The Powdery Mildews: A Comprehensive Treatise

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Preface

During the First International Powdery Mildew Conference in 1999 at Avignon, France, it became clear that our knowledge of powdery mildew fungi, and the plant diseases they cause, has advanced substantially since publication of The Powdery Mildews, edited by D. M. Spencer, in 1978. Spencer’s excellent book was the first and, until now, the only book devoted to this major group of plant diseases. Among participants at the First Powdery Mildew Conference, there was general consensus that a new book, comprehensively updating our understanding of powdery mildews, would be a welcome and valuable addition.

Over the last 20 years, powdery mildews have been investigated by an increasing number of specialists in diverse fields, including chemistry, biochemistry, molecular and cell biology, genetics, morphology and histology, taxonomy, host-parasite interaction, ecology, and epidemiology. With such expansion in understanding of powdery mildews, only a book combining the contributions of many specialist scientists could hope to achieve a comprehensive and thorough update. Accordingly, we solicited manuscripts from recognized experts in many aspects of powdery mildews research.

To complement Spencer’s 1978 book, which includes chapters devoted to powdery mildews of individual crops as well chapters organized by scientific discipline, we chose to concentrate on research areas in which the most dramatic advances have occurred in the last 20 years. We felt this would best add to the earlier knowledge while emphasizing recent research that has advanced our understanding.

This new book comprises 18 chapters, starting with an overview. The following chapters are organized in five sections: The Fungi, Techniques, Host-Parasite Interactions, Populations, and Control. The three chapters in “The Fungi” deal with taxonomy, genetics, and infection structures of the powdery mildew fungi. Some readers may be surprised by the new nomenclature used in this section. However, the new nomenclature and classification of species reflects the phylogeny of the fungi more accurately than the older, more familiar names. Therefore, under Dr. Uwe Braun’s guidance, we present both the ‘new’ and the ‘old’ names throughout the treatise. We intend that this will facilitate understanding and accelerate transition towards general use of the new taxonomy and nomenclature. With respect to nomenclature for powdery mildew fruitbodies, we have retained the traditional term ‘cleistothecium’ in most cases. However, we refer the reader to page 44, in the chapter by Braun et al., in which the shortcomings of the term are discussed and the general term ‘ascoma’ and a newly coined, more specific term, ‘chasmothecium,’ are presented as alternatives.

The “Techniques” section includes two chapters on research techniques especially pertinent to investigation of powdery mildews. Being strict biotrophs, the maintenance and study these fungi present unique challenges. In this section, the first chapter provides information on basic and more advanced biological methods used to maintain, handle and study the powdery mildews. Over the last decade, the power of molecular genetic analysis has become increasingly apparent, and hugely significant and rapid advances have resulted from the development of methods for transformation of many organisms. It is, however, only very recently that a technique for the stable transformation of a powdery mildew fungus has been devised.

This method, detailed in the second chapter of this section, may prove generally useful for the study of other obligate biotrophs as well as culturable fungi.

Section three, “Host-Parasite Interactions,” covers arguably the most dynamic area of powdery mildew research in recent years. Major aspects of host-parasite interactions including genetics, physiology, and molecular biology are treated in five chapters. The section includes discussions of both induced and race-specific resistance in host plants, as well as the chain of host defense responses leading to resistance. These chapters, detailing basic research on host-parasite interactions, provide insights into how the new information may be exploited practically to limit the devastating world-wide impact of powdery mildews.

The section “Populations” includes two chapters on epidemiology and population genetics of powdery mildews. The first deals with epidemics in agricultural systems, and the second documents the fascinating complexity of genetic interactions between host resistance and parasite virulence in natural plant pathosystems.

The final section is “Control Methods.” The five chapters cover breeding for resistance in crops as diverse as wheat and cucurbits, the development and use of fungicides, alternative controls employing biological agents and other environmental manipulations, and the potential for developing transgenic, mildew resistant. These chapters reflect the increasing complexity and sophistication of attempts to manage powdery mildews in the field and glasshouse. A brief comparison of this section and Spencer’s 1978 book reveals that strategies to combat powdery mildews have evolved in many significant ways, utilizing diverse new developments in the chemistry and deployment of fungicides, biological control methods, and use of genetic resistance.

To assemble a book representing the current ‘state of the art’ has proved challenging because knowledge is advancing so iii

iv

PREFACE

rapidly. We believe, however, that this book will continue to provide a useful and comprehensive source of information on powdery mildews to both established scientists and students, whether they are specifically interested in powdery mildews or more broadly interested in plant diseases. As editors, we sincerely thank the authors who have so generously shared their vast experience and knowledge, and devoted considerable time in preparing of their contributions. Special thanks are extended to Dr. Uwe Braun for going through the entire work to standardize powdery mildew nomenclature. The editing work of Dr. Tyler Avis was invaluable, and Genevieve Marchand’s con-

tribution in developing the index is greatly appreciated. We also thank all those who encouraged, helped and advised us throughout. Finally we thank the many scientists listed on pages v and vi, who reviewed individual chapters, provided scientific and editorial suggestions, and generally ensured the overall quality of the treatise.

May 9, 2002

R ICHARD R. B ÉLANGER W ILLIAM R. B USHNELL ALEID J. DIK TIMOTHY L. W. CARVER

Contributors

EDITORS

J. K. M. Brown Cereals Research Department, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom

R. R. Bélanger Centre de recherche en horticulture, Département de phytologie, Université Laval, Québec, Québec, G1K 7P4, Canada

W. R. Bushnell U.S. Department of Agriculture, Agricultural Research Service, Cereal Rust Laboratory, University of Minnesota, St. Paul, Minnesota 55108, United States

W. R. Bushnell U.S. Department of Agriculture, Agricultural Research Service, Cereal Rust Laboratory, University of Minnesota, St. Paul, Minnesota 55108, United States

T. L. W Carver Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed SY32 3 EB, United Kingdom

A. J. Dik Applied Plant Research, P.O. Box 8, 2670 AA Naaldwijk, The Netherlands

P. Chaure Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom

T. L. W Carver Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed SY32 3 EB, United Kingdom

H. T. Christensen Department of Plant Biology, Risø National Laboratory, Postbox 42, 4000 Roskilde, Denmark D. Clarke Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr Building, Glasgow University, Glasgow G12 8QQ, United Kingdom

ASSISTANT EDITORS M. Bardin INRA, Unité de Pathologie Végétale, Domaine St Maurice, B.P. 94, 84140 Montfavet, France

D. B. Collinge Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark

U. Braun Martin-Luther-Universität, FB. Biologie, Institut für Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21, D-06099 Halle (Saale), Germany

N. Collins Sainsbury Laboratory, John Innes Centre, Norwich, Norfolk NR47UH, United Kingdom

H. Kunoh Laboratory of Ecological Circulation, Faculty of Bioresources, Mie University, Tsu-city, 514-8507, Japan

R. T. A. Cook Central Science Laboratory (MAFF), Sand Hutton, York YO4 1LZ, United Kingdom

P. C. Nicot INRA, Unité de Pathologie Végétale, Domaine St Maurice, B.P. 94, 84140 Montfavet, France

A. J. Dik Applied Plant Research, P.O. Box 8, 2670 AA Naaldwijk, The Netherlands

A. Schmitt Federal Biological Research Centre for Agriculture and Forestry, Institute for Biological Control, 64287 Darmstadt, Germany

C. Frye Syngenta Agribusiness Biotechnology Research, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States J. R. Green School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

AUTHORS

P. L. Gregersen Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Department of Plant Biology, 4200 Slagelse, Denmark

A. Akhkha Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, Graham Kerr Building, Glasgow University, Glasgow G12 8QQ, United Kingdom

G. G. Grove Washington State University, Prosser, Washington 99350, United States

M. Bardin INRA, Unité de Pathologie Végétale, Domaine St Maurice, B.P. 94, 84140 Montfavet, France

W. G. Gubler University of California, Davis, California 95616, United States

R. R. Bélanger Centre de recherche en horticulture, Département de phytologie, Université Laval, Québec, Québec, G1K 7P4, Canada

S. J. Gurr Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom

U. Braun Martin-Luther-Universität, FB. Biologie, Institut für Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21, D-06099 Halle (Saale), Germany

D. W. Hollomon Department of Agricultural Sciences, University of Bristol, IACR Long Ashton Research Station, Long Ashton, Bristol BS41 9AF, United Kingdom v

vi

CONTRIBUTORS

S. L. K. Hsam Center of Life and Food Sciences, Division of Plant Breeding and Applied Genetics, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany J. Inman Central Science Laboratory (MAFF), Sand Hutton, York YO4 1LZ, United Kingdom

M. Oostendorp Syngenta Crop Protection, WST-540.E.69, CH-4332, Stein, Switzerland A. Sadanandom Sainsbury Laboratory, John Innes Centre, Norwich, Norfolk NR47UH, United Kingdom

M. Jahn Department of Plant Breeding, Cornell University, Ithaca, New York 14853-1902, United States

J. Salmeron Syngenta Agribusiness Biotechnology Research, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States

W. R. Jarvis Greenhouse and Processing Crops Research Centre, Harrow, Ontario, N0R 1G0, Canada

H.–D. Shin Department of Agricultural Biology, Korea University, Seoul 136-701, Korea

G. Knauf-Beiter Syngenta Crop Protection, WST-540.E.69, CH-4332, Stein, Switzerland

P. Schulze-Lefert Sainsbury Laboratory, John Innes Centre, Norwich, Norfolk NR47UH, United Kingdom

C. Kramer Syngenta Agribusiness Biotechnology Research, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States H. Kunoh Laboratory of Ecological Circulation, Faculty of Bioresources, Mie University, Tsu-city, 514-8507, Japan C. Labbé Centre de recherche en horticulture, Département de phytologie, Université Laval, Québec, Québec, G1K 7P4, Canada K. Lawton Syngenta Agribusiness Biotechnology Research, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States M. F. Lyngkjaer Plant Biology and Biogeochemistry Department, Risø National Laboratory, DK-4000 Rokskilde, Denmark J. D. McCreight United States Department of Agriculture, Agricultural Research Service, 1636 E. Alisal St., Salinas, California 93905, United States H. M. Munger Department of Plant Breeding, Cornell University, Ithaca, New York 14853-1902, United States P. C. Nicot INRA, Unité de Pathologie Végétale, Domaine St Maurice, B.P. 94, 84140 Montfavet, France

S. Somerville Carnegie Institution of Washington, Department of Plant Biology, Stanford, California 94305, United States P. Spanu Department of Biology, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, United Kingdom T. Staub Syngenta Crop Protection, WST-540.E.69, CH-4332, Stein, Switzerland B. Vernooij Syngenta Agribusiness Biotechnology Research, 3054 Cornwallis Road, Research Triangle Park, North Carolina 27709, United States J. Vogel Carnegie Institution of Washington, Department of Plant Biology, Stanford, CA 94305, United States I. E. Wheeler Department of Agricultural Sciences, University of Bristol, IACR Long Ashton Research Station, Long Ashton, Bristol BS41 9AF, United Kingdom F. J. Zeller Center of Life and Food Sciences, Division of Plant Breeding and Applied Genetics, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany R. J. Zeyen Department of Plant Pathology, 495 Borlaug Hall, University of Minnesota, St. Paul, Minnesota 55108, United States

Contents

1 The Role of Powdery Mildew Research in Understanding Host-Parasite Interaction: Past, Present, and Future ............................................................................................................................................................ 1 CHAPTER

W. R. Bushnell

2 The Taxonomy of the Powdery Mildew Fungi .......................................................................................................... 13 CHAPTER

U. Braun, R. T. A. Cook, A. J. Inman, and H.-D. Shin

3 Comparative Genetics of Avirulence and Fungicide Resistance in the Powdery Mildew Fungi .............................................................................................................................................. 56 CHAPTER

J. K. M. Brown

4 The Formation and Function of Infection and Feeding Structures ........................................................... 66 CHAPTER

J. R. Green, T. L. W. Carver, and S. J. Gurr

5 Basic Methods for Epidemiological Studies of Powdery Mildews: Culture and Preservation of Isolates, Production and Delivery of Inoculum, and Disease Assessment .......................................................................................................................................................... 83 CHAPTER

P. C. Nicot, M. Bardin, and A. J. Dik

6 DNA-Mediated Transformation of Blumeria graminis f.sp. hordei ......................................................... 100 CHAPTER

P. Chaure, P. Spanu, and S. J. Gurr

7 Epidermal Cell Papillae ........................................................................................................................................................... 107 CHAPTER

R. J. Zeyen, T. L. W. Carver, and M. F. Lyngkjaer

8 Localized Induction of Accessibility and Inaccessibility by Powdery Mildew ............................... 126 CHAPTER

H. Kunoh vii

viii

CONTENTS

CHAPTER

9

Genes and Molecular Mechanisms Controlling Powdery Mildew Resistance in Barley .........................................................................................................................

134

N. C. Collins, A. Sadanandom, and P. Schulze-Lefert

10 The Nature and Role of Defense Response Genes in Cereals ................................................................... 146 CHAPTER

D. B. Collinge, P. L. Gregersen, and H. Thordal-Christensen

11 Powdery Mildew of Arabidopsis: A Model System for Host-Parasite Interactions .................... 161 CHAPTER

J. Vogel and S. Somerville

12 Epidemiology of Powdery Mildews in Agricultural Pathosystems .......................................................... 169 CHAPTER

W. R. Jarvis, W. G. Gubler, and G. G. Grove

13 Population Genetics of Powdery Mildew–Natural Plant Pathosystems ............................................. 200 CHAPTER

D. D. Clarke and A. Akhkha

14 Breeding for Powdery Mildew Resistance in Common Wheat (Triticum aestivum L.) .............................................................................................................................................................. 219 CHAPTER

S. L. K. Hsam and F. J. Zeller

15 Breeding Cucurbit Crops for Powdery Mildew Resistance .......................................................................... 239 CHAPTER

M. Jahn, H. M. Munger, and J. D. McCreight

16 Controlling Powdery Mildews with Chemistry ..................................................................................................... 249 CHAPTER

D. W. Hollomon and I. E. Wheeler

17 Control of Powdery Mildews Without Chemicals: Prophylactic and Biological Alternatives for Horticultural Crops ........................................................... 256 CHAPTER

R. R. Bélanger and C. Labbé

18 Powdery Mildew Control Through Transgenic Expression of Antifungal Proteins, Resistance Genes, and Systemic Acquired Resistance .................................................................................... 268 CHAPTER

J. Salmeron, B. Vernooij, K. Lawton, C. Kramer, C. Frye, M. Oostendorp, G. Knauf-Beiter, and T. Staub

Index

..................................................................................................................................................................................................... 289

CHAPTER

1

The Role of Powdery Mildew Research in Understanding Host-Parasite Interaction: Past, Present, and Future W. R. Bushnell Cereal Disease Laboratory, Agricultural Research Service, U.S. Department of Agriculture, and Department of Plant Pathology, University of Minnesota, St. Paul

1.5.3.

1.5.4.

1.5.5.

1.5.6.

1.5.7.

1.1. Introduction 1.2. Factors favoring research on plant–powdery mildew interaction 1.3. Separating epidermis from leaves or coleoptiles 1.4. Factors affecting primary infection 1.4.1. Cell type 1.4.2. Leaf age 1.4.3. High-density spore inoculation 1.4.4. Monovalent cations 1.4.5. Wounding

Race-specific resistance Partial resistance mlo Resistance Local induced resistance Systemic acquired resistance

1.6. Compatible plant–powdery mildew combinations 1.6.1. Green island-related alterations 1.6.2. Alterations induced by sporulating mildew colonies 1.6.3. Role of haustoria

1.5. Incompatible plant–powdery mildew combinations 1.5.1. General background resistance 1.5.2. Nonhost resistance

1.7. The future

1.1. Introduction

search in powdery mildew–plant interactions (including the separation of mildewed epidermis from leaves for experimental purposes), factors that affect primary infection rates (especially in experimental systems), the broad diversity of available plant-mildew combinations which give a wide range in types of resistance for investigation, and the advantages of powdery mildew for investigating compatible host-parasite combinations.

Before us lies a promised land for investigating molecular interactions between plants and disease-causing microorganisms. Startling advances in techniques for high throughput genomic analysis, for screening large numbers of genes expressed in plants in response to pathogen attack, and for locating genes via mutant screens help fuel one of the most active areas of basic research in plant biology—the molecular basis of hostparasite interaction. What part will research on powdery mildew diseases play as we move into this new era of research?

This chapter provides an overview of the characteristics of powdery mildew–plant systems that put them in a competitive, and often a leading role, in the context of present and future research on host-parasite interaction. What part has powdery mildew played in the past and what are the prospects for a continued leading role?

The chapter also focuses largely on the much investigated powdery mildews of cereals (barley, oat, and wheat) with some inputs from mildews of dicotyledonous plants such as cucumber, pea, and, increasingly, Arabidopsis. Included are topics of personal interest, drawing in part from chapters of this book and, in some cases, dwelling longer on certain subjects that are not included elsewhere. Topics include factors favoring re-

1.2. Factors favoring research on plant– powdery mildew interaction The economic impact of powdery mildews drives research at all levels, including the need to understand the basis of hostparasite interaction in both resistant and susceptible plants.

Powdery mildew causes economic loss on a wide range of agricultural and ornamental plants, despite being caused by obligate, biotrophic parasites, each specialized for a limited range of host species. Powdery mildew fungi propagate vegetatively with short-lived spores which prefer high humidity and have an aversion to rain or immersion in water (Sivapalan 1993a,b). The mildew fungi overcome such limitations by producing abundant conidia, a trait that has its origin in natural 1

2

BUSHNELL

plant communities in which plants of a susceptible species may be distant from one another (Clarke and Akhkha Chapter 13).

The prodigious ability to sporulate provides potentially overwhelming amounts of inoculum in agricultural systems where susceptible plants are crowded close together. In turn, the demands made by the parasite for photoassimilates and other nutrients to support spore production (Ayres et al. 1996), coupled with decline in photosynthetic ability, limit the yield and quality of fruit, grain, and vegetables produced by mildewed plants. Clearly, there is an economic need to understand the remarkable ability of powdery mildew fungi to feed at the expense of host plants. Concomitantly, there is a need to understand and improve mechanisms of disease resistance.

Another major factor driving powdery mildew research on host-parasite interaction is the location of the parasite on the surface of leaves, stems, and sometimes fruits (Jarvis et al.

Chapter 12). Infection structures, mycelium, and sporophores of most powdery mildew fungi develop on the epidermal surface, receiving nutrients from host epidermal cells by way of haustoria, the only part of the fungus located within host cells.

Consequently, development of the fungus is easy to monitor by light microscopy. For example, entire leaves can be fixed and stained to view the fungus on the epidermal surface; it is not necessary to section the leaves as is commonly required for fungal parasites that invade leaf mesophyll. In addition, light microscopy of living epidermal monolayers can be used (as described later), and, by removing the mildewed epidermis from a leaf, the remaining uncolonized mesophyll can be sampled for alterations induced by the parasite.

As pointed out by Collinge et al. (Chapter 10), powdery mildew fungi have a high degree of synchrony in the development of infection structures by spore populations used as inoculum.

If enough spores are used, virtually all epidermal cells in the inoculated specimen can be challenged by one or more germinating spores (although high spore density can reduce infection rate, as discussed later).

Yet another advantage of powdery mildew is the diversity in available types of incompatible plant-parasite combinations.

This includes not only a large number of race-specific resistance genes (R-genes) that give a broad range of infection types, but also several types of race-nonspecific resistance. As described in more detail later, mildew-plant combinations variously exhibit general background resistance, nonhost resistance, and partial resistance, as well as the unique penetration resistance controlled by mlo resistance alleles. Separately, there is local and systemic resistance induced by preinoculation with mildew or other fungi. Finally, diversity is added by the availability of mutants in Arabidopsis (and to a lesser extent in barley) which have altered compatible or incompatible phenotypes (Vogel and Somerville Chapter 11).

Together, these several advantages have put powdery mildew in the forefront of research on host-parasite interaction.

The advantages outweigh the facts that powdery mildew fungi cannot be cultured on artificial media and their spores cannot be stored reliably for more than a few days. Powdery mildews are especially well suited as models for diseases caused by rust fungi, which induce similar physiological changes in plants but are more difficult to study because infection structures and hyphae develop within plant tissues. Likewise, powdery mildews can serve as models for diseases caused by hemibiotrophic pathogens such as Mycovellosiella fulva (syn. Cladosporium

fulvum) and Rynchosporium secalis, which develop biotrophically in early stages of infection. Resistance responses elucidated in powdery mildews, such as papillae deposited at sites of attempted penetration by the fungus or hypersensitive cell death, serve as general models for such events occurring in a wide variety of plant diseases.

1.3. Separating epidermis from leaves or coleoptiles It has become standard procedure to strip mildewed epidermis from leaves of barley or wheat to separate epidermal events from those in underlying tissue. The epidermis is easy to strip from cereal leaves, especially from the abaxial surface of barley leaves (Silcox and Holloway 1986), although the stripping becomes more difficult if leaves are heavily mildewed. Allen and Goddard (1938) were the first to remove mildewed epidermis for physiological study purposes. They measured respiration rates of mildewed wheat epidermis after the mesophyll was removed, concluding that host mesophyll contributed a large part of the respiratory increase that occurs in intact mildewed wheat leaves. Subsequent investigations confirmed this conclusion, relating respiratory increase to stages of mildew colony development and to loss of photosynthetic capacity in infected leaves (Allen 1942; Bushnell and Allen 1962b; Scott 1965; Scott and Smillie 1966). In recent years, the removal of epidermis reemerged as a method to distinguish between epidermal and mesophyll activities. The procedure was used, for example, to locate where individual defense-related genes are activated (Collinge et al. Chapter 10), where phenolic polyamines accumulate (as described later) (von RĂśpenack et al. 1998), and where invertase activity is enhanced (Wright et al. 1995).

Because living epidermis is difficult to strip from leaves without injury, Bushnell et al. (1967) introduced a procedure of stripping unwanted tissues from portions of barley coleoptiles, leaving an intact, living epidermal monolayer relatively undisturbed. When carefully prepared, the monolayer has excellent optical properties for light microscopy. The procedure was developed originally for investigation of haustoria but proved to be ideal for observation and experimental manipulation of infection structure development and cytoplasmic responses in epidermal cells (Bushnell 1971; Bushnell and Bergquist 1975).

The coleoptile epidermal monolayer has since been used intensively for this purpose (Aist and Bushnell 1991; Kunoh Chapter 8; Zeyen et al. Chapter 7). Few experimental systems have been developed in plant pathology that give a comparable view of host-parasite interaction in living specimens at the cellular level. Living epidermal tissues have been viewed successfully in cowpea (Mellersh and Heath 2001) and pea (LeeStadelmann et al. 1981; Silcox and Holloway 1986; Prithiviraj and Singh 1995).

However, the cereal coleoptile epidermal monolayer has inherent limitations including: (1) expression patterns of racespecific R-genes sometimes differ from those in leaves (Collins et al. Chapter 9); (2) the epidermal monolayer is not suited for resistance expressed later than 2–3 days after inoculation; (3) the mildew fungus rarely sporulates; and (4) events dependent on mesophyll are missing, e.g., the transfer of photoassimilates to mildewed epidermis. Nevertheless, the coleoptile

ROLE OF POWDERY MILDEW RESEARCH

epidermal monolayer remains useful and contributes to the continued importance of powdery mildews for investigation of host-parasite interaction.

1.4. Factors affecting primary infection Incompatibility in many mildew-plant combinations is expressed at least in part by reduction in infection as measured by the percentage of appressoria that produce haustoria. In addition, infection rates in nominally compatible plant–powdery mildew combinations can be increased by experimental treatment as will be described later. However, infection rates in powdery mildew of cereals are modulated by several factors that need to be kept in mind in designing and interpreting experiments where outcome is based on infection rate.

1.4.1. Cell type In leaves of barley, infection rates are usually recorded only for appressoria that challenge ‘short’ epidermal cells where rates are appreciably higher than when ‘long’ cells are challenged. The location and relative size of long (L) and short (S) cells on the adaxial surface of primary barley leaves is shown in Fig. 1, as described by Koga et al. (1990). Short cells (less than 450 µm long), which are generally located near stomatal files, covered 68% of the adaxial leaf surface but only 35% of the abaxial surface. Long cells averaged 1515 µm in length.

Infection rates were 73–79% in short cells, compared to 15– 16% in long cells. Papillae were generally present at sites of failed penetration in long cells. In sum, Blumeria graminis is well adapted to short cells but poorly adapted to long cells. The resistance of long cells is a major contributor to the general background resistance of cereals, as will be discussed later.

3

pared to 10% in fully expanded leaves 14 cm long. Leaves of intermediate length (8–11 cm long), which are often used for experimental purposes, sustained infection rates no more than half those of the newly emerging leaves. As the leaves matured, infection rates were always lower in long than in short cells, with the greatest difference in leaves of intermediate length (Fig. 2). Similar reduction in infection rate with age was reported by Nelson et al. (1989). By interchanging epidermal strips between leaves of different age, Nelson et al. (1990) showed that age-related resistance was a characteristic of the epidermis and not the underlying mesophyll. Background resistance can be minimized, but not eliminated, by use of nascent seedling leaves when they first emerge. Another possibility is to use etiolated leaves reported to sustain high infection rates (Nelson et al. 1989).

In contrast to cereals, leaves of some dicotyledonous crops, such as cucumber, tobacco, and tomato, tend to increase in susceptibility with age, as judged by mildew colony development on leaf surfaces (Jarvis et al. Chapter 12).

1.4.3. High-density spore inoculation Large numbers of spores per unit leaf area are routinely used in physiological experiments to maximize the percentage of epidermal cells that are challenged by appressoria. Spore densities of 100–400/mm2 are not uncommon, giving up to one appressorium per epidermal cell.

Unfortunately, infection rate typically decreases as spore density increases, whether on leaves or coleoptiles (Carver and Ingerson-Morris 1989; W. Bushnell, unpublished data). This can be attributed in part to resistance induced by primary germ tubes as described later although, at high spore densities, the few epidermal cells unchallenged by primary germ tubes had low infection rates. This showed that the spore crowding effect

1.4.2. Leaf age Infection rates in both long and short cells decline as cereal seedling leaves elongate and mature as shown by the data adapted from Lin and Edwards (1974) (Fig. 2). In newly emerging leaves 2.5 cm long, infection rates were 60–70%, com-

Fig. 1. Adaxial epidermis of barley primary leaf showing short (S) and long (L) cells. Infection rates are much higher in short than in long cells (see text). Short cells tend to be located near stomatal files; long cells are over vascular bundles, except that abaxial surfaces have additional long-celled regions apart from vascular bundles (not shown). (Adapted from Koga et al. 1990)

Fig. 2. Infection rate by spores of Blumeria graminis f.sp. hordei in relation to length of developing primary seedling leaves. Infection rate as percentage of germinated spores that produced haustoria.

Data from abaxial leaf surface. Short cells were in contact with the stomatal apparatus; long cells were the predominant cell type.

(Data adapted from Lin and Edwards 1974)

4

BUSHNELL

extended beyond cells exposed directly to primary germ tubes (Carver and Ingerson-Morris 1989). The resistance induced at high spore densities was reduced by inhibitors of phenylalanine ammonia lyase (PAL), indicating that phenolic substances have a role in blocking haustorium formation (Carver et al. 1992a).

1.4.4. Monovalent cations Monovalent cations reduce primary infection rates when applied as salts to soil or in solutions fed to detached leaves or epidermal monolayers. Thus, lithium salts applied to soil have long been under trial to control powdery mildew (Kent 1941; Smith and Blair 1950; Abood et al. 1991, 1992). In addition, Hirata (1971) found that either Li+ or NH4+ cations inhibited mildew development in detached leaves. He found that infection rates in detached leaves could be increased by either Ca++ or Sr++. Takamatsu et al. (1979), using epidermal monolayers from barley coleoptiles, showed that Ca++ was antagonistic to Li+ inhibition of mildew infection. Later, the data of Kunoh et al. (1985) indicated that K+ and Na+, like Li+, inhibited haustorium formation. Effectiveness of monovalent cations, in decreasing order, is Li+, NH4+, Na+, and K+ (Table 1). All can be counteracted by Ca++. The use of 10 mM Ca++ to incubate epidermal monolayers assures that the balance between monoand divalent ions is favorable for mildew development. (For a discussion of possible effects of Ca++ on primary infection, see Zeyen et al. Chapter 7.) Monovalent cations possibly enhance papilla formation or other defense responses, although high concentrations of Li+ act directly against the fungus, preventing normal appressorium development (W. Bushnell, unpublished data).

In any case, monovalent cations need to be avoided in experiments involving infection rate. For example, potassium or sodium phosphate pH buffers reduce infection rates. This can be avoided by use of Ca-MOPS organic buffers (Russo and Bushnell 1989).

Organic compounds with amine groups such as putrescine, glucosamine, and N-acetyl glucosamine also inhibit infection (Bushnell and Curran 1984). Like the effect of NH4+, the inhibition by N-acetyl glucosamine is reversed by Ca++.

1.4.5. Wounding Mechanical wounding of epidermal tissue can reduce infection rates considerably. For example, bombardment of coleoptiles with DNA-coated microparticles to introduce transgenes into cells reduces infection rate in surviving cells by as much as 25–50% (B. Bucciarelli and W. Bushnell, unpublished data).

Table 1. Monovalent cations reduce infection rate of Blumeria graminis f.sp. hordei on barley coleoptile epidermisa Cation (10 mM) Li+ NH4+ Na+ K+ Water a Data

Infection rateb 2 16 20 25 46

from W. Bushnell (unpublished) and Bushnell and Curran 1983. of appressoria that produced a haustorium. All values for cations are significantly different from the value for water.

b Percentage

Kobayashi et al. (2000) reported that puncture of living epidermal cells with microneedles reduced subsequent infection rates. Barley epidermal cells repair needle punctures by rapidly depositing papilla-like wound plugs (Russo and Bushnell 1989).

Wounding possibly leads to physical changes in cell walls that increase their resistance to fungal penetration. In addition, woundng is known to activate batteries of wound response genes with some similarities to defense-related response genes.

1.5. Incompatible plant–powdery mildew combinations Two principal types of resistance are utilized by plant breeders to improve powdery mildew resistance in agricultural crops: race-specific (gene-for-gene) or ‘vertical’ resistance and race-nonspecific polygenic partial resistance (also termed general or ‘horizontal’ resistance). Both types occur also in wild (noncultivated) host species; e.g., Hordeum spontaneum (used as a source of both race-nonspecific and partial resistance in barley), Senecio fischeri, and Arabidopsis thaliana (Clarke and Akhkha Chapter 13; Vogel and Somerville Chapter 11). In addition, recessive alleles at the Mlo locus in barley provide unique and highly effective race nonspecific resistance. However, several additional types of resistance phenomena are available for basic research on mildew-plant incompatibility, including general background resistance widely expressed in nominally compatible mildew-cereal combinations, nonhost resistance (including both true and conditional types as defined later), and resistance to inappropriate formae speciales. Separately, there are local and systemic resistance phenomena induced in compatible mildew-plant combinations by prior inoculation with an incompatible race, forma specialis, or species.

These diverse types of resistance often have plant responses in common: e.g., papillae (localized wall appositions) are almost universally produced at sites of attempted penetration by powdery mildew appressoria (Zeyen et al. Chapter 7). Papillae are thought to block penetration if they are deposited rapidly and contain antifungal phenolic substances in sufficient quantities. In a similar way, arrays of defense-related genes are activated in leaf tissues in response to attempted penetration of epidermal cells (Collinge et al. Chapter 10). That said, each type of resistance has distinctive features useful for understanding plant-mildew interaction.

1.5.1. General background resistance Leaves of cereals have general background resistance that can limit the percentage of B. graminis appressoria able to penetrate and produce haustoria in nominally susceptible plants. As described earlier, background resistance to penetration is stronger in long than in short epidermal cells (Fig. 1) and increases markedly as seedling leaves elongate and mature (Fig. 2).

Carver et al. (1991, 1992a) reported that infection rates in nominally susceptible barley or oats could be increased by inibitors of the phenylpropanoid biosynthetic pathway, with conomitant reduction in autofluorescence in papillae produced at sites of attempted penetration and in nearby cell walls. These results strongly suggest that phenolic substances contribute to

ROLE OF POWDERY MILDEW RESEARCH

penetration resistance. The experimental result was termed an increase in ‘quantitative susceptibility.’ Alternatively, the result could be described as a reduction in background resistance.

As discussed by Vogel and Somerville (Chapter 11), several mutants of nominally susceptible Arabidopsis lines (susceptible to Golovinomyces orontii, syn. Erysiphe orontii) exhibit increased susceptibility. Some of these mutants are known to be compromised in ability to activate defense-related genes.

This indicates that defense-related genes contribute to the background resistance of the parental, susceptible lines.

Another experimental method that increases infection rate in susceptible plants is to inoculate the plants with an ‘inducing’ compatible powdery mildew isolate. This increases the infection rates obtained with subsequent ‘challenge’ inoculations with the same fungus (Carver et al. 1999; Lyngkjaer and Carver 1999). The increased infection rate “appeared to be associated with suppression of localized autofluorescent host cell responses.” Carver and coworkers termed the mildew-induced increase in infection rate as an increase in ‘accessibility’ [following terminology used earlier for increases in infection rates in incompatible mildew-plant combinations induced by a prior inoculation with a compatible mildew isolate (Kunoh Chapter 8)]. As with other approaches to increasing susceptibility of nominally susceptible plants, the increased ‘accessibility’ of Carver and coworkers is possibly a consequence of reduced background resistance.

Although the level varies with leaf age as described earlier (Fig. 2), background resistance is probably omnipresent in nominally susceptible plants, always limiting penetration and haustorium formation by an attacking mildew population. Consequently, any comparison of responses between a resistant line with a nominally susceptible line (for example, a comparison of two near-isogenic lines differing in an R-gene for racespecific resistance) will always have a component of background resistance expressed in both lines. This is generally recognized in investigation of the expression of defense-related genes (Collinge et al. Chapter 10) but, nevertheless, the resistance generates background ‘noise’ which can obscure expression of genes that relate to other types of resistance being investigated.

1.5.2. Nonhost resistance Plant species that are nonhost to a given powdery mildew fungus are classified here at three levels of specificity: (1) true nonhosts, which are not host to any powdery mildew fungi (to any members of the Erysiphaceae), (2) conditional nonhosts, which are host to one or more powdery mildew species but are nonhost with respect to others, and (3) host species that are inappropriate for all but certain formae speciales of a given mildew species. In general, powdery mildews have more difficulty germinating and producing infection structures and penetrating true and conditional nonhosts than on hosts inappropriate for a forma specialis. This was demonstrated by Johnson and coworkers (Johnson 1977; Johnson et al. 1982) in a comprehensive investigation of infection structure development with 38 species in 11 plant families inoculated with B. graminis f.sp. hordei and (separately) Golovinomyces cichoracearum (syn. Erysiphe cichoracearum). Included were members of the Polypodiaceae (ferns) and Equisetaceae (horsetails) as well as dicotyledonous and monocotyledonous families in the Angio-

5

spermae. With respect to the fungus involved, the plants had the role of host in five plant-mildew combinations, of true nonhost in 32 combinations, of conditional nonhost in 24 combinations, and of nonhost inappropriate at the forma specialis level in 13 combinations. Combined data for each level of specificity are shown in Fig. 3.

Rates of germination and appressorium formation were reduced on all nonhosts, except for B. graminis at the forma specialis level. For example, on conditional nonhost species (designated D in Fig. 3) and certain true nonhost species (designated C), germination rates were about 50% of rates on host species. On other true nonhost species (designated E), however, germination rates were less than 10% of rates on hosts.

Clearly, the mildew spore finds the cuticular surface of true and conditional nonhosts relatively inhospitable. Since B. graminis does not depend on chemical or physical inducing factors for germination (Green et al. Chapter 4), reduced germination rates are probably the result of inhibitory factors on the cuticular surface. However, no signs of spore collapse or degradation were seen by Johnson and coworkers (1982). In any case, it follows that in the course of adapting to host surfaces, powdery mildew fungi evolve ways to mitigate cuticular factors that inhibit germination.

Of the spores that germinated on true and conditional nonhosts (C, D, and E, Fig. 3), less than half produced appressoria.

This may be a consequence of the factors that inhibit germination. However, differentiation of appressoria in B. graminis depends on interaction between a primary germ tube (a short tube initially produced by the spore) and the cuticular surface (Green et al. Chapter 4). An appressorial germ tube is not produced unless the required interaction takes place. For example, wax on the cuticular surface was postulated to interfere with triggering of appressorium formation by B. graminis on the glossy lower (abaxial) surface of ryegrass (Lolium spp.)

(Carver et al. 1990). Although ryegrass is a host for B. graminis, the lower leaf surface resembles true nonhost surfaces in limiting appressorium development. In this case, however, the fungus germinates at near-normal rates, indicating that the surface is not toxic. The cuticle of the lower surface is covered with amorphous sheets of wax in contrast to crystalline wax platelets on the susceptible upper surface. If the wax is removed, appressoria develop normally. Apparently the sheets of wax interfere with signalling interactions required for differentiation of the appressorium. In a similar way, true and conditional nonhosts may have wax or other factors that interfere with the triggering of appressorium formation by B. graminis.

No powdery mildew fungi other than B. graminis produce primary germ tubes. Is lack of an appropriate initial interaction with the nonhost surface part of the reason appressoria of G. cichoracearum often failed to develop and penetrate on nonhosts (Fig. 3)?

Virtually no haustoria or hyphae were produced on conditional (D) or true nonhosts (C and E) (Fig. 3). Concomitantly, the hypersensitive (cell death) response was rare (induced by 0.3–3% of spores). The hypersensitive response sometimes occurred in the absence of visible haustoria, although haustorial initials may have gone unseen due to the collapse of host cells.

Haustoria that were produced most often led to the hyperensitive response, although not always. Resistance factors unelated to the hypersensitive response may come into play after haustoria are produced.

6

BUSHNELL

In an investigation of Arabidopsis thaliana (a conditional nonhost for B. graminis), H. Thordal-Christensen (as described

by Vogel and Somerville Chapter 11) found that hypersensitive cell death occurred at the few infection sites at which haustoria were produced. A mutant of Arabidopsis was found in which

rates of haustorium formation were increased somewhat, but always leading to the hypersensitive response.

At the forma specialis level (B, Fig. 3), the data of Johnson and coworkers (1982) indicate that 3–8% of spores were able to produce a haustorium and 1–4% of spores produced a hypha, rates somewhat higher than on conditional and true nonhosts.

Rates of hypersensitive cell death were also higher (4–7% of spores).

In cereals, inappropriate formae speciales activate transcription of defense-related genes (Schweizer et al. 1989) much as appropriate formae speciales do. Carver et al. (1992b) found, however, that resistance to inappropriate formae speciales usually was not broken by AOPP, an inhibitor of phenylalanine ammonia lyase (PAL). AOPP reduces resistance in most other types of resistance, an indication that phenolic substances are involved (Zeyen et al. Chapter 7). Carver et al. concluded that resistance factors operate in inappropriate forma specialis– plant combinations which are not present in appropriate combinations.

The host range of B. graminis formae speciales on wild grass species is not as narrow as it appears from investigations of cultivated cereals (Clarke and Akhkha Chapter 13). Clearly, B. graminis as well as other powdery mildew species offer abundant experimental materials for investigation of this level of specificity.

1.5.3. Race-specific resistance

Fig. 3. Development of infection structures by species of Blumeria graminis (top) and Golovinomyces cichoracearum (bottom) on nonhost species at differing levels of specificity, and on host species. A, host species; B, nonhost species inappropriate at the forma specialis level; C, conditional nonhost species (host to some mildew species and nonhost to others); D, true nonhost species (nonhost to all mildew species), surface moderately incompatible for spore germination; E, true nonhost species, surface highly incompatible for spore germination. Data are from the indicated number of species (given in parentheses) within each plant family.

For B. graminis: A, Gramineae (1); B, Gramineae (3); C, Compositae (5), Cruciferae (2), Cucurbitaceae (4), Labiatae (2), Leguminosae (2), Solanaceae (3); D, Gramineae (4), Iridaceae (2), Liliaceae (4), Compositae (1), Polypodiaceae (3); E, Iridaceae (1), Equisitaceae (1). For G. cichoracearum: A, Cucurbitaceae (4); B, Compositae (5), Cruciferae (1), Labiatae (2), Solanaceae (2), Leguminosae (1); C, Cruciferae (1), Leguminosae (1), Gramineae (4); D, Gramineae (4), Compositae (1), Polypodiaceae (3); E, Iridaceae (3), Liliaceae (4), Equisitaceae (1). (Data adapted from Johnson 1977 and Johnson et al. 1982)

Barley, oat, and wheat each have large numbers of genes (R-genes) for race-specific resistance to powdery mildews. For example, Jørgensen (1994) estimated that barley had 85 such R-genes, located at 10 separate loci on several chromosomes.

Thus, cereals provide a rich genetic resource for investigation of the molecular genetic basis of R-gene specificity, including the structure of complex loci with multiple genes for resistance, the mechanisms of interaction with specific Avr genes in mildew parasites, and the downstream signaling events leading to expression of R-gene resistance (Collins et al. Chapter 9).

Depending on the R-gene involved, mildew resistance can be expressed over a wide range of visible phenotypes varying from apparent immunity (infection type 0, no visible response to infection), through increasing but limited amount of colony development (infection types 1–3), compared to full colony development on compatible plants (infection type 4). With most R-genes, germination and development of appressoria are normal on the epidermal surface. In virtually all resistant phenotypes (at least in infection types 0–2), some plant cells near infection sites undergo the hypersensitive cell death response. This apparently can occur before haustoria are initiated, as with Mlg (Görg et al. 1993), but usually happens only after one or more haustoria are produced. With Mla, which is widely used for physiological investigation of resistance, the first invaded epidermal cell at an infection site dies within a few hours after a haustorium is initiated in that cell, resulting in infection type 0 (Aist and Bushnell 1991). With genes giving infection type 1–3, visible colonies, each with many functional haustoria, may be produced. This leads to delayed hypersensitive death of many cells, both in the colonized epidermis and in underlying fungus-free mesophyll. The delayed expression of the hypersensitive response giving infection types 1–3 is more the rule than the exception in race-specific resistance in pow-

ROLE OF POWDERY MILDEW RESEARCH

dery mildews of cereals. The diversity of R-genes and associated patterns of expression will continue to make powdery mildews of barley, oat, and wheat attractive for research on race-specific (R-gene) resistance.

1.5.4. Partial resistance Partial resistance is general (race-nonspecific) and controlled polygenically (Clifford et al. 1985; Jørgensen 1987, 1994). It has pleiotropic effects, sometimes blocking the penetration that leads to haustorium formation, but, more often, reducing the size of haustoria and limiting the growth and sporulation of colonies. Reduced colony development usually occurs in the absence of hypersensitive responses visible to the naked eye.

Reduced haustorial efficiency in the flow of nutrients from host to parasite is postulated to limit fungus growth and sporulation.

Breeding lines of barley, oat, and wheat usually carry some partial resistance, although the resistance is difficult to measure and the genes involved are ill defined (Jørgensen 1987, 1994). Nevertheless, it is an extremely important and potentially durable type of resistance, in need of continued investigation to understand its genetic and molecular bases.

1.5.5. mlo Resistance Recessive mlo alleles at the Mlo locus in barley gives racenonspecific resistance to penetration which prevents haustorium formation by B. graminis in virtually all epidermal cells except subsidiary cells near stomates (Aist and Bushnell 1991; Collins et al. Chapter 9; Zeyen et al. Chapter 7). Colonies originating in the subsidiary cells develop normally without inducing the hypersensitive cell death response, indicating that mlo resistance limits only the primary infection process. Invariably, large papillae are produced at sites of attempted penetration by appressoria. Consequently, mlo plants have been widely used to investigate the physiology of papilla formation (Aist and Bushnell 1991; Zeyen et al. Chapter 7). Recently, a phenolic polyamine (p-CHA) was identified which accumulates in the epidermis of inoculated mlo plants and probably has a role in preventing infection (von Röpenack et al. 1998; Zeyen et al.

Chapter 7).

A dominant Mlo allele, recently cloned, codes for a membrane-spanning protein which is thought to regulate papillaassociated resistance (Collins et al. Chapter 9). Homologous genes in wheat open the possibility of developing mlo-like resistance there. Because mlo resistance in barley is highly effective, has been durable after widespread use in northern Europe, and has the potential of use in other cereals, it remains under intensive investigation in several laboratories. It will continue to be important in understanding the molecular basis of papillaassociated penetration resistance, which is prevalent in diverse incompatible mildew-plant combinations.

1.5.6. Local induced resistance As reviewed in detail by Kunoh (Chapter 8), resistance can be induced by an ‘inducer’ mildew inoculation prior to a subsequent ‘challenge’ inoculation. The resistance is expressed by failure of the fungus to produce haustoria, i.e., the inducer apparently enhances resistance to penetration. The result has been termed an increase in ‘inaccessibility,’ the converse of induced

7

‘accessibility’ as discussed earlier in connection with general background resistance. The examples given here involve B. graminis as the challenge inoculation on leaves of oat or barley or epidermal monolayers from barley. The inducer powdery mildew fungus can be compatible or incompatible as follows: 1. Failed attempts by appressoria of a compatible inducer fungus to produce a haustorium in an epidermal cell (which always happens with a portion of appressoria produced on nominally compatible hosts, as described earlier) induces resistance to haustorium formation in that cell by subsequent challenge inoculation. This is especially true if a papilla is produced by the inducer at the point of attack (Carver et al. 1999; Lyngkjaer and Carver 1999). The induced resistance is associated with increase in frequency of autofluorescence induced at sites of attack by the challenging fungus.

2. Inducer inoculation with an incompatible race of B. graminis induces resistance to subsequent challenge inoculation with a compatible race (Ouchi et al. 1974, 1976).

3. Inducer inoculation with a forma specialis inappropriate for barley (e.g., B. graminis f.sp. tritici) gives resistance to a challenge inoculation with an appropriate forma specialis (B. graminis f.sp. hordei) (Ouchi et al. 1976).

4. Inducer inoculation with a fungus (e.g., Erysiphe pisi) for which the plant is a conditional nonhost, induces resistance to a challenge inoculation with B. graminis (Kunoh Chapter 8).

5. In addition, a single inoculation with a compatible race of B. graminis can induce resistance effective against appressoria produced by that same inoculation. Primary germ tubes induce resistance in individual epidermal cells against appressoria subsequently produced on those cells mentioned earlier (Woolacott and Archer 1984).

Except for resistances induced by primary germ tubes, induced resistance spreads a few cells laterally from inducing infection sites. It should be emphasized that infection rate (usually the percentage of appressoria that produce haustoria) is used to assess local induced resistance. The hypersensitive response or other possible manifestations of resistance that might be present are not usually reported.

As a working hypothesis, infection attempts from inducer inoculations may modulate levels of background resistance, much as factors related to leaf age and cell type do. In any case, local induced resistance adds to the experimental systems available for investigating resistance, in particular, resistance expressed by reduced infection rate.

1.5.7. Systemic acquired resistance (SAR) Inoculation of a leaf of wheat or barley with an incompatible race of B. graminis induces systemic acquired resistance (SAR) in other leaves of the plant as detected by subsequent ‘challenge’ inoculation. For a thorough presentation of SAR, see Salmeron et al. (Chapter 18). Generally, as a result of SAR, certain characteristic defense-related genes are activated. The salicylic acid signaling pathway is known to be involved in the events leading to SAR, but other less well defined pathways also come into play. SAR-like resistance can be induced by chemicals such as BTH. Investigations of SAR in powdery mildew of cereals and related research in Arabidopsis (Sal-

8

BUSHNELL

meron et al. Chapter 18; Vogel and Somerville Chapter 11) play leading roles in learning what signalling pathways lead to SAP and the genetic controls involved.

In sum, the powdery mildew diseases offer a wealth of incompatible fungus-plant combinations for experimental purposes. At the same time, these resistances often grade into each other, sharing plant cell responses in common, such that more than one category of response may be present in a given experimental plant-mildew combination. The challenge will be to find out what signaling pathways and downstream molecular events are shared and which are unique to each type of resistance.

1.6. Compatible plant–powdery mildew combinations Research on processes associated with host-parasite compatibility have received far less attention than phenomena related to incompatibility. However, interest in compatibility in powdery mildews has been sparked by the recent finding of Arabidopsis mutants that exhibit increased or decreased mildew development in compatible host-parasite combinations (Vogel and Somerville Chapter 11). Interest is further increased by the ease with which large numbers of mRNAs transcribed at a given stage of disease development can be selected and their corresponding cDNAs sequenced. Because gene expression can be investigated separately in mesophyll or in fungal-colonized epidermis, powdery mildews provide ideal materials for such experiments. Furthermore, powdery mildew colonies in barley induce a sequence of physiological alterations in mesophyll tissues that are defined with respect to time and location in relation to colony development.

1.6.1. Green island-related alterations In powdery mildew of barley, a zone of influence develops in host mesophyll extending 0.5–1.0 mm beyond the border of individual mildew colonies (Bushnell and Allen 1962a; Bushnell 1967). The zone is marked by the presence of starch-like materials that stain with potassium iodine. The zone is detectable 2–3 days after inoculation and persists at colony borders as the colonies enlarge (if colonies are not crowded). Sugars tend to accumulate in infected leaves at the time that the zone of influence develops (Allen 1942; Wright et al. 1995). The zone is thought to act as a sink for translocation of photoassimilates from uninfected to infected parts of leaves (Bushnell 1967, 1984).

The zone of influence corresponds in size and location to ‘green islands’ that develop if leaves become senescent. (Senescence can be accelerated experimentally by detaching leaves or placing them under low light intensities.) Chlorophyll levels and photosynthetic activity are maintained in the green island as chlorophyll levels decline elsewhere. Green islands develop as a consequence of powdery mildew infection in many plant species.

The zone of influence is postulated to be produced by cytokinins emanating from the mildew fungus or produced by the host in response to the fungus (Bushnell 1967). Cytokinin solutions applied topically in droplets to wheat or barley leaves induce localized zones of starch deposition and green islands if leaves senesce. In cereal rusts, cytokinins have likewise been

implicated in green island formation. Cytokinins accumulating in infected leaves were judged to be of host origin, or possibly produced from fungal precursors (Dekhuijzen 1976). As Ashby (2000) has pointed out, the biosynthetic pathway for cytokinins in plants has proved elusive. Genes for enzymes involved in cytokinin biosynthesis are available only from microorganisms.

Nevertheless these genes can be introduced into plants to learn if they influence host-parasite interaction.

Polyamines, which share with cytokinins an ability to retard senescence, also have been proposed as a cause of the green island-related zone of influence (Walters 2000). The polyamines putrescine, spermidine, and spermine increase in amount in barley leaves infected by B. graminis (Coghlan and Walters 1990). Activity of two polyamine biosynthetic enzymes, arginine and ornithine decarboxylases, increased in a zone surrounding mildew colonies coincident with the location of green islands (Walters and Wylie 1986). As Walters (2000) concludes, genetic manipulation of polyamine levels would help evaluate their role in compatible host-parasite interaction.

1.6.2. Alterations induced by sporulating mildew colonies As mildew (or rust) colonies begin to sporulate about five days after inoculation, tissues beneath each colony center begin to lose chlorophyll and suffer net protein loss, as amino acids, sugars, and other soluble substances apparently move into the fungus. Rates of dark respiration increase two-fold or more (Bushnell and Allen 1962b). Rates of photosynthesis decline (Allen 1942; Bushnell 1984), a factor contributing to yield loss. The chlorotic mesophyll cells at colony centers usually remain alive, but tend to resemble highly senescent cells as organelles become degenerate. The causes of these changes are unknown, although growth hormones such as ethylene and abscissic acid are suspected to have a role. The changes in mesophyll at sporulation are especially important to understand since they occur at the stage of heavy nutrient assimilation by the parasite. Infected tissue acts as a sink, drawing photosynthate and other nutrients from other parts of the plant, especially in powdery mildews of cereals. However, Clarke and Akhkha (Chapter 13) point out that powdery mildew development by Golovinomyces cichoracearum var. fischeri (syn. Erysiphe fischeri) on wild groundsel does not disturb distribution of carbon assimilates as B. graminis does in cultivated barleys. Is this difference a consequence of a difference between the two mildew fungi, the hosts, or both?

Epidermal cells containing haustoria play a key role at the time of sporulation, since sugars and other substances must pass through the epidermal cell to reach the fungus. The plasma membrane of the epidermal cell apparently favors entry of substances from mesophyll. The physical properties of the plasma membrane are altered as indicated by change in plasmolysis form and reduced permeability to nonelectrolytes (Lee-Stadelmann et al. 1984, 1991). Apparently the membrane lipid bilayer undergoes structural change, although the consequences of these changes for nutrient flux are unknown. Other changes in the plasma membrane include decrease in fatty acid unsaturation, increase in membrane ATPase activity (except for the extrahaustorial membrane, a modified form of the plasma membrane), and increase in membrane potential (von Alten and Saile 1998). Possibly related to changes in epidermal cell

ROLE OF POWDERY MILDEW RESEARCH

membranes is an increase in synthesis of inositol phosphatides as reported for cucumber leaves infected with powdery mildew (LĂśsel et al. 1994). Also possibly related is upregulation of a gene for ‘14-3-3’ protein in epidermal tissues of mildewed barley (Collinge et al. Chapter 10). This type of protein is known to serve as a membrane-integrated transport protein.

Overall, the powdery mildews are ideally suited for investigating the physiological role of the haustorium-containing host cell in the flow of substances from host to parasite.

1.6.3. Role of haustoria Understanding the role of haustoria in nutrient flow to rust, powdery mildew, and other haustorium-forming biotrophic parasites has long been a major research objective, but the intracellular position of the haustorium and the complexities of the structures surrounding it [the extrahaustorial matrix and membrane (see Green et al. Chapter 4)] interfere with research on haustorial function. To be fully functional, the haustorial apparatus must not only remain connected to hyphae by way of the haustorial neck, but also be surrounded by living host cytoplasm (Sullivan and Bushnell 1974).

Use of fluorescent probes and antibodies, coupled with fluorescence microscopy has improved understanding of components of the haustorial apparatus, especially in combination with techniques for isolating the haustorial complex which retains the intact extrahaustorial matrix and membrane (Gil and Gay 1977; Manners and Gay 1977; Green et al. Chapter 4).

The complex can be isolated more successfully with Erysiphe s.lat. species than with Blumeria graminis because the extrahaustorial membrane of the latter is more fragile and distends more in hypotonic media. On the other hand, the haustorium of B. graminis in epidermal monolayers (as described earlier) remains useful for in situ observation of fully functional haustoria, e.g., to follow hyphal elongation in response to experimental treatment of host epidermal cells.

Research on haustoria will continue toward the challenging objective of understanding nutrient flow and the role of haustoria in signalling exchanges between host and parasite (Green et al. Chapter 4). Particularly intriguing is preliminary evidence that proteins are exchanged between the two organisms, presumably by way of haustoria. Immunocytological evidence indicated that a protein component (PST-D) of chloroplast thylakoids was present in haustorial complexes (Testut et al. 1999; Green et al. Chapter 4). Conversely, Van Roestel et al. (1991) showed the presence of fungal antigens in barley mesophyll 24 and 96 hr after inoculation with B. graminis. As SpencerPhillips (1997) pointed out in review, extracellular fungal proteins are postulated to have a role in pathogenicity of nonhaustorial parasites that grow intercellularly. Do proteins excreted by powdery mildew haustoria also mediate host-parasite interaction?

1.7. The future Powdery mildew diseases will continue in the forefront of research on host-parasite interaction, especially interactions involving biotrophic parasites, with powdery mildews of Arabidopsis and barley (and other small grains) leading the way.

Vogel and Somerville (Chapter 11) have compared and summa-

9

rized the experimental advantages of these two host-parasite systems. The ability to separate mycelium from the host and, in cereals, to remove haustorium-containing epidermis will allow the physical separation of host activities from those of the parasite. Sequential stages of physiological changes in host leaves are defined for powdery mildew of barley. Events in Arabidopsis may prove to be different, but they will be defined as part of the thrust to use Arabidopsis mildew as a model system.

In Arabidopsis, and to a lesser extent in barley, an array of mutants pointing to genes involved in host-parasite interaction will be a major force for progress. For both diseases, a rich diversity of incompatible combinations are available, not only involving R-gene race-specific resistance, but also a broad assortment of resistance phenomena at other levels of specificity, especially in barley. Recent cloning of both R-genes and genes required for R-gene function in both barley (Collins et al. Chapter 9) and Arabidopsis (Vogel and Somerville Chapter 11) demonstrates that powdery mildew research will contribute to the rapid progress being made in understanding the structure, function, and evolution of R-genes. Given the progress being made in mapping corresponding fungal genes for avirulence (Brown Chapter 3), we can expect that mildew research will be a major player in understanding the molecular basis of gene-for-gene interaction.

Progress in understanding resistance and other aspects of host-parasite interaction will increasingly be supplemented by research on powdery mildews other than those of cereals and Arabidopsis. Several chapters of this book attest to the vitality of research on powdery mildews of fruits, vegetables, ornamental, and wild species other than Arabidopsis. As the genetics and physiology of powdery mildew resistance becomes more thoroughly investigated in these species, features unique to certain host-parasite combinations will emerge, strengthening our ability to use the knowledge gained to control powdery mildews of diverse mono- and dicotyledonous plants.

On the compatibility side, powdery mildews also will be major contributors to understanding host-parasite interaction because the pronounced physiological changes induced in host mesophyll cells can be investigated separately from the colonized epidermis. Again, the combined advantages of Arabidopsis and barley mildews will lead the way, at least with respect to biotrophic fungal diseases. With the discovery of genes expressed in compatible hosts at specific stages of colony development, the promoters of these genes, in turn, can be used to drive expression of the genes in transformed plants to test their effects when expressed at given stages of development. Arabidopsis is easily transformed (Vogel and Somerville Chapter 11); barley is more difficult to transform (Salmeron et al. Chapter 18) but methods no doubt will improve. With respect to the parasite, methods recently developed for transformation of B. graminis (Chaure et al. Chapter 6) have overcome the problems inherent in transforming an obligate parasite and bode well for transformation of other powdery mildew fungi.

The powdery mildews seem to be ideal for investigation of changes in host cell membranes, whether in compatible or incompatible interactions. Alterations in haustorium-containing epidermal cells are readily investigated. Yet research in this area has lagged behind. What is needed is a more focused and comprehensive effort to investigate mildew-induced structural and functional alterations in host cell membranes and functional interactions between the membranes and cell walls.

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Another area ready for progress is the role of eliciting and signalling events in the interaction between hosts and mildew parasites as emphasized by Zeyen et al. (Chapter 7). For example, Toyoda et al. (1993) obtained an elicitor of temporary resistance and cytoplasmic responses from spores of E. pisi.

Schweizer et al. (1996) reported that cutin momomers from barley cuticle induced localized resistance. Exquisite physiological experiments, such as those by Carver and coworkers (see Green et al. Chapter 4) on differentiation of infection structures and by Kunoh and his predecessors on host cytoplasmic responses (Kunoh Chapter 8), have clearly defined reproducible, easily monitored host and parasite phenomena well suited for investigation of eliciting factors. Likewise, the epidermal cell is probably ideally suited for investigation of downstream signalling pathways leading to host responses, whether in compatible or incompatible interactions. The body of information on activation of defense-related genes (Collinge et al.

Chapter 10) provides a strong base for this area of research.

Seemingly more distant, perhaps, is understanding the basis of obligate parasitism through research on powdery mildews.

How do powdery mildew fungi suppress or avoid defense responses so that the host and parasite live together in a peaceful biotrophic relationship? Unlike the obligately parasitic rust fungi, which can be trained over long periods of repeated subculturing to grow on amino acid-rich media (Williams 1984), powdery mildew fungi have not been cultured. For both rusts and powdery mildews, we lack insight into the ways in which the parasites depend on living host cells. Transport proteins in the haustorial plasma membrane have been implicated in sugar uptake by the bean rust fungus (Voegele et al. 2001). Will research on the powdery mildew haustorium and its host epidermal cell help unlock the secrets of obligate parasitism?

ACKNOWLEDGMENTS

Thanks to Tim Carver, Kurt Leonard, and Dick Zeyen for many years of advice and counsel and for reviewing this chapter. Thanks also to Ann Holcomb Bushnell for her encouragement and for expert secretarial assistance.

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