of pathogen populations, at least with respect to the relative frequencies of different pathotypes. Pathotype surveys have been conducted on numerous pathogens, and are still part of the strategies for managing diseases with host plant resistance (e.g. Kolmer, 1989). Population genetic concepts received greater attention in relation to variation in pathotypes when pathologists began asking questions such as “Why is there more pathotype variation in some populations than others?”, or “Will complex pathotypes (super-races) evolve in response to resistance gene deployment?” Questions like these grew out of the need to manage disease, but could be addressed adequately only within a population genetics framework; in other words, there was a natural integration of epidemiology and population genetics. These questions generated studies on fitness, host specialization, sexuality and recombination, population structure, etc., in relation to pathotype evolution (reviewed in Wolfe and Caten, 1987). As a consequence, an awareness was established of the link between evolutionary processes and disease management. In the late 1970s to mid-l980s, a different perspective emerged as attention was turned towards variation in genetic markers besides pathotypes. Some of the earlier work was done with enzyme polymorphisms, vegetative incompatibility and mating types (e.g. Leonard, 1978; Burdon et al., 1982; Puhalla, 1985; Stenlid, 1985; Tooley et. al., 1985; Leung and Williams, 1986). Plant pathologists were not only applying well-known laboratory techniques to plant pathogen populations, but also began asking questions about evolutionary processes beyond selection for pathotypes by host plant resistance. By the late 1980s and early 1990s studies of molecular variation in plant pathogens all but eclipsed those of pathogenic variation in plant pathology. These recent studies were made possible by technological innovations in molecular biology that made genetic markers (e.g. RFLPs and RAPDs) more accessible.
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The advances in technology also brought about a marked change in emphasis in population genetics of plant pathogens. The biology of pathogens at the population level and processes other than selection have been emphasized. For example, studies have been conducted on population structure to make inferences on gene flow, genetic drift and recombination (reviewed in Fry et al., 1992; Leung et al., 1993; McDermott and McDonald, 1993; McDonald and McDermott, 1993; Anderson and Kohn, 1995; Milgroom, 1995a, 1996). This field has been exciting, resulting in new knowledge about the biology of pathogens. However, with few exceptions, little effort has yet been made to apply this new knowledge to epidemiology and disease management.
111. WHAT CAN POPULATION GENETICS CONTRIBUTE
TO EPIDEMIOLOGY AND PLANT DISEASE MANAGEMENT? The answer to this question is not simple. At present, there are few generalities about the contributions that population genetics can make. The obstacles to applying population genetics to epidemiological questions are both historical and conceptual. For some plant pathologists, the concepts of population genetics and evolutionary biology are foreign ideas, and conversely, few population geneticists have an understanding of plant pathology. Population genetics derives from general biology, in which organisms are considered primarily in natural environments (or experimental cages in the laboratory). The separate developments of population genetics in biology and in plant pathology have inhibited effective communication among researchers and delayed the integration of these disciplines. There are several fundamental concepts from population genetics that can be applied profitably to epidemiology and disease management. In the following sections we identify some concepts that seem most likely to integrate these fields. We also discuss some of the obstacles that need to be recognized in order to apply population genetics appropriately to epidemiological problems. Each concept is illustrated with relevant plant pathological examples. A . THE CONCEPT OF A POPULATION
A population is not simply a haphazard collection of individuals, but has biological and practical significance that is important from a research perspective. A fundamental concept in any research is that the samples or experiments are so structured that the inferences made can be applied to more general phenomena beyond the objects being observed. The key question is to what populations will inferences be made? Although this is
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often done implicitly, a more careful examination of this question is crucial in studies of population genetics - and indeed, this may be one of the defining features of population biology. The challenge is to direct research efforts towards an appropriate target population. A precise definition of a population is somewhat elusive. A restrictive definition of a population that has sometimes been used by ecologists is: a collection of individuals that are actually or potentially interbreeding (Pianka, 1988). For most microorganisms, such as many plant pathogens, the dependence of this definition on “interbreeding” makes it irrelevant because of their largely asexual life histories. As an alternative, a population may be viewed as “a group of organisms of the same species occupying a particular space at a particular time” (Krebs, 1985, p. 157). Although the potential to interbreed is not required in this definition, and is therefore more appropriate for most plant pathogen species, it is somewhat unsatisfying from a genetic perspective. The missing concept in genetic and evolutionary terms is that a population is a pool of individuals from which the next generation will be drawn. In addition, the concept of a population has to be extended further to include genetic composition: the spatial and temporal limits of a population are defined by uniform allele frequencies. Populations may sometimes have underlying genetic structure in which subpopulations have differences in allele frequencies. In this case, a population comprises multiple subpopulations, some or all of which have differences in allele frequencies. Accurate population definition is essential so that sampling is done in a manner which will enable inferences to be made about the population of interest. There are two practical problems that commonly limit the inferences that can be made appropriately to the population level; neither is unique to plant pathology. The first problem is that of recognizing and sampling populations. The second problem is the study of laboratory strains that are not representative of individuals present in field populations. Unfortunately, it is frequently difficult to define the target population, and therefore appropriate sampling strategies are not always clear. Because of these practical difficulties, analyses are sometimes done on laboratory strains. Each of these problems will be discussed in more detail. From an operational perspective, recognition of population structure is very important when estimating population genetic parameters. Wolfe and Knott (1982) discussed this problem in relation to pathotype surveys, but many of the same caveats apply to any population sampling. The most important caveat is to avoid pooling individuals belonging to genetically distinct subpopulations. This problem can arise by sampling over diverse areas or at different times. Knowledge of the biology of an organism can be useful for rationally delimiting the scale of a population. A population may comprise individuals within a given geographic region on a continental scale, e.g. Puccinia graminis f. sp. tritici, which migrates hundreds of kilometres in North America (Roelfs, 1985), or as small as a soil core, e.g.
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Fusarium oxysporum, where populations 10 m apart in undisturbed soil may be genetically distinct (Gordon et al., 1992). The defining feature is the subdivision of the population into genetically distinct subpopulations. In addition, the size of genetic individuals (genets) also varies greatly (see Brasier, 1992; Anderson and Kohn, 1995), which, in turn, influences the sampling strategy to be adopted. Although studies on characterized laboratory strains can be valuable for revealing biological phenomena, inferences made from them to natural populations may be misleading or inappropriate. For example, an important question for the management of fungicide resistance is the fitness of resistant individuals (Milgroom et al., 1989). Some studies have shown that laboratoryselected mutants resistant to fungicides have reduced fitness compared to wild type strains (e.g. De Waard and Van Nistelrooy, 1990). To apply this finding to management in the field, however, one needs to ask whether it is appropriate to make inferences from laboratory strains. Surveys of field populations have sometimes shown quite different results from those in the laboratory. For example, there were only two variable amino acid sites in the p-tubulin gene that conferred benomyl resistance in Venturia inaequalis and other fungal plant pathogens collected from field populations; in contrast, seven additional variable amino acid sites were found among resistant laboratory mutants (Koenraadt et al., 1992). A possible explanation for this discrepancy is that most mutants are at such a selective disadvantage that they are not likely to be found anywhere but in a laboratory. Inferences made about fitness of laboratory strains in this case would be very misleading. The problem of defining a population on which to make inferences is perhaps best exemplified by studies in which genetic variation of culture collections is described. These studies may be valuable for systematics or biogeography, or finding diagnostic markers, but they are less useful for population genetics because the target “population” is artificial, being a collection of disparate individuals which would normally be separated both in time and space. One example of different inferences from different samples is found with the chestnut blight fungus, Cryphonectriaparasitica. The effects of vegetative incompatibility (vic) genes on transmission of fungal viruses (hypoviruses), which cause hypovirulence in C. parasitica, were studied independently in two laboratories. One approach was to start with laboratory strains from various populations, but with characterized vic genes (Huber and Fulbright, 1994). The other approach was to sample a large number of uncharacterized isolates randomly from a single natural population (Liu and Milgroom, 1996). Although both studies found similar results, the choice of strains made a fundamental difference to the inferences that could be made. The strength of the Huber and Fulbright (1994) study was the valuable insight into genetic control and cellular-level interactions affecting virus transmission. However,
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inferences to the population level were not possible because of lack of knowledge of vic allele frequencies in specific populations. In contrast, Liu and Milgroom (1996) estimated the average effects of vic genes in a natural population, but without an understanding of the effects of each individual gene. The inference from this study was the average transmission of hypoviruses and the potential success of biological control with hypovirulence in a specific population. The weakness was that inferences could not be made to other populations without assuming that vic allele frequencies were similar to those in the sampled population. The important lesson is that the population to which inferences can be made depends on the population that is sampled for study. For epidemiology and management, where it is essential to make inferences beyond laboratory strains, the best approach is to sample directly from the target population of interest. Wolfe and Knott (1982) and Milgroom (1995a) discussed some of the practical constraints on sampling populations for population genetic studies; this issue will not be discussed further here. B. GENETIC VARIATION IN POPULATIONS
Almost every population has some degree of genetic diversity; the few exceptions are usually where single clones have colonized new areas (e.g. Correll et al., 1992; Goodwin et al., 1994b). There are two basic types of genetic variation: ecologically important variation and selectively neutral variation. Ecologically important variation refers to traits that affect fitness and, therefore, may be under selection. Selectively neutral variation refers to variation in traits that do not (or are assumed not to) affect fitness and are not under selection. Changes in selectively neutral traits are affected by evolutionary forces such as mutation, genetic drift and gene flow (although they may be affected indirectly by selection on linked genes). The different inferences that can be made from each type of genetic variation will become evident below. The challenge is to understand how each type of genetic variation relates to epidemiology and disease management. In many situations, ecologically important variation is the type of variation that is more relevant for disease management. Variation in pathogen populations in response to a management practice may have implications for the durability of that practice, since individuals may be selected for or against. The best-known examples in which genetic variation, and therefore selection, has a direct effect on disease management are pathotypes and fungicide resistance. However, variation in other traits may also be relevant to management and may help to explain some failures of bialogical or cultural controls, although these latter areas have been studied little to date. Similarly, variation in response to environmental conditions has also been hypothesized to affect the genetic composition of fungal populations (e.g. Koller et al., 1995).
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An understanding of variation in ecologically important traits is relevant to epidemiology and management because it helps predict how a population will respond to selection. The strength of selection (or more formally, the rate of change of the mean fitness in a population) is proportional to the variance in fitness in the population; this is known as the fundamental theorem of natural selection (Fisher, 1930). For example, the variance in fitness in a fungal population when a fungicide is applied is roughly equivalent to the variance in fungicide resistance phenotypes (Milgroom et af., 1989). Therefore, surveys of pathogen populations may be used to estimate this variance and to predict the rate of selection. If a pathogen population is uniformly sensitive to a fungicide, i.e. it exhibits no variation in fungicide resistance, then resistance cannot be selected and the use of that fungicide may provide effective disease management. Conversely, once there is variation for response to fungicide, selection will favour the resistant individuals, whose fitness will be greater than sensitive individuals when fungicide is applied; the resistant individuals will then increase in frequency. Estimates of variance need to be made from adequate samples, from defined target populations (e.g. Smith et af., 1991; Peever and Milgroom, 1993) in order to make inferences to the population level that are relevant to management. A culture collection is unlikely to represent a target population accurately. Successful breeding and deployment of durable host-plant resistance depends on an understanding of variation in pathogen populations. Proper resistance screening requires first defining the pathogen populations against which resistance is being bred, and then sampling the target populations adequately. In some cases a target population has been well sampled. For example, a collection of the most common clones of Sclerotinia sclerotzorurn that occur across the canola-growing regions of Canada (Kohli et af., 1992) is available for screening for resistance in Canada (L. M. Kohn, personal communication). The goal in screening for resistance against large samples of the pathogen is not only to find the best resistance, but also to assess the range of variation in the pathogen population for virulence on particular breeding lines. In other words, successful deployment of resistance relies on both breeding and screening against appropriate target populations of pathogens. From an evolutionary perspective, there are two main outcomes of a comprehensive resistance screening programme. First, interactions between breeding lines and pathogen isolates can be identified, which indicate specialization of pathogen genotypes to resistant lines. The magnitude of the interaction and variation on individual hosts are important features in interpreting the significance of interactions, Second, the variance in virulence and fitness among isolates on particular host lines can also be estimated, allowing the rate at which a population will shift to higher levels of virulence to be predicted.
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As biological control is used increasingly against plant pathogens, failures are likely to occur because target organisms evolve resistance to the introduced agents. There are few examples of plant pathogens in which resistance to biological control agents has been documented. The potential for resistance to some hypoviruses in C. parasitica was recently demonstrated in the laboratory (Polashock el al., 1994). The evolutionary processes leading to failures of biological control are similar to those for the evolution of fungicide resistance or pathotypes, but are rarely considered when biocontrol agents are deployed. Surveys of existing vaiiation (as with fungicide resistance and virulence) would be logical steps in estimating the evolutionary potential for resistance in target populations. An equally challenging evolutionary problem in biological control is the potential for declining effectiveness of introduced agents because of selection against more effective individuals. An outstanding example is myxoma viruses released in Australia to control rabbits (reviewed in Fenner and Myers, 1978). The myxoma virus caused nearly 100% mortality in the rabbit population when it was first introduced. However, several years later, moderately virulent strains increased in frequency, and the most virulent viruses all but disappeared. Simultaneously, viruses placed such intense selection pressure on rabbit populations that the average level of virus resistance increased. Needless to say, the effectiveness of biological control decreased dramatically. Comparable phenomena have yet to be described in plant pathology, but the example of the myxoma virus should not be ignored. The primary lesson from these examples is that an understanding of ecologically important variation is needed in order to predict the durability of any particular management practice. The evolutionary process most pertinent to management is selection, and direct measurement of the phenotype of interest is usually the best approach for studying ecologically important variation. C. THE USE OF NEUTRAL GENETIC MARKERS TO ESTIMATE ECOLOGICALLY IMPORTANT VARIATION
As selectively neutral genetic markers have become more accessible, there have been numerous attempts to substitute them for direct assessment of ecologically important characters. The motivation is quite reasonable: it is generally simpler to determine an allozyme or RAPD profile of a pathogen isolate than it is to inoculate a series of differential cultivars to determine its pathotype. If neutral genetic markers are good predictors of pathotypes, then their use could save valuablettime and expense. However, this approach must be undertaken cautiously; there is no general rule that variation in one marker can predict variation in another. The success of this approach depends on the type of marker used, or knowledge of the population structure of the
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pathogen. This issue will be discussed at the level of both individuals and populations. 1. Predicting variation among individuals Genetic markers can sometimes identify an ecologically important trait. A direct assay is sometimes possible by detecting a particular gene responsible for the trait of interest. For example, benzimidazole resistance can be assayed directly by selective polymerase chain reaction (PCR) amplification of alleles of the P-tubulin gene that confer resistance (Koenraadt and Jones, 1992). Alternatively, when a gene of interest cannot be assayed directly, neutral genetic markers tightly linked to it may be used if the association between loci is strong. Finding linked markers is becoming increasingly possible given the efforts in numerous laboratories to develop genetic maps of various pathogens, or the use of techniques such as bulked segregant analysis (Michelmore et al., 1991). For example, RAPD markers tightly linked to avirulence loci in barley powdery mildew, Erysiphe graminis f. sp. hordei, have been found to have strong non-random associations (cited in Wolfe and McDermott, 1994), but the association is not perfect and estimation of avirulence allele frequencies from these markers would include a measurable amount of error. Exploiting non-random associations is an approach that has been used successfully to predict ecological traits with neutral markers in some organisms. There are a number of examples of plant pathogens for which there are almost perfect associations between neutral markers and pathotypes or vegetative compatibility groups (e.g. Burdon and Roelfs, 1985; Kistler et at., 1991; Kohn et al., 1991; Goodwin et al., 1992b; Anderson and Kohn, 1995). Non-random association among alleles at different loci is referred to as “gametic disequilibrium”, or “gametic phase disequilibrium”, and is caused primarily by two different factors. First, simultaneous selection for different traits can result in non-random associations; this is exemplified by selection for pathotypes with combinations of avirulence genes compatible to host-plant resistance with two or more corresponding specific resistance genes (Wolfe and Knott, 1982; Hovmoller and 0sterglrd, 1991). Second, and more relevant to the use of neutral markers, gametic disequilibrium often occurs between tightly linked loci. An extreme case of gametic disequilibrium is when organisms are asexual such that the entire genome is effectively linked; all the organisms cited above for strong non-random associations among different markers are asexual for much or all of their life cycles (the exception above is S. sclerotiorum, which is homothallic and, therefore, no recombination occurs [Kohn et al., 19911). Conversely, gametic disequilibrium is minimal in sexually reproducing organisms because of recombination (e.g. Milgroom et al., 1992a; McDonald et al., 1994; Liu et al., 1996; Milgroom, 1996). Therefore, population structure and reproductive biology should be known before attempting to use neutral markers to predict other traits.
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Prediction of ecologically important variation from neutral markers has several serious drawbacks. First, different mutation rates among loci can result in uncorrelated variation. For example, in the rice blast fungus, Magnaporthe grisea, a single clonal lineage, defined by a multilocus RFLP fingerprint, may comprise many diverse pathotypes because the mutation rate for avirulence genes is apparently greater than for the RFLP marker (Levy et al., 1993; Zeigler et al., 1995). Therefore, in M . grisea, even though the range of pathotypes within a lineage may be limited, a lineage does not necessarily identify pathotype. A similar situation occurs with Phytophthora infestans, in which there is much pathotype variation within some clonal lineages (Goodwin et al., 1995~).As a contrasting example, there was no significant variation in virulence among clones of S . sclerotiorum from canola in Canada that differed in RFLP genotypes and mycelial compatibility (L. M. Kohn, personal communication), The problem of differential mutation rates is further highlighted in pathogens such as F. oxysporum in which host specialization is only sometimes associated with vegetative compatibility groups (Correll, 1991; Appel and Gordon, 1994). These discrepancies can be explained simply by variations in mutation rates between different loci. The restrictions on using neutral genetic markers to predict ecologically important variation should be considered carefully before attempting such a project. First, a decision has to be made whether a genetic marker is simpler than a direct assay of the phenotype of interest. For example, it may be simpler in some organisms to assay a fungicide resistance phenotype than it is to probe for a particular gene: compare a simple cultural technique (Lalancette et al., 1984) to amplifying P-tubulin genes (Koenraadt and Jones, 1992) to assay for benomyl resistance. On the other hand, the determination of a pathotype may require considerably more effort compared to determining simple molecular markers. However, a random search for markers associated with pathotypes may be fruitless unless the target population is clonal and markers can be found with appropriate levels of variation. Alternatively, finding tightly linked markers with predictive value in sexual populations may require extraordinary effort unless a genetic map has already been constructed. In addition, it may be necessary to verify an association between markers for each population to be studied because different degrees of association may be found within different populations. For example, the simple relationship between pathotypes and clonal lineages found in US populations of M . grisea (Levy et al., 1991) was more complex in populations in Colombia and the Philippines (Levy et al., 1993; Zeigler et al., 1995) (see also section IIIE, p. 18). When all these factors are considered, it may be more accurate, and less effort, to survey variation in the ecologically important trait of interest than it is to find reliable diagnostic genetic markers.
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2. Predicting variation among populations An important misconception about the use of neutral genetic markers is that knowledge of their variation can be used to predict variation in ecologically important traits. This misconception may derive from generalization of the special cases described above (clonal populations, use of linked markers, similar mutation rates, etc.). Unfortunately, there are surprisingly few studies comparing diversity of both types of markers among different populations. Diversity in pathotypes and allozymes showed a rough correlation in the asexual barley scald fungus, Rhyncosporium secalis (Goodwin et al., 1993); however, the correlation was not strong, and sample sizes were sometimes small. In contrast, variations in RAPD markers were poor predictors of variation in avirulence alleles in E . gruminis f. sp. hordei (Wolfe and McDermott, 1994), and in fungicide resistance in Pyrenophora teres (Peever and Milgroom, 1994b; see also section IVA). Conceptually, any attempt to correlate diversity of different markers is fraught with problems. First, as mentioned above, differential mutation rates confound any correlation. Second, and more important, is the effect of selection on ecologically important variation but not on unlinked neutral markers. For example, a population that is highiy diverse for both pathotypes and neutral markers could lose much of its pathotype diversity if exposed to highly selective host-plant resistance such that few pathotypes survive. This same population could maintain its diversity for neutral markers if the population is not highly clonal. Claims that knowledge of variation of neutral markers can predict variation in pathotypes or fungicide resistance should, therefore, be interpreted with caution. D. THE INTERFACE BETWEEN POPULATION GENETICS AND EPIDEMIOLOGY
We will discuss two entirely different ways in which neutral genetic variation can be applied to epidemiological questions. First, selectively neutral markers can be exploited directly for addressing epidemiological questions in which specific strains are tracked in the environment. Second, concepts of population genetics allow inferences to be made about evolutionary processes, which in turn may affect interpretations of epidemiology. Examples of how evolutionary inferences can be made to plant pathogen populations have been reviewed recently (see section 1, p. 1 for references) and will not be repeated here. In the following sections, we give examples of how genetic variation and population genetic concepts can be integrated with epidemiology and disease management. 1 . Direct tracking o j genotypes One of the simplest applications of genetic variation and genetic markers in an epidemiological context is the direct tracking of specific genotypes. This
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may be useful for determining the source of inoculum or for identifying different genotypes in competition studies. The ability to discriminate among genotypes can facilitate competition experiments (Ennos and McConnell, 1995; Legard et al., 1995), and may be used even for organisms that have regular sexual cycles if tracking is done during the asexual phase (Sierotzki et al., 1994). An excellent example of tracking specific genotypes to address epidemiological questions is the work summarized by Webber et al. (1987) on the saprophytic phase of the Dutch elm disease fungus, Ophiostoma novo-ulmi. Using benomyl-resistant isolates, Webber and colleagues showed that there were two distinct sources of inoculum contributing to the colonization of elm bark: the fungus present in the xylem from the pathogenic phase and spores introduced by bark beetle vectors from the saprophytic phase in dead elm trees. In addition, after inoculating trees with benomyl-resistant isolates of known vegetative compatibility types, they found recombinant genotypes on the emerging beetles. This result demonstrated that at least some ascospores contributed to bark colonization and that recombination maintained genotype diversity in the population. In another example of determining the source of inoculum, Shah et al. (1995) tested the hypothesis that a major source of Stagonospora nodorum on wheat in New York state was infected seed. They used two complementary approaches, one using classical epidemiological methods, the other taking advantage of genetic variation in S. nodorum populations. They infected wheat seeds with S. nodorum isolates that had known RFLP genotypes and then mixed these with clean seed to create seed lots with varying proportions of infected seed, which were then planted in the field. The severity of the ensuing foliar epidemics correlated to the proportions of infected seeds planted. In addition, the majority of the isolates recovered from foliage and seed in the next generation were the same genotypes as those used to infect seeds, although these same genotypes were not found in the resident background population. Results from both approaches clearly demonstrated a significant role of seed-borne inoculum in this pathosystem. As a source of inoculum, long-distance dispersal, or gene flow, is usually quantitatively insignificant, although there are exceptions. For example, the Puccinia pathway in North America has been described in detail (Roelfs, 1985) in which Puccinia graminis f. sp. tritici overwinters in Texas and Mexico and migrates annually throughout the midwestern USA and into Canada; migration occurs in the reverse direction in autumn. Another pathogen that overwinters far from an agricultural host population in the USA is the tobacco blue mould pathogen, Peronospora tabacina. Each year inoculum must be reintroduced into tobacco-growing regions in the USA from overwintering areas. However, the exact locations of the inoculum sources are not known despite attempts to identify them by trajectory studies (Davis and Main, 1988). Tobacco blue mould is an example where direct tracking of genotypes
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could potentially be applied to answer a basic epidemiological problem. An essential requirement would be to develop a set of polymorphic genetic markers and to characterize genetic variation in all possible source populations. This approach has been used successfully for tracking the sources of immigrant genotypes of Phytophthora infestans in the USA and Canada (Goodwin et a f . , 1994a,b; see also section IVB). Although long-distance dispersal is generally not important quantitatively as a source of inoculum, it can sometimes have significant qualitative effects, especially the introduction of novel pathotypes. A good example of this is barley powdery mildew in Europe. Analyses of neutral genetic markers made it possible to demonstrate that some genotypes have dispersed on a continental scale, introducing pathotypes compatible with host resistance genes that otherwise were effective against the resident mildew population (Brown et af., 1991; Wolfe and McDermott, 1994). By surveying genetic variation throughout many possible source populations and matching genotypes, the origin of dispersing inoculum could be identified confidently. 2.
Evolutionary inferences f r o m population genetic studies
Gene pow. The analysis of selectively neutral genetic variation among subpopulations by gene-diversity analyses provides another approach to studying gene flow and sources of inoculum (for methods see Leung et al., 1993; McDermott and McDonald, 1993; Milgroom, 1995a). Populations that are genetically subdivided are likely to have restricted gene flow among subpopulations, and to some degree may be managed as separate entities (Leung et a f . , 1993; Milgroom, 1995a). In contrast, a population that is genetically uniform over a large area may be experiencing widespread dispersal among subpopulations such that, in theory, the whole population must be considered as a management unit. Disease management for subdivided pathogen populations with host-plant resistance has been recently summarized (Leung et al., 1993) and will not be discussed further here. Management of fungicide resistance in subdivided populations is discussed below (section IV). Population structure due to various levels of gene flow may be a factor that can be exploited to a greater extent in future disease-management efforts. Estimates of gene flow from analyses of gene diversity have some inherent weaknesses, especially in agricultural systems that are highly disturbed by episodic introductions of plant pathogens. When populations are subdivided, it is an indication that gene flow is restricted to some degree. However, lack of genetic differentiation among populations is more problematic because it can be explained in at least two ways: by high levels of current gene flow, and/or by gene flow that occurred in the past (Slatkin, 1987). It is possible that populations with little genetic subdivision because of past gene flow
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actually experience low levels of current gene flow among subpopulations (although gene-diversity analyses would not show this). An example of historical gene flow is when a pathogen is introduced into a new area along with an agricultural crop. If the introduction occurred relatively recently it is unlikely that populations would have reached an equilibrium between gene flow and drift. To estimate current gene flow from gene-diversity data, one must assume that populations are in equilibrium for gene flow and genetic drift (Slatkin, 1987). Therefore, estimates of gene flow are likely to be fairly reliable when populations are highly subdivided, but claims of high levels of gene flow may be overestimated when subdivision is weak, and need to be supported by more information about the biology of an organism, especially its ability to disperse or be transported long distances. This problem was discussed in detail in relation to population subdivision in C. parasitica in North America (Milgroom and Lipari, 1995). Recombination. There are numerous plant-pathogenic fungi that reproduce sexually. Sexual reproduction not only contributes to recombination and the potential to generate new genotypic diversity, but equally important from an epidemiological perspective, it often involves structures that differ in dormancy, survival or dispersal characteristics from asexual propagules (see also section IVB). As an example, primary inoculum of Mycosphaerellu graminicola on wheat could be either sexual or asexual. Asexual pycnidiospores produced on crop debris are generally dispersed over short distances. However, ascospores of M. gruminicola can disperse over longer distances (Shaw and Royle, 1989). Identification of sexual or asexual inoculum sources for M . graminicola has been investigated both with epidemiological techniques and population genetics. The importance of airborne inoculum of M . graminicola, most likely ascospores, as primary inoculum was demonstrated by Shaw and Royle (1989) by excluding other sources of inoculum. A complementary population genetic approach was used by Chen et al. (1994) in the same system. They showed that, although allele frequencies did not change over 3 years, there was no carryover of multilocus genotypes from year to year; this finding is consistent with frequent recombination and annual recolonization by ascospores. Both approaches led to similar conclusions about the role of ascospores: one observed the effects of airborne spores on disease progress, while the other made inferences from patterns of genetic variation. In contrast to the above example, where airborne inoculum could be assayed directly, studies of genetic variation are sometimes the only approach available for determining if sexually produced inoculum is present. For example, until recently only the A1 mating type of Phytophthora infestans was found outside of central Mexico (Goodwin et al., 1994b). The discovery of the A2 mating type in Europe in the 1980s (Hohl and Iselin, 1984; Fry et al., 1993) signalled an important potential for oospore formation. Oospores
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would allow P. infestuns to survive in the soil between potato crops, potentially altering the source of inoculum and epidemiology of late blight. The increase in genotypic diversity of P. infestuns populations in Europe in recent years has been attributed to immigration of new genotypes and the occurrence of sexual reproduction (Drenth et uf., 1994; Sujkowski et uf., 1994). Using genetic markers in an epidemiological context, Drenth et uf. (1995) confirmed that oospores survived in soil and that recombinant genotypes infected potato crops the following year. Recombination also has significant effects on the diversity of multilocus genotypes (reviewed in Milgroom, 1996), although recombination per se does not affect gene diversity, which is a function of the number and frequencies of different alleles. The importance of recombination to the diversity of pathotypes is well documented for Pucciniu gruminis f. sp. tritici in North America (Roelfs and Groth, 1980). Following the eradication of barberry, on which P. graminis reproduces sexually and produces overwintering teliospores, the diversity of pathotypes decreased significantly. Similarly, the diversity of pathotypes west of the Rocky Mountains in the USA, where the barberry was not eradicated, was significantly greater than in areas to the east where barberries were eradicated (Roelfs and Groth, 1980). The population structure of this pathogen was significantly altered such that it is now almost exclusively asexual with few clones (Burdon and Roelfs, 1985). Recombination and multilocus population structure are highly relevant to resistance gene deployment. For example, non-random associations among avirulence alleles in P. gruminis f. sp. tritici sparked a debate about the causes and significance of gametic disequilibrium. Vanderplank (1982) speculated that non-random associations were caused by selection against particular combinations of avirulence alleles. The implication of such selection is that certain combinations of resistance genes would be durable because compatible pathotypes would have reduced fitness. However, a simpler explanation for the observed gametic disequilibrium in populations of P. gruminis f. sp. tritici is clonal population structure (Knott, 1986). This explanation has fundamentally different implications for resistance gene deployment, since cultivars with particular resistance gene combinations would be vulnerable to compatible pathotypes if they occurred in the population, for example by immigration or mutation. The importance of gametic disequilibrium and gene deployment has been studied in much detail for barley powdery mildew, Erysiphe gruminis f. sp. hordei. Hovmcdller and 0stergbrd (1991) and Hovmdler et uf. (1993) demonstrated both positive and negative associations among avirulence alleles, depending on which resistance genes occurred together in common barley cultivars; their findings were similar to theoretical predictions for gametic disequilibrium (0stergird and Hovm~ller,1991). However, they also found that selection was not entirely predictable because of hitch-hiking selection, in which the frequency of avirulence alleles not under selection
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increased (or decreased) as a result of selection for other avirulence alleles in clonal populations. Brown (1995) modelled gametic disequilibrium among alleles at different avirulence loci in relation to the extent of recombination occurring and initial genotype distributions, and showed that hitch-hiking selection is difficult to predict in asexually reproducing populations. The dynamics of gametic disequilibria among avirulence alleles offers promise for understanding and improving resistance gene deployment in response to knowledge of pathotype distributions in populations. A unique example of disease management that required a thorough understanding of recombination was described by Crute (1989) for lettuce downy mildew, caused by Bremia Zacfucae. During the 1980s, resistance to the fungicide metalaxyl occurred in B. lucfucae in the UK. However, metalaxyl resistance initially arose only in a single clone, with the B2 mating type, which was avirulent on cultivars with the D m l l resistance gene. In order for metalaxyl resistance genes to be recombined into pathotypes virulent on Dmll cultivars, resistant and sensitive isolates had to occur on the same cultivar to mate successfully. Mating between resistant and sensitive isolates could only occur on cultivars that lacked D m l l (to allow metalaxylresistant isolates to colonize) and were not treated with metalaxyl (to allow metalaxyl-sensitive isolates to colonize). Therefore, to prevent recombination, growers were recommended to apply metalaxyl to cultivars lacking D m l l , even though control against the resistant isolates would be ineffective. Although this strategy was short lived due to the appearance of metalaxyl resistance in other pathotypes (Crute, 1992), it provides insight into the direct integration of population genetics and disease management.
E. POPULATION CHARACTERISTICS VARY AMONG POPULATIONS
It is important to recognize that different populations of the same species sometimes have completely different population structures. This concept is important because management strategies applicable to one population may not apply to other populations. Unfortunately, this means that studies need to be replicated in several different populations in order to understand the range of variation that occurs within a species. Populations may vary in allele frequencies, or their overall genetic structures may be completely different. Differences in allele frequencies may result from restricted gene flow and genetic drift (Goodwin et al., 1993; Peever and Milgroom, 1994b; Milgroom and Lipari, 1995), founder effects (Milgroom et uZ., 1992b) or selection (Kolmer, 1989; Wolfe and McDermott, 1994). The two case studies discussed below (section IV) demonstrate some of the management implications of different allele frequencies among populations. Populations may also differ in the extent to which recombination occurs,
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affecting the multilocus structure of populations. There are several striking examples of both clonal and sexual population structure in the same species: Puccinia graminis f. sp. tritici (Roelfs and Groth, 1980), Magnaporthe grisea (Kumar et al., 1995), Ophiostoma novo-ulmi (Brasier, 1988), Phytophthora infestans (Goodwin et al., 1992b; Drenth et al., 1994) and Cryphonectria parasitica (Liu et a f . , 1996). Other examples are likely to emerge as more investigations are done.
IV. EXAMPLES OF THE INTEGRATION OF POPULATION GENETICS AND EPIDEMIOLOGY A. FUNGICIDE RESISTANCE IN PYRENOPHORA TERES
Population genetics has made a significant contribution to managing resistance to sterol biosynthesis-inhibiting fungicides (SBIs) in Pyrenophora teres, which causes net blotch of barley (Peever and Milgroom, 1992, 1993, 1994a,b). The questions addressed in this system were familiar in fungicide resistance management: (1) is there cross-resistance among chemically related fungicides, and (2) are there fitness costs associated with fungicide resistance? Approaching these questions from the perspective of population and ecological genetics yielded some useful information about population structure and disease management. The approach used to address questions of cross-resistance and fitness costs was to sample discrete populations of P. teres. Samples of 22-35 isolates were taken from each of four populations in North America and one in Germany; SBIs had been used previously for disease control in only two of the five populations. The growth rate of each isolate was measured in vitro in the presence of five different SBIs. There was significant variation in SBI resistance within populations, indicating that SBI applications would potentially result in selection for higher frequencies of resistant phenotypes. Cross-resistance among different SBIs was analysed in terms of genetic correlations, to determine whether the same genes controlled resistance to different SBIs (Peever and Milgroom, 1993). Resistance to many pairs of SBIs was correlated in some populations, but not in others. In other words, there were different cross-resistance relationships in different populations. A similar approach to estimating genetic correlations was used to determine if genes that control SBI resistance also affect fitness (Peever and Milgroom, 1994a). Latent period and sporulation were estimated in isolates from two populations. However, there was no correlation between SBI resistance phenotype and fitness. These results must be interpreted somewhat cautiously, as it is difficult to make inferences from greenhouse studies to fitness in natural populations. In addition to studying SBI resistance and fitness, Peever and Milgroom
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(1994b) also analysed population structure using RAPD markers in the same samples. The diversity of RAPDs was not correlated with variation in SBI resistance between populations, i.e. variation in neutral genetic markers was not a good predictor of ecologically important variation for these populations. In addition, there was no discernible association between RAPDs and SBI resistance within populations. Analysis of the multilocus genetic structure revealed that four of five populations did not deviate from what was expected under random mating. Therefore, recombination may be sufficient to uncouple selection for SBI resistance from neutral genetic markers (contrast this with the Phytophthoru infestans example below; section IVB). The analysis of population structure among Pyrenophora teres populations using RAPDs showed moderately high genetic subdivision, with 33-46% of the variation attributable to differences among populations (Peever and Milgroom, 1994b). Genetic subdivision is most likely a result of restricted gene flow among populations; therefore, each population may have to be considered individually in terms of fungicide resistance management. Interestingly, when phenotypic variation in fungicide resistance was partitioned within and among populations, the result was similar to that found with RAPDs: 38% of the variation was due to differences among populations. The similar degree of genetic differentiation for both ecologically important and selectively neutral traits suggests that selection has not strongly affected population structure (Spitze, 1993), even though SBIs had been applied occasionally in two of the populations studied. A result that merits closer examination is the finding that different populations had different cross-resistance relationships (Peever and Milgroom, 1993). The cause of these differences is not understood entirely, but the simplest explanations are that there are different alleles conferring SBI resistance, or allele frequencies are different in each population. An alternate hypothesis is that fungicide resistance genes are non-randomly associated. However, the lack of gametic disequilibrium among RAPD loci suggests that populations may experience a significant amount of recombination. In addition, selection from fungicide use appears minimal since only two populations had experienced these fungicides prior to sampling, and, as shown above, differentiation among populations was similar for both RAPDs and resistance phenotypes. These studies have some clear lessons for SBI resistance management in P. teres. First, because of significant phenotypic variation in SBI resistance in each population, it can be predicted that resistance would evolve if fungicides were applied intensely; and, because there are no detectable fitness costs associated with SBI resistance in P. teres, the frequencies of resistant phenotypes would not necessarily decline if fungicide use were subsequently discontinued. Second, differences in cross-resistance among populations mean that SBI resistance has to be managed independently in each population; this conclusion is further supported by strong genetic differentia-
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tion among subpopulations. Third, by assaying for cross-resistance in different populations, we can predict which combinations of fungicides would be effective in each population. Fourth, differences in allele frequencies for SBI resistance may strongly affect genetic correlations for cross-resistance. Therefore, no single model of cross-resistance among SBIs is appropriate for P. teres. Whether similar conclusions apply to other populations of P. teres, or to different species, awaits further studies of this type. An important feature of this research is the sampling and, therefore, the populations to which inferences can be made. Inferences that can be made from the population approach are simple to understand and relevant for management. B.
EPIDEMIOLOGY AND POPULATIONS GENETICS OF PHYTOPHTHORA INFESTAN S
Recent studies of Phytophthora infestans have revealed unexpected population structure that in turn has led to hypotheses concerning migrations, and insights into management of late blight. The initial motivation for studying the population genetics of P. infestans came from disease management problems in Europe in the early 1980s. At that time, there were two factors that signalled some unexpected variation in the pathogen population: disease control failures caused by metalaxyl resistance (Davidse et al., 1981), and the detection of A2 mating types (Hohl and Iselin, 1984). In addition, there was a profound lack of knowledge concerning geographic distribution, population structure, and the evolutionary processes shaping populations of f. infestam. Population genetic studies in North America have provided the bases for short-term management strategies, and may eventually lead to long-term management strategies. 1. Population structure
Prior to the 1980s there were probably three geographically defined populations around the world. First, the source population in central'Mexico is unique among P. infestans populations. This population has characteristics of a randomly mating sexual population (Tooley ef al., 1985; Goodwin et al., 1992b): approximately equal frequencies of A1 and A2 mating types (Tooley et ul., 1985), allozyme genotypes in Hardy-Weinberg equilibrium (Goodwin et al., 1992b), and a huge diversity of multilocus genotypes in any small geographic area (e.g. a single field) (Matuszak et al., 1990). Second, populations in the USA and Canada were derived from the population in Mexico, and were asexual, but with several clonal lineages. Third, on other continents, populations were dominated by a single clonal lineage (termed US-l), with most genetic diversity caused by mutation or mitotic recombination within that lineage (Goodwin et al., 1994b). The hypothesized pathways of migrations of f. infestans were recently summarized (Fry et al., 1993).
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Recent immigration of P. infestans into the USA and Canada illustrates the importance of population genetics to epidemiology and management. A significant migration occurred into the USA and Canada, probably from northern Mexico beginning in the late 1970s, but became most noticeable in the early 1990s (Goodwin et al., 1994a, 1995b). As in Europe, these migrations were first signalled by metalaxyl resistance (Deahl et al., 1993) and then by detection of individuals with the A2 mating type (Deahl et al., 1995; Goodwin et al., 1995b). In the early 1990s, populations in the USA and Canada were dominated by four clonal lineages: the resident lineage (US-l), and three recent immigrant lineages (US-6, US-7 and US-8) (Goodwin et al., 1994a, 1995b). The recent immigrant lineages represented A1 (US-6) and A2 (US-7 and US-8) mating types; all were resistant to metalaxyl (Fry et al., 1993). In addition, in most locations, the individuals in a single field and often over a vast region were in the same lineage (Goodwin et al., 1994a, 1995b). Analysis of pathotypes has generally not been as useful as analysis of neutral markers in deciphering relationships among populations. Individuals sampled in Mexico generally represent complex pathotypes, which were virulent on cultivars with multiple specific resistance genes (Tooley et al., 1985; Rivera-Pena and Molina-Galan, 1989). In addition, clonal lineages found in the USA and Canada that were recently derived from Mexico had greater pathotype complexity than resident pathotypes (Goodwin et al., 199%). Selection for complex pathotypes may be important in some locations, e.g. Mexico, but in the USA and Canada most potato cultivars lack specific resistance. In the absence of selection, rare pathotypes (and virulence factors) are likely to be lost because of genetic drift: relatively small numbers of individuals survive over winter in asexual populations, i.e. there are annual bottlenecks (Goodwin et al., 1995~).Further confounding our understanding of pathotypes, it has also been shown that pathotype diversity can develop rapidly within a clonal lineage (Goodwin et al., 199%). 2. Late blight management The strong clonal population structure of P. infestans in the USA and Canada during the early 1990s has led to some interesting management options. Knowledge of the widespread occurrence of immigrant genotypes in 1994 stimulated plant pathologists to launch an intensive effort to educate growers and extension personnel concerning the attributes of immigrant genotypes. At the same time, plant pathologists began to petition for the use of fungicides that otherwise would not have been available. Convincing documentation that the significant late blight problems were caused by immigrant genotypes was instrumental in convincing growers and regulatory personnel that new measures were needed. These efforts helped avert major epidemics in 1995. Knowledge of genetic variation and population structure made it possible
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to develop rapid diagnostic tests to identify P. infestuns lineages. Because field populations usually consisted of a single clonal lineage, and because the four major lineages in the USA and Canada could be distinguished via allozymes (Gpi), a rapid assessment of the allozyme genotype (Goodwin et al., 1995a) enabled practitioners to learn (sometimes within hours) which lineage was causing the problem in a particular field. This was especially important in predicting the efficacy of metalaxyl. Metalaxyl was extremely effective against US-1, but was much less effective against the immigrant lineages US-6, US-7 and US-8. The observation that certain lineages appeared to be associated mainly with potatoes and that other lineages were associated with both potatoes and tomatoes motivated investigations of host specialization. It became clear that US-6 and US-7 were pathogenic on both tomatoes and potatoes, whereas US-8 was primarily a pathogen of potatoes (Legard et ul., 1995). Host specialization was polymorphic in US-1 (Legard et al., 1995). Because P. infestuns is aerially dispersed, tomato growers needed to know if late blight in nearby potato fields was caused by US-6 or US-7. If so, then the fungus in neighbouring fields posed a serious threat to tomatoes, and management efforts were needed. Variation in aggressiveness was another characteristic that has been important to late blight management. Because clonal lineages could be easily distinguished, it became possible to investigate whether the immigrant lineages posed a more significant threat than did the resident US-1. During 1994 and 1995, several investigators began testing the hypothesis that US-8 was more aggressive than US-1 on potatoes. If the immigrant lineages are more aggressive, an overall intensification of management efforts will be needed. In contrast to earlier populations where only A1 mating type individuals were present, the occurrence of both A1 and A2 individuals in a few fields signals the possibility of sexual reproduction. The presence of oospores could alter the life history of P. infestans in the USA and Canada, consequently altering the epidemiology of the disease. The potential for sexual reproduction has two significant implications for late blight management. First, oospores survive as dormant propagules in soil and may serve as a significant additional source of inoculum. Previously, the soil had not been a source of inoculum for this fungus; in its asexual phase P. infestuns is essentially an obligate parasite and cannot persist in the absence of living hosts. Second, recombinant progeny could result in greater genotypic diversity in the population of P. infestuns in the USA and Canada, destroying the simple clonal structure that exists. Recombination will eliminate the association between allozyme genotype and pathogenic traits, and destroy the potential for using neutral genetic markers to predict epidemiologically important traits. Knowledge that populations of P. infestuns in the USA and Canada are in flux signals the need for significant new investigations in epidemiology and
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management. Probably most urgent is the need to investigate the role of oospores in the epidemiology of potato and tomato late blight in the USA and Canada; whether oospores will contribute significantly to initial inoculum is now unknown. Quantification of the greater aggressiveness of recent immigrant genotypes should lead to appropriate intensifications of late blight management strategies. Some opportunities for adjustments in late blight management include development and use of more resistant plants and new chemistries of fungicides. Knowledge that a single clone can be broadly and rapidly dispersed emphasizes the need to determine factors affecting dispersal of P. infesruns and to determine if all genotypes are similarly capable of such dispersal.
V. FUTURE CONTRIBUTIONS OF POPULATION GENETICS TO PLANT PATHOLOGY Population genetics can enhance our understanding of pathogen biology and, therefore, has much to offer plant pathology. Although much of the recent research on population genetics of plant pathogens has involved the description of genetic variation at various scales, some research has progressed to asking questions about evolutionary processes. This is a critical step beyond the descriptive phase because it allows greater insight into pathogen evolution and biology. Like any basic science, the utility of population genetics in plant pathology may at first seem negligible. Rather than letting its fundamental nature deter us, we need to broaden our perspectives to examine how this information can be applied. The most significant applications of population genetics to epidemiology and disease management are likely to derive from practical needs to understand pathogen variation and evolution. Descriptive studies of variation without questions focused on evolutionary processes are not sufficient. Similarly, elegant evolutionary inferences made about pathogen populations for which there are few associated management problems contribute little to the integration of population genetics and disease management. To apply population genetics to epidemiology and disease management, the burden is on plant pathologists to increase their understanding of population genetics and evolution. This educational development is essential, and fortunately is well underway. Education on evolutionary concepts will enhance communication with geneticists and lead to a better integration of population genetics into plant pathology in the future.
ACKNOWLEDGEMENTS We thank Jim Anderson, Clive Brasier, Tom Gordon and Bob Marra for making helpful suggestions on an earlier draft of this chapter.
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REFERENCES Anderson, J . B. and Kohn, L. M. (1995). Clonality in soilborne, plant pathogenic fungi. Annual Review of Phytopathology 33, 369-391. Appel, D. J. and Gordon, T. R. (1994). Local and regional variation of Fusarium oxysporum from agricultural field soils. Phytopathology 84, 786-791. Brasier, C. M. (1988). Rapid changes in genetic structure of epidemic populations of Ophiostoma ulmi. Nature 332, 538-541. Brasier, C. M. (1992). A champion thallus. Nature 356, 382-383. Brasier, C. M. (1995). Episodic selection as a force in fungal microevolution with special reference to clonal speciation and hybrid introgression. Canadian Journal of Botany 73(Suppl. l ) , S1213-S1221. Brown. J. K . M. (1995). Recombination and selection in populations of plant pathogens. Plant Pathology 44, 279-293. Brown, J. K. M., Jessop, A. C. and Rezanoor, H. N. (1991). Genetic uniformity in barley and its powdery mildew pathogen. Proceedings of the Royal Society of London, Series B Biological Sciences 246, 83-90. Burdon, J . J. (1992). Host population subdivision and the genetic structure of natural pathogen populations. Advances in Plant Pathology 8, 81-94. Burdon, J. J. and Roelfs, A. P. (1985). Isozyme and virulence variation in asexually reproducing populations of Puccinia graminis and P. recondita on wheat. Phytopathology 75, 907-913. Burdon, J. J . , Marshall, D. R., Luig, N. H. and Gow, D. J. S. (1982). Isozyme studies on the origin and evolution of Puccinia graminis f. sp. tritici in Australia. Australian Journal of Biological Science 35, 231-238. Chen, R.-S., Boeger, J. M. and McDonald, B. A. (1994). Genetic stability in a population of a plant pathogenic fungus over time. Molecular Ecology 3, 209-2 18. Correll, J. C. (1991). The relationship between forma speciales, races, and vegetative compatibility groups in Fusarium oxysporum. Phytopathology 81, 1061-1064. Correll, J. C., Gordon, T. R. and McCain, A. H. (1992). Genetic diversity in California and Florida of the pitch canker fungus Fusarium subglutinans f. sp. pini. Phytopathology 82, 415-420. Crute, I. R. (1989). Lettuce downy mildew: a case study in integrated control. In “Plant Disease Epidemiology”, Vol. 2 (K. J . Leonard and W. E. Fry, eds), pp. 30-53. McGraw-Hill, New York. Crute, I. R. (1992). The role of resistance breeding in the integrated control of downy mildew, Bremia luctucae, in protected lettuce. Euphytica 63, 95-102. Davidse, L. C., Looijen, D., Turkensteen, L. J. and Van der Wal, D. (1981). Occurrence of metalaxyl-resistant strains of Phytophthora infestans in Dutch potato fields. Netherlands Journal of Plant Pathology 87, 65-68. Davis, J . M. and Main, C . E. (1988). Applying atmospheric trajectory analysis to problems in epidemiology. Plant Disease 70, 49W97. Deahl, K. L . , DeMuth, S. P., Sinden, S. L. and Rivera-Pena, A. (1995). Identification of mating types and metalaxyl resistance in North America populations of Phytophthora infestans. American Potato Journal 72, 35-49. Deahl, K. L., Inglis, D. A. and DeMuth, S. P. (1993). Testing for resistance to metalaxyl in Phytophthora infestans isolates from northwestern Washington. American Potato Journal 70, 779-795. De Waard, M. A. and Van Nistelrooy, J. G. M. (1990). Stepwise development of laboratory resistance to DMI-fungicides in Penicilliurn italicurn. Netherlands Journal of Plant Pathology 96, 321-329.
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Drenth, A., Tas, I. C. Q . and Govers, F. (1994). DNA fingerprinting uncovers a new sexually reproducing population of Phytophthora infestans in the Netherlands. European Journal of Plant Pathology 100, 97-107. Drenth, A., Janssen, E. M. and Govers, F. (1995). Formation and survival of oospores of Phytophthora infestans under natural conditions. Plant Pathology 44, 86-94. Ennos, R. A. and McConnell, K. C. (1995). Using genetic markers to investigate natural selection in fungal populations. Canadian Journal of Botany 73(Suppl. l), S302-S310. Fenner, F. and Myers, K. (1978). Myxoma virus and myxomatosis in retrospect: the first quarter century of a new disease. I n “Viruses and Environment”, 3rd International Conference on Comparative Virology, Mont Gabriel, Quebec (E. Kurstak and K. Maramorosch, eds), pp. 539-570. Academic Press, New York. Fisher, R. A. (1930). “The Genetical Theory of Natural Selection.” Clarendon Press, Oxford. Fry, W. E., Goodwin, S. B., Matuszak, J. M., Spielman, L. J., Milgroom, M. G. and Drenth, A. (1992). Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review of Phytopathology 30, 107-129. Fry, W. E., Goodwin, S. B., Dyer, A. T., Matuszak, J. M., Drenth, A., Tooley, P. W., Sujkowski, L. S . , Koh, Y. J., Cohen, B. A., Spielman, L. J., Deahl, K. L., Inglis, D. A. and Sandlan, K. P. (1993). Historical and recent migrations of Phytophthora infestans: chronoIogy, pathways, and implications. Plant Disease 17, 653-661. Goodwin, S. B., Allard, R. W., Hardy, S. A. and Webster, R. K. (1992a). Hierarchical structure of pathogenic variation among Rhynchosporium secalis populations in Idaho and Oregon. Canadian Journal of Botany 70, 810-817. Goodwin, S. B., Spielman, L. J., Matuszak, J. M., Bergeron, S. N. and Fry, W. E. (1992b). Clonal diversity and genetic differentiation of Phytophthora infesrans populations in northern and central Mexico. Phytopathology 82, 955-961. Goodwin, S. B., Saghai-Maroof, M. A., Allard, R. W. and Webster, R. K. (1993). Isozyme variation within and among populations of Rhynchosporium secalis in Europe, Australia and the United States. Mycological Research 97, 49-58. Goodwin, S. B., Cohen, B. A., Deahl, K. L. and Fry, W. E. (1994a). Migration from northern Mexico was the probable cause of recent genetic changes in populations of Phytophthora infestans in the United States and Canada. Phytopathology 84, 553-558. Goodwin, S. B., Cohen, B. A. and Fry, W. E. (1994b). Panglobal distribution of a single clonal lineage of the Irish potato famine fungus. Proceedings of the National Academy of Sciences, USA 91, 11591-11595. Goodwin, S. B., Schneider, R. E. and Fry, W. W. (1995a). Cellulose-acetate electrophoresis provides rapid identification of allozyme genotypes of Phytophthora infestans. Plant Disease 79, 1181-1185. Goodwin, S. B., Sujkowski, L. S., Dyer, A. T., Fry, B. A. and Fry, W. E. (1995b). Direct detection of gene flow and probable sexual reproduction of Phytophthora infestans in northern North America. Phytopathology 85, 473-479. Goodwin, S. B., Sujkowski, L. S. and Fry, W. E. (1995~).Rapid evolution of pathogenicity within clonal lineages of the potato late blight disease fungus. Phytopathology 85, 669-676. Gordon, T. R., Okamoto, D. and Milgroom, M. G. (1992). The structure and interrelationship of fungal populations in native and cultivated soils. Molecular Ecology 1, 241-249.
GENETICS AND PLANT DISEASE EPIDEMIOLOGY AND MANAGEMENT
27
Hohl, H. R. and Iselin, K. (1984). Strains of Phytophthora infestans from Switzerland with A2 mating type behaviour. Transactions of the British Mycological Society 83, 529-530. Hovmdler, M. S. and 0sterglrd, H. (1991). Gametic disequilibria between virulence genes in barley powdery mildew populations in relation to selection and recombination. 11. Danish observations. Plant Pathology 40, 178-189. Hovmdler, M. S., Munk, L. and Bsterglrd, H. (1993). Observed and predicted changes in virulence gene frequencies at 11loci in a local barley powdery mildew population. Phytopathology 83, 253-260. Huber, D. H. and Fulbright, D. W. (1994). Preliminary investigations on the effect of individual vic genes upon the transmission of dsRNA in Cryphonectria parasitica. In “Proceeding of the International Chestnut Conference”, 1&14 July 1992 (M. L. Double and W. L. MacDonald, eds), pp. 15-19. West Virginia University Press, Morgantown, VA. Kistler, H. C., Momol, E. A. and Benny, U. (1991). Repetitive genomic sequences for determining relatedness among strains of Fusarium oxysporum. PhytopatholOgy 81, 331-336. Knott, D. R. (1986). The genetic structure of populations of Puccinia graminis f. sp. tritici. Phytopathology 76, 1149-1 151. Koenraadt, H. and Jones, A. L. (1992). The use of allele-specific oligonucleotide probes to characterize resistance to benomyl in field strains of Venturia inaequalis. Phytopathology 82, 1354-1358. Koenraadt, H., Somerville, S. C. and Jones, A . L. (1992). Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. Phytopathology 82, 1348-1354. Kohli, Y., Moral], T. A. A., Anderson, J. B. and Kohn, L. M. (1992). Local and trans-Canadian clonal distribution of Sclerotinia sclerotiorum on canola. Phytopathology 82, 875-880. Kohn, L. M. (1995). The clonal dynamic in wild and agricultural plant pathogen populations. Canadian Journal of Botany 73(Suppl. l ) , S1231-S1240. Kohn, L. M., Stasovski, E., Carbone, I., Royer, J. and Anderson, J. B. (1991). Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum. Phytopathology 81, 480485. Koller, W., Smith, F. D., Reynolds, K. L., Wilcox, W. F. and Burr, J. A. (1995). Seasonal changes of sensitivities to sterol demethylation inhibitors in Venturia inaequalis populations. Mycological Research 99, 689-692. Kolmer, J. A. (1989). Virulence and race dynamics of Puccinia recondita f. sp. tritici in Canada during 1956-1987. Phytopathology 79, 349-356. Krebs, C. J. (1985). “Ecology”, 3rd edn. Harper & Row, New York. Kumar, J., Nelson, R. J. and Zeigler, R. S. (1995). Population analysis of Magnaporthe grisea at a high diversity site in the Himalayas. Phytopathology 85, 1132 (abstract). Lalancette, N., Jr, Russo, J. M. and Hickey, K. D. (1984). A simple device for sampling spores to monitor fungicide resistance in the field. Phytopathology 74, 1423-1425. Legard, D. E., Lee, T. Y. and Fry, W. E. (1995). Pathogenic specialization in Phytophthora infestans: aggressiveness on tomato. Phytopathology 85, 13561361. Leonard, K. J . (1978). Polymorphisms for lesion type, fungicide tolerance, and mating capacity in Cochliobolus carbonum isolates pathogenic to corn. Canadian Journal of Botany 56, 1809-1815. Leonard, K. J. and Fry, W. E. (eds) (1989). “Plant Disease Epidemiology: Genetics,
28
M. G . MILGROOM and W. E. FRY
Resistance and Management”, Vol. 2. McGraw-Hill, New York. Leslie, J. F. (1995). Gibberella fujikuroi: available populations and variable traits. Canadian Journal of Botany 73(Suppl. l), S282-S291. Leung, H. and Williams, P. H. (1986). Enzyme polymorphism and genetic differentiation among geographic isolates of the rice blast fungus. Phytopathology 76, 778-783. Leung, H., Nelson, R. J . and Leach, J. E. (1993). Population structure of plant pathogenic fungi and bacteria. Advances in Plant Pathology 10, 157-205. Levy, M., Correa-Victoria, F. J., Zeigler, R. S., Xu, S. and Hamer, J. E. (1993). Genetic diversity of the rice blast fungus in a disease nursery in Colombia. Phytopathology 83, 1427-1433. Levy, M . , Romao, J . , Marchetti, M. A. and Hamer, J . E. (1991). DNA fingerprinting with dispersed repeated sequence resolves pathotype diversity in the rice blast fungus. Plant Cell 3, 95-102. Liu, Y.-C. and Milgroom, M. G . (1996). Correlation between hypovirus transmission and the number of vegetative incompatibility (vic) genes different among isolates from a natural population of Cryphonectria parasitica. Phytopathology 86, 79-86. Liu, Y.-C., Cortesi, P., Double, M. L., MacDonald, W. L. and Milgroom, M. G. (1996). Diversity and multilocus genetic structure in populations of Cryphonectria parasitica. Phytopathology (in press). Matuszak, J . M., Goodwin, S. B., Fry, W. E. andvillarreal-Gonzalez, M. J. (1990). Changes in the genetic diversity of Phytophthora infestans during an epidemic in central Mexico as determined by DNA fingerprints. Phytopathology 80, 965 (abstract). McDermott, J. M. and McDonald, B. A. (1993). Gene flow in plant pathosystems. Annual Review of Phytopathology 31, 353-373. McDonald, B. A. and McDermott, J. M. (1993). Population genetics of plant pathogenic fungi. BioScience 43, 311-319. McDonald, B. A., Miles, J . , Nelson, L. R. and Pettway, R. E. (1994). Genetic variability in nuclear DNA in field populations of Stagonospora nodorum. Phytopathology 84, 250-255. McDonald, B. A., Pettway, R. E., Chen, R. S., Boeger, J . M. and Martinez, J. P. (1995). The population genetics of Septoria tritici (teleomorph Mycosphaerella graminicola). Canadian Journal of Botany 73(Suppl. l), S292-S301. Michelmore, R. W., Paran, I. and Kesseli, R. V. (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences, USA 88, 9828-9832. Milgroom, M. G. (1995a). Analysis of population structure in fungal plant pathogens. In “Disease Analysis through Genetics and Molecular Biology: Interdisciplinary Bridges to Improved Sorghum and Millet Crops” (J. F. Leslie and R. A. Frederiksen, eds), pp. 213-229. Iowa State University Press, Ames, IA. Milgroom, M. G. (1995b). Population biology of the chestnut blight fungus, Cryphonectria parasitica. Canadian Journal of Botany 73(Suppl. l), S311S319. Milgroom, M. G. (1996). Recombination and the multilocus structure of fungal populations. Annual Review of Phytopathology 34, 457477. Milgroom, M. G. and Lipari, S. E. (1995). Population differentiation in the chestnut blight fungus, Cryphonectria parasitica, in eastern North America. PhytopatholOgy 85, 155-160. Milgroom, M. G., Levin, S. A. and Fry, W. E. (1989). Population genetics theory
GENETICS AND PLANT DISEASE EPIDEMIOLOGY AND MANAGEMENT
29
and fungicide resistance. In “Plant Disease Epidemiology”, Vol. 2 (K. J . Leonard and W. E. Fry, eds), pp. 340-367. McGraw-Hill, New York. Milgroom, M. G . , Lipari, S. E. and Powell, W. A. (1992a). DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica. Genetics 131, 297-306. Milgroom, M. G., Lipari, S. E. and Wang, K. (1992b). Comparison of genetic diversity in the chestnut blight fungus, Cryphonectria (Endothia) parasitica from China and the US. Mycological Research 96, 1114-1120. Bsterglrd, H. and Hovmejller, M. S. (1991). Gametic disequilibria between virulence genes in barley powdery mildew populations in relation to selection and recombination. I. Models. Planr Pathology 40, 166-177. Peever, T. L. and Milgroom, M. G. (1992). Inheritance of triadimenol resistance in Pyrenophora teres. Phytopathology 82, 821-828. Peever, T. L. and Milgroom, M. G . (1993). Genetic correlations in resistance to sterol biosynthesis-inhibiting fungicides in Pyrenophora teres. Phytopathology 83, 1076-1082. Peever, T. L. and Milgroom, M. G. (1994a). Lack of correlation between fitness and resistance to sterol biosynthesis-inhibiting fungicides in Pyrenophora teres. Phytopathology 84, 5 15-5 19. Peever, T. L. and Milgroom, M. G. (1994b). Genetic structure of Pyrenophora teres populations determined with RAPD markers. Canadian Journal of Botany 72, 9 15-923. Pianka, E. R. (1988). “Evolutionary Ecology”, 4th edn. Harper & Row, New York. Polashock, J. J., Anagnostakis, S . L., Milgroom, M. G. and Hillman, B. I. (1994). Isolation and characterization of a virus-resistant mutant of the chestnut blight fungus. Current Genetics 26, 528-534. Puhalla, J . E. (1985). Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Canadian Journal of Botany 63, 179-183. Rivera-Pena, A . and Molina-Galan, J. (1989). Wild tuber-bearing species of Solanum and incidence of Phytophthora infestans (Mont.) de Bary on the western slopes of the volcano Nevado de Toluca. 1. Solanum species. Potato Research 32, 181-195. Roelfs, A. P. (1985). Epidemiology in North America. In “The Cereal Rusts”, Vol. 2 (W. R. Bushnell and A. P. Roelfs, eds), pp. 403-434. Academic Press, New York. Roelfs, A. P. and Groth, J. V. (1980). A comparison of virulence phenotypes in wheat stem rust populations reproducing sexually and asexually. Phytopathology 70, 855-862. Shah, D., Bergstrom, G. B. and Ueng, P. P. (1995). Initiation of Septoria nodorum blotch epidemics in winter wheat by seedborne Stagnospora nodorum. Phytopathotogy 85, 452-457. Shaw, M. W. and Royle, D. J. (1989). Airborne inoculum as a major source of Septoria tritici (Mycosphaerella graminicola) infections in winter wheat crops in the UK. Plant Pathology 38, 35-41. Sierotzki, H., Eggenschwiler, M., Boillat, O., McDermott, J. M. and Gessler, C. (1994). Detection of variation in virulence toward susceptible apple cultivars in natural populations of Venturia inaequalis. Phytopathology 84, 1000-1009. Slatkin, M. (1987). Gene flow and the geographic structure of natural populations. Science 236, 787-792. Smith, F. D., Parker, D. M. and Koller, W. (1991). Sensitivity distribution of Venturia inaequalis to the sterol demethylation inhibitor flusilazole: baseline sensitivity ~~
~
30
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and implications for resistance monitoring. Phytopathology 81, 392-396. Spitze, K. (1993). Population structure in Daphnia obtusa: quantitative genetic and allozyme variation. Genetics 135, 367-374. Stenlid, J. (1985). Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility, and isoenzyme patterns. Canadian Journal of Botany 63, 2268-2273. Sujkowski, L. S., Goodwin, S . B., Dyer, A. T. and Fry, W. E. (1994). Increased genotypic diversity via migration and possible occurrence of sexual reproduction of Phytophthora infestans in Poland. Phytopathology 84, 201-207. Tooley, P. W., Fry, W. E. and Villarreal Gonzalez, M. J. (1985). Isozyme characterization of sexual and asexual Phytophthora infestans populations. Journal of Heredity 76, 431-435. Vanderplank, J. E. (1963). “Plant Diseases: Epidemics and Control.” Academic Press, New York. Vanderplank, J. E. (1982). “Host-Pathogen Interactions in Plant Disease.” Academic Press, New York. Vilgalys, R. and Cubeta, M. A . (1994). Molecular systematics and population biology of Rhizoctonia. Annual Review of Phytopathology 32, 135-155. Webber, J. F., Brasier, C. M. and Mitchell, A. G. (1987). The role of the saprophytic phase in Dutch elm disease. In “Fungal Infection of Plants” (G. F. Pegg and P. G . Ayres, eds), pp. 298-313. Cambridge University Press, Cambridge. Wolfe, M. S. and Caten, C. E. (eds) (1987). “Populations of Plant Pathogens: Their Dynamics and Genetics.” Blackwell, Oxford. Wolfe, M. S. and Knott, D. R. (1982). Populations of plant pathogens: some constraints on analysis of variation in pathogenicity. Plant Pathology 31, 79-90. Wolfe, M. S. and McDermott, J. M. (1994). Population genetics of plant-pathogen interactions: the example of the Erysiphe graminis-Hordeum vulgare pathosystem. Annual Review of Phytopathology 32, 89-1 13. Zeigler, R. S . , Cuoc, L. X., Scott, R. P., Bernardo, M. A., Chen, D. H., Valent, B. and Nelson, R. J. (1995). The relationship between lineage and virulence in Pyricularia grisea in the Philippines. Phytopathofogy 85, 44-51.
A Molecular View Through the Looking Glass: the Pyrenopeziza brassicae-Brasska Interaction
A . M . ASHBY
Department of Plant Sciences. University of Cambridge. Downing Street. Cambridge CB2 3EA. UK
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I1 . The P. brassicae-Brassica Interaction ......................................... A . The Fungus ..................................................................... B . Pathogenesis .................................................................... C . Sexual Morphogenesis ....................................................... D . Disease Epidemiology .......................................................
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111. Molecular Techniques in the Analysis of the P . brassicae-Brassica Interaction .............................................................................
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I . Introduction
IV . Molecular Analysis of Pathogenesis ........................................... A . Surface Growth and Penetration: the Role of Cutinase .......... B . Subcuticular Growth: the Role of Protease ........................... C . Using Reporter Genes to Measure Fungal Biomass In Pfanra . . D . Proposed Role of Extracellular Protease in Pathogenicity ....... E . Implications for Disease Control .........................................
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V. Analysis of the Hemibiotrophic Phase: the Role of Cytokinins ....... A . Biochemical Analysis of Cytokinin Production by P . brassicae . B . Molecular Analysis of P . brassicae Cytokinins ....................... C . The Role of Cytokinins in Pathogenicity .............................. D . Implications for Disease Control .........................................
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VI . Analysis of Sexual Morphogenesis ............................................. A . Biochemical Analysis: Identification of a Post-Mating Factor ... B . Molecular Analysis ........................................................... C . Sexual Morphogenesis in P . brassicae: a Speculative Summary . D . Implications ....................................................................
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VII. A Molecular View through the Looking-glass: the P. brassicaeBrassica Interaction .................................................................
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VIII. Concluding Remarks ............................................................... Acknowledgements ................................................................. References ............................................................................
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I. INTRODUCTION Molecular biology has had a profound influence on all of the biological sciences. Over the last decade the use of molecular techniques in plant pathology has advanced to such a degree that we are now able to analyse complex plant-microbe interactions at the molecular level and to establish the role of certain genes and gene products in these interactions. This approach, encompassing biochemistry, molecular biology and conventional plant pathology, involves the dissection of an interaction into its component parts and investigates how the manipulation of these component parts may alter the interaction as “a whole”. This type of multidisciplinary approach will lead to great advances in our understanding of plant pathology in the decade to come. We are using such an approach to address three key questions concerning the Pyrenopeziza brassicae-Brassica interaction: 1. How does P. brassicae pathogenize Brassica species? 2. How does P. brassicae derive nutrients from host tissue during its biotrophic phase of growth? 3. What are the mechanisms controlling sexual morphogenesis in P. brassicae? By addressing and beginning to obtain answers to these questions through molecular analysis, a greater understanding of this fungus-host interaction will be realized, which will ultimately allow the development of novel strategies for controlling light leaf spot disease of brassicas. This review focuses initially on our understanding of the P. brassicae-Brassica interaction from a conventional plant pathological standpoint. Current progress on the use of molecular and biochemical techniques to analyse pathogenicity and sexual morphogenesis are then discussed and, finally, using the information gained from our molecular analyses together with a little speculation, a view of the interaction “through the molecular looking-glass’’ is proposed.
11. THE P. BRASSICAE-BRASSZCA INTERACTION A. THEFUNGUS
Pyrenopeziza brassicae Sutton and Rawlinson (anamorph Cylindrosporium concentricum) is a haploid, heterothallic discomycete and is the cause of light
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leaf spot disease of brassicas, which is considered one of the most damaging diseases of winter oilseed rape (Brassica napus L. subsp. ofeifera (Metzger) sink) in the UK (Lacey et al., 1987; Yarman and Giltrap, 1989; Hardwick et al., 1991). B . PATHOGENESIS
During pathogenesis the fungus displays a mode of nutrition characteristic of the hemibiotrophic plant pathogens within the Ascomycotina. After spore germination and initial infection (Fig. lA), a biotrophic phase of growth within the host is established. Here a relatively balanced physiological relationship exists between host and pathogen, and only limited visible signs of infection are evident (Courtice et a f . , 1988). These include thickening and stiffening of infected tissue and the formation of “green islands”, which result from the redirection of host nutrients to the site of infection leading to preferential retention of chlorophyll around the lesion as a consequence of growth substance imbalance (Maddock, 1979). During this early phase of development, the fungus obtains all its nutritional requirements from the plant, but little is known about how this is achieved. The first and most characteristic visible symptom of the disease is the formation of minute, snow-white spots erupting through the leaf surface. Each spot or conidiomata consists of numerous, unicellular, cylindrical spores, formed within the host in an acervulus beneath the cuticle (Fig. 1B,C). Sporulation ultimately results in rupture of the cuticle, with the lesion enlarging in a concentric fashion (Fig. 1D). The central chlorotic region becomes cracked and blistered, possibly as a result of toxin production by the fungus or simply as a result of separation of the cuticular layer from the upper epidermal membrane, rendering the lower surface susceptible to desiccation. In cases of severe infection the lesions will coalesce and infected areas may eventually wither and die. Asexual conidia are then dispersed to other susceptible hosts primarily by rain-splash, although atmospheric dispersal of conidia is possible (McCartney el al., 1986). C. SEXUAL MORPHOGENESIS
P. brussicae is heterothallic, having two mating types designated MAT 1-1 and MAT 1-2 (Mot et al., 1984). Reproduction occurs by two mutually exclusive pathways of development (Fig. 2). In the absence of a compatible mating type, spores germinate and differentiate by a process of enteroblastic conidiation to form asexual conidia (Fig. 1C). Conversely, in the presence of the opposite mating-type, an early interaction of opposite mating type hyphae results in suppression of asexual sporulation and initiation of a complex, co-ordinated, pathway of development culminating in the formation
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of apothecia (Fig. 3A-F). The fungus is competent to undergo sexual development during the first 6 days of conidial growth. After this period commitment to asexual sporulation is irreversibly established. Early interaction between opposite mating types is therefore required for successful induction of the sexual cycle, after which hyphae are destined to reproduce asexually (Illot, 1984; Siddiq, 1989; Ashby, unpublished observations). The production of fertile ascocarps in vitro was first described by Maddock and Ingram (1981), and subsequently the sexual system has been used to analyse fungicide-resistant, auxotrophic and pathogenicity mutants (Illot et al., 1987; Courtice et al., 1988; Ball et al., 1991a,b). The clear delineation of this state is now an important marker for a molecular analysis of sexual morphogenesis and pathogenicity in the fungus (Ball et al., 1991b; Ashby and Johnstone, 1994). The teleomorph of the fungus has been shown to occur naturally on oilseed rape in the UK and Ireland (Staunton and Kavanagh, 1966; Lacey et al., 1987; McCartney and Lacey, 1990) and is commonly found on vegetable brassicas in New Zealand (Cheah and Hartill, 1985). A survey of isolates of P . brassicae from 27 winter oilseed rape crops in East Anglia in 1987 revealed that both mating types were represented amongst the isolates from 74% of the fields tested, suggesting that there is a possibility that the teleomorphic stage may appear regularly (Ball et al., 1990). D. DISEASE EPIDEMIOLOGY
Oilseed rape is now a well-established and important “break crop”, and the increased acreage under cultivation may potentiaIly act as a source of wind-borne inoculum of light leaf spot disease of other brassica crops (Gladders, 1984). Most damage occurs after severe infection of new oilseed rape crops by P. brassicae in the autumn, although visible symptoms often go unnoticed until the following spring (Jeffery et al., 1989). Recent work by Figueroa et al. (1995) using a number of isolates of P. brassicae and a range of double-low (low erucic acid and low glucosinolate) cultivars of Brassica napus, demonstrated that low-temperature regimes increased both the incubation period (the time from inoculation until 50% of the lesions were produced) and the latent period (the time until the first lesion with
Fig. 1. Scanning electron micrographs illustrating the pathogenesis of P. brassicae. (A) Hyphae of P.brassicae JH26 (MAT 1-2) germinatingon a leaf surface and growing subcuticularly within B. napus (L. ssp. oleifera cv. Shogun). (B) An acervular conidioma rupturing the epidermis. (C) Enteroblastic conidiation (scale bar represents 10prn). (D) Typical symptoms induced in planta and signifying the late stages of infection; concentric rings of erumpant conidiornata surrounding a central necrotic lesion (scale bar represents 12mm). S. Batish, K . Johnstone and A . Ashby, unpublished observations.
36
A. M.ASHBY
Fig. 2. Mutually exclusive pathways of development in P. brussicue and the proposed role of sex factor (SF) in controlling these pathways. Open and closed conidia depict different mating types of the fungus. The sex factor inhibits asexual sporulation, resulting in all available resources being diverted into fuelling sexual morphogenesis. From Ashby and Johnstone (1994).
acervuli appeared) of infection and also decreased the rate at which conidia were able to germinate. The low temperatures prevailing during the winter months may therefore account for the decrease in the rate of progression of epidemics of light leaf spot disease on oilseed rape. However, it is equally probable that the incidence and severity of the disease during the winter months of some years may be sufficiently low to go unnoticed (Figueroa et al., 1995). The significance of light leaf spot is such that substantial reductions in green leaf area and plant dry weight at flowering were observed when fungicide applications were delayed, resulting in a 46% loss of seed yield (Jeffery et al., 1994).
Fig. 3. Sexual morphogenesis in P. brussicue. (A) Scanning electron micrograph (SEM) showing the interaction between JH26 (MATl-2) and NHlO (MAT1-1) after 4 days (scale bar represents 100pm). T. Cole and A. Ashby, unpublished observations. (B) Mycelia of a MAT 1-1GUS isolate (NHlOpNOM102/18) interacting with wild type MAT 1-2 (JH26) mycelia after 4 days on CMM medium stained with X-Gluc (scale bar represents 1 mm). Reproduced from Ashby and Johnstone (1994). (C) SEM showing a mature fertile apothecium from a cross of JH26 and NHlO (scale bar represents 100pm). T. Cole and A. Ashby, unpublished observations. (D) Crushed apothecium from a cross of a GUS transformant of P. brassicae MAT 1-2 (JH26pNOM102/4) and a wild-type MAT 1-1 isolate (NH10). The apothecia were stained with X-Gluc to show the differential contribution made by each mating type to fruiting body formation (scale bar represents 25 pm). Reproduced from Ashby and Johnstone (1993). (E, F) SEMs showing magnification of the apothecium to reveal the ectal explicium (E), paraphyses (P) and asci (A) (scale bar represents 10 pm). T. Cole and A. Ashby, unpublished observations.
P YRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
37
38
A. M. ASHBY
Annual crop losses in the UK have recently been estimated at over 25 million (Thomas and Walker, 1994). The disease has greatest impact in oilseed rape crops sown in consecutive years (Figueroa et al., 1994), which suggests that inoculum may be carried over from one season to the next on infected rape stubble as well as volunteer plants, probably as a result of overwintering by the teleomorphic stage (McCartney and Lacey, 1990). It has also been suggested that the release of ascospores from apothecia may provide an important inoculum source later in the season, reducing the efficacy of fungicide application (Figueroa et al., 1994). The occurrence of wind-borne ascospores sheds new light on disease dissemination, and, in particular, the fact that P. brassicae has, by this route, the potential to spread beyond the immediate vicinity of oilseed rape into other brassica crops, thus providing an effective mechanism for continuing infection from season to season. Occurrence of the sexual stage provides the opportunity for enhagced genetic variability which may affect both pathogenicity and the development of tolerance to fungicides. Such factors are likely to become more predominant as oilseed rape is exploited by more intensive agricultural practice (Maddock and Ingram, 1981). A survey carried out by Maddock et al. (1981) revealed considerable heterogeneity of resistance between a range of brassica varieties and subspecies. This was the case both between cultivars within these groups and between individuals of a single cultivar. Although differential interactions between cultivars and isolates were shown, they were not sufficiently defined to establish clear physiological races of the pathogen. It has been suggested that as agricultural exploitation of the host proceeds, as a result of breeding for resistance, P. brassicae may face more selection pressure than ever before and that by monitoring changes in the population of P. brassicae it may be possible to follow the evolution of a host-pathogen relationship from a state of relatively balanced coexistence (Maddock et al., 1981). To this end, Simons and Skidmore were able to demonstrate that a differential interaction between several host genotypes of Brassica oleracea and P. brassicae isolates can occur and that resistance to P. brassicae appears, in most cases, to be expressed as a dominant character (Simons and Skidmore, 1988). A survey of host-pathogen interactions is required, allowing a differential set of host cultivars to be defined for the identification of key virulence factors in P. brassicae.
111. MOLECULAR TECHNIQUES IN THE ANALYSIS OF THE P. BRASSICA E-BRASSICA INTERACTION Over the last decade a range of molecular techniques has been developed for analysing the P. brassicae interaction (Table I; Skidmore et al., 1984; Ashby and Johnstone, 1994). The extraction of high-molecular-weight DNA is an essential prerequisite for the cloning of fungal genes. Using the method of Raeder and Broda (1985) up to 1pg DNA per milligram of fungal tissue
TABLE I Molecular techniques developed to analyse the P. brassicae-Brassica interaction Molecular technique Generation of mutants DNA extraction Fungal transformation Co-transformation Reporter genes GUS Luciferase Genomic library complementation cDNA library construction Differential display PCR AFLP analysis
Status in P. brassicae research Auxotrophic Pathogenicity Developmental 1 p g per mg dry weight 2-200 transformants per pg DNA 70% co-transformation with pAN7-1 Mitotically stable GUS transformants
4 luciferase vectors for expression in fungi have been constructed Auxotrophic mutants Pathogenicity mutants Developmental mutants Sex factor induced in preparation Casein induced Technique currently being developed for P. brassicae Analysis of P. brassicae population substructure
Reference Courtice and Ingram (1987) Ball et al. (1991b) Siddiq et nl. (1992) Ball et al. (1991a) Ball et al. (1991a) Ashby and Johnstone (1993)
0
2
5 !2 ta
a
R
Ashby and Johnstone (1993) 7 M. Chadwick and A. Ashby (unpublished observations) Ball et al. (1991a) Ball et al. (1991b) Ashby and Johnstone (1994) M. Robb (unpublished observations) A. J. Clark (unpublished observations) ga A. J. Clark (unpublished observations) @
z R
3 2
Majer et a1 (1995)
E
40
A. M. ASHBY
with a molecular weight >50 kDa is obtained (Ball et al., 1991a). There are several vectors available for fungal transformation, but those most suitable for this system are the PAN series of vectors (Punt et al., 1987). Both PAN 7-1 and PAN 7-2 are shuttle vectors which encode ampicillin resistance for expression in Escherichia coli and carry the hygromycin B-phosphotransferase gene flanked by the gpd promoter and trp C terminator sequences from Aspergillus for expression in the fungus. Since P. brassicae is highly sensitive to hygromycin (growth is inhibited at concentrations above 5 pg ml-’) these vectors were useful in establishing a transformation system for this fungus. A third vector, pNOM102 (Roberts et al., 1989) based on the PAN vectors but containing a P-glucuronidase gene under the control of a constitutive promoter, has been used as a reporter gene in P. brassicae (Ashby and Johnstone, 1993). A second reporter gene vector has been constructed based on “click beetle” luciferases (Chadwick and Ashby, unpublished observations). There are four luciferases, each emitting light at a different wavelength (Wood et al., 1989). Each of the four click beetle cDNAs was cloned into pAN52-1 (Punt et al., 1987) and they are currently being evaluated as reporter genes in P. brassicae and other fungi. Using such reporter genes in combination will enable both spatial and temporal expression of a number of fungal genes to be analysed at any one time. Fungal protoplasts can be prepared by a method based on that of Yelton et al. (1984), and are transformed using a modified method of Vollmer and Yanofsky (1986), as described by Ball et al. (1991a). Transformation frequencies in the region of 50 transformants per microgram of transforming DNA are routinely achieved. A genomic library has been constructed in pAN7-2 and used successfully in transformation experiments to complement auxotrophic mutants to restored prototrophy (Ball et al., 1991a), a protease positive pathogenic phenotype to a protease negative non-pathogenic m:itant (Ball et al., 1991b; see section IVB) and a sex factor responsive fertile phenotype to a sexual development mutant (Ashby and Johnstone, 1994; see section VIB). cDNA libraries have also been constructed (M. Robb and A. Clark, unpublished observations; Table I). Recently, the technique of amplified fragment length polymorphism (AFLP) analysis has been developed to study both P. brassicae population substructure and the evolution of a relationship between P. brassicae and B . napus (Majer et al., 1995).
IV. MOLECULAR ANALYSIS OF PATHOGENESIS With a view to understanding the complexities of plant-microbe interactions, the “molecular dialogue” between pathogens and their hosts has been intensively studied over the last decade. For biotrophic and hemibiotrophic plant pathogens, a number of criteria have to be met to establish successful pathogenesis. Pathogenicity factors must be expressed to enable penetration of host tissue by mechanical or enzymatic degradation and to allow the
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
41
ingressing pathogen to access nutrients from the host without initiating any visible disease symptoms. The successful biotrophic pathogen must also resist activating host defence mechanisms which may result in a hypersensitive reaction (HR), thereby culminating in localized cell death and the consequent localization of the pathogen. Since P. brassicae lacks a clear race structure and no differential interaction with the host is evident, the fungus does not appear to elicit an H R response. The fungus does, however, appear enzymatically to degrade the cuticle prior to ingress (Maddock, 1979), and recently, and in view of the controversy surrounding the role of cutinase in the pathogenicity of Fusarium solani f. sp. pisi on pea (Stahl and Schifer, 1992; Rogers et al., 1994), we have begun to analyse the role of an extracellular cutinase in penetration of B. napus by P. brassicae. The importance of cell-wall-degrading enzymes in pathogenesis is well documented (Cooper, 1984). An initial strategy to look for such determinants in the P. brassicae interaction involved screening UV mutants of P. brassicae for deficiencies in cell-wall-degrading enzyme production. Mutants that were deficient in endopolygalacturonase (EndoPG), pectin methyl esterase (PME) and protease production in vitro were also found to be non-pathogenic in detached cotyledon tests (Ball et al., 1991b). Our research has focused primarily on the role of the protease in pathogenicity (see sections IVB and IVD).
A . SURFACE GROWTH AND PENETRATION: THE ROLE OF CUTINASE
Suspensions of conidia of P. brassicae artificially inoculated onto a leaf surface tend to be deposited preferentially over the anticlinal walls of epidermal cells (Rawlinson et al. , 1978). Upon germination conidia become septate forming relatively short germ tubes, which may swell slightly at the apex prior to penetration. Appressoria are never formed and hyphae d o not enter through stomata but directly penetrate through the cuticle. From microscopical analysis showing sites of pathogen ingress it appears that enzymatic degradation of the cuticle may be important in facilitating penetration (Maddock, 1979). P. brassicae has been shown to produce methyl esterase activity (Ball, 1989) and quantification of enzyme production was obtained by hydrolysis of p-nitrophenylbutyrate. Degradation of tritiated cutin confirmed that this activity was consistent with that of a cutinase (K. Davies, unpublished observations). The cutinase was also inhibited by PMSF, and ebelactones A and B (A. Jones and K. Davies, unpublished observations). Identification of the cutinase gene(s) by heterologous probing and polymerase chain reaction (PCR) is currently ongoing within the laboratory and our long-term aim is to perform gene knockout experiments on the P. brassicaecutinase(s) to assess the role played by cutinase in fungal penetration of brassica species.
42
A . M. ASHBY B. SUBCUTICULAR GROWTH: THE ROLE OF PROTEASE
Upon penetration, the fungus forms a hypomycelium of long thin septate infection hyphae which extend through the subcuticular space between the cuticle and walls of the epidermal cells (Rawlinson et a f . , 1978; Maddock, 1979). The hyphae then branch and proliferate below the cuticle to form a mycelial plate, beneath which hyphae begin to penetrate into leaf tissue along anticlinal walls and between cells of the upper mesophyll, but never through into cell lumina (Rawlinson et al., 1978; Maddock, 1979). The mechanisms by which the fungus is able to occupy space beneath the cuticle and to derive nutrients from within the plant are still unknown, and are therefore of great scientific interest. A UV mutant, designated NHlO 247, was found to be deficient in its ability to produce an extracellular protease and was also found to be nonpathogenic in a cotyledon-based pathogenicity test (Ball et al., 1991b, 1992). When crossed with the opposite wild type mating type, both protease and pathogenicity phenotypes co-segregated, suggesting that protease is a determinant of pathogenicity or that both protease and pathogenicity genes are closely linked. The resulting protease minus pathogenicity minus progeny from the cross (NH10 247:JH26; Ball et al., 1991b) were functionally complemented by a single integration event from a bulk library transformation (Fig. 4) and cloned sequences resulting in the acquired phenotype were recovered by the technique of cosmid rescue (Perucho et al., 1980). Approximately 4.5 kb of genomic DNA which flanked the original cosmid insert in NHlO 247:JH26 T was rescued in pAN7-2 and designated pPROTl (Fig. 5A). PCR using a primer reverse translated from the N-terminal amino acid sequence and a primer flanking the 4.5 kb insert in pPROTl gave a unique product (Fig. 5B,C), suggesting that the N-terminus of protease was located on the 4.5 kb fragment from pPROT1. Sequencing identified the unique ClaI site; however, analysis of open-reading frames (ORFs) on either side revealed no ORF with significant sequence homology to proteases. When pPROTl was transformed into P. brassicae, 40% of the resulting transformants displayed a loss of protease and pathogenicity phenotypes. Fig. 4. Complementation of the protease minus pathogenicity minus mutant by bulk cosmid library transformation. (A) Proteolytic transformant recovered from transformation of P. brussicue NH10247:JH26 (MAT 1-2) with a bulk cosmid library. A cleared zone of enzyme activity is evident around the complemented transformant (arrowed) (scale bar represents 2 cm). (B) Southern blot of NH10247:JH26T (MAT 1-2). Genomic DNA restricted with ClaI (which does not cut within the vector pAN7-2, track 1) and restricted with EcoRV (which linearizes pAN7-2, track 2) was probed with radiolabelled pAN7-2. (C) Symptoms induced by the protease transformant NH10247:JH26T (MAT 1-2) in a detached cotyledon test. Small white conidiomata are visible on the cotyledon (scale bar represents 2 mm). Reproduced with permission from Ball et al. (1991b).
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
43
44
A.
M. ASHBY
Barn HI
Fig. 5. Molecular analysis of the functionally complemented transformant, NH10247:JH26T which displayed a restored protease and pathogenicity phenotype. (A) pAN7-2 with 4.5 kb of rescued flanking sequence extending to the first ClaI site on either side of the original insert in NH10247:JH26T and designated pPROTl. (B) The unique product derived from a PCR reaction using the N-terminal protease primer and a primer flanking the 4.5kb insert in pPROTl. A. Ashby, unpublished observations.
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
45
Similarly, all subcloned fragments were able to elicit some effect on protease production by the fungus. It is possible that the pPROTl fragments rescued from the transformant contain regulatory sequences, rather than encoding the protease structural gene. Fragments from either side of the ClaI site are currently being used to chromosome walk through the genomic library of P. brussicue and 10 putative positive genomic clones are currently being analysed (A. Hunter, A. Clark and A. Ashby, unpublished observations). The extracellular protease was purified by FPLC separation and a biochemical profile of the protease obtained as well as sequence from its N-terminus (Batish, 1992). Biochemical analysis of the purified protease showed a pH optimum of 8.0, temperature optimum of 40°C and 100% inhibition with HgC12 confirming the findings of Ball et ul. (1991b) using a crude preparation. However, further biochemical analysis and information from the N-terminal amino acid sequence suggests that the protease is likely to belong to the serine family of proteases rather than cysteine (S. Batish, A. Clark, A. Hunter, A. Ashby and K. Johnstone, unpublished observations). A parallel approach has involved the construction of a cDNA library to casein-induced mycelium. The primer derived from the N-terminus was used in a PCR reaction against messenger RNA from induced mycelium to generate a unique RT-PCR product as a probe for library screening. cDNA clones are currently being analysed (A. Clark and A. Hunter, unpublished observations). C . USING REPORTER GENES TO MEASURE FUNGAL BIOMASS IN PLANTA
Reporter genes such as P-galactosidase, P-glucuronidase (GUS) and luciferase are widely used in plant and bacterial systems, but are relatively new tools for use in fungi. The enzymes encoded by the reporter genes hydrolyse specific chromogenic and fluorogenic substrates, liberating a specific colour or fluorescence indicative of reporter gene activity. Co-transformation of P. brussicae isolates NHlO (MAT 1-1)and JH26 (MAT 1-2) with pAN7-1 (for hygromycin selection; Punt et ul., 1987) and the constitutively expressing GUS vector pNOM102 (Roberts et ul., 1989) resulted in a range of transformants of both mating types expressing GUS activity (Ashby and Johnstone, 1993). One transformant, designated JH26pNOM102/13, was also found to be non-pathogenic and deficient in its ability to produce extracellular protease in vitro. In pluntu studies using the GUS expressing, protease minus, pathogenicity minus mutant and a wild-type GUS expressing isolate revealed that, although penetration and growth of infection hyphae were achieved in both cases, the mutant was unable to build up sufficient biomass to elicit the formation of acervular conidiomata and, therefore, visual symptoms of disease progression (Fig. 6).
C
0
2
4
6
8
10
12
Days after inoarlaton
Fig. 6. Thin sections through detached cotyledons of B . napus L. ssp. oleifera cv. Shogun showing the extent of fungal biomass after 15 days. (A) A MAT1-1 GUS expressing wild-type P. brassicae (NH10 pNOM102/18) (scale bar represents 150 Wm). (B) The MAT 1-2 protease minus pathogenicity minus GUS expressing isolate (JH26 pNOM102/13) (scale bar represents 150 pm). (C) Quantification of fungal biomass from NHlO pNOM 102/18 (MAT 1-1) and JH26 pNOM102/13 (MAT 1-2) using GUS expression mutant JH26 pNOM102/13. S. Batish, K . Johnstone and as a direct measurement: (0)wild-type NHlO pNOM102/18; (.)protease A. Ashby, unpublished observations.
14
16
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
47
D. PROPOSED ROLE OF EXTRACELLULAR PROTEASE IN PATHOGENICITY
From our findings one might expect that a key role for protease is the degradation of matrix glycoproteins which decrease the association between cells of the epidermis, facilitating growth of fungal mycelium both subcuticularly and, eventually, between the cells of the mesophyll. In support of this hypothesis, we have recently identified a brassica glycoprotein that is preferentially degraded by the protease ( A . Clark, A. Hunter, A . Ashby and K. Johnstone, unpublished observations). Secondly, localized degradation may result in release of nutrients from the apoplast, thus allowing the fungus to establish sufficient biomass prior to sporulation. There are other possible roles for a protease, however, including degradation of host cell matrices during the necrotrophic and saprophytic phase of growth and the degradation of host pathogenesis-related proteins. E.
IMPLICATIONS FOR DISEASE CONTROL
Establishing that cutinase is required for penetration of the cuticle of brassicas may allow the formulation of a number of strategies to effect disease control. These may include the spraying of ebelactones or other cutinase inhibitors on brassica crops at the beginning of the growing season or the use of genetically manipulated brassicas which express a cutinase inhibitor constitutively throughout their developmental cycle. Equally, cultivars with thicker cuticles could be generated transgenically or by conventional plant-breeding methods. Subcuticular growth of the fungus may be limited by expression of protease inhibitors in plants. Equally, once the natural substrate of the protease is established, subtle modification may prevent proteolytic cleavage, thereby reducing the space available for fungal growth in pluntu. Both strategies might be easily implemented through brassica transformation.
V. ANALYSIS OF THE HEMIBIOTROPHIC PHASE: THE ROLE OF CYTOKININS One of the major gaps in our current knowledge of plant pathology is an understanding of the physiological mechanisms of biotrophy and hemibiotrophy in fungal plant pathogens (Brian, 1967). In particular, several key questions remain unanswered, namely: how this delicate state of co-existence is maintained, what differentiates it from the necrotrophic mode of nutrition and, in a broader context, how these interactions relate to host specificity and disease resistance. There is substantial evidence that plant-growth regulators play a role in bacterial and fungal diseases of plants, including abnormal plant growth as
48
A . M. ASHBY
a result of infection, for example in formation of galls, hypertrophies, stem elongation and premature senescence. In addition, it has been established that many plant pathogenic micro-organisms are able to synthesize plantgrowth regulators including auxins and cytokinins (Greene, 1980). Cytokinins are N-6 substituted derivatives of adenine and in healthy plants are synthesized in the roots and transported to other regions of the plant through the xylem. They are responsible for stimulating metabolism and transportation of nutrients concomitant with a general stimulation of metabolic activity and cell division. In pathogenic interactions where hormonal imbalance does not result in prolific cell division in the host, but where the host is metabolically responsive to changes in endogenous levels of growth regulators, fungal cytokinins could function as key determinants of pathogenicity by increasing host metabolism, diverting nutrients and retaining chlorophyll to establish localized “metabolic sinks”, thus reducing the expression of senescence symptoms (Elstner, 1983). Fungal cytokinins may by themselves, or through the activation of enzymes such as superoxide dismutase, catalase and peroxidase, defend against the deleterious effects of free radicals, scavenging these highly active oxygen species, and therefore limit the processes which collectively contribute to the HR (Elstner, 1983; Beckman, 1990). A classic symptom of biotrophic rust infections is the presence of “green islands” around the point of infection which have been attributed to an imbalance in plant-growth regulators, caused directly or indirectly by the pathogen. In addition, aqueous extracts have been taken from spores of several fungi and have been shown to induce “green island” effects, suggesting that cytokinin-like molecules can be produced by both host and pathogen (Sequeira, 1973; Moore, 1979; Skoog, 1980). In the P. brassicae-Brussica interaction, “green islands” are often observed during the early stages of infection, indicative of plant-growth regulator imbalance (Maddock, 1979). This effect may be a result of either overproduction of cytokinins by the host in response to pathogen ingress, or synthesis of fungal cytokinins. There are a number of disparate reports in the literature on the ability of plant pathogenic fungi and mycorrhiza to synthesize cytokinins in vitro (Laloue and Hall, 1973; Miura and Hall, 1973; Mills and Van Staden, 1978; Kraigher et al., 1991), but definitive evidence for a role for fungalderived cytokinins in these interactions is yet to be established. Since both plant and pathogen may be capable of synthesizing plant-growth regulators and can influence each other’s ability to produce plant-growth regulators, it is experimentally difficult to identify the contribution of each partner to this phenomenon. In the cases of Agrobacterium turnefuciens and Pseudomonas savastanoi there is direct molecular evidence that bacterial genes are required for the synthesis of plant-growth regulators, and mutation of these genes either causes loss of or a change in disease symptoms (Nester and Gordon, 1991; Surico and Iacobellis, 1992). In order for the role of pathogen-derived plant-growth regulators to be
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
49
definitively established in a plant-microbe interaction, it is essential to produce direct molecular evidence. This is best provided by insertional inactivation of gene(s) essential for biosynthesis of the pathogen-derived plant-growth regulators and comparing disease symptoms of mutants with near-isogenic counterparts. To date, no such molecular evidence has been produced for a fungal plant pathogen. One of the major difficulties associated with the analyses of most obligate biotrophs is their dependence on the host for growth and development. Conveniently, P. brussicue is a hemibiotroph and can be cultured in vitro on nutrient medium. We are therefore well placed to test the hypothesis that cytokinins of fungal origin are responsible for maintaining the physiological balance between host and pathogen during the biotrophic phase of growth. This hypothesis draws together the ideas of early workers (Brian, 1967; Lewis, 1973) who, frustrated by the complexity of the interactions displayed by obligate parasites, were unable to investigate this phenomenon further. This source of control over plant metabolism maintained by the pathogen, if a widespread phenomenon, may be a fundamental determinant of biotrophy in plant-pathogen interactions and may be a significant contributing factor towards host specificity and disease resistance. A.
BIOCHEMICAL ANALYSIS OF CYTOKININ PRODUCTION BY P. BRASSICAE
Initial studies have shown that cytokinins are synthesized and exuded by P. brussicae during the early stages of its development, and cytokinin presence can be qualitatively visualized using a detached barley cotyledon assay (Fig. 7). A high-performance liquid chromatography (HPLC) enzyme-linked immunosorbent assay (ELISA) for cytokinins has been established (Huntley , 1995) and has been used both to analyse levels of cytokinins from aqueous spore washes of P. brassicae and to quantify the levels of cytokinins found in extracts of mycelium and culture filtrate from. P. brassicae grown under a range of different culture conditions. Cytokinins with cross-reactivity to zeatin riboside (ZR) antibodies were identified in spore washes from P. brussicae, and when grown under different culture conditions the fungus produced differing levels of predominantly ZR cross-reactive cytokinins, with most ZR being produced under conditions comparable to those experienced by the fungus in planta (Table 11; A. Murphy, K . Johnstone and A. Ashby, unpublished observations). B. MOLECULAR ANALYSIS OF P. BRASSICAE CYTOKININS
From comparison of deduced amino acid sequence homologies between the p t z gene from Pseudomonas savastanoi and the ipt and tzs genes from A .
50
A . M. ASHBY
Fig. 7. Detached barley leaf assay. “Green island” inducing activity from 5 PI of aqueous spore extracts from: (A) P. brassicae JH26 (MAT 1-2); (B) P. brassicae NHlO (MAT 1-1); (C) Venturin inaeqclalis (positive control); (D) Erysiphe graminis (positive control); (E, F) Water (control), spotted onto detached barley leaves. 5/11 of concentrated extract was equivalent to approximately lo5 spores (A. Murphy, K. Johnstone and A. Ashby, unpublished observations). (Scale bar represents 1 cm.)
TABLE 11 Cytokinin production (pmol gfwt-’) by P. brassicae isolate JH26 (MAT 1-2) Culture filtrate Medium Malt extract Murshige and Skoog
Mycelium
z type
IPA type
z type
IPA type
9.6 13.4
ND 19.4
1.8 118.0
-
+
+ +
t
+
+
+>-
+
+
+
+
+
+
+
i
+
P. P. P. P. P.
rapsicr cinnomomi eryrhroseprica infesrans megaspermu var. sojae P. palmivora
P. parasirica Pyrh. aphanidermarum Pyrh. proliferum Bremia lacrucae Pwudopernnnspora ruhenric Sclerospora graminicola
Williams and Webster, 1970 Hardham. 1987a. Hyde er nl . 1WIa Chapman and Vujicic, 1965 King er a / . , 1968; Elsner er al. , 1Y70 Ho er a / . . 1968 Hohl and Hamamoto, 1967; Bimpong and Hickman, 1975; Barlnicki-Garcia and Hemmes. 1976 Reichle, 1969 Grove and Bracker, 1978 Lunney and Bland, 1976a,b Sargent and Payne. 1974 Lange el a / . . lYX9 Lange er a / . . 1984
Pyrh. proliferum Bremia lacmcae Pseudoperonospora cubensis Sclerospora graminrcob
Williams and Webster, 1970 Hardham, 1987a; Hyde e r a / . , 1991a Chapman and Vujicic, 1965 Ehrlich and Ehrlich. 1966; King er a / . , 1968; Elsner ec a l . , 1970 Hohl and Hamamoto, 1967; Bimpong and Hickman, 1975; Bartnicki-Garcia and Hemmes, 1976 Ehrlich and Ehrlich. 1966 Grove and Bracker, 1978 Lunney and Bland, 1976a, l976b Sargent and Payne, 1974 Lange el ol.. 1989 Lange er al.. 1984
P. capsici
Williams and Webster. 1970
P. P P. P. P.
cnppsici cinnamomi eryrhrosepnca infescans palmivora
P. parasirica 0 t h . aphanrdermarum
Fihrillar/large peripheral vesicles
+
-
+
+
+
+
+
+
+
+ +
+
+
+
+ +
+
+>-
P. cinnamomi
+
+>-
P. megasperma var. sojae P. palmrvora
+ +
+>-
P. parasirica Pyrh. aphonidermarum Pyrh. proliferum Pyrhium spp Sclerospora graminicolo
Hardham, 1987a; Gubler and Hardham, 1988. 1990: Hyde el a / . , 1991a; Hyde and Hardham. 1993; Deamaley and Hardham. 1994; Dearnaley el al.. 1996 Ho er o f . , 1968 Hemmes and Hohl, 1969; Bimpong and Hickman. 1975; Bartnicki-Garcia and Hemmes, 1976 Reichle. 1%9 Grove and Bracker, 1978; Estrada-Garcia er a / . 1990 Lunney and Bland. 1976a.b Cope el
01..
1996
Lange er al., 1984 (Fig. 5 )
Encystmentidorsal cyst coal vesicles -
+
+ +
I
L
-
-
+
P. megarperma var. sojae Pyrhrum spp.
+
K-hodiedventral vesicles -
+
+ +
+
+ +
+
-
~
L
+
Pyth. aphanidermanrm 0 t h . proliferum Pythium spp. Pseudopcronospora cubensh
t
+ Mastigonemes -
+
+
+ +
+
+
+
+
+
+ + +
+
P. capsici P. cinnamoni
P. infesfans P. megarperma var. sojae P. pdmivom
+ I
P. capsici P. cinnamomi
+
+
P. P. P. P.
cqsici
cinnamomi infesrans poraririca Pyrh. apJianidermatum Pyth. proliferum
Williams and Webster. 1970 (Fig. 26) Gubler and Hardham, 1988, 19w; Hardham and Gubler. 1990; Hyde er a/.. 1991a; Hyde and Hardham. 1993: Dearnaley and Hardham. 1994: Dearnaley er al., 1996 Ho el al.. 1%8 (Fig. 23) Cope er al., 1996 Williams and Webster, 1970 (Fig. 26) Gubler and Hardham, 1988, 1990; Hardham and Gubler. 1990: Hyde et a / . , IWIa; Hyde and Hardham. 1993; Dearnaley and Hardham, 1994; Dearnaley cr al.. IW6 King er al.. 1%8 Ho et al., 1964 (Figs 25 and 26) Hohl and Hamamoto, 1967; Hemmes and Hohl, 1969; Bimpong and Hickman, 1975 (Figs I, 3 and 4): Powell and Bracker, 1986 Grove and Bracker, 1978 (Figs 17-19) Lunney and Bland, 1976b (Fig. 1) Cope er al.. 19% Lange er a/., 1989
Williams and Webster,
1WO (Figs 25, 27 and 28)
Cope and Hardham, 1994 Elsner er al.. 1970
Reichle, 1%9 Grove and Bracker, 1978 Lunney and Bland, 1976a.b
w o\ w
TABLE I-continued Vegetative Sporulating Post-septum Cleaving hyphae hyphae sporangia sporangia Flagella
Zmspores
Cysts
+
+ -
Germinating cysts
Species
+
P. apsici P. c*mcunomi
+ +
P. infestm P. pdmivora Pyth. proliferum Pseudopronospara cubensis Sclerospora graminicola
+ + +
References
Williams and Webster, 1970 Hyde el d.,1991a; Cope and Hardham, 1994 C o l b U O , 1%; King er d.,1%. Elsner et d.,1970 HoM and Hamamoto, 1%7; Hernmes and HoM, 1969 Lunney and Bland, 1976a.b Lange et a/., 1989 Lange d d.,1%
Peripheral cisternae
P. cinluvnomi
Water expulsion vacuole
+
+ + +
+
+ +
+>-
P. palm'vora P. parmifica Pyth. aphonidcrmnrum Pyih. proliferum
Hardham, 1 W a ; Hyde er d.,1991b Bimpong and Hickman. 1975; Bartnicki-Garcia and Henunes, 1976 Reichle, 1969 Grove and Bracker, 1968 Lunney and Bland, 1976b
P. cinluvnom' P. mcgmperma P. patm'wra Pyrh. aphami&rmahun Pyth. prdifwum Sckrospom graminicola
Hardham, 1981, Ho d d.,1968 Bimpong and Hickman, 1975 Grove and Bracker, 1978 Lunney and Bland, 1976b Lange el d..1984
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. + , Character present; - , character absent, +>-, character initially present but subsequently disappearing.
ASEXUAL SPORULATION IN THE OOMYCETES
365
allowing zoospore vesicles to be grouped into five main categories (Beakes, 1989, 1995; Hardham, 1995): (1) dense-body or fingerprint vesicles; (2) fibrillar or large peripheral vesicles; (3) encystment, cyst coat or dorsal vesicles; (4) K-bodies or ventral vesicles; and ( 5 ) peripheral cisternae. Justification for the interpretations of vesicle homologies basic to this categorization is given in the following section. Summaries of alternative names for these vesicles as used in the older literature can be found in earlier reviews (Lunney and Bland, 1976b; Holloway and Heath, 1977b; Hemmes, 1983; Hardham, 1987a; Beakes, 1989, 1995; Hardham, 1995). Detailed studies of P. cinnamomi (Cope and Hardham, 1994; Dearnaley et al., 1996) and collation of data from a diverse range of oomycete species (Tables 1-111) indicate that the five different categories of vesicles (Figs 1-4 and 6-18), mastigonemes (Figs 19-21), flagella (Fig. 22) and the water expulsion vacuole (Fig. 1) show three different temporal patterns of synthesis. Dense-body or fingerprint vesicles are present throughout the asexual life cycle; large peripheral or fibrillar vesicles, encystment or dorsal vesicles, K-bodies or ventral vesicles and packets of mastigonemes are synthesized after the onset of sporulation, and peripheral cisternae, the flagella (with two possible exceptions) and water expulsion vacuoles are synthesized during zoosporogenesis (summarized diagrammatically in Fig. 23). The reasons for the synthesis of different components at specific times are as yet unknown, however they may relate (as in the case of the precise arrangement of nuclei within newly formed sporangia) to the advantage gained by the ability of the organism to complete zoosporogenesis very rapidly. Some species can cleave within 10-15 min (P. cinnamomi: Byrt and Grant, 1979; P. nicotianae: Y. Gautam and A. R. Hardham, unpublished observations), and even if sporangia already contained the appropriate mRNA transcripts, it would be likely to take much longer than this to synthesize, process and package the contents of all three types of peripheral vesicle and the mastigonemes. Preformation might not be needed for the other three components that are produced only after cleavage has begun (the plasma membrane, peripheral cisternae and water expulsion vacuole) since their predominantly membranous nature probably allows them to be manufactured more quickly. I. Large peripheral vesicles or jibrillar vesicles Most ultrastructural studies of oomycete zoospores report the presence, in the cortical cytoplasm, of large ellipsoidal vesicles with a diameter of 0.2-0.5 wm in the plane parallel to the zoospore surface and approximately 0.8 p m in the plane perpendicular to the surface (Figs 1,6,7,13, 14) (Lunney and Bland, 1976b; Grove and Bracker, 1978; Hemmes, 1983; Cerenius et al., 1984; Beakes, 1987; Hardham et al., 1991a). Their contents appear electron lucent, granular or fibrillar, a difference in morphology which is likely to be due to variability in the preservation of their contents by different chemical
TABLE I1 Occurrence of zoospore components throughout the asexual life cycle in the Saprolegnialesa Vegetative hyphae
Sporulating Post-septum hyphae sporangia
Dense-bcdylfingerprint vesicles
+ + +
-
+
+
+ +
+
+ + +
Cleaving sporangia
Primary zoospores
Primary cysts
+
+ +
+
+
+ +
+
+
+
+
+
+
+>-
+
+
+
+ +
+ +
+
+
+
+ + +
Fibrillarnarge peripheral vesicles
+
-/+
+
EncystrnentldorsaVcyst coat vesicles
+
+
+
+ +
+
+ +
+
-/+
+
+
+ + +
Species
t
Beakes, 1987, 1989 Armbruster, 1982d Hoch and Mitchell, 197Za.b. 1975 Armbruster, 1982a,b Armbruster, 1982a Gay and Greenwood, 1966; Holloway and Heath, 1977b; Lehnen and Powell, 1989
A . pageUara A . debaryanwn Aphanomyces eufeiches
Beakes, 1987 Armbruster, 1982a Hoch and Mitchell. 1972a.b; Sadowski and Powell, 1990 Armbruster. 1982a.b Armbruster, 1982a Gay and Greenwood, 1966; Holloway and Heath, 1977b
A . pagellafa Aphanomyces eufezches
+
+
-
+
-I+
+
References
A . flageUala A . debaryanwn Aphanomyces eureiches Brevilegnia spp. Dictyuchlcr spp. Saprolegnu spp.
Brevilegnw spp. Dictyuchus spp. Saprolegnia spp
+ +
+
+
Secondary Germinating cysts cysts
+
Lipid
+
Secondary zoospores
Saprolegnia spp
A . pagellafa Aphanomyces eureiches Brevilegnia spp. Saprolegnia spp
Beakes. 1987 Hoch and Mitchell. 1972b; Sadowski and Powell. 1990 Heath and Greenwood, 1971; Holloway and Heath, 197%; Beakes, 1983; Lehnen and Powell, 1989; Burr and Beakes, 1994
Beakes, 1987 Hoch and Mitchell, 1972a.b: Sadowski and Powell, 19w Armbruster, 1982a,b Gay and Greenwood. 1966; Holloway and Heath, 3977b; Beakes, 1983; Lehnen and Powell, 1989; Burr and Beakes, 1994
K-hodiedventral vesicles
+ -
+
+
+ +
+
-/t
+
+
-/+
+ Mastigonemes
+ Flagella
-
+
+
+
+
+
+
+ +
+ -
-
+
+
+
+ +
+ Water expulsion vacuole
+ +
-
-
Brevilegnia spp. Saprolegnia spp.
Thrausrorheca
+
Peripheral vesicles
A . pugelluiu A . umbibr~e+uu/rs Aphanomyces eurerches
+
-
Beakes. 1987 Powell er ol.. 1985 Hoch and Mitchell. 1972a; Powell er u/ 1985; Sadowski and Powell, 1990 Armbruster, 1982b Holloway and Heath, 1977h; Beakes. 1983; Lehnen and Powell, 1989. 1991; Burr and Beakes, 1994 Powell er a / . , 1985
,
Aphunomym eureiches Brevilegniu Dicryuchus Saprolegnw spp
Hoch and Mitchell. 1972a.b Armbuster, 1982b Armbuster, 1982b Heath PI a / . , 1970; Holloway and Heath, 1977b
Achlyu spp. Aphanomyres eureiches Suprolegniu spp.
Cotner, 1930 (cited in Colhoun, 1966) Hoch and Mitchell, 1972a Heath and Greenwood, 1971
A . flagellorn Aphanomyres eureiches
Beakes. 1987 Hoch and Mitchell. 1972a.b; Sadowski and Powell, 1990 Beakes, 1983; Lehnen and Powell. 1989; Burr and Beakes, 1994
Saprolegnia spp.
+
-
A . flage/[otu Aphanomyces euieiches
Beakes, 1987 Powell,and Hoch 1990 Mitchell. 1972a; Sadowski and
+
-
Suprolegnia spp.
Holloway and Heath, 1977b; B u n and Beakes, 1994
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. +, Character present; -, character absent, +>-, character initially present but subsequently disappearing; +/-, character reported in different studies to be present or absent.
'8 z
0
0
5
' 0
2
w
m
4
TABLE 111 Occurrence of zoospore components throughout the asexual life cycle in the Lagenidialesa Vegetative hyphae
Sporulating hyphae
Dense-bodylfingerprintvesicles
+
+
Post-septum sporangia
Cleaving sporangia
+
+
+ + Lipid
+ +
+
Zoospores
QSS
+
+ + + +
+
+
+
+
+
+
+
+ +
+
FibrillarAarge peripheral vesicles
+
+
+ + Encystment/dorsaVcyst coat vesicles
+
+
+
+
-
References
Species
Lagenidium callinectcs Lagenidium giganfeum Lagena radicola Olpidiopsir sapmlegniae Olpidwpsis varians Petersenia palmariae
Gotelli. 1')74a,b Domnas er 111.. 1986 Ban and Waulnien. 1987, 1990 Bortnick et PI., 1985 Martin and Miller. 1986 Pueschel and van der Meer, 1985
Lagenidium callinecfes Lagenidium giganfeum Lagena radicola Olpidiopsis saprolegniae Olpidwpsir v a b Perersenia palmariac Lagenisma coscinodisci
Gotelli, 1974a.b
Domnas er el., 1986 Barr and h u l n i e n . 1987, 1990 Bortnick cr al.. 1985 Martin and Miller, 1986 Pueschel and van der Meer,1985 Schnepf el al., 1978a
Lagenidium callinecres Lagenidium giganfeum Lagena radicola Olpidbpsis saprolegniae
Gotelli, 19741, Domnas er al., 1986 Barr and Desaulnien, 1987, 1990 Bortnick er al., 1985
Lagenirma coscinodisci
Schnepf el 01.. 1978b
?
P 3:
$U
3:
s J
n.
P ?
3: 4 U
rn
K-bodieslventral vesicles Lagenidiwn callinecres
Lngena radkoln OIpidiopsis saprolegniae Olpidiopsis varians Mastigonemes
+
+
+ +
+ Flagella
Peripheral vesicles
+
+
+
+ +
+ -
+ +
+
+ +
+
+
+
+
+ +
Water expulsion vacuole
+
+ +
Gotelli, 1974b Ban and Desauloien. 1987, 1990 Bortnick er a/., 1985 Martin and Miller, 1986
Lngenidiwn caUinecvs Lagenidium gigantcum Lagena radicoln Olpidiopsis saprolcgniae Olpidiopsis varians Petemenio palmariae Lagenisma coscinodisci
Gotelli, 1914a
Lagenidium callinecres Perersenia palmariae Lagenisma coscinodisci
Gotelli, 1974b Pueschel and van der Meer, 1985 Schnepf et al., 1978a
Lagenidiwn giganleum Olpidiopsis saprolegniae
Domnas er al., 1986 Bortnick er al., 1985
Lagenidium giganreum Olpidiopsir saprolegniae Lagena radicola
Domas et d.,1986 Bortnick er a/.,1985 Barr and Desaulniers, 1987, 1990
Domnas er d.,1986
b m X C cn
F
Barr and Dhaulnien, 1987, 1990 Bortnick et al., 1985 Martin and Miller, 1986 Pueschel and van der Meer, 1985 Schnepf er al., 1978a
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. +, Character present; -, character absent.
0 Z
0 0
5 m
370
A. R. HARDHAM and G. J. HYDE
fixation regimes (Hardham, 1987a). They tend to be called large peripheral vesicles in Phytophthora and Pythium and fibrillar vesicles in Sapro legnia. Early studies of P. palmivora (Pinto da Silver and Noguiera, 1977; Sing and Bartnicki-Garcia, 1975a), Pythium aphanidermatum (Grove and Bracker, 1978) and Saprolegnia (Beakes, 1983), led to the suggestion that the contents of the large peripheral vesicles were secreted during zoospore encystment. However, it was later shown that this was an artefact arising during chemical fixation (Hardham, 1985). More recently, immunolabelling of Pyth. aphanidermatum zoospores was also interpreted as giving evidence of secretion of large peripheral vesicle contents (Estrada-Garcia et al., 1990). This interpretation has also been challenged, and it has been suggested that ambiguity may have arisen because the antibodies recognize an epitope common to large peripheral vesicles and to other vesicles whose contents are secreted (Cope et al., 1996). We believe that there is good evidence that the contents of the large peripheral vesicles are not secreted during encystment in species of any of these genera (Lunney and Bland, 1976b; Holloway and Heath, 1977b; Gubler and Hardham, 1988; Cope et al., 1996). Biochemical analyses of their contents have shown that they contain high-molecular-weight glycoproteins (Sing and Bartnicki-Garcia, 1975b; Gubler and Hardham, 1988; Estrada-Garcia et al., 1990). The large peripheral vesicles in Phytophthora and Pythium disappear from cysts (see Table I) and it has been shown in P. cinnamomi that they are broken down several hours after cyst germination (Gubler and Hardham, 1990). This observation has led to the suggestion that their glycoprotein contents form protein stores for use during germling growth before the pathogen has established access to an exogenous source of nutrients (Gubler and Hardham, 1990).
Figs 6-12. Transmission electron micrographs showing vesicles in zoospores of Phytophthora (Figs 6 and 7) and Saprolegnia (Figs 8-12) species. Fig. 6: Zoospore of P.megasperma showing groove (g), large peripheral vesicles (L) and ventral vesicles (v). From morphology alone, it is not possible to identify with certainty the two vesicles that are unlabelled. Peripheral cisternae (arrowheads) lie beneath the plasma membrane (scale bar = 0.5 pm). Fig. 7: Zoospores of P. cactorum showing large peripheral vesicles (L), a dorsal vesicle (d) and ventral vesicles (v) (scale bar = 0.5 pm). Fig. 8: Primary encystment vesicle in sporangium of Saprolegnia parasitica (scale bar = 0.1 pm). Fig. 9: Primary encystment vesicle in sporangium of S. ferax (scale bar = 0.1 pm). Fig. 10: Secondary encystment vesicle in primary cyst of S. diclinu-parusitica (scale bar 0.5 pm). Fig. 11: K,-bodies and vesicles similar to primary bars in primary zoospores of Saprolegnia (scale bar = 0.5 pm). Fig. 12: K2-body in Saprolegnia (scale bar = 0.5 pm). (Fig. 7 is reproduced with permission from Hardham et al. 1991a; Figs 8-10 are reproduced with permission from Beakes, 1983; and Figs 11 and 12 are reproduced with permission from Holloway and Heath, 1977b).
ASEXUAL SPORULATION IN THE OOMYCETES
371
372
A. R. HARDHAM and G . J. HYDE
Figs 13-18. Immunogold (Figs 13, 15 and 17) and immunofluorescence (Figs 14, 16 and 18) labelling of the contents of large peripheral vesicles (Figs 13 and 14), dorsal vesicles (Figs 15 and 16) and ventral vesicles (Figs 17 and 18) in zoospores or P. cinnamomi. Large peripheral and dorsal vesicles occur on the dorsal surface and tend to avoid the groove (g) on the ventral surface. Ventral vesicles occur predominantly close to the ridges of the ventral groove (scale bar in Figs 13, 15 and 17 = 0.5 pm. Figs 14, 16 and 18 are at the same magnification. Scale bar in Fig. 18 = 10pm) (Figs 13 and 15 courtesy of Dr Frank Gubler. Figs 14 and 17 reproduced with permission from Hardham et al., 1991a).
Large peripheraYfibrillar vesicles are absent from vegetative hyphae (Dearnaley et al., 1996), but have been described in sporulating hyphae in Phyrophrhora species (Dearnaley et al., 1996) and in sporangia of species of Phytophthoru (Hemmes and Hohl, 1969; Williams and Webster, 1970; Bartnicki-Garcia and Hemmes, 1976; Dearnaley et al., 1996), Pythium (Lunney and Bland, 1976a), Lagena (Barr and DCsaulniers, 1990) and
ASEXUAL SPORULATION IN THE OOMYCETES
373
Figs 19-22. The flagellar apparatus of zoospores of P. cinnamomi. Fig. 19: Transmission electron micrograph of a packet of tubular mastigonemes (scale bar = 0.5 pm). Fig. 20: Immunofluorescent labelling of packets of mastigonemes within a zoospore. (Reproduced with permission from Hardham et al., 1994.) Fig. 21: Immunofluorescent labelling of mastigonemes distributed in two rows on the surface of the anterior flagellum of a zoospore. Fig. 22: Immunofluorescent labelling of microtubules in the flagellar axonemes, in the flagella roots and cytoplasm of a zoospore. (Scale bar in Figs 20-22 = 10pm.)
Saprolegnia (Beakes, 1983). Immunocytochemical studies of P. cinnamomi have shown that they begin to form about 5 h after the induction of sporulation by transfer of mycelia from nutrient broth to mineral salts solution (Dearnaley et al., 1996). Immunogold labelling with a monoclonal antibody specific for the contents of large peripheral vesicles in P. cinnamomi has demonstrated the appearance of these antigens within Golgi cisternae, indicating that the vesicles are formed by the Golgi apparatus (Dearnaley and Hardham, 1994). 2. K-bodies or ventral vesicles Zoospores of members of the Saprolegniales, Leptomitales and Lagenidiales contain a distinctive type of vesicle in the groove region on the ventral surface close to the basal bodies (kinetosomes) (Figs 11 and 12) (Hoch and Mitchell, 1972b; Holloway and Heath, 1977b; Powell et al., 1985; Bortnick et al., 1985; Gotelli and Hanson, 1987; Lehnen and Powell, 1989, 1991). The structure of these kinetosome-associated organelles, or K-bodies, differs in primary and secondary zoospores: K1-bodies are about 0.4 pm in diameter and have
374
A . R. HARDHAM and G. J. HYDE
Fig. 23. Diagram summarizing data on the occurrence of zoospore components in cells throughout the life cycle of oomycete species. WEV, Water expulsion vacuole.
ASEXUAL SPORULATION IN THE OOMYCETES
375
a granular matrix and a narrow osmiophilic cortex (Fig. 11); K2-bodies are about 0.8 pm in diameter and (except in Thraustotheca; Powell et al., 1985) have a complex internal structure including a hemispherical cavity containing tubules, a granular matrix and cortical shell (Fig. 12) (Lehnen and Powell, 1991). There are typically one to six K-bodies per zoospore (Holloway and Heath, 1977b; Powell et al., 1985). Vesicles of similar morphology have not been observed in members of the Peronosporales; however, the occurrence of a special type of organelle near the ventral region (Figs 6 and 7) was noted in early studies (Ho et al., 1968; Lunney and Bland, 1976b; Grove and Bracker, 1978). Immunolabelling of Phytophthora (Figs 17 and 18) (Hardham and Gubler, 1990; Hardham et al., 1991a; Hardham, 1995), Pythium (Cope et al., 1996) and Plasmopara species (A. R. Hardham and L. Lange, unpublished observations) has now confirmed the presence of a distinct type of vesicle that is confined predominantly to the groove on the ventral surface of zoospores. They are about 0.3 pm in diameter and usually contain electron-dense plate-like structures. They have been called ventral vesicles. A similar distribution within the zoospore is not the only feature shared by K-bodies and ventral vesicles. The contents of both K2-bodies and ventral vesicles are secreted during encystment, and in both cases there is evidence that these ventrally located vesicles give rise to an adhesive pad that sticks the cysts to the adjacent substratum (Lehnen and Powell, 1989; Hardham and Gubler, 1990). We thus propose that K-bodies and ventral vesicles are equivalent organelles serving homologous functions. Lectin labelling of Saprolegnia zoospores indicates that the tubular portion of K2-bodies consists of glycoconjugates containing N-acetylglucosaminosyl residues (Lehnen and Powell, 1989). Ventral vesicles in P. cinnamomi contain proteins over 220 kDa in molecular weight (Hardham and Gubler, 1990). K-bodies and ventral vesicles are formed after the onset of sporulation (Tables 1-111). K1-bodies have been observed in differentiating sporangia; K2-bodies develop in primary cysts (Holloway and Heath, 1977b; Lehnen and Powell, 1991) and may arise from a smooth endomembrane system, possibly the endoplasmic reticulum (Lehnen and Powell, 1991). Ventral vesicles in P. cinnamomi first appear in hyphae about 5 h after the induction of sporulation (Dearnaley et al., 1996). They have been observed in developing sporangia and are thought to be formed in the Golgi apparatus (Dearnaley and Hardham, 1994). 3. Encystment vesicles, cyst coat vesicles or dorsal vesicles The peripheral cytoplasm of oomycete zoospores contains a second population of vesicles that are secreted during encystment. In species of Phytophthora these vesicles are spherical and approximately 0.3 pm in diameter (Fig. 7); in species of Saprolegnia, they are cylindrical in shape and 0.2-0.5pm in diameter and 0.3-1.5pm in length (Figs 8-10) (Beakes, 1983). In
376
A. R. HARDHAM and G. J. HYDE
Saprolegnia, they have been recognized by their inclusions of spines and hairs, and have been described as barbodies, encystment vesicles or cyst coat vesicles (Beakes, 1983, 1987, 1989). In Phytophthora they have a marbled appearance and have been identified by labelling with monoclonal antibodies (Figs 15 and 16) (Gubler and Hardham, 1988; Hardham et al., 1991b). Immunofluorescence microscopy reveals that they occur predominantly on the dorsal surface of the zoospores, and avoid the ventral groove region (Fig. 16). They have been called small peripheral vesicles or dorsal vesicles (Hardham, 1987a; Hardham and Gubler, 1990). The morphological and immunological markers have allowed the fate of the vesicles to be followed during encystment and it is clear that in both Phytophthora and Pythium species they are secreted, their contents giving rise to a layer of material that coats the surface of the cyst (Beakes, 1983, 1987; Gubler and Hardham, 1988; Beakes, 1989; Hardham and Suzaki, 1990; Hardham et at., 1994). The cyst wall subsequently forms between the cyst coat and the plasma membrane. Thus, while their morphology varies, their similar role in the formation of the cyst coat during encystment suggests to us that cyst coat vesicles (first suggested by Beakes, 1987) would be the most appropriate name for these vesicles. The function of the cyst coat is not altogether clear, although the spines and boathooks that adorn it in species of Saprolegnia may aid attachment to the host surface (Beakes, 1983). In Phytophthora the cyst coat first forms on the side of the cyst facing away from the host surface (Hardham and Gubler, 1990), but subsequently spreads to cover surrounding surfaces (Hardham et al., 1994). In P. cinnamomi the cyst vesicles contain high-molecular-weight glycoproteins that possess N-acetylgalactosaminosyl residues (Gubler and Hardham, 1988). The ontogeny of cyst coat vesicles has been studied in Lugenisma (Schnepf et al., 1978a), Saprolegnia (Holloway and Heath, 1977b; Beakes, 1983) and P. cinnamomi (Dearnaley and Hardham, 1994). Ultrastructural observations have been interpreted as indicating an origin in the endoplasmic reticulum in Saprolegnia (Beakes, 1983) and in the Golgi apparatus in Lagenisma coscinodisci (Schnepf et al., 1978a). Immunogold labelling has unambiguously demonstrated that cyst coat vesicles form in the Golgi apparatus in P. cinnamomi (Dearnaley and Hardham, 1994). In fact, individual Golgi cisternae process cyst coat vesicle and large peripheral vesicle glycoproteins simultaneously, sorting and packaging them into separate categories of vesicles. Cyst coat vesicles have been observed in sporulating hyphae of P. cinnamomi (Dearnaley et al., 1996), and in developing sporangia of Lagenisma coscinodisci (Schnepf et al., 1978a) and Saprolegniacean species (Armbruster, 1982a; Beakes, 1983) (Tables 1-111). In P. cinnamomi, they are not present in vegetative hyphae, and appear about 5.5-6h after the induction of sporulation; this is about 30-60 min after the appearance of large peripheral and ventral vesicles (Dearnaley et al., 1996).
ASEXUAL SPORULATION IN THE OOMYCETES
377
4. Mmtigonemes Mastigonemes are tripartite, tubular hairs about 1.5 pm in length that are attached in two rows to the anterior flagellum of zoospores of the oomycete and heterokont algae (Fig. 21) (Heath et al., 1970; Bouck, 1972; Holwill, 1982; Hardham, 1987b; Inouye, 1996). It was proposed from theoretical considerations that mastigonemes reverse the thrust of flagellar beat (Jahn et al., 1964), an hypothesis that has recently received experimental verification (Cahill el af., 1996). Mastigonemes form in rectangular, dilated regions of the endoplasmic reticulum or nuclear envelope (Fig. 19) (Bouck, 1969; Reichle, 1969; Heath et al., 1970; Leedale et al., 1970; Deason, 1971; Hoch and Mitchell, 1972a; Loiseaux, 1973; Hill and Outka, 1974; Hardham, 1987b). They are arranged within the packets in an antiparallel fashion with the base of each hair attached to the membrane at one end of the packet (Heath et al., 1970). Packets of mastigonemes are easily recognized (Figs 19 and 20) and have been observed in the cytoplasm of zoospores in members of the Peronosporales, Lagenidiales and Saprolegniales (Tables 1-111). They become associated with the flagella surface in late cleavage (Heath and Greenwood, 1971; Cope and Hardham, 1994). There is little information on the biochemical composition of oomycete mastigonemes, although one mastigoneme antigen in P. cinnamomi is a glycoprotein (Cope and Hardham, 1994). Evidence that mastigonemes are synthesized during sporangiogenesis comes from ultrastructural observations of their presence in developing sporangia in Lagenisma coscinodisci (Schnepf et al., 1978a) and in post-septum (but before cleavage) sporangia in species of Phytophthora (King et al., 1968; Hemmes, 1983), Pythium (Lunney and Bland, 1976a) and Saprolegnia (Heath et al. , 1970). Immunolabelling with monoclonal antibodies specific for mastigonemes in P. cinnamomi have shown that mastigonemes form in hyphae and developing sporangia after sporulation is induced (Cope and Hardham, 1994).
111. ZOOSPOROGENESIS A. INDUCTION
A reduction in temperature causes multinucleate sporangia to cleave into uninucleate zoospores. Nothing is known about the molecules that perceive this signal but, by analogy with other plant and animal systems, it is likely that within minutes the receptors initiate a signal transduction cascade which leads to changes in gene expression and cellular organization. Two common components of this cascade are Ca2+ and H+. Recently, changes in the cytoplasmic concentration of these ions has been investigated during cleavage of P. cinnamomi sporangia. The concentration of Ca2+ and H + in the cytoplasm has been measured
378
A. R. HARDHAM and G. J. HYDE
by microinjecting fluorescent indicator dyes into sporangia before cleavage is induced by a cold shock treatment (Suzaki et al., 1996; Jackson and Hardham, 1996). Fura-2 is a ratiometric dye whose characteristics of fluorescence are indicative of [Ca2+] (Tsien, 1989); BCECF (2’,7’-bis(2carboxethyl)-5(6)-carboxyfluorescein) is a ratiometric dye indicative of pH. Dextran-conjugated dyes were used because they are unable to cross cell membranes and thus remain in the cytosol and are not sequestered into organelles. Microinjection of fura-Zdextran revealed that there are two rises in [Ca2+] during cleavage (Jackson and Hardham, 1996). The first is rapid and transient: the [Ca2+]increases by 25-130% from a resting level of 104 f 54 nM in the first minutes of the cold shock treatment. The concentration of Ca2+ returns to near resting levels and then rises slowly during the course of cleavage. If dibromo-BAPTA, a Ca2+ buffer, is microinjected into sporangia before the cold shock, cleavage is inhibited; if it is injected after cold shock, most sporangia cleaved. Artificially raising the concentration of Ca2+ by incubating sporangia in the Ca2+ ionophore (A23187) in the presence of 10 mM Ca2+ induced cleavage in about 37% of sporangia in the absence of a cold shock. Both internal and external sources of Ca2+ contribute to the initial rise in [ ~ a * + ] . Injection of sporangia with BCECF-dextran revealed that cytoplasmic pH increases rapidly by about 0.2 pH units from a resting level of 6.84f0.05 during an inductive cold shock treatment (Suzaki et al., 1996). If this rise in pH is inhibited by injection of a pH buffer such as HEPES, the sporangia failed to cleave. These results indicate that a rise in the cytoplasmic concentration of Ca2+ and H+ not only accompanies cleavage, but is also required for cleavage to occur. It seems likely that these factors are part of the signal transduction pathway operating during the onset of cleavage, but additional details of their role and of other molecules involved await further studies.
B . THE PROCESS OF CLEAVAGE
1. Patterns of cleavage membrane formation: a reappraisal The sporangium is subdivided into uninucleate domains by the elaboration of partitioning membranes that give rise to the plasma membrane of the zoospore. The formation of these membranes has been much studied and many different developmental patterns have been proposed. In other eukaryotes, differing patterns of cleavage, and cytokinesis in general, have long been recognized and are typically considered as evolutionary variations (e.g. Pickett-Heaps, 1972). Thus, until recently, there had been no suggestion that the various patterns of oomycete cleavage represented anything more than a set of possibly useful taxonomic characters. However, as will be
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Fig. 24. Diagram showing models proposed to explain cleavage of oomycete sporangia. The upper portion of the diagram shows, clockwise, vesicle pre-alignment, expansion of a large central vacuole, invagination of the plasma membrane and expansion of a cytoplasmic vacuole. In the lower portion of the diagram, which is based on studies of cleavage in Phytophthora species, the cleavage vacuoles are shown to develop as a ramifying network in the sporangial cortex and between nuclei.
described in detail below, recent rapid-freezing and freeze-substitution (RF-FS) studies have now raised the possibility that much of the diversity reported for oomycete zoosporogenesis may be artefactual and derive from inadequate fixation of the developing partitioning membranes (Hyde et al., 1991b,c). It is therefore worthwhile to now reconsider all the existing data on cleavage in this taxon in the light of the recent RF-FS studies of oomycete and other sporogenic fungi. Five main patterns of zoosporogenic membrane development have been described in the oomycetes (see Fig. 24). For any one species, several of these processes may have been proposed.
Vesicles. Vesicles of one or more types, either pre-existent within the sporangium or induced to form by some event, redistribute in planes around the nuclei; after alignment the vesicles fuse, thus forming the cleavage vacuoles (Fig. 24). Images of arrays of aligned vesicles are found in studies of most of the Phytophthora species examined so far (Hohl and Hamamoto, 1967; Elsner et al., 1970; Williams and Webster, 1970; Hemmes, 1983; Hyde et al., 1991a) and of Pythium (Lunney and Bland, 1976a), Pseudoperonospora (Lange et al., 1989), Dictyuchus and Brevilegnia (Armbruster, 1982b). All these studies have, however, relied upon chemical fixation, and when P. cinnamomi and P. palmivora were re-examined with RF-FS no vesicular arrays were apparent. Instead, cleavage appeared to follow from the extension of partitioning membranes in a progressive fashion (Fig. 24; Hyde et al., 1991b). It is likely that the RF-FS results represent more
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faithfully the events that take place in the living cell: numerous studies have indicated that elongated membranous systems are preserved in material prepared by RF-FS, but become highly vesiculated by chemical fixation (e.g. McCully and Canny, 1985; Shepherd et al., 1993). This indicates that any images showing vesicles lined up neatly along “future” planes of cleavage are to be treated with suspicion. In hindsight it is easy to understand the misinterpretation of these arrays. In the final stages of cleavage, as the zoospores round-up, the interzoosporic spaces widen so much that they are not prone to the chemical fixation-induced vesiculation typical of the narrow, early stage vacuoles. Hence, in chemically fixed material a plausible, but incorrect, story suggests itself: the vesicular network that forms from the disrupted early vacuoles is an intermediate stage of a process completed by fusion of the vesicles. Some reports of vesicular fusion in zoosporogenesis in the Saprolegniales require further mention because they involve the fusion of dense body vesicles with (Armbruster, 1982b) or without (Gay and Greenwood, 1966; Gay et al., 1971) the participation of other vesicle types. Dense-body, or fingerprint, vesicles are common in other oomycete sporangia but are not involved in the cleavage process, and it is possible that their apparent involvement in the cases above also results from misinterpretation of poorly preserved, chemically fixed material. In chemically fixed sporangia of Phytophthora, not only cleavage vesicles but also other vesicles at the periphery of developing zoospores (e.g. large peripheral vesicles) are prone to rupture, and often appear fused with the cleavage membrane (G. 1. Hyde and A. R. Hardham, unpublished observations; see also section IICl). It should be noted that chemical fixation is not intrinsically incapable of preserving progressively extending cleavage vacuoles: the basic form of ascosporogenic membranes, for example, is very similar in material preserved by chemical fixation or RF-FS, although the latter technique has revealed some previously undetected features (Mims et al., 1990; Van Wyk et al., 1991). Why the cleavage vacuoles of some taxa are more resistant to vesiculation than others is unknown; it is not their size, since early cleavage vacuoles of ascomycetes (Mims et al., 1990) are as narrow as those of P. cinnamomi. Work with P. cinnamomi has, however, indicated that the fixation protocol itself can affect the degree and type of vesiculation. When fixative solutions were made up with high concentrations of buffer, the cleavage system was less vesiculated and those vesicles that did form were large (Hyde et al., 1991a). In the “preserved” sections of the cleavage system, the paired membranes were much further apart than in RF-FS material. This suggests that contraction of the sporangial domains (a likely effect of the more concentrated, highly-buffered solutions) may have pulled sections of the cleavage membranes too far apart to permit vesiculation. As discussed by Hyde et al. (1991b) it is also probable that fixation problems have led to misinterpretations of cytokinesis in the many other
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eukaryotic organisms for which fusion of pre-aligned vesicles has been reported. Indeed it is possible that this process does not occur at all: to our knowledge not one RF-FS study has yet confirmed its existence and the first RF-FS study of plant cytokinesis has now indicated that membrane formation in these organisms also involves progressive elongation of partitioning membranes (Samuels et al., 1995). The use of variations in the cytokinetic process as taxonomic indicators is now open to question.
Involvement of a large central vacuole. Some studies of oomycete zoosporogenesis report the incorporation or expansion of a large central vacuole (Fig. 24) in cleavage (Gay and Greenwood, 1966; Hohl and Hamamoto, 1967; Williams and Webster, 1970; Gay et al., 1971; Beakes, 1995). Mature sporangia often contain one or more large central vacuoles which disappear during zoospore formation, but recent use of monoclonal antibody markers in P. cinnamomi and P. palmivora has indicated that in these species the vacuoles do not contribute to the cleavage process (Hyde et al., 1991a,b). Their role in cleavage is uncertain. In S . ferax, numerous smaller vacuoles in the sporangial cortex fuse to form a central vacuole which then expands centrifugally, and preferentially, between the nuclei (Gay and Greenwood, 1966). The mature Saprolegnia sporangium also sometimes contains a pre-existing central vacuole, but micrographs indicate that these have different contents to the newly forming ones, and that the two types often abut each other over large areas without signs of fusion (Gay and Greenwood, 1966; Gay et al., 1971). It is possible that as in Phytophthora, the pre-existing vacuoles play no part in cleavage, but disappear coincidentally. Involvement of the sporangial plasma membrane. Centripetal invaginations of the plasma membrane to form cleavage vacuoles between nuclei (Fig. 24) have only been reported in Pyth. middletoni (Heintz, 1970; Beakes, 1995). More common are reports in which other parts of the developing cleavage system fuse with the plasma membrane as in Saprolegnia ferax, Pyth. proliferum and P. cinnamomi (Gay and Greenwood, 1966; Gay et al., 1971; Lunney and Bland, 1976a; Hyde et al., 1991b). Cortical cleavage vacuole. A cortical cleavage vacuole forms parallel to the sporangial wall (Fig. 24; Elsner et al., 1970; Williams and Webster, 1970; Armbruster, 1982b; Hyde et al., 1991b). Apart from their peripheral location such vacuoles, reported in some Phytophthora and Saprolegnia species, appear to behave similarly to the central vacuole of S . ferax. For example, in Phytophthora, once having formed, the cortical vacuole expands preferentially between the nuclei, and fuses with the sporangial plasma membrane (Hyde et al., 1991b). The cortical vacuole itself forms by the fusion of smaller vacuoles initiated at the narrow poles of the cortical nuclei. The relationship between the cortical and internal planes of the Saprolegnia species is perhaps obscured by
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chemical fixation artefacts (Armbruster, 1982b). Other cases of cortical cleavage plane formation in the oomycetes may have also been missed or misinterpreted because of poor preservation and/or insufficient sampling.
Progressive extension of cleavage vacuoles around or between nuclei. This process (Fig. 24) is mostly clearly seen in the two Phytophthora species studied by RF-FS and is also described as one aspect of cleavage in S. ferax and Pyth. middletoni (Gay and Greenwood, 1966; Heintz, 1970; Gay et al., 1971; Hyde et al., 1991b; Beakes, 1995). For nuclei in the sporangial cortex of ~ ~ y t ~ ~ h tthese ~ o rcleavage u , vacuoles are mostly, or completely, composed of the cortical vacuole and its ingrowths. If a sporangium is large enough to have non-cortical nuclei, then vacuoles initiated near their narrow poles also contribute to the cleavage network (Fig. 24). Progressive extension of cleavage vacuoles is likely to be a cleavage process occurring in most oomycete, and indeed fungal, sporogenesis. In support of this, two ongoing RF-FS studies of chytrid zoosporogenesis report that cleavage occurs by progressive extension, and not by vesicular fusion as thought previously (D. Lowry and J. Shields, personal communications); also, RF-FS and chemical fixation studies of ascosporogenesis describe a comparable process to that seen in Phytophthora (Mims et al., 1990; Van Wyk et al., 1991; Van Wyk and Wingfield, 1991a,b). There are two accounts of sporogenesis in the oomycetes which do not fit into our schema. One, that of zoosporogenesis in L . coscinodisci (Schnepf et al., 1978a), will not be discussed here because we feel that the model proposed needs verification. The other, that of spore (not zoospore) formation in Aphanomyces euteiches (Hoch and Mitchell, 1972b, 1975), involves a possibly more primitive mechanism than that seen in true zoosporogenesis. Spores form within a non-swollen hypha, not by active subdivision, but by the aggregation of originally dispersed cytoplasm around regularly spaced nuclei, and the concomitant passive redistribution of a central vacuole between the spore units (Hoch and Mitchell, 1972b, 1975). If it is accepted that organisms that were thought to cleave by the fusion of aligned vesicles actually do so by vacuolar extension, then a number of topological commonalities in the cleavage processes of oomycete sporangia emerge. The first, most basic feature, shared possibly by all species, is that the development of the paired cleavage membranes and the interzoosporic space they enclose proceeds in a progressively ramificatory fashion. Second, the “outside” space between the membranes establishes a continuity with the true extracellular space of the sporangium, either immediately (in the case of plasma membrane invaginations) or when internally generated cleavage planes fuse with the sporangial plasma membrane. Third, many of the ramifications derive, in a systematic fashion, from one communal “pool” of future outside space, either a central or cortical vacuole, or the
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extracellular space between the sporangial plasma membrane and wall. The remainder of the cleavage system arises from discrete, often paranuclear, sites of initiation and these may originally be the source of the larger communal pools. A final shared feature, and one which distinguishes cleavage in the oomycetes from that in other taxa (e.g. ascosporogenesis), is the degree of “co-operation” between adjacent domains: they do not cleave independently but utilize common furrows. The ramificatory sporangial cleavage process described above can usefully be likened to dynamic branching systems in which energy becomes channelled along paths of least resistance (Rayner et al., 1994). As suggested previously (Olson et al., 1981; Heath and Harold, 1992), the driving energy for sporangial cleavage development probably derives mainly from osmotic expansion of the cleavage vacuoles. RF-FS has shown that the vacuoles are filled with a very dense matrix material and that the leading edges of cleavage vacuoles are swollen (Hyde et al., 1991b), both of which are consistent with this idea. Another aspect of vacuolar expansion is the addition of the new membrane and matrix material required by the developing network. With an osmotically driven, interconnected system these elements would not need to be transferred to the developing edges of the cleavage system. Thus, without any change in the end result, new material could be added at any point in the network, for example near the narrow nuclear poles where the dictyosomes are concentrated.
2. Spatial regulation of cleavage If the developing cleavage network is a dynamic branching system, then how are the paths of least resistance defined? That is, what causes the ramifications to occur along such neatly prescribed courses? Considerable evidence indicates that both microtubules and actin microfilaments are involved. Treatment of sporangia with antimicrotubule or antimicrofilament drugs results in grossly abnormal cleavage (Schnepf et al., 1978a; Oertel and Jelke, 1986; Heath and Harold, 1992; Hyde and Hardham, 1993). Structural studies have shown that both microtubules and microfilaments are present during the cleavage process. In P. cinnamomi microtubule arrays persist in similar form to that seen in the uncleaved sporangium. Microfilament arrangements in cleaving sporangia have recently been studied, using fluorescence microscopy, in Saprofegnia, Achlya and P. cinnarnomi (Heath and Harold, 1992; S. L. Jackson and A. R. Hardham, unpublished observations). In each species microfilament arrays show a similar pattern of development to that of the cleavage vacuoles, but unfortunately the data do not clarify whether the actin arrays precede o r follow after the cleavage membranes with which they finally associate. As yet we do not understand how microtubules or microfilaments control cleavage, but there are at least two broad possibilities. In the first model, microtubules act indirectly, determining where the actin arrays will form. The
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actin arrays, which would need to form prior to the cleavage system, would then be directly responsible for ensuring the extension of the network along the correct lines. The actin arrays might, for example, form the equivalent of canal banks that direct the expansive “flow” of the system between and around the zoosporic nuclei. In the second model, microtubules are the more important element, stabilizing domains of cytoplasm around the nuclei: the paths of least resistance for vacuolar expansion lie between, and around the domains, where there are the least number of microtubules. This role for microtubules was first proposed by Heath (Heath and Greenwood, 1971). In this model the role of actin might not be to guide the vacuoles into new regions but to brace in situ those that have already formed. They would thus be the equivalent of levee banks added to rivers in danger of flooding. In their absence the microtubules may not be sufficient to hold back the formation of inappropriate ramifications, leading to abnormal cleavage. One of these two models will be eliminated when it is known which comes first: actin or the vacuoles. C. SYNTHESIS OF ZOOSPORE-SPECIFIC COMPONENTS DURING ZOOSPOROGENESIS
Two cell components, the water expulsion vacuole and peripheral cisternae, are consistently reported to develop during zoosporogenesis. In most, but not all, cases flagella are also reported to form at this time (Tables 1-111). I . Water expulsion vacuoles The water expulsion vacuole complex consists of a central vacuole surrounded by numerous vesicles and tubular cisternae. The whole complex is flanked by stacks of Golgi cisternae. The water expulsion vacuole is believed to be responsible for zoospore osmoregulation, although the molecular basis of its operation is not known. Its cycle of dilation and contraction can be observed in living cells and takes about 4-6s (Grove and Bracker, 1978; A. R. Hardham, unpublished observations). Ultrastructural examination of fixed material (Lange et al., 1984) and observations of living material (A. R. Hardham, unpublished observations) indicate that the complex forms and begins operating during late cleavage. After zoospore encystment, the pulsation cycle time increases (A. R. Hardham, unpublished observations) and within about 10 min the water expulsion vacuole disappears (Holloway and Heath, 1977b; Grove and Bracker, 1978; Hemmes, 1983). 2. Peripheral cisternae The plasma membrane of oomycete zoospores is lined by a system of flattened membranous discs, the peripheral cisternae. In chemically fixed material, the
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disks tend to be dilated at the edges (e.g. Hemmes, 1983; Hardham et al., 1991a) but in RF-FS material the cisternae are of uniform thickness throughout their length (Cho and Fuller, 1989). The cisternae line the plasma membrane except in the groove region (see Grove and Bracker, 1978; Hemmes, 1983). The membrane of the peripheral cisternae is more similar to that of the plasma membrane than to the endoplasmic reticulum (Grove and Bracker, 1978; Hardham, 1987a), and during encystment, peripheral cisternae vesiculate and are believed to fuse with the plasma membrane of the zoospore. Their function is not known, although a reaction with an antibody that also labels the cyst wall has led to the suggestion that they play a role in cyst wall formation (Hardham et al., 1991a). Whether or not the possession of this common epitope is a true indication of a functional relationship remains to be determined, but one thing is clear: their fusion with the plasma membrane during encystment has the potential to bring about rapid and dramatic changes in plasma membrane properties. Peripheral cisternae first appear during cleavage plane formation (Hyde el al., 1991b). Short segments of cisternae become aligned next to the new plasma membrane of the zoospores even before cleavage is complete. Their site and mode of formation are unknown, although the Golgi apparatus is a likely candidate. 3. Flagellar axonema Oomycete zoospores are biflagellate, the two flagella arising at basal bodies near the centre of the groove on the ventral surface. Microtubule flagellar roots and cytoplasmic arrays are associated with the flagellar apparatus (see Fig. 22) (Barr, 1981; Barr and Allan, 1985; Hardham, 1987b). Flagella are responsible for zoospore motility, and some components of the flagellar apparatus may also maintain zoospore shape. During encystment the flagella are detached at the level of the terminal plate (Hardham, 1987b). The basal body acts as a template for axoneme formation and nine microtubule doublets assemble by addition of tubulin dimers to two microtubules in each of the nine triplets in the basal bodies. The central pair of microtubules has the opposite polarity and apparently assembles by tubulin addition at the proximal end (Lefebvre and Rosenbaum, 1986). In oomycete sporangia, in all but two cases, flagella have been observed to form during cleavage (Heath, 1976; Lange et a f . , 1984, 1989; Cope and Hardham, 1994). However, in P. infestam (King et al., 1968; Elsner et al., 1970) and Lagenidium callinectes (Gotelli, 1974b), flagella are reported to be present in sporangia that have not been induced to cleave. Although flagella formation during direct germination in P. parasitica (Hemmes and Hohl, 1969) also suggests that flagella assembly may be independent of cleavage events, sporangia are very sensitive to changes in environmental conditions and cleavage may be induced by mild manipulations, for example, by a 5°C decrease in temperature (Byrt and Grant, 1979; Suzaki et al., 1996). It may
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be worthwhile to re-examine the timing of flagella formation using immunofluorescence microscopy with anti-tubulin antibodies. This technique allows the examination of large numbers of cells and can thus produce more accurate assessment of modes of development. D. POLARIZATION OF ZOOSPORIC ORGANELLES
Oomycete zoospores are highly polarized cells. Many of the components described in previous sections are more concentrated at one or the other end of an axis running from the centrioles back through the broad pole of the nucleus (see Figs 1,14,16,18,25A).This organellar polarity is highly defined in the secondary zoospore which is also morphologically polarized about the same axis, with grooved ventral, and convex dorsal, surfaces. While a great deal is known about the arrangement of zoosporic components, only recently has any work addressed how it develops. With the aid of monoclonal antibodies raised against various vesicles of P. cinnamomi, Hyde et al. (1991a) showed that three of the vesicles attained their characteristic distributions along the zoospore periphery in a sequential manner. If abnormal cleavage were induced by elimination of the sporangial microtubule arrays (by use of the drug oryzalin), the three vesicles still managed to reach the periphery of the disrupted cleavage vacuoles, but were not concentrated along any particular portions of it, as occurs normally (Fig. 25B) (Hyde and Hardham, 1993). Abnormal cleavage per se was not responsible for these effects, since this could also be induced with the anti-actin drug cytochalasin D without inhibiting either vesicle migration to the cortex or localized concentration along it (Fig. 25C). It is therefore likely that, under normal conditions, vesicles do not require either microtubules or microfilaments to reach the zoospore periphery, but that microtubules somehow regulate where along the periphery the vesicles will concentrate. A possible model is that vesicles reach the zoospore membrane by Brownian motion and then dock with plasma membrane receptors with polarized distributions maintained by microtubules. Mitochondria1 redistribution was also examined in this study, and both mitochondria1 movements and polarization were inhibited by the antimicrotubule drug. E. ZOOSPORE DISCHARGE
Zoospores may be released directly into the surrounding medium by rupture of the papilla at the apex of the sporangium (as in Saprolegnia and Aphanomyces: Gay and Greenwood, 1966; Hoch and Mitchell, 1972a), or they may be temporarily confined within an evanescent vesicle derived from papillar material (as in Phytophthoru, Pythium and Lagenidium: Webster and Dennis, 1967; Gotelli, 1974b; Gisi et al., 1979). In those species in which
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Fig. 25. Diagram showing (A) the distribution of (a) microtubules, (b) nuclei, (c) mitochondria, (d) ventral vesicles, (e) dorsal vesicles and (f) large peripheral vesicles in zoospores within a recently cleaved sporangium of Phyfophthoru. The effects of (B) microtubule and ( C ) microfilament inhibitors on cleavage planes and on the distribution of the components during cleavage are shown. In (B), loss of microtubules causes abnormal cleavage and misalignment and rounding up of the nuclei; mitochondria remain randomly distributed; peripheral vesicles concentrate along the abnormal cleavage planes but do not become segregated into discrete cortical domains. In (C), loss of microfilaments causes abnormal cleavage, however mitochondria and peripheral vesicles are both transported to the cortex and segregated as normal. (Reproduced with permission from Hyde and Hardham, 1993.)
a vesicle forms, zoospores or sporangial cytoplasm rapidly flow into the vesicle as it expands. The time at which this occurs relative to the cleavage process varies, a feature that is used to distinguish taxa of Phytophthora and Pythium (de Bary, 1887, cited in Dick, 1990). In Phytophthora, movement into the vesicle occurs after cleavage is more or less complete. Thirty to seventy per cent of the zoospores move into the vesicle before it ruptures and the zoospores swim away (Gisi et u l . , 1979). Zoospores remaining in the sporangium swim out through the activity of their flagella. In Pythium, the uncleaved mass of cytoplasm flows into the vesicle where cleavage subsequently occurs (Webster and Dennis, 1967). In Lagenidium an apparently intermediate situation exists whereby partially cleaved cytoplasm is expelled into the vesicles (Gotelli, 1974b). Interpretations of the extent of cleavage in electron microscopic images of discharging sporangia may, however, be confused by fixation artefacts. Studies of the release of zoospores or cytoplasm from sporangia indicate that a difference in the osmotic pressure between the inside of the sporangium and the external solution is required for discharge (Gisi, 1983). Reduction of this difference by depression of external water potential retards or inhibits spore release in Aphanomyces euteiches, and Phytophthora species (Hoch and Mitchell, 1973; Gisi et al., 1979). There are two current theories as to how
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this pressure differential could be utilized to bring about papillar expansion and release of sporangial contents. One theory suggests that extracellular material that surrounds the zoospores (or sporangial protoplast) acts as a gel that takes up water and swells, thereby building up pressure within the sporangium (Lunney and Bland, 1976a; MacDonald and Duniway, 1978; Gisi and Zentmyer, 1980). The other theory suggests that extracellular solutes in the medium surrounding the spores act as an osmoticum to build up turgor pressure within the sporangium (Gisi, 1983; Money and Webster, 1985; Money et al., 1988). Until recently, one of the main shortcomings of the swelling gel theory was the fact that there was little evidence of extracellular material surrounding spores or uncleaved cytoplasm. However, in freeze-substituted sporangia of P. cinnamomi and P. palmivora the extracellular space was found to be filled with electron-dense material that was labelled by an antibody that reacted with the contents of the cleavage vacuoles in pre-cleavage sporangia (Hyde et al., 1991b). It seems likely that cryofixation studies could reveal the presence of similar material in sporangia of other species of oomycetes. One of the requirements of the osmotic pressure theory is that the permeability of the wall of the sporangium is such that osmotically active solutes remain inside the sporangium while water enters along the osmotic potential gradient. Plant and fungal cell walls are semipermeable structures which allow the passage only of molecules whose molecular dimensions are less than the sizes of the smallest pores within the wall (Nobel, 1991). Sporangial walls could thus retain osmotically active molecules of an appropriate size within the sporangial extracellular space. Studies of Achlya and Saprolegnia sporangia have demonstrated that the pore size of the walls is of similar magnitude to that of higher plants and fungi (Money and Webster, 1985; Money et al., 1988). The pore size for Achlya is of the order of about 2 nm (Money et al., 1988). This compares with a value of 4-5 nm for walls of a range of higher plant cells (Carpita et al., 1979). A pore size of 2nm would mean that molecules with a molecular weight above about 1000 Da would be trapped inside the sporangium. Dense-body/fingerprint vesicles were reported to fuse with developing cleavage furrows thus transferring their p-1,3-glucan contents, the mycolaminarans, into the extracellular space (Gay and Greenwood, 1966; Gay et al., 1971; Annbruster, 1982b) and it has thus been suggested that these molecules could act as the osmoticum (Money et al., 1988). However, it now seems unlikely that dense-body vesicle contents are secreted at any stage of zoosporogenesis, and therefore could not play this role. Western blotting with a monoclonal antibody that reacts with the extracellular material within Phytophthora sporangia indicates that these polypeptides have a range of apparent molecular weights between 60 and 330 kDa (Hyde et al., 1991a). Molecules of this size would not be able to move across a wall with a porosity
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of the order of 2 nm, but it remains to be determined if they could accumulate to sufficient concentration to generate the required osmotic potential difference. To date there have been no definitive experiments that demonstrate which of the two proposed mechanisms operates during release of zoospores or protoplast from oomycete sporangia. Increasing the osmotic potential of the external solution would inhibit the operation of either mechanism. Similarly, the extracellular material visualized in Phytophthoru sporangia could play a role in either mechanism. One possible approach might be to examine if the extracellular material remains within or near the sporangia after spore release. A gel-like material could be expected to persist at least temporarily, while solutes would be expected to quickly disperse. Immunolabelling of the extracellular material in Phytophthora sporangia could give evidence of the longevity of the material after zoospore release. In some respects, it is easier to envisage either of these two mechanisms operating in sporangia in which cleavage is complete within the sporangia before release. In this case, spaces (in which the gel or the solute can accumulate) are observed between the spores and between spores and the sporangial wall (e.g. Hyde et al., 1991b). But in Pythium the cytoplasmic mass appears to be appressed to the sporangial wall right up to the time of release (see Webster and Dennis, 1967, Plate 11). There thus seems to be little space for the extracellular gel or solute to accumulate before release begins. Perhaps in Pythium the secretion of gel-like material or solutes is very rapid and coincides with the onset of release.
F. CONCLUSIONS
Ten years ago sporulation in the oomycetes might have been cited as a telling example of biological diversity. For the sporangium and zoospores of each species, there seemed to be a unique assortment of vesicles; for each species, a different way for how the sporangium cleaved to produce the zoospores. But as the recent research reviewed in this chapter demonstrates, what is emerging now are similarities in the processes of sporulation across this taxon. By following the fate of vesicles through into encystment, with the help of immunolocalization techniques, it has become apparent that sporangial vesicles that vary morphologically from species to species have homologous future functions. The adoption of this more function-oriented approach has reduced the perplexing multitude of zoospore vesicles down to a manageable handful of five types. Likewise, use of RF-FS to study development of the partitioning membranes has indicated that progressive ramification of cleavage vacuoles may be a common feature of the cleavage process throughout the oomycetes. Such clarifications indicate that, as long as adequate methods of study are used, observations of sporulation in
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different oomycete species can be compared and contrasted in order to discover the underlying principles in operation. The principles that emerge may have direct relevance for our understanding of biological processes fundamental to the growth and development of other eukaryotic organisms.
ACKNOWLEDGEMENTS We would like to thank David Lowry, John Shields and Gordon Beakes for providing unpublished results and helpful comments during the preparation of the manuscript. We would also like to thank Frank Gubler for allowing us to use some of his unpublished micrographs and Gordon Beakes and Brent Heath for supplying copies of their previously published micrographs.
REFERENCES Agrios, G. N. (1988). “Plant Pathology”. Academic Press, San Diego. Andrianopoulos, A. and Timberlake, W. E. (1994). The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Molecular and Cellular Biology 14, 2503-2515. Armbruster, B. L. (1982a). Sporangiogenesis in three genera of the Saprolegniaceae. I: Pre-sporangium hyphae to early primary spore initial stage. Mycologia 74, 433459. Armbruster, B. L. (1982b). Sporangiogenesis in three genera of the Saprolegniaceae. 11: Primary spore initial to secondary spore inital stage. Mycologia 74, 975-999. Axelrod, D. E., Gealt, M . and Pastushok, M. (1973). Gene control of developmental competence in Aspergillus nidulans. Developmental Biology 34, 9-15. Barr, D. J. S. (1981). The phylogenetic and taxonomic implications of flagellar rootlet morphology among zoosporic fungi. BioSysrems 14, 359-370. Barr, D. J. S. (1983). Appendix 111. The genera of phytopathogenic zoosporic fungi. I n “Zoosporic Plant Pathogens. A Modern Perspective” (S. T. Buczacki, ed.), pp. 293-302. Academic Press, London. Barr, D. J. S. and Allan, P. M. E. (1985). A comparison of the flagellar apparatus in Phytophthora, Saprolegnia, Thraustochytrium and Rhizidiomyces. Canadian Journal of Botany 63, 138-154. Barr, D. J . S. and Dtsaulniers, N. L. (1987). Ultrastructure of the Lagena radicola zoospore, including a comparison with the primary and secondary Saprolegnia zoospores. Canadian Journal of Botany 65, 2161-2176. Barr, D. J. S. and Dtsaulniers, N. L. (1990). The life cycle of Lagena radicola, an oomycetous parasite of wheat roots. Canadian Journal of Botany 68, 813824. Barstow, W. E. and Lovett, J. S. (1978). Ultrastructure of a reduced developmental cycle (minicycle) in Blastocladiella emersonii. Experimental Mycology 2 , 145-155. Bartnicki-Garcia, S. (1987). The cell wall: a crucial structure in fungal evolution. I n “Evolutionary Biology of the Fungi” (A. D. M. Rayner, C. M. Brasier and D. Moore, eds), pp. 389403. Cambridge University Press, Cambridge.
ASEXUAL SPORULATION IN THE OOMYCETES
39 1
Bartnicki-Garcia, S . and Hemmes, D. E. (1976). Some aspects of the form and function of Oomycete spores. In “The Fungal Spore” (D. J. Weber and W. M. Hess, eds), pp. 593639. Wiley, New York. Beakes, G. W. (1983). A comparative account of cyst coat ontogeny in saprophytic and fish-lesion (pathogenic) isolates of the Saprolegnia declina-parasitica complex. Canadian Journal of Botany 61, 603-625. Beakes, G. W. (1987). Oomycete phylogeny: ultrastructural perpectives. In “Evolutionary Biology of the Fungi” (A. D. M. Rayner, C. M. Brasier and D. Moore, eds), pp. 405421. Cambridge University Press, Cambridge. Beakes, G. W. (1989). Oomycete fungi: their phylogeny and relationship to chromophyte algae. In “The Chromophyte Algae: Problems and Perspectives” (J. C. Green, B. S. C. Leadbeater and W. L. Diver, eds), pp. 325-342. Clarendon Press, Oxford. Beakes, G. W. (1995). Sporulation of lower fungi. In “The Growing Fungus’’ (N. A. R. Gow and G. M. Gadd, eds), pp. 339-366. Chapman & Hall, London. Bimpong, C. E. and Hickman, C. J. (1975). Ultrastructural and cytochemical studies of zoospores, cysts, and germinating cysts of Phytophthora palmivora. Canadian Journal of Botany 53, 1310-1327. Bortnick, R. N., Powell, M. J. and Bangert, T. N. (1985). Zoospore fine structure of the parasite Olpidiopsis saprolegniae variety Saprolegniae (Oomycetes, Lagenidiales). Mycologia 77, 861-879. Bouck, G. B. (1969). Extracellular microtubules. The origin, structure, and attachment of flagellar hairs in Fucus and Ascophyllum antherozoids. Journal of Cell Biology 40, 446-460. Bouck, G . B. (1972). Architecture and assembly of mastigonemes. In “Advances in Cell and Molecular Biology”, vol. 2 (E. J. Du Praw, ed.), pp. 237-276. Academic Press, New York. Burr, A. W. and Beakes, G. W. (1994). Characterization of zoospore and cysts surface structure in saprophytic and fish pathogenic Saprolegnia species (oomycete fungal protists). Protoplasma 181, 142-163. Byrt, P. and Grant, B. R. (1979). Some conditions governing zoospore production in axenic cultures of Phytophthora cinnamomi Rands. Australian Journal of Botany 27, 103-115. Cahill, D. M., Cope, M. and Hardham, A. R. (1996) Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores of Phytophthora cinnamomi. Protoplasma (in press). Carpita, N., Sabularse, D., Montezinos, D. and Delmer, D. P. (1979). Determination of the pore size of cell walls of living plant cells. Science 205, 1144-1147. Cavalier-Smith, T. (1981). Eukaryote kingdoms: seven or nine? BioSysfems 14, 461-481. Cerenius, L., Olson, L. W. and Soderhall, K. (1984). The secondary zoospore of Aphanomyces astaci and A . laevis (Oomycetes, Saprolegniales). Nordic Journal of Botany 4, 697-706. Chapman, J. A. and Vujicic, R. (1965). The fine structure of sporangia of Phytophthora erythroseptica Pethyb. Journal of General Microbiology 41, 275-282. Cho, C. W. and Fuller, M. S . (1989). Ultrastructural organization of freeze-substituted zoospores of Phytophthora palmivora. Canadian Journal of Botany 67, 14931499. Christen, J. and Hohl, H. R. (1972). Growth and ultrastructural differentiation of sporangia in Phytophthora palmivora. Canadian Journal of Microbiology 18,
392
A. R. HARDHAM and G .
J. HYDE
1959-1964. Clark, M. C., Melanson, D. L. and Page, 0. T. (1978). Purine metabolism and differential inhibition of spore germination in Phytophthora infestans. Canadian Journal of Microbiology 24, 1032-1038. Colhoun, J. (1966). The biflagellate zoospore of aquatic Phycomycetes with particular reference to Phytophthora spp. In “The Fungus Spore” (M. F. Madelin, ed.), pp. 85-92. Butterworths, London. Cope, M. and Hardham, A. R. (1994). Synthesis and assembly of flagellar surface antigens during zoosporogenesis in Phytophthora cinnamomi. Protoplasma 180, 158-1 68. Cope, M., Webb, M. C., O’Gara, E . T., Philip, B. A. and Hardham, A. R. (1996). Immunocytochemical comparison of peripheral vesicles in zoospores of Phytophthora and Pythium species. Mycologia 88, 523-532. Dahlberg, K. R. and Van Etten, J. L. (1982). Physiology and biochemistry of fungal sporulation. Annual Review of Phytopathology 20, 281-301. Dearnaley, J. D. W. and Hardham, A. R. (1994). The Golgi apparatus of Phytophthora cinnamomi makes three types of secretory vesicles concurrently. Protoplasma 182, 75-79. Dearnaley, J. D. W., Maleszka, J. and Hardham, A. R. (1996). Synthesis of zoospore peripheral vesicles during sporulation of Phytophthora cinnamomi. Mycological Research 100, 39-48. Deason, T. R. (1971). The origin of flagellar hairs in the xanthophycean alga Pseudobumilleriopsis pyrenoidosa. Transactions of the American Microscopical Society 90, 441-448. Dick, M. W. (1990). Phylum Oomycota. In “Handbook of Protoctista” (L. Margulis, J. 0. Corliss, M. Melkonian and D. J. Chapman, eds), pp. 661-685. Jones and Bartlett, Boston, MA. Domnas, A., Jaronski, S. and Hanton, W. K. (1986). The zoospore and flagellar mastigonemes of Lagenidium giganteum (Oomycetes, Lagenidiales). Mycologia 78, 810-817. Ehrlich, M. A. and Ehrlich, H. G . (1966). Ultrastructure of the hyphae and haustoria of Phytophthora infestans and hyphae of P. parasitica. Canadian Journal of Botany 44, 1495-1508. Elsner, P. R., VanderMolen, G. E., Horton, J. C. and Bowen, C. C. (1970). Fine structure of Phytophthora infestans during sporangial differentiation and germination. Phytopathology 60, 1765-1772. Estrada-Garcia, M. T., Callow, J. A. and Green, J. R. (1990). Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognise 200 X lo3 M, glycoproteins stored within large peripheral vesicles. Journal of Cell Science 95, 199-206. Forster, H., Coffey, M. D., Elwood, H. and Sogin, M. L. (1990). Sequence analysis of the small subunit ribosomal RNAs of three zoosporic fungi and implications for fungal evolution. Mycologia 82, 306-312. Gay, J. L. and Greenwood, A. D. (1966). Structural aspects of zoospore production in Saprolegnia ferax with particular reference to the cell and vacuolar membranes. In “The Fungal Spore” (M. F. Madelin, ed.), pp. 95-108. Butterworths, London. Gay, J. L., Greenwood, A. D. and Heath, I. B. (1971). The formation and behaviour of vacuoles (vesicles) during oosphere development and zoospore germination in Saprolegnia. Journal of General Microbiology 65, 233-241, Gisi, U . (1983). Biophysical aspects of the development of Phytophthora. In “Phytophthora. Its Biology, Taxonomy, Ecology and Pathology” (D. C.
ASEXUAL SPORULATION IN THE OOMYCETES
393
Erwin, S . Bartnicki-Garcia and P. H. Tsao, eds), pp. 109-119. American Phytopathological Society, St Paul, MN. Gisi, U. and Zentmyer, G. A. (1980). Mechanism of zoospore release in Phytophthora and Pythium. Experimental Mycology 4, 362-377. Gisi, U., Hemmes, D. E. and Zentmyer, G . A. (1979). Origin and significance of the discharge vesicle in Phytophthora. Experimental Mycology 3, 321-339. Gotelli, D. (1974a). The morphology of Lagenidium callinectes. I: Vegetative development. Mycologia 66, 639-647. Gotelli, D. (1974b). The morphology of Lagenidium callinectes. 11: Zoosporogenesis. Mycologia 66, 846-358. Gotelli, D. and Hanson, L. C. (1987). An ultrastructural investigation of the zoospore of Sapromyces androgynus (Oomycetes, Leptomitales). Mycologia 79, 745752. Griffin, D. H. (1994). “Fungal Physiology”. Wiley-Liss, New York. Griffin, D. H. and Breuker, C. (1969). Ribonucleic acid synthesis during the differentiation of sporangia in the water mold Achlya. Journal of Bacteriology 98, 689-696. Grove, S . N. and Bracker, C. E. (1978). Protoplasmic changes during zoospore encystment and cyst germination in Pythium aphanidermatum. Experimental Mycology 2, 51-98. Gubler, F. and Hardham, A. R. (1988). Secretion of adhesive material during encystment of Phytophthora cinnamomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles. Journal of Cell Science 90, 225-235. Gubler, F. and Hardham, A. R. (1990). Protein storage in large peripheral vesicles in Phytophthora zoospores and its breakdown after cyst germination. Experimental Mycology 14, 393404. Gunderson, J. H., Elwood, H., Ingold, A., Kindle, K. and Sogin, M. L. (1987). Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes. Proceedings of National Academy of Sciences, USA 84, 5823-5827. Gwynne, D. I. and Brandhorst, B. P. (1982). Changes in gene expression during sporangium formation in Achlya ambisexualis. Developmental Biology 91, 263-277. Hardham, A. R. (1985). Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: the influence of fixation on patterns of lectin binding. Journal of Histochemistry and Cytochemistry 33, 110-1 18. Hardham, A. R. (1987a). Ultrastructure and serial section reconstruction of zoospores of the fungus Phytophthora cinnamomi. Experimental Mycology 11, 297-306. Hardham, A. R. (1987b). Microtubules and the flagellar apparatus in zoospores and cysts of the fungus Phytophthora cinnamomi. Protoplasma 137, 109-124. Hardham, A. R. (1995). Polarity of vesicle distribution in Oomycete zoospores: development of polarity and importance for infection. Canadian Journal of Botany 73 supplement 1, S4OCbS407. Hardham, A. R. and Gubler, F. (1990). Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube. Cell Biology Znternational Reports 14, 947-956. Hardham, A. R. and Suzaki, E. (1990). Glycoconjugates on the surface of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry. Canadian Journal of Microbiology 36, 183-192. Hardham, A. R., Gubler, F. and Duniec, J . (1991a). Ultrastructural and immunological studies of zoospores of Phytophthora. In “Phytophthora” (J. A. Lucas,
394
A. R. HARDHAM and G . J. HYDE
R. C. Shattock, D. S . Shaw and L. R. Cooke, eds), pp. 50-69. Cambridge University Press, Cambridge. Hardham, A. R., Gubler, F., Duniec, J. and Elliott, J. (1991b). A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens. Journal of Microscopy 162, 305-318. Hardham, A. R., Cahill, D. M., Cope, M., Gabor, B. K., Gubler, F. and Hyde, G. J. (1994). Cell surface antigens of Phytophthora spores: biological and taxonomic characterization. Protoplasma 181, 213-232. Heath, I. B. (1976). Ultrastructure of freshwater Phycomycetes. In “Recent Advances in Aquatic Mycology” (E. B. Gareth-Jones, ed.), pp. 603-650. Elek Science, London. Heath, I. B. and Greenwood, A. D. (1971). Ultrastructural observations on the kinetosomes, and Golgi bodies during the asexual life cycle of Saprolegnia. Zeitschrift fiir Zellforschung 112, 371-389. Heath, I. B. and Harold, R. L. (1992). Actin has multiple roles in the formation and architecture of zoospores of the oomycetes, Saprolegnia ferax and Achlya bisexualis. Journal of Cell Science 102, 611-627. Heath, I. B., Greenwood, A. D. and Griffiths, H. B. (1970). The origin of flimmer in Saprolegnia, Dictyuchus, Synura and Cryptomonas. Journal of Cell Science 7 , 445-461. Heintz, C. E. (1970). Zoosporogenesis in Pythium middletoni. American Journal of Botany 57, 760. Hemmes, D. E. (1983). Cytology of Phytophthora. In “Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology” (D. C. Erwin, S . Bartnicki-Garcia and P. H. Tsao, eds), pp. 9-40. American Phytopathological Society, St Paul, MN . Hemmes, D. E. and Hohl, H. R. (1969). Ultrastructural changes in directly germinating sporangia of Phytophthora parasitica. American Journal of Botany 56, 300-313. Hemmes, D. E. and Hohl, H. R. (1973). Mitosis and nuclear degeneration: simultaneous events during secondary sporangia formation in Phytophthora palmivora. Canadian Journal of Botany 51, 1673-1675. Hill, F. G. and Outka, D. E. (1974). The structure and origin of mastigonemes in Ochromonas minute and Monas sp. Journal of Protozoology 21, 299-312. Ho, H. H., Zachariah, K. and Hickrnan, C. J . (1968). The ultrastructure of zoospores of Phytophthora megasperma var. sojae. Canadian Journal of Botany 46, 37-40. Hoch, H. C. and Mitchell, J. E. (1972a). The ultrastructure of Aphanomyces euteiches during asexual spore formation. Phytopathology 62, 149-160. Hoch, H. C. and Mitchell, J. E. (1972b). The ultrastructure of zoospores of Aphanomyces euteiches and of their encystment and subsequent germination. Protoplasma 75, 113-138. Hoch, H. C. and Mitchell, J. E. (1973). The effects of osmotic water potentials on Aphanomyces euteiches during zoosporogenesis. Canadian Journal of Botany 51, 413-420. Hoch, H. C. and Mitchell, J. E. (1975). Further observations on the mechanisms involved in primary spore cleavage in Aphanomyces euteiches. Canadian Journal of Botany 53, 1085-1091. Hohl, H. R. (1990). Nutrition. Advances in Plant Pathology 7 , 53-83. Hohl, H. R. and Hamarnoto, S. T. (1967). Ultrastructural changes during zoospore formation in Phytophthora parasitica. American Journal of Botany 54, 11311139.
ASEXUAL SPORULATION IN THE OOMYCETES
395
Holloway, S. A. and Heath, I. B. (1977a). Morphogenesis and the role of microtubules in synchronous populations of Saprolegnia zoospores. Experimental Mycology 1, 9-29. Holloway, S. A. and Heath, I . B. (1977b). An ultrastructural analysis of the changes in organelle arrangement and structure between the various spore types of Saprolegnia. Canadian Journal of Botany 55, 1328-1339. Holwill, M. E. J. (1982). Dynamics of eukaryotic flagellar movement. In “Prokaryotic and Eukaryotic Flagella” (W. B. Amos and J. G . Duckett, eds), pp. 289-312. Cambridge University Press, Cambridge. Hyde, G. J. (1991). The ultrastructural basis of zoosporogenesis in Phytophthora. Ph.D. Thesis, Australian National University. Hyde, G. J. and Hardham, A. R. (1992). Confocal microscopy of microtubule arrays in cryosectioned sporangia of Phytophthora cinnamomi. Experimental Mycology 16, 207-218. Hyde, G . J. and Hardham, A. R. (1993). Microtubules regulate the generation of polarity in zoospores of Phytophthora cinnamomi. European Journal of Cell Biology 62, 75-85. Hyde, G . J., Gubler, F. and Hardham, A. R. (1991a). Ultrastructure of zoosporogenesis in Phytophthora cinnamomi. Mycological Research 95, 577-591. Hyde, G. J., Lancelle, S . , Hepler, P. K. and Hardham, A. R. (1991b). Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes. Journal of Cell Science 100, 735-746. Hyde, G. J., Lancelle, S . , Hepler, P. K. and Hardham, A. R. (1991~).Sporangial structure in Phytophthora is disrupted after high pressure freezing. Protoplasma 165, 1-3. Inouye, I. (1996). Flagella and flagellar apparatuses of algae. In “Ultrastructure of Microalgae” (T. Berner, ed.), pp. 99-128. CRC Press, Boca-Raton, FL. Jackson, S. L. and Hardham, A. R. (1996). A transient rise in cytoplasmic free calcium is required for the induction of cytokinesis in zoosporangia of Phytophthora cinnamomi. European Journal of Cell Biology 69, 180-188. Jahn, T. L., Landman, M. D. and Fonseca, J. R. (1964). The mechanism of locomotion of flagellates. 11: function of the mastigonemes of Ochromonas. Journal of Protozoology 11, 291-296. Jaworski, A. J. and Harrison, J. A. (1986). RNA synthesized during late sporulation is required for germ tube formation in Blastocladiella emersonii. Experimental Mycology 10, 42-51. Jelke, E., Oertel, B., Bohm, K. J. and Unger, E. (1987). Tubular cytoskeletal elements in sporangia and zoospores of Phytophthora infestans (Mont.) de Bary (Oomycetes, Pythiaceae). Journal of Basic Microbiology 27, 11-21. Kevorkian, A. G. (1935). Studies in the Leptomitaceae. 11: Cytology of Apodachlya brachynema and Sapromyces reinschii. Mycologia 27, 274-285. King, J. E., Colhoun, J. and Butter, R. D. (1968). Changes in the ultrastructure of sporangia of Phytophthora infestans associated with indirect germination and ageing. Transactions of the British Mycological Society 51, 269-281. Lange, L. and Olson, L. W. (1983). The fungal zoospore. Its structure and biological significance. In “Zoosporic Plant Pathogens. A Modern Perspective” (S. T. Buczacki, ed.), pp. 2 4 2 . Academic Press, London. Lange, L., Olson, L. W. and Safeeulla, K. M. (1984). Pearl millet downy mildew (Sclerospora graminicola): zoosporogenesis. Protoplasma 119, 178-187. Lange, L., Eden, U. and Olson, L. W. (1989). Zoosporogenesis in Pseudo-
396
A . R. HARDHAM and G . J . H Y D E
peronospora cubensis, the causal agent of cucurbit downy mildew. Nordic Journal of Botany 8, 497-504. Leedale, G . F., Leadbeater, B. S. C. and Massalski, A. (1970). The intracellular origin of flagellar hairs in the Chrysophyceae and Xanthophyceae. Journal of Cell Science 6, 701-719. Lefebvre, P. A. and Rosenbaum, J. L. (1986). Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration. Annual Review of Cell Biology 2 , 517-546. Lehnen, L. P. Jr. and Powell, M. J. (1989). The role of kinetosome-associated organelles in the attachment of encysting secondary zoospores of Saprolegnia ferax to substrates. Protoplasma 149, 163-174. Lehnen, L. P. Jr. and Powell, M. J. (1991). Formation of K2-bodies in primary cysts of Saprolegnia ferax. Mycologia 83, 163-179. Loiseaux, S. (1973). Ultrastructure of zoidogenesis in unilocular zoidocysts of several brown algae. Journal of Phycology 9, 277-289. Lunney, C. Z. and Bland, C. E. (1976a). An ultrastructural study of zoosporogenesis in Pythium proliferum de Bary. Protoplasma 88, 85-100. Lunney, C . Z. and Bland, C. E. (1976b). Ultrastructural observations of mature and encysting zoospores of Pythium proliferum de Bary. Protoplasma 90, 119137. MacDonald, J. D. and Duniway, J. M. (1978). Influence of the matric and osmotic components of water potential on zoospore discharge in Phytophthora. Phytopathology 68, 751-757. Margulis, L., Corliss, J. O., Melkonian, M. and Chapman, D. J. (1990). “Handbook of Protoctista”. Jones and Bartlett, Boston, MA. Martin, R. W., Jr. and Miller, C. E. (1986). Ultrastructure of zoosporogenesis in the endoparasite Olpidiopsis varians. Mycologia 78, 230-241. McCully, M. E. and Canny, M. J. (1985). The stabilization of labile configurations of plant cytoplasm by freeze substitution. Journal of Microscopy 139, 27-33. McNaughton, E. E. and Goff, L. J. (1990). The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae. Protoplasma 157, 19-37. Mims, C. W., Richardson, E. A. and Kimbrough, J . W. (1990). Ultrastructure of ascospore delimitation in freeze substituted samples of Ascodesmis nigricans (Pezizales). Protoplusma 156, 94-102. Money, N. P. and Webster, J. (1985). Water stress and sporangial emptying in Achlya (Saprolegniaceae). Botanical Journal of the Linnean Society 91, 319-327. Money, N. P., Webster, J. and Ennos, R. (1988). Dynamics of sporangial empyting in Achlya intricata. Experimental Mycology 12, 13-27. Nobel, P. S. (1991). “Physicochemical and Environmental Plant Physiology”. Academic Press, San Diego, CA. Oertel, B. and Jelke, E. (1986). Formation of multinucleate zoospores in Phytophthora infestans (Mont.) de Bary (Oomycetes, Pythiaceae). Protoplasma 135, 173-179. Olson, L. W., Eden, U. M. and Lange, L. (1981). Zoosporogenesis: model systems-problems-possible approaches. In “The Fungal Spore: Morphogenetic Controls” (G. Turian and H. R. Hohl, eds), pp. 43-70. Academic Press, London. Pickett-Heaps, J. D. (1972). Variation in mitosis and cytokinesis in plant cells: its significance in the phylogeny and evolution of ultrastructural systems. Cytobios 5 , 59-77. Pinto da Silver, P. and Noguiera, M. L. (1977). Membrane fusion during secretion.
ASEXUAL SPORULATION IN THE OOMYCETES
397
A hypothesis based on electron microscope observation of Phytophthora palmivora zoospores during encystment. Journal of Cell Biology 73, 161181. Powell, M. J. and Bracker, C. E. (1986). Distribution of diaminobenzidine reaction products in zoospores of Phytophthora palmivora. Mycologia 78, 892-900. Powell, M. J., Lehnen, L. P. Jr. and Bortnick, R. N . (1985). Microbody-like organelles as taxonomic markers among Oomycetes. BioSystems 18, 321334. Pueschel, C. M. and van der Meer, J. P. (1985). Ultrastructure of the fungus Petersenia palmariae (Oomycetes) parasitic on the alga Palmaria mollis (Rhodophyceae). Canadian Journal of Botany 63, 409-418. Rayner, A. D. M., Griffith, G. S. and Ainsworth, A. M. (1994). Mycelial interconnectedness. In “The Growing Fungus” (N. A. R. Gow and G. M. Gadd, eds), pp. 21-40. Chapman & Hall, London. Reichle, R. E. (1969). Fine structure of Phytophthora parasitica zoospores. Mycologia 61, 30-51. Ribeiro, 0. (1983). Physiology of asexual sporulation and spore germination in Phytophthora. In “Phytophthora. Its Biology, Taxonomy, Ecology, and Pathology” (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, eds), pp. 55-70. American Phytopathological Society, St Paul, MN. Sachay, D. J., Hudspeth, D. S. S., Nadler, S. A. and Hudspeth, M. E. S. (1993). Oomycete mtDNA: Phytophthora genes for cytochrome c oxidase use an unmodified genetic code and encode proteins most similar to those of plants. Experimental Mycology 17, 7-23. Sadowski, L. A. and Powell, M. J. (1990). Cytochemical detection of polysaccharides in zoospores of Aphanomyces euteiches. Canadian Journal of Botany 68, 1379-1 388. Samuels, A. L., Giddings, T. H. and Staehelin, L. A. (1995). Cytokinesis in tobacco BY-2 and root tip cells - a new model of cell plate formation in higher plants. Journal of Cell Biology 130, 1345-1357. Sansome, E. R. (1987). Fungal chromosomes as observed with the light microscope. In “Evolutionary Biology of the Fungi” (A. D. M. Rayner, C. M. Brasier and D. Moore, eds), pp. 97-113. Cambridge University Press, Cambridge. Sargent, J. A. and Payne, H. L. (1974). Effect of temperature on germination, viability and fine structure of conidia of Bremia lactucae. Transactions of the British Mycological Society 63, 509-518. Schnepf, E., Deichgraber, G. and Drebes, G. (1978a). Development and ultrastructure of the marine, parasitic Oomycete, Lagenisma coscinodisci Drebes (Lagenidiales): formation of the primary zoospores and their release. Protoplasma 94, 263-280. Schnepf, E., Deichgraber, G. and Drebes, G . (1978b). Development and ultrastructure of the marine parasitic Oomycetc, Lagenisma coscinodisci (Lagenidiales): encystment of primary zoospores. Canadian Journal of Botany 56, 13091314. Schnepf, E . , Deichgraber, G. and Drebes, G. (1978~).Development and ultrastructure of the marine, parasitic Oomycete, Lagenisma coscinodisci Drebes (Lagenidiales). Thallus, zoosporangium, mitosis, and meiosis. Archive of Microbiology 116, 141-150. Shepherd, V. A., Orlovich, D. A. and Ashford, A. E. (1993). A dynamic continuum of pleomorphic tubules and vacuoles in growing hyphae of a fungus. Journal of Cell Science 104, 495-507. Sing, V. 0. and Bartnicki-Garcia, S. (1975a). Adhesion of Phytophthora palmivora
398
A. R. HARDHAM and G. J. HYDE
zoospores: electron microscopy of cell attachment and cyst wall fibril formation. Journal of Cell Science 18, 123-132. Sing, V. 0. and Bartnicki-Garcia, S. (1975b). Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A receptor sites appearing during encystment. Journal of Cell Science 19, 11-20. Suzaki, E., Suzaki, T., Jackson, S. L. and Hardham, A. R. (1996). Changes in intracellular pH during zoosporogenesis in Phytophthora cinnamomi. Protoplasma 191, 79-83. Timberlake, W. E., McDowell, L., Cheney, J. and Griffin, D. H. (1973). Protein synthesis during the differentiation of sporangia in the water mold Achlya. Journal of Bacteriology 116, 67-73. Trigano, R. N. and Spurr, H. W. J. (1987). The development of the multinucleate condition of Peronospora tabacina sporangia. Mycologia 79, 353-357. Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. Methods in Cell Biology 30, 127-156. Van Wyk, P. W. J. and Wingfield, M. J. (1991a). Ultrastructure of ascosporogenesis in Ophiostomia davidsonii. Mycological Research 95, 725-730. Van Wyk, P. W. J. and Wingfield, M. J. (1991b). Ascospore ultrastructure and development in Ophiostoma cucullatum. Mycologia 83, 698-707. Van Wyk, P. W. J., Wingfield, M . J. and Van Wyk, P. S. (1991). Ascospore development in Ceratocystis moniliformb. Mycological Research 95, 96-103. Webster, J. and Dennis, C. (1967). The mechanism of sporangial discharge in Pythium middletonii. New Phytologist 66, 307-313. Williams, W. T. and Webster, R. K. (1970). Electron microscopy of the sporangium of Phytophthora capsici. Canadian Journal of Botany 48, 221-227. Wilson, J. G. M. (1976). Immunological aspects of fungal disease in fish. In “Recent Advances in Aquatic Mycology” (E. B . Gareth-Jones, ed.), pp. 573-601. EIek Science, London. Wolters, J. and Erdmann, V. A. (1988). Cladistic analysis of ribosomal RNAs - the phylogeny of eukaryotes with respect to the endosymbiotic theory. BioSystems 21. 209-214. Youatt, J . (1976). Sporangium formation in Allomyces throughout the growth cycle. Transactions of the British Mycological Society 67, 159-161.
Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution?
J. WOSTEMEYER, A WOSTEMEYER and K. VOIGT
Friedrich-Schiller-UniversistatJena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany
I. Horizontal or Lateral Gene Transfer: where does it Occur?
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IV. Plants as Recipients of Foreign DNA ........................................... A . Agrobacterium tumefaciens .................................................... B. Rhizobium Interactions with Legumes ....................................
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VI . Relevance of Lateral Gene Transfer for Evolution .......................... References ...............................................................................
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11. Bacteria as Recipients of Foreign DNA 111. Fungi as Recipients of Foreign DNA
V. Interkingdom Gene Transfer
I. HORIZONTAL OR LATERAL GENE TRANSFER: WHERE DOES IT OCCUR? Since modern biotechnology enables the construction of defined genotypes of prokaryotic and eukaryotic micro-organisms, plants and animals, the question of uncontrolled spread of artificially manipulated genes over species boundaries has provoked the interest of many ecologists, geneticists and the general public. Very often, artificial gene transfer by in vitro techniques is regarded as a taboo, forbidden either due to religious considerations or the argument that gene exchange in nature is limited to sexual o r parasexual Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN &12-005924-X
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recombination systems within a single defined species. Geneticists know that there is no such strict confinement to intraspecific gene transfer. Initially, bacterial conjugation based on the F-factor was detected as a parasexual system within the single species Escherichia cofi (Lederberg and Tatum, 1946). However, as soon as the nature of F as a DNA molecule was detected, it was observed that F can be easily transferred from E. coli to Serratia marcescens (Marmur et al., 1961). Both species belong to the enteric bacteria and are phylogenetically related, but certainly no bacteriologist will ever doubt that these species are distinct enough to be assigned to different genera. Apart from F, a huge variety of different plasmids, especially colicinogenic factors (Bhaskaran, 1958) and resistance transfer factors (RTFs) (Mitsuhashi et a f . , 1960) were described, many of which were transmissible among many different species of enterobacteria. Very early during the development of bacterial genetics, it became evident that interspecies gene transfer is more the rule than the exception. Later it was found that conjugation is also encountered in bacteria which are often found in soil: Gram-positive bacteria (Clewell and Flannagan, 1993) and streptomycetes (Hopwood and Kieser, 1993). We discuss the bacterial systems in more detail in section 11. When looking at organismic communities in soil and, in the immediate vicinity of plant roots, at rhizosphere and rhizoplane systems, we have to take into account fungi, bacteria, plants and also animals. Relationships between soil organisms may be of different kinds: there is in any case a competition for nutrients. The least-fit organisms will die and their DNA becomes available. Close cell-to-cell contact is a starting point for most symbioses. In these, genetic exchange may be facilitated. Genetic exchange is less probable in parasitic o r predator-prey relationships, but even here gene transfer is possible. In this chapter we will not consider animal systems, although we suspect that at least protozoa are good candidates for incorporating foreign DNA into their chromosomal complement. We should not forget that even in vertebrates DNA fragments survive the intestinal tract to some degree and can be found in the bloodstream by using sensitive polymerase chain reaction (PCR) methods (Schubert et al., 1994). Analyses of this type do not prove a true gene transfer. But, on an evolutionary scale, the occasional appearance of foreign DNA molecules outside the organs or, in the case of protozoa, the organelles for food processing may represent the starting point for recombination events. Evolution does not need frequent events. The incorporation of a single highly recombinogenic or replicative molecule into a pre-existing genome may subsequently lead to considerable rearrangements. On the whole, very little research has been done in animals with regard to naturally occurring horizontal gene transfer. In fungi, the situation is somewhat different. Although actual genetic investigations on interspecies gene transfer are rare, appropriate mechanisms are known. Many fungi have the ability to form anastomoses; we will discuss the possibilities for genetic hybridization via this mechanism. Apart from this
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more conventional and widespread option, several mycoparasitic systems are known which involve the formation of plasma bridges between host and parasite. Such parasites are named “fusion biotrophs” (Jeffries and Young, 1994). For two of these mycoparasitic systems, experimental evidence has been obtained that the cytoplasmic fusion is accompanied by the invasion of genetic material of the parasite into the host (Kellner et a f . , 1993; A . Wostemeyer, unpublished). We discuss fungal gene transfer in section 111. Possibly the best-described gene transfer system in the rhizosphere involves the soil bacterium Agrobacterium turnefaciens, which transfers a special region of its Ti-plasmid to many different plant species. This interkingdom system is based on a conjugational mechanism (see section IV). Are there other interkingdom gene transfers in nature? The answer is “possibly”. We have some evidence that DNA from decaying plants may be introduced into soil fungi (Hoffmann et a f . , 1994). We also have an idea that host DNA may show up in a plant pathogen, at least transiently (Bryngelsson et al., 1988). Taking into account that conjugational gene transfer systems have also been observed between E. coli and yeasts (Sprague, 1991), we should perhaps address the questions of frequency and importance of genetic exchange between kingdoms more seriously (see section V). Other questions of major importance for the evolutionary impact of horizontal gene transfer are addressed in section VI: Is horizontal gene transfer a rare event, limited to rather specialized organismic associations, or do certain biotopes represent a genetic continuum, where gene pools are shared by many different species? Is there a reasonable probability for permanent gene acquisition via interspecies gene transfer? Are there mechanisms that remove foreign DNAs or suppress their activity? By necessity, due to sparse experimental information, the attempts to answer these questions are speculative.
11. BACTERIA AS RECIPIENTS OF FOREIGN DNA During evolution several types of mechanisms for horizontal gene transfer in bacteria have been evolved. Diverse independent lines of research have provided convincing evidence that these processes occur in the environment (Cresswell and Wellington, 1992; Pickup, 1992; Saunders, 1992). All of them are essentially infection processes, in which DNA passes from a donor to a recipient, integrates into the prokaryotic chromosome by recombination and expresses its genetic information in the phenotype. Unlike the common case in eukaryotes, this recombination followed by natural gene flow is not linked to reproduction (Young, 1992). It is not the intention of this general overview to give a fully detailed compilation of the biology of the three main mechanisms and their genetic regulation, although general aspects are discussed. Details may be found in a number of excellent reviews (Schmidt,
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1992; Stewart, 1992; Dreiseikelmann, 1994; Lorenz and Wackernagel, 1994). The aim of this overview is to give a general picture of bacterial gene flux based on recent experimental concepts and its putative role in long-term evolution. Natural genetic transformation seems to be the easiest way for genetic exchange in the environment. Bacteria enter a physiologically regulated state of competence, bind, and take up actively extracellular DNA (plasmid and chromosomal) from the surrounding medium and express this exogeneous DNA after heritable incorporation. This process depends on the function of several genes located on the bacterial chromosome and involves many not easily recognizable regulation mechanisms (for a review see Lorenz and Wackernagel, 1994). For a systematic experimental investigation and for development of a general model for natural transformation, it was previously proposed to divide the complex process of gene transfer by free DNA into separate steps which are common to all bacteria. Lorenz and Wackernagel (1994) distinguish various phases between the release of donor DNA, development of competence and the final recombination event into the recipient’s chromosomal complement. Many conditions have to be fulfilled for a natural transformation in the rhizosphere to occur. The first question is: Is a pool of free DNA available for soil micro-organisms in interacting in situ systems? One reason for the presence of free DNA in the environment may be the continuous production of DNA by micro-organisms. Indeed, naked DNA of microbial origin can be released during growth of Streptomyces in nature by autolysis following cell death. In certain phases of their development, Streptornyces vegetative hyphae could lyse, supplying not only nutrients but also free DNA to the environment (Schmidt, 1992). The main cause for the pickup of external DNA might be of a trophic nature. Like other biopolymers, nucleic acids serve as nutrients for many degradative micro-organisms. DNA-degrading bacteria can be readily isolated from environmental samples. In fact, nucleases which are ubiquitously present in the environment attack free DNA. The action of such dissolved, not cell-associated DNA-degrading enzymes may cause a decrease in stability and availability of naked DNA in soil habitats. On the other hand, several factors have been identified that contribute to the persistence and protection of extracellular DNA (Lorenz and Wackernagel, 1994). It has been demonstrated that DNA forms complexes with several particulate constituents of soils and sediments, such as quarz, feldspar and clay minerals, which also have sorptive capacities for other organic material. This adsorption on soil particles decreases the fluctuation of DNA in soil caused by free nucleases and reduces or abolishes its immediate utilization by soil micro-organisms. Thus, the degree of exogenous nucleolytic degradation is an important factor in determining the relative efficiency of transformation in the environment. The relative concentration and the heterogeneity of free DNA will also influence natural
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transformation. In addition, distribution of free DNA and local contact between transforming DNA and bacterial cells will influence natural transformation in nature. Moreover, the cells must be transformable and this is determined by competence. So the second important question is: Are soil bacteria able to take up and process foreign DNA? Competence is a regulated state in transformable bacteria, the development of which requires the influence of certain physiological stress factors (e.g. nutrient limitation, varying pH, temperature, fluctuation of available water in soil, oxygen radicals and other cytotoxic agents, ionic strength). The ability of Azotobacter vinelandii, and similarily of the cyanobacterium Anacystis nidulans, to become competent in soil has been estimated to be influenced by the mobilization of iron from soil minerals (for a review see Lorenz and Wackernagel, 1994). The production of siderophores coincided with a decreased appearance of transformants. Interestingly, the rhizosphere of plants is an iron-limited habitat (O’Sullivan and O’Gara, 1992). Consequently, the competence development may be increased in this case. Competence is internally regulated among most transformable bacteria (for a review see Stewart, 1992). Moreover, in Bacillus subtilis and in some species of Streptococcus the induction of competehce is controlled by the concentration of a competence factor, a low-molecular-weight polypeptide which is synthesized constitutively and secreted in the surrounding medium. At a critical concentration the exogenous protein stimulates the expression of genes involved in competence induction. Bacteria grow in soil by formation of colonies (Creswell and Wellington, 1992). This kind of growth provides the high densities of bacterial cells required for accumulation of competence factors for competence induction. Competent cells of B. subtilis and Streptococcus pneumoniae bind any kind of double-stranded DNA noncovalently and without base-sequence specificity, although only homologous DNA can be integrated into the genome by homologous recombination. The purity of the extracellular DNA may not affect its transforming activity. The DNA may be complexed with material from lysed cells or other environmental substances, which can lead to reduction of transforming activity (Romanowski et al., 1992). These data suggest that in maintenance of transforming activity of stable DNA, there is no simple rule which is common to all variations of bacteria-DNA interaction. The efficiency of transformation also depends on the molecular size of the transforming DNA molecule. In B. subtilis, the shearing of DNA from 28.5 to 4.5kb decreased the transformation by 100-fold. Further shearing to 2 kb reduced it a further 100-fold. DNA of about 1kb mean molecular size was inactive (Morrison and Guild, 1972). Table I indicates similarities and differences between Gram-positive and Gram-negative bacteria in some aspects of the mechanism of transformation (according to data reviewed by Dreiseikelmann, 1994). Despite the differences in the cell surface of Gram-positive and Gram-negative organisms
TABLE I Comparison of natural transformation in Gram-positive and Gram-negative bacteria (adapted from Dreiseikelmann, 1994) ~
~~
Gram-positive
~~~~
~
Gram-negative L.
Model systems Nature of transforming DNA Sensitivity of free DNA to extracellular nucleases Sequence-specific DNA uptake Existence of transformasomes Receptor proteins for DNA binding Existence of transmembrane channeldpores Place of resistance development of DNA against nucleolytic degradation, including cellular restriction enzymes Conversion of ds DNA to single-strand DNA with the help of membrane-bound translocase (endonuclease I) probably by degradation of the 5'-strand
Polarity of DNA entry: 3'-end ahead (exceptions might be possible) Specialized pores or channels formed by membrane proteins involved in the transformation mechanism Existence of polyhydroxybutyrate (PHB) channels Protection of single-strand DNA by a competence-specific SSB protein Existence of DNA-containing membrane vesicles (blebs)
Bacillus subtilis Streptococcus pneumoniae Double strand Yes No No At the cell surface Yes Transmembrane channel Yes
Haemophilus spp. Neisseria spp. Double strand Yes Yes Yes At the surface of transformasomes Yes Transformasome
Yes Yes
Yes (hypothetical; not shown for Haemophilus or Neisseria, but shown for Acinetobacter calcoaceticus) Yes Not identified
Yes (hypothetical) Yes No
No Probably Yes
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and the differences in the early steps of the transformation processes of the model organisms, which have been manifested in DNA binding, and early DNA passage through the different membrane systems, the DNA transformation machineries seem to be similar. Modifications occur in specific details of transformation steps in dependence on differences in components of the bacterial cell surfaces. These modifications suggest that they follow separate mechanisms in some aspects and therefore have unique characteristics. So Gram-negative bacteria such as Neisseria gonorrhoeae take up DNA into membrane vesicles called “blebs” (Dorward and Garon, 1990), which are a kind of mobile transformasomes analogous to the situation in Haemophilus. These membrane vesicles may be transport carriers to other cells and reach the cytoplasm of the recipient cell by fusion. This special case of transformation would not include DNA transfer across membranes, and may be advantageous in overcoming competence problems. Speculatively, the range of recipient cells may be expanded into interspecific, intergeneric or perhaps transkingdom regions, just in case the DNA uptake is not homospecific (sequence-specific DNA binding see Table I). The third important question concerning DNA exchange between soil bacteria is: Do recipient bacteria express the acquired foreign DNA? Model experiments have shown that resistance against mercury and the ability to degrade a herbicide are expressed after plasmid transfer from Alcaligenes eutrophus to Variovorax paradoxus in soil (Neilson et al., 1994). So the third main prerequisite for a successful gene transfer may be fulfilled. Besides transformation there are two more mechanisms of DNA transfer: conjugation and transduction. Conjugation is the only form of bacterial sex that involves direct contact, the donor producing a pilus to which the recipient becomes attached and transfers a copy of its F-plasmid as a single strand with the 5‘-end ahead. The whole conjugative machinery, including the pilus, is normally plasmid encoded. The transferred DNA is, in some cases, not only that of the plasmid. DNA regions of chromosomal origin integrated into the plasmid flanking regions by incorrect recombinational excision from the genome (F’-plasmids) have also been observed. In this way conjugative plasmids can assist in the transfer of both chromosomal DNA and other plasmids mediated by formation of a co-integrate between the plasmid and part or all of the other replicon. Streptomycetes are widely distributed among rhizosphere bacteria. They contain different types of plasmids: normal closed circular double-stranded DNA, single-stranded DNA and integrative elements. The conjugative plasmid PIG 101 is especially interesting. It is a closed circle of doublestranded DNA with a size of 8.8 kb. A Streptomyces bacterium can carry up to 300 copies of this plasmid. It can be transferred in vitro into Micromonospora, Thermonospora, Saccharopolyspora and Amycolatopsis, so perhaps it can also experience this wide host range in vivo (Hopwood and Kieser, 1993). The DNA consists of 73% GC, which is normal for
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Streptomyces DNA, and 9 ORFs can be deduced from the DNA sequence. A search for homologies to corresponding protein sequences in a data base showed no obvious similarities with known proteins. Where does this plasmid DNA come from? Did it travel through many organisms until it came to an intermediate stop in Streptomyces? The plasmid carries genes for its own replication (rep coding for a 456 amino acid protein) and a transfer protein (tru coding for a protein of about 621 amino acids). A regulatory gene on this plasmid is Kor B which limits plasmid copy number. The second regulatory gene, Kor A , represses the promoter of the tra gene and three other genes belonging to the same operon. These three other genes code for hydrophobic proteins which may be localized at the cell membrane. During plasmid transfer chromosomal genetic markers are mobilized to a small percentage. In vitro insertion of the tru region of the plasmid into a Streptomyces chromosome mobilizes this chromosome. The plasmid pIJ 101 proves to be a good carrier for a horizontal gene transfer between bacteria. Another type of DNA element which can move and rearrange the DNA of the recipient is a transposon. Transposable elements, which may have several copies per genome scattered across the chromosome and plasmids, frequently provide homology that can lead to co-integrate formation by recombination. Transposons occur in Gram-negative and Gram-positive bacteria, as well as in eukaryotes. Transposons can integrate into chromosomes and plasmids, their integration and excision creating short duplications at the respective sites. They carry their own enzyme genes for integration and excision, and in prokaryotes usually antibiotic resistance genes as well. The frequency of transposition varies from element to element, and for some eukaryotic elements there is no clear evidence for their “jumping”. Generally, plasmids and transposons appear to be restricted in host range. Thus, for example, each type of a Sym plasmid is found only in a restricted range of chromosomal genotypes within the species Rhizobium leguminosurum. Likewise the insertion sequence Rml is widespread in Rhizobium meliloti but is not found in other species. Also the insertion sequences in E. coli, which tend to be confined to a part of the genotypic range of this species, provide evidence for rather limited chromosomal gene exchange (for reviews see Schmidt, 1992; Young, 1992). At first sight the accessory element-mediated genetic traffic seems to be intraspecific or even narrower. But some accessory elements have been transferred across much greater taxonomic distances. The best known examples are the antibiotic resistance transposons and the plasmids that carry them (for a review see van Elsas, 1992). Inc P-group plasmids, for example, can be transferred between and maintained in bacteria from both the y (enterics, pseudomonads) and the a (rhizobia) subdivisions of proteobacteria, which are by molecular sequence divergence as distant from each other as whales are from worms (Young, 1992).
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Several in vitro studies show conjugational gene transfer which permits genetic elements to cross the Gram barrier. Bertram et al. (1991) demonstrate the genetic transfer of the conjugative streptococcal transposon Tn 916 from the Gram-positive Enterobacter faecalis to the Gram-negative Alcaligenes eutrophus, Citrobacter freundii and E. coli and from E. coli to the Gram-positive B . subtilis, Clostridium acetobutylicum, Enterobacter faecalis and Streptococcus lactis. Such heterogramic transfers have been shown to occur in vitro. However, data about their occurrence under natural conditions are lacking. Kreps et al. (1990) conjugatively transferred an Inc Q plasmid from E . coli to the cyanobacterium Synechocystis PCC 6830. Transduction is the transfer of bacterial genes between bacteria by bacteriophages. Since most phages have a narrow host range, this reduces the probability of extensive gene transfer within a mixed bacterial population. Another group of virus-like elements may play a bigger role in genetic exchange processes, these are retrotransposons and retrons (Garfinkel, 1992). Retrotransposons occur in yeast and other fungi, like Ty in Saccharomyces cerevisiae and Tf in Schizosaccharomyces pombe. Besides their passive replication with the chromosome, these elements can replicate via an RNA intermediate and integrate at a new site. At the target site they cause short duplications. In a few bacteria a multicopy element with resemblance to retroviruses is found: the retron. It consists of a single strand DNA covalently bound to RNA forming a stem-loop structure. In E . coli the retron Ec 67 has been demonstrated to generate a 26 nt target-site duplication and to carry 34 kb novel sequences into the target site. So this retron serves as carrier for a gene transfer. Another retron, Ec 73, is part of an E . coli phage. Transposons may assist in increasing the probability of survival and dispersal in new ecological niches. Class I1 transposons are widespread and contain systems for gene integration and dispersal among bacteria. They might be based originally on drug-producing (antibiotics) micro-organisms like streptomycetes and fungi, and may have been evolved for self-protection from autotoxicity (Schmidt, 1992). Table I1 gives a short overview of the differences between several forms of horizontal gene transfer in bacteria. However, with new answers from investigations and increased understanding the boundaries which distinguish the transfer processes become less defined and fuse to a supermechanism containing different stages, steps and machineries of genetic exchange pathways. In addition, like sequence comparisons in search of homologies at the nucleic acid or protein level, retrospective studies could be generalized and summarized to a more complete picture of the main pathways of DNA transfer. The requirements for homology by the recombination machinery will ensure that the great majority of successful genetic exchange events are intraspecific. Interspecific and intergeneric gene transfer may thus be one of the reasons for
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TABLE I1 Comparison of the four main mechanisms (transformation, transduction, transposition, conjugation) belonging to horizontal gene transfer (adapted from reviews by Pickup, 1992; Lorenz and Wackernagel, 1994; Dreiseikelmann, 1994) Conjugative processes involving direct contact Transformation Cell-cell contact Carrier of transversing DNA Nature of DNA to be transversed
Nature of DNA passing the cytoplasm of the recipient In vitro sensibility of the process to DNases Transmembrane channels formed by proteins encoded by genes dispersed over the genome Limiting factor for long-term evolutionary steps Interbacterial genetic exchange
No Free DNA (associated with organic and inorganic particles) Double strand (linear)
Single strand
Transduction No Phages
Transposition Yes Transposons
Conjugation Yes Plasmids