Rust Diseases of and Poplar We dedicate this book to our children Michael and Jeffrey, Sarah and Philippa Diseases of Willow and Poplar

Edited by

Ming Hao Pei

Rothamsted Research, Harpenden, Hertfordshire, UK

and

Alistair R. McCracken

Applied Science Division, Department of Agriculture and Rural Development, Belfast, UK

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Library of Congress Cataloging-in-Publication Data Rust diseases of willow and poplar / edited by Ming Hao Pei & Alistair R. McCracken. p. cm. ISBN 0-85199-999-9 (alk. paper) 1. --Diseases and pests. 2. Poplar--Diseases and pests. 3. Rust diseases. I. Pei, Ming Hao. II. McCracken, Alistair R. III. Title.

SB608.W65R87 2005 634.9′7236--dc22 2004018883

ISBN 0 85199 999 9

Typeset by AMA DataSet Ltd, UK. Printed and bound in the UK by Biddles Ltd, King’s Lynn. Contents

Contributors ix

Preface xi

Abbreviations xv

Part I: Taxonomy and Overview of Rusts

1. Phylogenetic Position of in Rust Fungi Inferred from Ribosomal DNA Sequences 1 Ming Hao Pei, Carlos Bayon and Carmen Ruiz

2. A Brief Review of Melampsora Rusts on Salix 11 Ming Hao Pei

3. The Species of Melampsora on Salix () 29 Gaddam Bagyanarayana

4. A Brief Summary of Melampsora Species on Populus 51 Ming Hao Pei and Yan Zhong Shang

Part 2: Occurrence and Population Biology of Melampsora

5. Variability and Population Biology of Melampsora Rusts on Poplars 63 Pascal Frey, Pierre Gérard, Nicolas Feau, Claude Husson and Jean Pinon

6. Genetic Diversity of Melampsora Willow Rusts in Germany 73 Mirko Liesebach and Irmtraut Zaspel

7. Genetic Structure of Melampsora larici-epitea Populations in North-western 91 Berit Samils

8. Current Taxonomic Status of Melampsora Species on Poplars in 99 Cheng-Ming Tian and Makoto Kakishima

v vi Contents

9. Current Status of Poplar Leaf Rust in India 113 R.C. Sharma, S. Sharma and K.R. Sharma

10. Melampsora Willow Rust in Chile and Northern Europe: Part of a Metapopulation? 119 Mauritz Ramstedt and Sergio Hurtado

Part 3: Rust Resistance and Infection Process

11. Disease Scoring by Taking Inoculum Densities into Consideration in Leaf Disc Inoculations with Poplar and Willow Rusts 131 Ming Hao Pei and Tom Hunter

12. Interactions Between Poplar Clones and Melampsora Populations and their Implications for Breeding for Durable Resistance 139 Jean Pinon and Pascal Frey

13. Transgenic Hybrid Aspen with Altered Defensive Chemistry: a Model System to Study the Chemical Basis of Resistance? 155 Johanna Witzell, Marlene Karlsson, Marisa Rodriguez-Buey, Mikaela Torp and Gunnar Wingsle

14. Basidiospore-derived Penetration by Species of Cronartium and Melampsora: an Outline 161 Alessandro Ragazzi, Nicola Longo, Biancamaria Naldini, Salvatore Moricca and Irene Dellavalle

Part 4: Rust Management

15. Host Diversity, Epidemic Progression and Pathogen Evolution 175 Chris C. Mundt

16. Short-rotation Coppice Willow Mixtures and Rust Disease Development 185 Alistair R. McCracken, W. Malcolm Dawson and Diane Carlisle

17. Short-rotation Coppice Willow Mixtures and Yield 195 W. Malcolm Dawson, Alistair R. McCracken and Diane Carlisle

18. Effect of Preventative Fungicide Sprays on Melampsora Rust of Poplar in the Nursery 209 R.C. Sharma, S. Sharma and A.K. Gupta

Part 5: Rust Mycoparasites and their Potential for Biological Control

19. Biocontrol of Rust Fungi by Cladosporium tenuissimum 213 Salvatore Moricca, Alessandro Ragazzi and Gemma Assante

20. Biology and Genetic Diversity of the Rust Hyperparasite Sphaerellopsis filum in Central Europe 231 Mirko Liesebach and Irmtraut Zaspel Contents vii

21. Mycoparasite Sphaerellopsis filum and its Potential for Biological Control of Willow Rust 243 Ming Hao Pei and Zhiwen W. Yuan

Index 255 This page intentionally left blank Contributors

Gemma Assante, Istituto di Patologia Vegetale, Università di Milano, Via Celoria 2, 20133 Milano, Italy Gaddam Bagyanarayana, Department of Botany, Osmania University, Hyderabad 500 007 (A.P), India Carlos Bayon, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Diane Carlisle, Department of Applied Plant Science, Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK Irene Dellavalle, CNR, Istituto per la Protezione delle Piante, Area della Ricerca del CNR di Firenze, Via Madonna del Piano, 50019 – Sesto Fiorentino (FI), Italy Malcolm Dawson, Applied Plant Science Division, Northern Ireland Horticulture and Plant Breeding Station, Department of Agriculture and Rural Development, Loughgall, Co. Armagh, BT61 8JB, UK Nicolas Feau, Centre de Recherche en Biologie Forestière, Université Laval, Sainte-Foy (QC), G1K 7P4, Canada Pascal Frey, UR Pathologie Forestière, INRA, F-54280 Champenoux, France Pierre Gérard, Laboratoire Ecologie, Systématique et Evolution, UMR ENGREF-UPXI- CNRS 8079, Université Paris-Sud, 91405 Orsay, France A.K. Gupta, Department of Mycology and Plant Pathology, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan – 173 230 Himachal Pradesh, India Claude Husson, UR Pathologie Forestière, INRA, F-54280 Champenoux, France Tom Hunter, 4 Wally Court Road, Chew Stoke, Bristol BS40 8XL, UK Sergio Hurtado, Plant Pathology and Biocontrol Unit, Swedish University of Agricultural Sciences, PO Box 7035, S-75007 Uppsala, Sweden Marlene Karlsson, Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden Makoto Kakishima, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Mirko Liesebach, Federal Office and Research Centre for Forests, Department of Forest Genetics, Hauptstrasse 7, A-1140 Vienna, Austria Nicola Longo, Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira 4, 50121 Firenze, Italy Alistair McCracken, Applied Plant Science Division, Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, UK Salvatore Moricca, Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira 4, 50121 Firenze, Italy

ix x Contributors

Chris C. Mundt, Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, OR 97331-2902, USA Biancamaria Naldini, Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira 4, 50121 Firenze, Italy Ming Hao Pei, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Jean Pinon, UR Pathologie Forestière, INRA, F-54280 Champenoux, France Alessandro Ragazzi, Dipartimento di Biotecnologie Agrarie, Sezione di Patologia Vegetale, Università di Firenze, Piazzale delle Cascine 28, 50144 Firenze, Italy Mauritz Ramstedt, Plant Pathology and Biocontrol Unit, Swedish University of Agricultural Sciences, PO Box 7035, S-75007 Uppsala, Sweden Marisa Rodriguez-Buey, Umeå Plant Science Center, Department of Plant Physiology, Umeå University, S-901 87 Umeå, Sweden Carmen Ruiz, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Berit Samils, Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden Yan Zhong Shang, College of Forestry, Inner Mogolia Agricultural University, Huhehot, China K.R. Sharma, Department of Forest Products, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan – 173 230 Himachal Pradesh, India R.C. Sharma, Department of Mycology and Plant Pathology, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan – 173 230 Himachal Pradesh, India S. Sharma, Department of Mycology and Plant Pathology, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan – 173 230 Himachal Pradesh, India Cheng-Ming Tian, College of Resource and Environment, Beijing Forestry University, 35 Tsinghua Eastern Road, Beijing 100083, China Mikaela Torp, Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden Gunnar Wingsle, Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden Johanna Witzell, Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden Zhiwen W. Yuan, Institute of Applied Ecology, Academia Sinica, PO Box 417, Shenyang, China Irmtraut Zaspel, Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Eberswalder Chaussee 3A, D-15377 Waldsieversdorf, Germany Preface

The plant family Salicaceae comprises two major genera, willow (Salix) and poplar (Populus). Some 300–500 species, according to different authorities, are recognized in Salix and 30–100 species in Populus. Willows and poplars are among the most common woody in the northern hemisphere and, through centuries of human intervention, they have been widely planted in various regions of the world. Traditionally, willows were used for an age-old practice – basket-making. Willows are also common as amenity trees in urban areas and as garden shrubs. More recently, increasing demand for energy from renewable sources has provided a new impetus to growing willows in short-rotation coppice (SRC) plantations for biomass energy production. Poplars, on the other hand, have long been cultivated for timber and pulp production, shelterbelts and amenity purposes. Rust caused by the members of the genus Melampsora is the most widespread and damaging disease of willow and poplar. Conventionally, willow and poplar rusts are regarded as nursery diseases. However, as SRC plantations sustain dense, young canopy during the growing season, the rust can cause serious damage. Up to 40% yield loss due to rust has been recorded in SRC willow. In Europe, poplar rusts did not cause serious damage until the 1980s and 1990s, when the rise of new pathotypes had a serious impact on poplar cultivation. Concerns were raised because poplar rust outbreaks severely affected not only SRC plantations but also single-stem tree plantations. In some cases, the rust resulted in stem dieback and even death of coppice stools. In Europe, early research on willow and poplar rusts was represented by the series of studies carried out by Klebahn in the late 19th and the early 20th centuries. In the Far East, Japanese mycologists such as Takashi Matsumoto and Naohide Hiratsuka conducted much of the early work. These early studies established the foundation for understanding the morphology, host alternation and host range in willow and poplar rusts. The work of the Canadian mycologist W.G. Ziller, in the mid-20th century, contributed much to the knowledge of host range and life cycle of willow and poplar rusts in North America. Over the past two decades, Melampsora rusts on poplar and willow have been subject to extensive study, partly because of the severe damage caused by the occurrence of new, virulent types of rusts and partly because of the growing interest in SRC for biomass energy. As well as conventional plant pathological and mycological methods, DNA profiling techniques have been used widely in willow and poplar rust research. These studies have had a profound impact on our understanding of willow and poplar rust diseases, especially in the fields of population biology, genetics of resistance and functions of host–genotype mixtures.

xi xii Preface

Current state-of-the-art knowledge of poplar rusts would not have been achieved without the outstanding contributions over the past two decades by Jean Pinon and others at the Institut National de la Recherche Agronomique, Nancy, France. George Newcombe, now based at the University of Idaho, played a leading role in poplar rust research in North America. George declined our invitation to contribute a chapter on North American poplar rusts, simply because he had just written a review on the subject published elsewhere. Colleagues from India, China and Japan also made valuable contributions to the progress of research on poplar rusts. Compared to poplar rusts, little work had been done on willow rusts until the late 1980s, when growing willows for biomass gained momentum. Over the years, the Swedish Agricultural University, the Department of Agriculture and Rural Development Northern Ireland and Long Ashton Research Station (the team moved to Rothamsted Research due to the closure of the Long Ashton Research Station in 2003) have played important parts in driving forward the research on willow rusts. Swedish colleagues contributed a great deal to the knowledge of population biology of willow rusts. The outstanding achievements in mixture plantation research in Northern Ireland would not have been possible without dedicated efforts and long-term commitment by the colleagues involved in the past 15 years. During 2000–2004, the Swedish Agricultural University, Queens University Belfast, Long Ashton Research Station and the Institute of Forestry and Tree Breeding, Germany, jointly carried out an EU project on integrated control of willow rusts in Europe. Although relatively new to the subject, German colleagues forwarded remarkable efforts in the studies of pathogenic and molecular variation of willow rusts and the mycoparasite Sphaerellopsis filum in central Europe. In terms of the quality of science and practical usefulness, the project was hugely rewarding. The project was truly memorable for the genuine teamwork and unreserved support among the participants. The primary purpose of this book is to summarize the progress of research on Melampsora rust on willows and poplars, and to provide insights into how this important group of plant pathogens behaves and interacts with their hosts. Such knowledge is essential in developing strategies and means of maintaining healthy crops and reducing disease impacts. The content of the book is based mainly on the papers presented at the ‘International Symposium on Melampsora on Salicaceae’ held in Belfast, Northern Ireland on 11–13 September 2003. The Department of Applied Plant Science, Queen’s University Belfast, hosted and chaired the meeting. This meeting was a part of the activities of the EU project QLK5-1999-01585 ‘Integrated, non-fungicidal control of Melampsora rusts in biomass willow plantations’ which was conducted during 2000–2004 in England, Northern Ireland, Sweden and Germany. Colleagues from various countries contributed to the meeting on taxonomy and population biology, rust resistance in the hosts, the use of host mixtures as a means of rust control and the potential of biological control using mycoparasites. Inspiring background lectures presented by Professor Chris Mundt, Editor-in-Chief of Phyto- pathology, and Dr Mogens Hovmøller were greatly appreciated by all the participants. The Department of Agriculture and Rural Development, Northern Ireland, and the Society of Irish Plant Pathologists offered organizational and financial support for the meeting. The symposium provided a forum in which participants were able to exchange ideas, highlight common ground and explore new directions for research. All the participants felt that it was a truly useful and enjoyable meeting. Several colleagues who were not able to attend the meeting also kindly agreed to contribute chapters for this book. The book comprises five parts: Part 1, Taxonomy and overview of rusts; Part 2, Occurrence and population biology of Melampsora; Part 3, Rust resistance and infection process; Part 4, Rust management; and Part 5, Rust mycoparasites and their potential for biological control. Few books addressing specific tree diseases are available. This book should give research workers, teachers and students a very comprehensive review on the Preface xiii

progress and current knowledge of rust diseases of willow and poplar. Thus, it also presents an overview of a woody plant/rust pathogen system. We would like to thank all the contributors for completing their chapters in time and at a high standard. M.H.P. would like to express his deep gratitude to Professor Li-Ping Shao, Northeastern Forestry University, China, who guided him into rust disease research in the early days of his career.

Ming Hao Pei Alistair R. McCracken July 2004 This page intentionally left blank Abbreviations

AFLP amplification fragment length polymorphism AMOVA analysis of molecular variance AUDPC area under the disease progress curve BCA biocontrol agent EBI ergosterol-biosynthesis inhibiting EtOAc CEs ethylacetate crude extracts GUA genotype unit area ITS internal transcribed spacer IPF incubation period for flecking LD M. larici-daphnoides LET M. larici-epitea typica LM light microscopy LPU latent period for eruption of first uredinium LR M. larici-retusae LSU large subunit NMR nuclear magnetic resonance PCR polymerase chain reaction RAPD random amplified polymorphic DNA rDNA ribosomal DNA RFLP restriction fragment length polymorphism SEM scanning electron microscopy SIF stem-infecting form SNP single nucleotide polymorphism SRC short-rotation coppice SRF short-rotation forest SSR single-sequence repeat SSU small subunit TEM transmission electron microscopy ULD uredinia per leaf disc UPGMA unweighted pair group method with arithmetic mean VAF % variance accounted for

xv This page intentionally left blank 1 Phylogenetic Position of Melampsora in Rust Fungi Inferred from Ribosomal DNA Sequences

Ming Hao Pei, Carlos Bayon and Carmen Ruiz Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Rust Fungi and their Classification genera of rust fungi, i.e. , and Cronartiaceae to The rust fungi (Uredinales) are one of include those forming sessile teliospores, the largest groups of fungi, accounting and Pucciniaceae for those having pedi- for one-third of the teleomorphic species of celled teliospores. Later, Dietel (1928) Basidiomycetes (Hawksworth et al., 1995). reduced the number of families to two, by Over 5000 species of rust fungi have been placing those having sessile teliospores in recognized and it is estimated that the order Melampsoraceae and those having pedi- Uredinales may contain more than 7000 celled teliospores in Pucciniaceae. The two species (Hawksworth et al., 1995). The families were widely accepted for a long species in Uredinales are grouped into time by various workers (Arthur, 1929; more than 100 genera (Cummins and Bessey, 1950; Azbukina, 1974). While Hiratsuka, 1983). accepting Puccinaceae, Gäumann (1959) Rust fungi are unique in many aspects. added a few more families for those tradi- They thrive only on living tissues of plants tionally grouped under Melampsoraceae. and produce up to five different types of Wilson and Henderson (1966) adopted three spores during their life cycle. Rust species families, Coleosporiaceae, Melampsoraceae are either heteroecious or autoecious. and Pucciniaceae. Further modifications Heteroecious species infect taxonomically were proposed by Leppik (1972) and Savile very different plants to complete their (1976). More recently, largely based on life cycle. Autoecious species, on the other spermogonial morphology, Cummins and hand, complete their life cycle on the same Hiratsuka (1983, 2003) proposed a 13- to hosts. The hosts of rust fungi are immensely 14-family system. In their treatment, only a diverse, including ferns, conifers, dico- single genus, Melampsora, is included in tyledonous and monocotyledonous plants. Melampsoraceae. Another important feature of rust fungi is their host specificity, each group being capable of infecting a certain range of plant taxa. Genus Melampsora Traditionally, teliospore morphology provided the basis for rust taxonomy. Dietel The genus Melampsora was established (1900) proposed four families to group the by Castagne in 1843 based on the rust on ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 1 2 Ming Hao Pei et al.

Euphorbia, M. euphorbiae (Scheb.) Cast. Salix and others on various dicotyledonous (Castagne, 1843). The main characteristic of plants, including Euphorbiaceae and the genus is formation of a crust of sessile, Linaceae. laterally adherent single-celled teliospores on the host surface. Melampsora species are either heteroecious or autoecious. Hetero- ecious species of Melampsora occur on Some Viewpoints on the Evolution willows (Salix) and poplars (Populus), both of Rust Fungi belonging to Salicaceae. The majority of heteroecious Melampsora species alternate The evolution of rust fungi has long been on conifers, but some have dicotyledonous a fascinating subject of speculation among or monocotyledonous plants as their alter- mycologists. Savile (1955) speculated that nate hosts (Fig. 1.1). Most of the autoecious the rust fungi have evolved from an species of Melampsora occur on dicotyle- ascomycetous Taphrina-like ancestor. In donous plants, including Euphorbiaceae contrast, Leppik (1965) supported Jackson’s and Linaceae. (1935) views that an auriculariaceous (jelly- The taxonomy of Melampsora is fungi-like) , which occurred on some notoriously difficult (Chapter 2). Numerous primitive ferns, gave rise to rusts. Savile species have been described under Melamp- (1976) modified these ideas and suggested sora but with many, it is not certain whether that a certain Taphrina-like fungus parasitic they should be treated as valid species or on plants gave rise to ancestral Basidiomy- merely as synonyms. The number of species cetes which, in turn, produced Uredinales, in Melampsora is estimated to be some- which remained parasitic on plants, and where between 80 (Hawksworth et al., 1995) Auricularis (jelly fungi), which became and 100 (Hiratsuka and Sato, 1982). To date, saprophytic (see Hennen and Buriticá, volume III of Sydow and Sydow’s Mono- 1980). graphia Uredinearum (1915) remains the In regard to the origin of the heteroecism only monograph available for Melampsora. among the rusts, Fischer (1898) suggested It appears that more than half of Melampsora that the ancestors of present heteroecious species occur on Salicaceae. For example, of rusts had possessed multi-spore forms and 51 species compiled by Sydow and Sydow were capable of living indiscriminately on a (1915), ten were described on Populus,22on wide range of hosts (cited by Arthur, 1929).

Fig. 1.1. Host specificity of some rust genera related to Melampsora. Phylogenetic Position of Melampsora 3

Each species gradually narrowed its host range of plants, including the members of range and became confined to fewer hosts. Pinaceae. Usually, aecial hosts of rust genera During the process, the haploid stage was capable of infecting Pinaceae are confined to adapted to one or a group of hosts, and the a single genus. For example, Uredinopsis, dikaryotic/diploid stage to another. This Milesina, Hyalopsora and Melampsorella early explanation was modified by Klebahn occur on Abies at their aecial stage, Coleo- (1904) and Dietel (1904, cited by Arthur, sporium and Cronartium on Pinus, Melam- 1929) by assuming that the early forms of psoridium on Larix, and Chryxomyxa on rusts were more or less restricted and fixed, Picea (Fig. 1.1). In contrast, the species in and that the change from an autoecious Melampsora occur on several genera of to a heteroecious condition was brought Pinaceae, i.e. Abies, Pinus, Picea, Larix, about by some internal change, possibly in Pseudotsuga and Tsuga. For this reason, the nature of mutation, which led to the Durrieu (1980) suggested that Melampsora acceptance of a very unlike host. could have been the first rust to attack As obligate parasites of plants, rusts Pinaceae. are considered to have coevolved with their hosts. The species in Uredinopsis, Milesina and Hyalopsora have uredinial and telial stages on ferns and spermogonial and aecial Ribosomal DNA Sequences and stages on firs (Abies). They have sessile Phylogenetic Studies of Fungi teliospores and produce five spore stages (macrocyclic) during their life cycle. Many The ribosome is an organelle responsible species of Uredinopsis and Hyalopsora also for protein synthesis, occurring in every liv- produce an extra kind of urediniospores, ing cell. Because of its universal existence called amphispores. These rusts have long and phylogenetically informative nature, been regarded as the most primitive because ribosomal DNA (rDNA) is widely used to the two groups of host are considered to be study evolutionary relationships in living more primitive than other plants serving as organisms, including fungi. The rDNA is a hosts to rust fungi (Ando, 1984). Other rust multigene family with nuclear copies genera producing aecia on conifers also bear arranged in tandem arrays in eukaryotes sessile teliospores, and predominantly have (organisms having cells with a true nucleus macrocyclic and heteroecious life cycles. and other organelles). Each unit within In contrast, autoecious and shortened life a single array consists of the genes coding cycles become common in the rusts on more for the small subunit (SSU) and large sub- advanced hosts (Laundon, 1973; Hiratsuka unit (LSU) rRNAs. The internal transcribed and Sato, 1982). Therefore, it is generally spacer (ITS) region (comprises ITS1, 5.8S believed that the heteroecious, macrocyclic rRNA gene and ITS2) lies embedded life cycle is the primitive feature and that between the SSU and LSU regions (Fig. 1.2). various reduced life cycles are derived from The rDNA sequences encoding the SSU and it. However, as Hiratsuka and Sato (1982) the LSU rRNAs are highly conserved, while pointed out, ‘it was a vexing problem to the sequences of ITS region evolve much imagine that the most complicated life- faster. The ribosomal genes undergo con- cycles (as found in Uredinopsis) are the most certed evolution and, therefore, sequence primitive forms’. A possible explanation is heterogeneity among repeats within a that the extraordinarily complex hetero- nucleus is rare. ecious macrocyclic life cycle evolved only In recent years, phylogenetic studies once (Mendgen, 1997). Reductions could using SSU rDNA data suggested that have easily occurred at any stage of Ascomycetes and Basidiomycetes originated evolution (Laundon, 1973). from a common ancestor but evolved sepa- Melampsora species form relatively rately (Berbee and Taylor, 2001; Tehler et al., simple aecia (no peridia or bearing only 2003). Thus, the molecular data did not rudimentary peridia) and occur on a diverse support the theory that rust fungi evolved 4 Ming Hao Pei et al.

Fig. 1.2. Structure of ribosomal DNA. from the Taphrina-like ancestor. Gene trees somewhat separated (bootstrap value = derived from the LSU (Berres et al., 1995; 62%) from all other genera, which included Begerow et al., 1997) and SSU (Berbee and those having sessile teliospores and those Taylor, 2001; Tehler et al., 2003) regions also having pedicelled teliospores. suggested that rust fungi are monophyletic, We further examined the phylogenetic forming a distinct group in . position of Melampsora among the rust Such studies also supported the recognition fungi, using rDNA sequence data from of the three major groups within Basidio- willow and poplar rusts and the sequence mycota, i.e. Urediniomycetes (rusts and information available in World Wide Web related fungi), Ustilaginomycetes (smuts databases. The partial LSU region of ten and related fungi) and Hymenomycetes species/forms of Melampsora on willows, (mushrooms and related fungi). Auricularia three species on poplar, Puccinia recondita (jelly fungi) was placed within Hymenomy- f.sp. tritici and Melampsoridium betulinum cetes in the gene trees, which showed that was sequenced using the primers described the split between Urediniomycetes and in Berres et al. (1995). The rDNA sequence Hymenomycetes occurred long before the information for other rusts was retrieved split between jelly fungi and mushrooms. from the GenBank database (http://www. Such results contradict the theory that ncbi.nlm.nih.gov/). there was a close phylogenetic association When the partial LSU sequences (about between rusts and jelly fungi. Additional 1000 bases of the 5′ end of LSU) of 53 species data from multiple genes are probably belonging to Uredinales (four other species needed to resolve the deepest divergences in related to Uredinales were used as the the Basidiomycota. outgroup) were examined, the neighbour- joining tree placed those having pedicelled teliospores into one group (bootstrap value = 75%) (Fig. 1.3). Among the rest, Melampsora Phylogenetic Position of Melampsora formed a distinct clade (bootstrap value = in Uredinales 100%). The remaining rusts having sessile teliospores were placed together, but this Cullings and Vogler (1998) included M. clade was not supported by bootstrap value epitea and M. lini in their 5.8S rDNA (< 50%). Within Melampsora, M. occiden- sequence analysis and found that the talis, which occurs on Populus in North Melampsora spp. formed a clade (bootstrap America, was more distant from other value = 95%) before the divergence among Melampsora species (Fig. 1.3). other genera, such as Pucciniastrum, When the SSU and LSU regions of 16 Cronartium, , Puccinia and Uredinales species (see Fig. 1.4; five species Uromyces. Recently, Maier et al. (2003) related to Uredinales were used as an examined partial DNA sequences of the outgroup) were examined, the average p LSU region of rDNA from some 50 rust distance (percentage difference/100) for the species belonging to 25 genera. Three SSU region among the Uredinales species Melampsora species, M. helioscopiae, M. was 0.034 (standard error = 0.003) and euphorbiae and M. hypericorum were that for LSU region was 0.098 (standard included in their study. From their results, error = 0.008). The neighbour-joining tree the genus Melampsora also appeared to be constructed based on the combined SSU and Phylogenetic Position of Melampsora 5

Fig. 1.3. Neighbour-joining tree based on 57 partial large subunit (LSU) sequences. Figures on branches indicate bootstrap values and asterisks (*) indicate the species used as an outgroup.

LSU data placed Gymnosporangium and between Melampsora and other rusts may be Kuehneola (having pedicelled teliospores) greater than that among other rusts. into one clade and the rest (having sessile teliospores) into another (Fig. 1.4). There was strong support (bootstrap value = 96%) for the grouping of those having sessile Implications for the Evolution of the teliospores, including Melampsora. This Rust Life Cycle somewhat differed from the results of Cullings and Vogler (1998) and of Maier The molecular evidence from rDNA et al. (2003), that the genetic distance sequences of rusts shows that the rust fungi 6 Ming Hao Pei et al.

Fig. 1.4. Neighbour-joining tree based on combined small subunit (SSU) and large subunit (LSU) rDNA sequence data. Figures on branches indicate bootstrap values and asterisks (*) indicate the species related to Uredinales and used as an outgroup. are monophyletic (Berbee and Taylor, 2001; Angiosperms are also considered to be Tehler et al., 2003) and that the complex, monophyletic (Doyle, 1998). One can multi-spore form life cycle may have assume that there had been a close host– evolved before the ancestral rust diverged parasite association between the ancestral into different groups (having either sessile Pinaceae and the ancestral rusts before or pedicelled teliospores) (Figs 1.3 and 1.4). flowering plants–rust fungi relationships The neighbour-joining trees derived from were established. rDNA sequences placed the rust genera which do not infect conifers at their aecial stage (they also have pedicelled teliospores) into a distinct clade (Maier et al., 2003; Figs Implications for the Relationships of 1.3 and 1.4). Gymnosperms have a much Melampsora with Other Rusts older fossil record (~320 million years; Doyle, 1998) than angiosperms (at most 130 In explanation of the distant relationships million years; Crane et al., 1995). Results between Melampsora and other rusts (based from molecular studies suggested that gym- on LSU data), Maier et al. (2003) pointed nosperms are monophyletic, evolved from out that Melampsora either split from other a common ancestor (Chaw et al., 1997). rust genera a long time ago or its mutation Phylogenetic Position of Melampsora 7

rate has been considerably higher than that well-preserved fossil ascomycetous fungus of the others. The life cycle of Melampsora from 400 million-year-old Rhynie Chert is similar to that of other rusts having ses- fossil beds in Scotland (Taylor et al., 1999). sile teliospores, i.e. producing up to five Using SLU rDNA sequence data and a spore stages, the majority forming aecio- re-calibrated molecular clock, Berbee and spores on different hosts and completing a Taylor (2001) estimated that Ascomycetes single sexual life cycle annually. Therefore, diverged from Basidiomycetes some 600 it is unlikely that the rate of mutation has million years ago. According to their gene been much higher in Melampsora than in tree, the pine stem rust Cronartium ribicola other rusts. It appears that Melampsora split and the mushroom fungus Boletus satanas from ancestors of other rust genera at early split sometime around 500 million years ago stages of evolution of rust fungi. Our results (Ordovician Period). If such a time scale is from the combined LSU and SSU data (Fig. applied to Fig. 1.3, we can roughly estimate 1.4) suggest that the split between the rusts that: (i) Melampsora may have split from having sessile teliospores and those having other rusts some 250 million years ago; (ii) pedicelled teliospores had occurred before many rust genera differentiated between 100 the split between Melampsora and other and 50 million years ago; and (iii) speciation rusts having sessile teliospores. Phylogen- in Melampsora occurred over the past 50 etic studies carried out by Sjamsuridzal million years. et al. (1999), using SSU sequences of rDNA, suggested that the fern rusts are not the most basal or ‘primitive’ rust fungi. Both Conclusions the molecular evidence and the broad host specificity of the genus at the aecial stage Some 20 years ago, Hennen and Buriticá indicate that the genus Melampsora arose (1980) commented that there was ‘little from the earliest line of ancestral rusts. direct knowledge of the evolution of rusts and few ways of testing evolutionary hypo- theses’. Since then, the rapid advances of Implications on the Timing of DNA technology have revolutionized our Divergence understanding of the evolutionary paths of the living organisms on Earth. So far, rDNA sequence information has provided some The molecular clock concept is one of the unprecedented insights into the evolution- most intriguing, yet hotly debated, topics in ary relationships among the rust fungi. The evolutionary biology. It was first proposed rDNA sequence data suggest that the multi- by Zuckerkandl and Pauling (1965), based spore form of life cycle is likely to have on the approximate constancy of amino- evolved at the very early stages of rust evo- acid substitution in the make-up of human lution. Molecular evidence also indicates haemoglobin. The molecular clock hypo- that the genus Melampsora evolved early thesis assumes that the rate of amino-acid as a distinct group from ancestral rusts. or nucleotide substitution is approximately Further molecular data from more and constant over evolutionary time. Much of wider regions of the rust genome will pro- the controversy surrounding the molecular vide answers to many intriguing questions clock approach is whether the clock is on the evolution of the rust fungi. accurately calibrated. Fossil evidence can be crucial in calibration of the molecular clock. Fungal fossils are rare in nature. For- tunately, some early fossils discovered so Acknowledgements far provide precious clues to the evolution- ary path of the fungi over the geological This study was funded by the Department time span (see Berbee and Taylor, 2001). for the Environment, Food and Rural One such example is the amazingly Affairs (DEFRA), UK and the European 8 Ming Hao Pei et al.

Union. Rothamsted Research receives Dietel, P. (1900) Uredinales. In: Engler, A. and Prantl, grant-aided support from the Biotechnology K. (eds) Die Natürlichen Pflanzenfamilien, 1(1). and Biological Sciences Research Council Verlag von Wilhelm Engelmann, Leipzig, of the UK. pp. 24–81. Dietel, P. (1928) Hemibasidii (Ustilagiales und Uredinales). In: Engler, A. and Prantl, K. (eds) Die Natürlichen Pflanzenfamilien, 2(6). Verlag von References Wilhelm Engelmann, Leipzig, pp. 24–98. Doyle, J.A. (1998) Molecules, morphology, Ando, K. (1984) Phylogeny of the fern rusts fossils, and the relationship of angiosperms (Uredinopsis, Milesina and Hyalopsora). Trans- and Gnetales. Molecular Phylogenetics and actions of the Mycological Society of Japan 25, Evolution 9, 448–462. 295–304. Durrieu, G. (1980) Phylogeny of Uredinales on Arthur, J.C. (1929) The Plant Rusts (Uredinales). John Pinaceae. Report of Tottori Mycological Institute Wiley & Sons, New York. 18, 283–290. Azbukina, Z.M. (1974) Rust Fungi of the Soviet Far Fischer, E. (1898) Entwicklungsgeschichtliche East [in Russian]. Nauka Publishers, Moscow. Untersuchungen über Rostpilze. Beiträge zur Begerow, D., Bauer, R. and Oberwinkler, F. (1997) Kryptogamenflora Schweiz 1, 1–120. Phylogenetic studies on nuclear large subunit Gäumann, E. (1959) Die Rostpilze Mitteleuropas. ribosomal DNA sequences of smut fungi and Beiträge zur Kryptogamenflora Schweiz 12. related taxa. Canadian Journal of Botany 75, Buchdruckerei Büchler and Co., Bern, 2045–2056. Germany. Berbee, M.L. and Taylor, J.W. (2001) Fungal Hawksworth, D., Kirk, P., Sutton, B. and Pegler, D. molecular evolution: gene trees and geologic (1995) Ainsworth’s and Bisby’s Dictionary of the time. In: McLaughlin, D.J., McLaughlin, E.G. Fungi, 8th edn. CAB International, Wallingford, and Lemke, P.A. (eds) The Mycota, VIIB, UK. Systematics and Evolution. Springer-Verlag, Hennen, J.F. and Buriticá, P.C. (1980) A brief Berlin, pp. 229–245. summary of modern rust taxonomic and Berres, M.E., Szabo, L.J. and McLaughlin, D.J. (1995) evolutionary theory. Reports of Tottori Phylogenetic relationships in auriculariaceous Mycological Institute 18, 243–256. basidiomycetes based on 25S ribosomal DNA Hiratsuka, Y. and Sato, S. (1982) Morphology and sequences. Mycologia 87, 821–840. taxonomy of rust. In: Scott, K.J. and Charkravorty Bessey, E.A. (1950) Morphology and Taxonomy of A.K. (eds) The Rust Fungi. Academic Press, Fungi. The Blakiston Co., Philadelphia. London, pp. 1–36. Castagne, L. (1843) Observations sur quelques Jackson, H.S. (1935) The nuclear cycle in Herpo- plantes acotylédonées et dans les soustribus des basidium filicinum with a discussion of the Nemasporées et des Aecidinées, recueillies dans significance of homothallism in Basidiomycetes. le Départment des Bouches-du-Rhône 2, 18. Aix. Mycologia 27, 553–572. Chaw, S.M., Parkinson, C.L., Cheng, Y.C., Vin- Klebahn, H. (1904) Die wirtswechselnde Rostpilze. cent, T.M., and Palmer, J.D. (1997) Seed plant Versuch einer Gesamtdarstellung ihrer phylogeny inferred from all three plant genomes: biologischen Verhältnisse. Borntraeger, Berlin. monophyly of extant gymnosperms and origin Laundon, G.F. (1973) Uredinales. In: Ainsworth G.C., of Gnetales from conifers. Proceedings of the Sparrow, F.K. and Sussman, A.S. (eds) The Fungi, National Academy of Sciences 97, 4086–4091. an Advanced Treatise, IVB, A Taxonomic Review Crane, P.R., Friis, E.M. and Pederson, K.R. (1995) The with Keys: Basidiomycetes and Lower Fungi. origin and early diversification of angiosperms. Academic Press, London, pp. 247–279. Nature 374, 27–33. Leppik, E.E. (1965) Some viewpoints on the Cullings, K.W. and Vogler, D.R. (1998) A 5.8S nuclear phylogeny of rust fungi. V. Evolution of ribosomal RNA gene sequence database: appli- biological specialization. Mycologia 57, 6–22. cations to ecology and evolution. Molecular Leppik, E.E. (1972) Evolutionary specialization of Ecology 7, 919–923. rust fungi (Uredinales) in the Leguminoaceae. Cummins, G.B. and Hiratsuka, Y. (1983) Illustrated Annales Botanici Fennici 9, 135–148. Genera of Rust Fungi, revised edn. APS Press, Maier, W., Begerow, D., Weiß, M. and Oberwinkler, St Paul, Minnesota. F. (2003) Phylogeny of the rust fungi: an Cummins, G.B. and Hiratsuka, Y. (2003) Illustrated approach using large subunit ribosomal DNA Genera of Rust Fungi, 3rd edn. APS Press, sequences. Canadian Journal of Botany 81, St Paul, Minnesota. 12–23. Phylogenetic Position of Melampsora 9

Mendgen, K. (1997) Uredinales. In: Carroll, G. Sydow, P. and Sydow, H. (1915) Monographia and Tudzynski, P. (eds) The Mycota, VB, Uredinearum III. Fratres Borntraeger, Leipzig. Plant Relationships. Springer, Heidelberg, Taylor, T.N., Hass, H. and Kerp, H. (1999) The oldest pp. 79–94. fossil ascomycetes. Nature 399, 648. Savile, D.B.O. (1955) A phylogeny of Basidiomy- Tehler, A., Little, D.P. and Farris, J.S. (2003) cetes. Canadian Journal of Botany 33, 60–104. The full-length phylogenetic tree from 1551 Savile, D.B.O. (1976) Evolution of the rust fungi ribosomal sequences of chitinous fungi, Fungi. (Uredinales) as reflected by their ecological Mycological Research 107, 901– 916. problems. Evolutionary Biology 9, 137–207. Wilson, M. and Henderson, D.M. (1966) The Sjamsuridzal, W., Nishida, H., Ogawa, H., British Rust Fungi. Cambridge University Press, Kakishima, M. and Sugiyama, J. (1999) Phylo- Cambridge. genetic positions of rust fungi parasitic on ferns: Zuckerkandl, E. and Pauling, L. (1965) Molecules as evidence from 18S rDNA sequence analysis. determinants of evolutionary history. Journal of Mycoscience 40, 21–27. Theoretical Biology 8, 357–366. This page intentionally left blank 2 A Brief Review of Melampsora Rusts on Salix

Ming Hao Pei Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Melampsora on Willow Pallas), is similar to Salix in many charac- teristics but resembles Populus in being Rust fungi found on willows (and also on wind-pollinated and having pendulous poplars) belong to the genus Melampsora. catkins. The main characteristic of Melampsora Willow species are dioecious, tend species is formation of crust-like telia to hybridize relatively easily, and often which comprise sessile, laterally adherent vary morphologically at different stages single-celled teliospores. In fact, the name of growth. Probably for these reasons, Melampsora originated from the Greek Salix taxonomy is notoriously difficult. words Melas (black) and psora (scab). Of According to different authorities, there some 80–100 Melampsora spp. (Hiratsuka are between 300 and 500 Salix species and Sato, 1982; Hawksworth et al., 1995), worldwide. Within the genus, the species more than half have been described on are grouped into 2–4 subgenera and, within Salicaceae. a subgenus, they are further grouped into various sections (Skvortsov, 1968; Dorn, 1976; Argus, 1986). There is a general consensus among taxonomists on the Host Genus Salix recognition of subgenus Salix, which com- prises the species typically forming trees The family of Salicaceae consists of two and large shrubs. The rest of Salix species major genera: Populus L. (poplar) and Salix (more than two-thirds of the total) range L. (willow). Populus is wind pollinated, from dwarf shrubs to small trees and are has loose and pendulous catkins, bears extremely variable in their morphology. numerous stamens (5–80) and produces Within these willows, Skvortsov (1968) rec- thin-walled pollen grains. Its flowers also ognized two subgenera: subgenus Vetrix and lack glands. Salix, on the other hand, is subgenus Chamaetia, but stated that bound- insect-pollinated, forms compact and erect aries can be drawn between them only with catkins, usually has two stamens (1–7) and stipulations and conditionally, and that they produces thick-walled pollen grains. Flow- are considerably more closely related to ers of Salix usually have nectar-secreting each other than to the subgenus Salix. glands. The genus Chosenia Nakai, which Dorn (1976) recognized only one subgenus, has only a single species Ch. arbutifolia Vetrix, to include all the species outside the (Pallas) A. Skvortsov (syn. Salix arbutifolia subgenus Salix. In this chapter, we adopt

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 11 12 Ming Hao Pei

Dorn’s (1976) treatment at subgenus level Aecial hosts of willow Melampsora and Bean’s (1980) treatment in classification include conifers, dicotyledonous and of sections and species. The names of willow monocotyledonous plants. Alternative hosts species, which were not included in Bean can be identified by inoculating suspected (1980), were adopted from Skvortsov (1968), plants in the spring, using overwintered Dorn (1976) and Wang and Fang (1984). telia. However, such work can be very demanding because, even if the potential alternate hosts have been identified, Geographic distribution of Salix species maturity of the teliospores, conditions of overwintering and teliospore germination, Species diversity of willow is richest in and growing conditions of the plants may China (c. 270 species) and then in the still affect successful outcome. With many former Soviet Union (c. 120 species) (Argus, willow rust species, only uredial and telial 1997). There are some 65 willow species stages have been described to the present in Europe and over 100 species in North day, and it is not known whether, and America. Willows are not native to which, aecial hosts are involved in their life Australasia, and only a single species, cycle. S. humboldtiana, is naturally distributed Facing the problems in identification of in South America. Melampsora on Salix, Hylander et al. (1953) In general, the natural distribution of recognized M. epitea Thüm. as a complex a Salix species is confined to a certain species to include those species (sensu geographical region and the willow species stricto) similar in morphology. Wilson and occurring in Europe usually differ from Henderson (1966) adopted M. epitea as a those native to the Far East and to North collective species made up of various races America. For the willows occurring in arc- and groups of races which are morpho- tic, subartic or alpine regions, there are often logically indistinguishable, but alternate no distinct boundaries between continents. on different hosts. They distinguished two varieties: var. reticulatae alternates on Saxifraga and has larger urediniospores and paraphyses than var. epitea, which includes Taxonomy of Willow Rusts all other forms alternating on a number of different hosts. Hiratsuka and Kaneko (1982) The taxonomy of Melampsora on Salix has and Pei et al. (1993, 1996) also adopted long been in a state of confusion. Many Wilson and Henderson’s treatment. How- Melampsora species on willows were ever, a remaining problem is that, because described in the late 19th to early 20th M. epitea contains many widely different century. By 1915, a total of 22 Melampsora groups, the name means very little in terms species on Salix were complied in the of the biological characteristics and genetic Monographia Uredinearum III by Sydow identity for the group or, in a narrow sense, and Sydow (1915). Willow Melampsora the species. species were mainly established based on In this chapter, for the convenience of their morphology, alternate hosts and the summarizing existing information, the spe- telial host range. Some of the great difficul- cies names are used in the narrow sense ties in identification of the species on wil- (sensu stricto) and the complex species M. low arise from the fact that the host ranges epitea is specified as ‘M. epitea complex’. of different rust species often overlap and there is no clear morphological distinction between those that are separated by having different alternate hosts. Usually, only one Morphology of Willow Rusts or two spore stages can be found at the time of observation and there is no information A summary of the morphological character- on the alternate hosts. istics of willow rust is listed in Table 2.1. Melampsora Rusts on Salix 13 m m m m

continued 7–14; wall 9–12; wall 5–11; wall 8–15; wall 11–14; 10–17; 7–14; wall 7–14; wall mthick × × × × × × × × m thick at m m mthick m thick at m m mthick mthick mthick mthick m m m m 30–45 porous and up to 10 apex and 1 thick at sides 19–30 1 18–60 0.8 25–45 1 20–45 wall 1 25–45 wall porous and up to 10 apex and 1 thick at sides 30–48 1 Size and wall of teliospores 18–42 1 Epiphyllous; sub-cuticular; 1 mm or more across Hypophyllous; sub-epidermal Mainly epiphyllous; sub-cuticular Mainly epiphyllous; sub-cuticular; 0.25 mm to, rarely, 1mmacross Epiphyllous; sub-cuticular; Epiphyllous; sub-epidermal Mainly epiphyllous; sub-cuticular; 0.25–1.5 mm Position and size of telia Hypophyllous; sub-epidermal: 0.3–0.5 mm m; m m; m; m; m; m; m m m m m mthick m mthick mthick mthick mthick 16–24 m m m m 18–26 18–26, 17–24 17–23 15–20 10–18 m m × m m × × × × × × m m 50–60 thickened at apex, 5–6 Thickened at apex, up to 7 wall 3–7 wall 2–5 50–70 thickened at apex, up to 6 wall 1.5–3 55–60 50–70 38–410 50–70 wall 3–5 Size and wall of paraphyses 35–80 wall 2–5 m; m; m; m; m m m m mthick mthick m m mthick mthick mthick mthick m m m m mthick m 13–15 15–22; 12–17 12–16; 14–23 12–16 13–15; 11–15; m m × × × × × × × × . evenly echinulate; wall 2–2.5 evenly echinulate; wall 1.5–2 14–21 evenly echinulate; wall 2.5–4 evenly echinulate; wall 1.5 17–25 evenly echinulate; wall 2.5–4 16–26 25–38 smooth at apex; wall up to 3 Size and wall of urediniospores 16–26 Salix 13–20 22–33 smooth at apex; wall up to 3 19–32 smooth at apex; wall 1.5 species/forms on .0.5mm .0.5mm .0.5mm Mainly hypophyllous; Hypophyllous; 1–2 mm or up to 5mmonjuvenile leaves Hypophyllous; Hypophyllous; Mainly hypophyllous Mainly hypophyllous Mainly hypophyllous; 0.5–1 mm Position and size of uredinia c c c Mainly hypophyllous m) m

Melampsora 12–19 12–17 12–14; 14–19 13–24 10–16 14–19 × × × × × × × 18–25 wall thickened at the apex in N. American population 15–25 Size of aeciospores ( 13–20 1 × 0.5–1 × 0.25–0.75 19–21 2 Up to 10 18–23 0.25 Size of aecia (mm) 0.3–0.6 16–17 1–2 17–22 Morphological characteristics of Rostr. Matsumoto Kleb.

Table 2.1. Species/form M. abieti- caprearum Tubeuf M. allii-fragilis Kleb. M. amygdalinae Kleb. M. arctica M. galanthi- fragilis M. caprearum Thüm. M. caprearum Thüm. (Japanese form) M. chelidonii- pierotii 14 Ming Hao Pei . m mat c m m mthick m 10–14; 7–14; wall 7–12.5; 6.5–15; 6.5–15; 7.5–13.5, 7–13; wall mthick, 16–30; wall m thick at × × × × × × × m m × mthick m mthick m or slightly mthick m m m 6–14 1 wall 1.5–2.5 thick 25–50 2 thicker at apex 22–50 porous and 3.5–5 apex, 0.5–1 sides 13–33 wall 1 sometimes slightly thicker at apex 20–45 wall 0.5–1 20–50 occasionally septate; wall 25–40 1 Size and wall of teliospores 1.5 .0.5mm Mainly epiphyllous; 28–64 Hypophyllous; sub-epidermal; 0.15–0.5 mm Hypophyllous; sub-epidermal; Mainly hypophyllous; sub-epidermal Mainly epiphyllous; sub-cuticular Mainly hypophyllous; sub-epidermal Hypophyllous; sub-epidermal; 0.2–0.5 mm Hypophyllous; mainly sub- epidermal; 0.5–1 mm Position and size of telia c m m m; m m m m; m; m; m m m m mthick m m mthick mat m m m wide, mthick 14–20 22–30 m m 12–18 20–45 18–25 × × × × × 15–24 × thick sides wall 2–2.5 wall up to 3 at apex, 2 25–35 35–80 (occasionally 90) (occasionally 35) wall 2–2.5 at sides, slightly thickened at apex 10–18 35–450 35–103 wall 3–4 Up to 22 50–70 wall up to 8 Size and wall of paraphyses m; m; m; m; m; m; m; m; m m m m m m m m mthick m mthick mthick mthick mthick m m m m 14–18 13–19 15–25 10–18 10–15 13–18 14–20 14–19 mthick mthick mthick × × × × × × × × m m m evenly echinulate; wall 1.2–2 evenly echinulate or occasionally smooth at apex; wall 1.5–2 evenly echinulate or verrucose; wall 2.5–3 evenly echinulate; wall 1.5–3 evenly echinulate; wall 1.5–2 evenly echinulate; wall 0.6–1 evenly echinulate; wall 2 evenly echinulate; wall up to 5 Size and wall of urediniospores 14–25 18–29 19–30 12–25 12–20 16–20 12–17 18–23 .0.5mm Position and size of uredinia Hypophyllous; Hypophyllous; 0.3–0.8 mm Mainly hypophyllous Mainly hypophyllous; 0.2–1 mm Amphigenous; 0.5–1.5 mm Mainly hypophyllous Mainly hypophyllous Hypophyllous; 0.05–0.15 mm c m) m 14–19; 10–21 13–18 15–24, × × × × wall up to 5 15–25 Size of aeciospores ( 15–20 wall thickened at apex . Up to 1.5 18–23 Size of aecia (mm) 2–4 14–21

Continued Dietel Kleb. Dietel Thüm. f. sp.

M. epitea Table 2.1. Species/form Azb. et Kov. Dietel Kaneko and Hiratsuka complex) M. choseniae M. coleosporioides M. dimorphospora M. epiphylla M. epitea ( M. epitea tsugae M. euonymi- caprearum M. humilis Melampsora Rusts on Salix 15 m m m m

continued 10–16; 7–14; wall 10–15; 8.5–20; 6–12; wall 7–14; wall 8–16; wall 7–10; wall 9–12; wall 29–43; mthick mthick mthick × × × × × × × × × × m m m m thick at m mthick mthick mthick m thick, slightly mthick m m m m m 25–53 wall 1 16–48 1 30–37 wall 0.5–0.7 thick 20–35 wall 1 30–50 1 20–50 1 thick at base and apex thicker at apex 35–70 1–2 sides, 2.5–3.5 30–62 1 19–30 1 11–14 wall 1 .0.5mm .0.5mm Amphigenous; sub-epidermal; 0.3–0.8 mm Amphigenous; sub-epidermal; Mainly hypophyllous; sub-epidermal; 0.2–0.5 mm Epiphyllous; sub-epidermal; 0.25–0.5 mm Hypophyllous, sub-epidermal Mainly epiphyllous; sub-epidermal; 0.2–0.5 mm Hypophyllous; sub-epidermal; Mostly hypophyllous; sub-epidermal; 0.25–1 mm Hypophyllous; sub-epidermal Mostly epiphyllous; sub-epidermal c c m; m; m; m m; m m m m m m m m m mthick m mthick mthick m, m m m 15–25 22–26 16–20 12–22 15–24 m long; wall m wide: wall mthick m at sides m m × × × × × m m mthick m mthick m at sides at apex, 2–2.5 2–2.5 wall 5–6.5 thick at apex, 1.5–2 wall 3–5 wall 3–5 Wall up to 8 1–4 17–20 wall 3–5 sometimes up to 10 15–30 40–60 40–75 50–70 40–70 Slightly thickened at apex 35–80 m; m; m; m; m; m; m; m; m; m m m m m m m m m m m m; m m m mthick m mthick m m m mthick mthick mthick m m m mthick mthick 11–15 15–16 12–16 15–18 10–12 17–24 12–14 9–19 13–17 10–15 m m × × × × × × × × × × evenly echinulate; wall 1.5–2 evenly echinulate; wall smooth at apex evenly echinulate; wall 2–2.5 at sides, 5–6.5 thick at apex evenly echinulate; wall up to 2 evenly echinulate; wall 2.5–3.5 evenly echinulate; wall 1.5 evenly echinulate; wall 2.5 16–26 evenly echinulate; wall 2–2.5 at sides, 4–7.5 at apex 20–21 26–44 20–33 10–15 12–25 15–19 13–17 evenly echinulate; wall 1.5–3.0 thick .0.25mm .1mm Mainly hypophyllous; Hypophyllous; less than 0.2 mm Hypophyllous; 0.25–1.5 mm Mainly hypophyllous; 0.25–0.5 mm Amphigenous; 0.3–0.5 mm Hypophyllous 11–17 Hypophyllous; 0.25–0.5 mm Hypophyllous; c c Hypophyllous 15–27 Mainly hypophyllous; 0.3–0.5 mm m m 18–27 13–21 18–20, 13–20 10–21 × × × × × 15–25 thick 19–27 wall up to 3 18–26 1 × 0.25 0.5–1.5 15–25 0.1–0.2 15–220 Kleb.

M. kamikotica Kaneko et Hiratuska M. kiusiana Hiratsuka M. kupreviczii Zenk. M. lapponum Linfors M. larici- pentandrae M. larici-epitea Kleb. M. larici- urbaniana Matsumoto M. microsora Dietel M. paradoxa Diet. & Holw. M. repentis Plowr. 16 Ming Hao Pei m m 8–15; wall 7–10; wall 7–11; wall 10–13; 11–14 7–10; wall 7–10; wall 6.5–12.5; 7–11; wall mthick mthick mthick × × × × × × × × × m m m mthick mthick mthick mthick m m m m 1–1.8 1 1 25–45 wall 1 20–30 35–44 1 25–40 1 28–39 25–30 wall 0.5–0.8 thick 1–1.5 20–35 36–69 Size and wall of teliospores Mostly hypophyllous 34–42 Mainly hypophyllous; sub-epidermal Hypophyllous; sub-epidermal; up to 0.5 mm Mainly epiphyllous; sub-epidermal; 0.3–0.5 mm Epiphyllous, sub-cuticular; 0.25–0.5 mm Hypophyllous; sub-epidermal Mainly hypophyllous; sub-epidermal; 0.25–0.5 mm Mostly hypophyllous; mostly sub-cuticular Position and size of telia Epiphyllous; sub-epidermal; 0.2–0.5 mm m; m; m; m; m; m; m m m m m m m; m m m m m m m mthick mthick m m mthick mthick m m 12–16 15–20 18–41 16–24 18–25 15–21 mthick 24–35 × × × × × × m × thick at apex wall up to 7 wall 2–2.5 wall up to 10 thick wall 2.5–4 wall 1–2 30–65 wall 3–5 wall 7 50–70 60–90 55–70 50–70 Wall up to 8 thick at apex 40–70 Size and wall of paraphyses c. 98 m; m; m; m; m; m; m; m m m m m m m mthick mthick mthick mthick mthick m m m m m mthick 13–28 11–18; 15–23 14–18 11–17 14–19 13–18 14–16 17–18; m mthick mthick mthick × × × × × × × × × m m m evenly echinulate; wall 2–2.5 18–37 angular? wall 1.5–2 15–18 17–35 evenly echinulate; wall 2–3.5 16–20 evenly echinulate; wall 3–3.5 20–36 smooth at apex; wall 2 15–23 evenly echinulate; wall 2.5 evenly echinulate; wall 2 Size and wall of urediniospores 18–29 evenly echinulate; wall 2–2.5 15–19 wall 1.5–2 .0.25mm Amphigenous; 0.1–0.3 mm Epiphyllous; 0.2–0.5 mm Position and size of uredinia Hypophyllous; Hypophyllous; 0.5–1 mm Hypophyllous; 0.5–1 mm Mainly hypophyllous; 0.5 mm; up to 5 mm on young twigs and up to 2 mmyoung on leaves Mainly hypophyllous; upto5mmon juvenile leaves Hypophyllous 21–23 c Mainly hypophyllous m) m 13.5–18.5 × 15–18 12–19 11–15 14–20 14–17 15–24 × × × × × × Size of aeciospores ( 15–20 17.5–29 . 1 17–26 0.5–1.5 15–23 In groups, 10–20 0.5–1 16–25 1.5 18–23 0.5–1.5 17–24 Size of aecia (mm) 0.2–1.5

Continued Kaneko Kleb. Tai Sawada Wang Kleb. Table 2.1. Species/form Blytt Kleb. Kleb. Miyabe et Matsumoto M. reticulatae M. ribesii-epitea M. ribesii- purpureae M. ribesii- viminalis M. salicis-albae M. salicis- cavaleriei M. salicis- cupularis M. salicis- warburgii M. yezoensis Caeoma salicis- miyabeana et Hiratsuka Melampsora Rusts on Salix 17

Uredinia and urediniospores apex. Several species, such as M. arctica and M. euonymi-caprearum, produce uredinio- Broadly, there are two types of uredinio- spores having even, but slightly thick spores in willow Melampsora, one type walls. Helfer (1992) described the spine having relatively small urediniospores density on the surface of urediniospores with an evenly echinulate surface and the in M. lapponum as considerably higher other having relatively large urediniospores than that of other willow rusts recorded with a smooth apex. In Europe, all the in Britain. Melampsora species occurring on tree wil- lows (subgenus Salix), i.e. M. allii-fragilis, M. amygdalinae, M. galanthi-fragilis, M. larici-pentandrae, produce urediniospores Telia and teliospores having a smooth surface at the apex. In con- trast to the European Melampsora species Historically, teliospore morphology formed on subgenus Salix, the species occurring the basis of classification at family and on tree willows in Japan and the Far East, sub-family level under the order of M. chelidonii-pierotii, M. larici-urbaniana, Uredinales. A classic example is the separa- M. microsora, M. salicis-warbugii and tion of two major groups, Pucciniaceae and M. yezoensis, produce evenly echinulate Melampsoraceae, based on the presence urediniospores. The urediniospores of and absence of a pedicel (Arthur, 1934). M. coleosporioides are mostly evenly With willow rusts, the position of telia on echninulate, but the rust also occasionally the host and the thickness of spore walls are produces uredinospores that are smooth at important features in species identification. the apex. From the existing description, it The species forming epiphyllous telia is not clear whether urediniospores of include M. allii-fragilis, M. arctica, M. M. salicis-cavaleriei, which was found on gallanti-fragilis, M. choseniae, M. a tree willow in China, have an unevenly caprearum, M. epiphylla, M. kiusiana, M. echinulate surface. In M. dimorphospora kupreviczii, M. lapponum, M. reticulatae, Kaneko and Hiratsuka, urediniospores are M. ribesii-viminalis and M. salicis- either echinulate or verrucose. In North cavalariei. The remainder are either America, all the rusts on willows have hypophyllous or amphigeneous (on both evenly echinulate urediniospores (all could sides of leaves). Some variation in the be placed under the M. epitea complex). appearance of telia was observed within The size and position of uredinia, M. larici-epitea. For example, telia of f. thickness of spore walls and, to a lesser sp. larici-daphnoides on S. daphnoides extent, the morphology of paraphyses also were button-shaped, round and confluent, serve as criteria to distinguish the species usually with a sunken centre, compared of willow rusts. Some species, such as M. with irregularly shaped telia of the other caprearum and M. ribesii-purpureae form M. larici-epitea samples (Pei et al., 1993). In uredinia up to 5 mm in diameter on juvenile some species, teliospores form above the leaves. In contrast, M. microsora produces epidermal cells of the hosts (subcuticular), uredinia less than 0.2 mm in diameter while in others teliospores are produced (Hiratsuka and Kaneko, 1982). The majority beneath the epidemal cells (subepidermal). of the species produce uredinia mainly on One of the most prominent morpho- the lower sides of leaves (hypophyllous). logical features in willow rusts is the Melampsora salicis-cupularis is the only thickness of teliospore walls. The majority species described as forming uredinia on the of willow rust species have teliospores with upper sides of host leaves (epiphyllous). For thin, evenly thickend walls. However, M. most species, the walls of urediniospores are caprearum and M. epiphylla have teliospore evenly thick, measuring 1.5–2 mm. However, walls thickened at the apex. Teliospore urediniospore walls in M. kamikotica and walls in M. larici-urbaniana are slightly M. larici-urbaniana are thickened at the thicker both at the apex and at the base. 18 Ming Hao Pei

Basidiospores and spermogonia appear on the alternate host larch 6–7 days after inoculation with basidiospores (Pei Basidia and basidiospores are produced et al., 1996, 1999). Spermogonial formation from overwintered teliospores in spring. is associated with production of light- Each basidium becomes four-septate and brown honeydew and a strong fragrance produces four spherical basidiospores, of incense which attracts insects and facili- which are vigorously discharged into the tates fertilization between spermogonia. air. Basidia and basidiospores are thin Fertilization between spermogonia on walled and relatively short lived. Spermo- aecial hosts results in formation of aecio- gonia are either subcuticular or subepi- spores (in the case of M. larici-epitea, aecia dermal, and often form as groups, espe- start to form 2–3 days after the appearance cially on dicotyledonous plants. Most of spermogonia). Studies (Pei et al., 1999) willow Melampsora species lack descrip- have shown that in M. larici-epitea, there tions of basidia, basidiospores and spermo- are two mating types among the spermogo- gonia, and therefore these features are not nia, and only the combination between one considered to be taxonomically significant. and the other leads to formation of aecia. The aeciospores infect the telial host willow to produce urediniospores. The Aeciospores basidiospores and spermatia are mono- karyotic, having a single haploid nucleus, while aeciospores, urediniospores and The surface of aeciospores of willow rusts early phases of teliospores are dikaryotic, is ornamented with numerous warts, each containing two nuclei. resembling stacked rings (verrucose). The For 11 species/forms of willow rusts, aeciospore walls of most species are evenly only uredinio- and teliospore stages are thick. Exceptions can be found in M. epitea known. In recent years, the stem-infecting f. sp. tsugae and the North American form (SIF) on S. viminalis has been studied populations of M. abieti-caprearum, both in some detail, due to its importance forming aeciospores having a thickened in short-rotation coppice plantations for wall at the apex. renewable energy. The SIF is highly specialized in its pathogenicity, being found only on willow genotypes belonging Life Cycle, Host Alternation and to S. viminalis L., which also serves as the Overwintering telial host of M. larici-epitea (Pei et al., 1995, 1996). M. larici-epitea infects only expanded Life cycle willow leaves and readily completes a full sexual life cycle, during which five spore Typically, Melampsora species are macro- stages are produced (Pei et al., 1993, 1996). cyclic, producing five different spore stages, Unlike M. larici-epitea, SIF infects shoot- i.e. basidiospores, spermatia, aeciospores, tips and young stems, and causes stem uredinospores and teliospores, during their cankers (Pei et al., 1995). In the field, SIF life cycle. The rusts spread on the willow occurs as the uredinial stage and over- hosts through repeated cycling of uredinio- winters in buds or stems of infected willows. spores (usually within 2 weeks from one Although SIF can produce teliospores cycle to the next) during the growing morphologically similar to those of M. season. They develop teliospores in late larici-epitea under laboratory conditions, summer and autumn, and overwinter on field observations and molecular evidence fallen willow leaves. In spring, teliospores showed that it is an asexual population and germinate to produce basidiospores that may have a clonal lineage (Pei et al., 1995; infect aecial hosts. Meiosis takes place in Pei and Ruiz, 2000). Our recent result the early stages of teliospore germination. showed that the stem-infecting form shares In M. larici-epitea, spermogonia start to the same sequences with M. larici-epitea and Melampsora Rusts on Salix 19

M. caprearum in the large subunit (LSU) miyabeana, only spermogonia and aecia region of ribosomal DNA (rDNA) and only were described on S. miyabeana but it was differs slightly in the internally transcribed assumed that this rust completes its life spacer (ITS) region of rDNA (M.H. Pei and C. cycle on the same willow host. Bayon, unpublished results). This indicates Of 14 species of willow Melampsora that the SIF is likely to have evolved rela- in Japan, four were shown by artificial tively recently from its heteroecious ances- inoculation to be capable of alternating on tor by shifting its niche from fully expanded Larix, one on Corydalis and one on both leaves to very young leaves and shoots, a Corydalis and Chelidonium (Hiratsuka and process known as ‘sympatric speciation’. Its Kaneko, 1982). In North America, Larix, ability to overwinter as an asexual uredinial Abies, Tsuga and Saxifraga are involved as stage on the telial host may have facilitated alternate hosts (Ziller, 1974). Pei et al. (1993) the shortening of its life cycle. reported that M. caprearum and most of the telial collections of M. larici-epitea readily infected the European larch, L. decidua, but not the Japanese larch L. kaempferi. In con- Host alternation trast, Hiratsuka (1932) infected L. kaempferi without difficulty using four species of In a review of the rust fungi occurring Melampsora, including several samples in central and western Europe, Gäumann of M. larici-epitea and M. caprearum.It (1959) summarized that most Melampsora is possible that, within the same species, species infecting willows are hetero- host–parasite coevolution in geographically ecious, with Abies, Allium, Euonymus, isolated populations may also have resulted Larix, Ribes, Saxifraga, Viola and some in the differences in selection of the Orchidaceae as alternate hosts. Some alternate hosts. willow Melampsora species were separated For some willow rust species in which solely on the basis of having different alter- only uredinio- and teliospore stages have nate hosts, often identified in early inocula- been described, their existence may depend tion experiments. For example, both M. on host alternation, but alternate hosts are allii-fragilis and M. galanthi-fragilis share yet to be found. For others, host alternation the same morphology, telial host range (on may not be vitally important for their S. fragilis and S. pentandra) and distribu- survival or may no longer be required, as tion, but they differ in their alternate hosts. seen in the case of SIF. Among willow rusts, The connection of the spore stages between asexual populations with a shortened life the willow hosts and Allium spp. in M. allii- cycle may not be unique to SIF. Pei et al. fragilis was first demonstrated by Klebahn (1993) inoculated Ribes spp. with two at the beginning of the 20th century and UK teliospore collections of M. ribesii- later confirmed by Mayor (1933, 1936). The purpureae. As a result, only one of the col- occurrence of the aecial stage of M. lections formed poorly developed spermo- galanthi-fragilis on snowdrop Galanthus gonia on Ribes. However, despite prolonged nivalis was first studied by Schröter (1893) incubation, no aecia were produced. M. and confirmed by Klebahn (1902). ribesii-purpureae has a doubtful record M. amygdalinae Kleb., which was of the aecial stage in the UK (Wilson and described on the tree willows S. triandra and Henderson, 1966) and the rust appears to be S. pentandra, is the only known autoecious, capable of overwintering as the uredinial macrocyclic species of willow Melampsora stage (Pei et al., 1993). Similar observations (Sydow and Sydow, 1915; Gäumann, 1959). with M. ribesii-purpureae were made by In addition to this, a rust apparently belong- Scaramella (1932) in Italy. A possible ing to Melampsora was found on S. miya- speculation is that some populations of beana in Japan and named Ceoma salicis- M. ribesii-purpureae may have lost the miyabeana (Hiratsuka and Kaneko, 1982; ability to infect the alternate host Ribes to Hiratsuka et al., 1992). With C. salicis- complete a full macrocyclic life cycle. 20 Ming Hao Pei

Overwintering the pioneering work on host specificity in willow rusts was conducted by Klebahn The rusts with a shortened life cycle, such and others in the late 19th to early 20th as SIF on S. viminalis, must overwinter as centuries (see Gäumann, 1959). In the Far the uredinial stage and re-infect the telial East, most studies on willow Melampsora hosts in next growing season to continue were carried out by Japanese mycologists their existence. In contrast, the typical life (Matsumoto, 1915, 1919, 1926; Hiratsuka, cycle of the heteroecious, macrocyclic rusts 1927, 1932; Hiratsuka and Kaneko, 1982). In involves overwintering as teliospores and North America, Ziller conducted a number producing basidiospores in the next spring of experiments to determine the alternate to infect their alternate hosts. However, hosts and telial host range of North overwintering as the uredinial stage American willow rusts around the mid- on willow has been reported with 20th century (see Ziller, 1974). many heteroecious species, including M. caprearum, M. larici-pentandrae (Hylander et al., 1953), M. arctica (Savile, 1953; Wil- son and Henderson, 1966), M. lapponum Rusts on tree willow (Wilson and Henderson, 1966), M. ribesii- purpureae (Scaramella, 1932), M. ribesii- Some 11 species of Melampsora occur viminalis (Scaramella, 1932), M. paradoxa exclusively on the subgenus Salix (Table (Ziller, 1970) and M. salicis-albae (Klebahn, 2.2). Three species, M. allii-fragilis, M. 1905). Thus, their continuation may not galanthi-fragilis and M. ari-salicina, have depend on the availability of alternate been described on S. fragilis in central hosts. In Britain, the autoecious M. Europe. Five species, M. larici-pentandrae, amygdalinae can also overwinter as the M. salicis-albae, M. allii-fragilis, M. uredinial stage in buds and stem cankers galanthi-fragilis and M. amygdalinae have of the host (Ogilvie, 1932; M.H. Pei, been described on S. pentandra. Contrary unpublished observations). For willow to the experience of Mayor (1933), inocula- rusts, the ability to infect willow buds/ tion attempts with urediniospores of M. stems is essential to overwintering as the amygdalinae from England failed to pro- uredinial stage. duce symptoms on S. pentandra (M.H. Pei, unpublished results). Also, the record of S. pentandra as the host of M. salicis-albae (see Gäumann, 1959) is doubtful. Over the Host Specificity in Willow Rusts past 12 years of observation, neither M. amygdalinae nor M. salicis-albae have Historical studies been found on S. pentandra in the national willow collection, south-west England, Melampsora rusts are specialized in their where both the rust species occur annually pathogenicity, and their telial host range (M.H. Pei, unpublished observations). In can be useful in distinguishing different Scandinavia, there have been no records of Melampsora species. At sub-species level, either M. amygdalinae or M. salicis-albae the host range provides the basis for on S. pentandra. the identification of formae speciales or M. coleosporioides occurring on tree pathotypes (races). The host range of willow is native to the Far East, from willow rusts is determined by inoculations maritime provinces of Russia to Taiwan, of willows with urediniospores or aecio- but was spread to Australasia in the spores. Since the late 19th century, exten- 1970s (Latch, 1980). In North America, sive inoculation experiments conducted by M. abieti-caprearum has been recorded on various workers have contributed much to S. exigua and M. paradoxa on S. lasiandra. the present knowledge of the host range of These two rusts occur mainly on shrub Melampsora species on Salix. In Europe, willows. Melampsora Rusts on Salix 21

continued Wilson and Ziller (1974) Wilson and Hiratsuka and Kaneko (1982) References Gäumann (1959), Sydow and Sydow (1915), Gäumann (1959) Gäumann (1959), Henderson (1966), Azbukina (1974) Sydow and Sydow (1915), Hiratsuka and Kaneko (1982), Spiers and Hopcroft (1996) Sydow and Sydow (1915), Gäumann (1959), and Kaneko (1982), Parmelee (1989) Sydow and Sydow (1915), Gäumann (1959), Henderson (1966) Azbukina (1974) Hiratsuka and Kaneko (1982) Japan Hiratsuka (1932), Hiratsuka Eurasia Gäumann (1959) Eurasia Gäumann (1959) Northern and central Europe, North and South America Distribution Central Europe Eurasia Maritime provinces of Russia, China, Japan, Taiwan, Australasia China Tai (1979) Arctic, subarctic and alpine regions of the northern hemisphere Japan and maritime provinces of Russia (V., Shrank

pentandra (V., Vetrix); (V.,

reinii

mackenzieana (S., Glaucae) . , incana (V., Vetrix)

sinica (S., Longifoliae) (V., Nigricantes),

repens pentandra Salix (Chosenia) Maritime provinces of Russia

chilensis var. (V., Vetrix) (S., Glandulosae)

exigua (V., Psilostigmatae)

commutata

nigricans (V., Retusae), (V., Vetrix),

(S., Amygdalinae), auilonia, groenlandica, (V., Vetrix) (S., Subalbaceae), (S., Salix), (V., Helix), species/forms on Telial host (subgenus, section) (V., Cordatae), (S., Pentandrae) (S., Pentandrea) (S., Subalbaceae), (S., Humboldtianae) Incubaceae), Glabrella), S. appendiculata, aurita, caprea, cinerea, scouleriana (V., Canae), purpurea S. fragilis S. triandra S. herbacea subreniformis, yezoalpina S. caprea S. aurita, caprea S. bakko, leucopithecia S. caprea, caprea S. spathulifolia S. pierotii chaenomeloides Chosenia arbutifolia S. babylonica, matsudana

Melampsora spp. spp. spp. spp. Aecial host

Abies Allium S. triandra, S. pentandra Saxifraga Larix Corydalis incisa ? Kleb.) Tubeuf Speg.) Schneid. Dietel * * Kleb. Thüm. Kleb. Azb. Et Kov. ? Kleb. sp. List of hosts and distribution of Juel) Rostr.

typica grandifoliae

M. humboldtiana M. alpina M. larici-caprearum ‘Chinese form’ f.sp. ‘Japanese form’ f.sp. (= Table 2.2. Matsumoto (= Melampsora M. abieti-caprearum M. allii-fragilis M. amygdalinae M. arctica M. caprearum (= M. chelidonii-pierotii M. choseniae M. coleosporoides 22 Ming Hao Pei .

et al Pei . (1953), Wilson

et al (1996) Hiratsuka and Kaneko (1982) References Gäumann (1959) Sydow and Sydow (1915), Gäumann (1959) Sydow and Sydow (1915), Gäumann (1959), and Henderson (1966) Hiratsuka and Kaneko (1982) Gäumann (1959) Hiratsuka and Kaneko (1982) Gäumann (1959) Hylander Japan, North-East Asia Azbukina (1974), Tai (1979), Northern and central Europe Gäumann (1959) Japan Hiratsuka and Kaneko (1982) Japan Hiratsuka and Kaneko (1982) Eurasia, North Africa Distribution North AmericaEurope Ziller (1974) Central Europe Gäumann (1959) Amur River, Russia Azbukina (1974) Eurasia and North Africa Northern and central Europe Sydow and Sydow (1915), Eurasia, North and South Americas, Australasia

argentea (V., Helix) var.

pet-susu

, sitchensis (V., Vetrix) (V., Vetrix),

(V., Helix) Sakhalin, Taiwan, Japan repens gilgiana pentandra brachypoda (Chosenia) Japan (V., Daphnella) var. (V., Incubaceae) (V., Vetrix), (V., Helix)

sachalinensis (S., Pentandrae) (V., Villosae) rorida , Shrank. (V., Canae) (S., Salix), (V., Vimen), (V., Vetrix),

incana Telial host (subgenus, section) (V., Vimen), (V., Sitchenses) (S., Pentandrae) (V., Incubaceae) (V., Incubaceae) S. koriyanagi S. kinuynagi S. scouleriana S. aurita, caprea, cinerea S. S. integra, koriyanagi Chosenia arbutifolia S. subopposita S. rosmarinifolia S. lapponum S. pentandra S. aurita, caprea, cinerea viminalis spp. spp. spp. ? Aecial host ? ? Larix kaempferi Euonymus europaeus Galanthus nivalis S. fragilis Larix ? Viola epipsila Larix Larix Kleb. . Kleb. Kleb. Kaneko Miyabe

tsugae Tsuga Kleb. Kaneko et Zenk. Linfors Kleb.

Continued sp. Dietel Hiratsuka Dietel f. sp.

larici-epitea typica typica euonymi-incanae

M. larici-opaca f.sp. f. sp. f.sp. Schneid. Kleb. et Hiratsuka Table 2.2. Hiratuska et Matsumoto) Melampsora M. dimorphospora M. epiphylla (= M. epiteaM. epitea M. euonymi-caprearum M. galanthi-fragilis M. humilis M. kamikotica M. kiusiana M. kupreviczii S. futura M. lapponum M. larici-pentandrae M. larici-epitea Melampsora Rusts on Salix 23 . .

et al et al

continued ,Pei ,Pei Gäumann (1959) (1996) (1996) Gäumann (1959) Ziller (1974) Gäumann (1959) and Kaneko (1982) Sydow and Sydow (1915), Gäumann (1959) and Kaneko (1982) Sydow and Sydow (1915), Gäumann (1959) and Kaneko (1982) Arctic, subarctic or alpine regins of the northern hemisphere Europe Gäumann (1959) North and South America Sydow and Sydow (1915), Japan Hiratsuka (1932), Hiratsuka Europe Sydow and Sydow (1915), Europe Japan Hiratsuka and Kaneko (1982) Europe Sydow and Sydow (1915), Europe Gäumann (1959) Japan Hiratsuka (1932), Hiratsuka Europe Japan and Kurile Islands Azbukina (1974), Hiratsuka

lasiandra (C., Retusae) , hastata (V., Vimen), glaucops, aurita

reticulata (V., Helix) (V., Sitchensis) (V., Daphnella) foetida, (V., Villosae),

daphnoides (V., Nigricantes), (V., Glaucae), mackenzieana dasyclados

purpurea (V., Arbuscella) helvetica sitchensis (V., Helix) (V., Arbuscella), (V., Lanatae), (S., Amygdalinae) (S., Urbanianae) (V., Vimen) (V., Vetrix), (V., Chamaetia) (V., Cordatae), (V., Incubaceae), (V., Vetris), (V., Nigricantes), S. acutifolia, daphnoides S. glabra, nigricans hegetschweileri S. aurita (V., Daphnella), S. alpicola, hibernica, nigricans S. disperma waldsteiniana (V., Cordatae), herbacea, retusa, serphyllifolia (V., Chamaetia) S. schwerinii S. miyabeana S. subfragilis S. barrattiana petrophila, vestita (S., Pentandrae), (V., Cordatae), (V., Vetrix) S. reticulata spp. spp.

Larix kaempferi S.Larix urbaniana OrchidaceaeSaxifraga S. repens Kleb.) Fischer Thüm.) Blytt Dietel Diet. & Holw. Plowr.

larici-daphnoides larici-nigricantis larici-purpureae larici-retusae larici-reticulata S. hastata

M. bigelowii M. orchidi-repentis f. sp. f. sp. ‘Japanese race 2’ f. sp. ‘Japanese race 1’ f. sp. f. sp. Kleb. Schnei. Schneid. Matsumoto (= M. larici-urbaniana M. microsora M. paradoxa M. repentis (= M. reticulatae 24 Ming Hao Pei , Tai (1979) , Tai (1979), , Wilson and . (1995)

et al Pei References Gäumann (1959) Gäumann (1959) Ziller (1974) Henderson (1966) Eurasia Gäumann (1959) North-west China Tai (1979) Distribution JapanBritish Isles Hiratsuka and Kaneko (1982) Taiwan, Japan Hiratsuka and Kaneko (1982) Eurasia and North America Gäumann (1959) Japan Hiratsuka and Kaneko (1982) Europe Gäumann (1959) South-west China Tai (1979) Europe

pierotii (V., Vetrix)

arbuscula

subfragilis (V., Helix) (V., Scleophylla) (S., Wilsonianae) (S., Glandulosae), (V., Helix) (V., Vimen) (V., Vetrix), (S., Salix)

S. miyabeana

Telial host (subgenus, section) S. aurita (V., Arbuscella) S. purpurea S. viminalis S. alba S. cavaleriei S. cupulariris S. warburgii (S., Subalbaceae), (S., Amygdalinae) S. viminalis ? spp. spp. spp. spp. Aecial host ? – ? Ribes Ribes alpinaRibes S.Ribes appendiculata, aurita Allium ? Corydalis ambigua S. yessoensis (S., Subalbaceae) S. miyabeana . Kleb.) Kleb. Kleb. * Tai Sawada Wang Kleb. Kleb. Kleb. Miyabe et

Continued sp.

ribesii-auritae ribesii-grandifoliae

M. allii-salicis-albae f. sp. Schneid. f. sp. Matsumoto ‘Stem-infecting form’ Table 2.2. Kaneko et Hiratsuka *Tentative name Melampsora M. ribesii-epitea M. ribesii-purpureae M. ribesii-viminalis M. salicis-albae (= M. salicis-cavaleriei M. salicis-cupularis M. salicis-warburgii M. yezoensis Caeoma salicis-miyabeana Melampsora Rusts on Salix 25

Rusts on shrub and dwarf willow 1959). However, Schneider’s inoculations produced only a trace infection (a single Of all the Melampsora species on willow, uredinium) on S. cinerea and, therefore, M. larici-epitea is the most widespread and cannot be regarded as sufficient evidence most complex in its host range. Much of the to prove that S. cinerea is a natural host of knowledge on the pathogenic specialization M. caprearum. There have been no records in this rust came from inoculation experi- of M. caprearum on S. cinerea in Scandina- ments carried out in continental Europe via, where both M. caprearum and S. cinerea (reviewed by Gäumann, 1959), Japan commonly occur (Hylander et al., 1953). (Hiratsuka, 1932) and the UK (Pei et al., Also, in the UK, M. caprearum has not been 1996). Six formae speciales within M. observed on any S. cinerea clones in the larici-epitea were recognized in Europe past 12 years of observation (M.H. Pei, (Sydow and Sydow, 1915; Gäumann, 1959). unpublished observations). Therefore, it While there is no clear morphological seems unlikely that S. cinerea serves as telial distinction between the formae speciales, host of M. caprearum in nature. In Europe, the host range of a forma specialis appears two formae speciales have been recognized largely confined to certain sections of wil- in M. caprearum; f. sp. typica, infecting only low. For example, f. sp. larici-epitea typica S. caprea, and f. sp. grandifoliae, infecting has been recorded on willows belonging to both S. aurita and S. caprea. In Japan, Secs Vimen, Vetrix and Incubaceae, while f. M. caprearum occurs on two Japanese sp. larici-daphnoides occurs on Sec. Daph- willow species of section Vetrix, S. bakko nella. M. larici-epitea f. sp. larici-retusae and S. leucopithecia. M. caprearum has also has been recorded on about a dozen willow been recorded on S. caprea var. sinica Hao species belonging to several sections of and S. spathulifolia Seem. (section Psilo- the subgenus Vetrix. In Japan, two ‘races’ stigmatae of subgenus Vetrix) in central have been recognized, out of four reported China (Tai, 1979). originally (Hiratsuka, 1932; Hiratsuka and M. abieti-caprearum, occurring in Kaneko, 1982). From their host range, these Europe and in North and South America, two races appear to be equivalent to and the native American species M. different formae speciales, as treated by paradoxa have also been recorded on a European mycologists. In the UK, three wide range of Salix species. M. paradoxa, formae speciales – larici-epitea typica, the American counterpart of M. larici-epitea, larici-retusae and larici-daphnoides – were can be distinguished from other willow rusts identified within M. larici-epitea (Pei et al., in the Americas by having relatively large 1996). In New Zealand, a new rust morpho- aeciospores. The early account of the host logically similar to M. epitea caused severe range of M. paradoxa included some 37 infections on S. viminalis and its hybrids in willow species belonging to various sections 1985 (Spiers and Hathaway, 1987; Spiers of the subgenus Vetrix or subgenus Salix and Hopcroft, 1996). From the host lists (Sydow and Sydow, 1915). Later, Ziller given by these authors, the M. epitea on (1974) stated that it was futile to try to iden- S. viminalis in New Zealand corresponds tify a North American willow rust without with the host range of f. sp. larici-epitea knowing its aecial state, and listed seven typica (Pei et al., 1996). willow species belonging to subgenus Vetrix M. caprearum is also one of the most or Salix as the telial hosts of M. paradoxa. widespread species in Eurasia. , Several species, such as M. arctica, M. S. caprea and S. cinerea (section Vetrix) lapponum and M. reticulatae mainly infect have been recorded as the telial hosts of M. small shrub or dwarf willows occurring in caprearum (Wilson and Henderson, 1966). arctic, subarctic or alpine regions in the S. cinerea was included in the host range northern hemisphere. Two morphologically of M. caprearum based on the inoculation distinct species, M. choseniae and M. experiment conducted by Schneider at the kamikotica, occur on Chosenia arbutifolia, turn of the 20th century (see Gäumann, the unique species in Salicaceae by having 26 Ming Hao Pei

characteristics resembling both Salix and infected by several isolates of M. larici- Populus. epitea in laboratory inoculation experi- ments. However, no rust was found on this species in the field (national willow collection, south-west England) where Historical Records and Host Range all the pathotypes occurred. In leaf disc inoculation experiments, young leaves of Host records of willow rusts in the litera- S. daphnoides also supported weak ture are based largely on the morphology at pathogen development after inoculations uredinial and telial stages. For many willow with f. sp. larici-epitea typica and f. sp. Melampsora species (sensu stricto) which larici-retusae. However, over a period of are morphologically indistinguishable, host 12 years, S. daphnoides has never been records without the backing of inoculation infected by the two formae speciales in the experiments are often doubtful. Misidentifi- field (M.H. Pei, unpublished observations). cation of the host species may add further confusion. Therefore, for many willow rusts, historical records on the host range Concluding Remarks need to be treated with caution. It is not dif- ficult to understand that such complexities In the past decade, the application of led to the wide acceptance of M. epitea as molecular tools in mycology has revolution- complex species to encompass immensely ized our understanding of the phylogeny diverse groups of willow rusts. and population biology of the fungi. It needs to be mentioned that whether Recently, several species of Melampsora on M. epitea is the legitimate name is question- willows collected from central Europe able. M. epitea was originally described by and the British Isles were examined for Thümen (1879) based on the specimen on S. variation in rDNA sequences (Chapter 1). It alba. However, to date, there have been no appears that M. larici-epitea, M. caprearum, credible records of M. epitea, which has SIF and M. epiphylla share a common evenly echinulate urediniospores, occurring ancestral lineage. In contrast, M. larici- on S. alba. It is likely that either the host spe- epitea and M. ribesii-purpureae, both of cies was misidentified in the type specimen which can be grouped under the M. epitea or the rust described was not the M. epitea complex, are phylogenetically distinct. that we recognize today, probably because Urgent revision of the taxonomy of Mel- microscopes available at the time may have ampsora on Salix has long been called for been too primitive to reveal morphological (Ziller, 1974; Helfer, 1992; Spiers and Hop- details. In fact, Thümen (1879) proposed the croft, 1996). The purpose of this chapter was name M. vitellinae Thüm. to include the to give a brief review of some of the existing rusts on S. alba var. vitellina, S. fragilis and information on the Melampsora species on S. pentandra. All the rust species on these Salix. Because of the extent of complexity willows are known to produce uredinio- and the lack of sufficient understanding of spores having a smooth surface at the apex. many aspects, appropriate revision of the However, this feature was not included in taxonomy and nomenclature of willow rusts Thümen’s description of M. vitellinae. cannot be achieved until further knowledge Occasionally, results obtained under of the genetic and phylogenetic relation- experimental conditions do not reflect the ships among the rusts is available. situation in the field because, in a protected environment, the pathogenicity of a given rust isolate is likely to be wider than in its natural habitat (Samborski et al., 1958; Acknowledgements Anikster, 1984; Helfer, 1989). For example, Pei et al. (1996) reported that S. discolor, This study was funded by the Department a North American willow species, became for Environment, Food and Rural Affairs Melampsora Rusts on Salix 27

(DEFRA), UK, and the European Union. Hiratsuka, N. and Kaneko, S. (1982) A taxonomic revi- Rothamsted Research receives grant-aided sion of Melampsora on willows in Japan. Reports support from the Biotechnology and Biolog- of the Tottori Mycological Institute 20, 1–32. ical Sciences Research Council of the UK. Hiratsuka, Y. and Sato, S. (1982) Morphology and taxonomy of rust. In: Scott, K. J. and Charkravorty A. K. (eds) The Rust Fungi. Academic Press, London, pp. 1–36. References Hiratsuka, N., Sato, T., Katsuya, K., Kakishima, M., Hiratsuka, Y., Kaneko, S., Ono, Y., Sato, S., Anikster, Y. (1984) The formae speciales. In: Bushnell, Harada, Y., Hiratsuka, T. and Nakayama, K. W.R. and Roelfs, A.P. (eds) The Cereal Rusts, (1992) The Rust Flora of Japan. Tsukuba Vol. I. Origins, Specificity, Structure and Shuppankai, Ibaraki, Japan, pp. 271–300. Physiology. Academic Press, Orlando, Florida, Hylander, N., Jøstad, I. and Nanfeldt, J.A. (1953) pp. 115–130. Enumeratio Uredinearum Scandinavicarum. Argus, G.W. (1986) The genus Salix (Salicaceae) in the Opera Botanica 1, 1–102. southeastern United States. Systematic Botany Klebahn, H. (1902) Kulturversuche mit Rostpilzen. X. Monographs, Vol. 9. The American Society of Zeitschrift für Pflanzenkrankhreiten 12, 17–44. Plant Taxonomists, Michigan. Klebahn, H. (1905) Kulturversuche mit Rostpilzen. Argus, G.W. (1997) Infrageneric Classification of XII. Zeitschrift für Pflanzenkrankhreiten 15, Salix (Salicaceae) in the New World. Systematic 65–108. Botany Monographs, Vol. 52. The American Latch, B.J. (1980) Weeping willow rust in New Society of Plant Taxonomists, Michigan. Zealand. New Zealand Journal of Agricultural Arthur, J.C. (1934) Manual of the Rusts in the Research 23, 535–538. United States and Canada. Purdue Research Matsumoto, T. (1915) Impfversuche mit Melampsora Foundation, Lafayette, Indiana. auf Japanischen Weiden. Transactions of the Azbukina, Z.M. (1974) Rust Fungi of the Soviet Far Sapporo Natural History Society 6, 22–37. East [in Russian]. Nauka Publishers, Moscow, Matsumoto, T. (1919) Some experiments with pp. 120–140. Melampsora in Japan. Annals of Missouri Bean, W.J. (1980) Trees and Shrubs Hardy in the Botanical Garden 6, 309–316. British Isles, Vol. IV, 8th edn. John Murray, Matsumoto, T. (1926) On the relationship between London, UK, pp. 246–312. Melampsora on Salix pierotii Miq. and Caeoma Dorn, R.D. (1976) A synopsis of American Salix. on Chelidonium majus L. and Corydalis incissa Canadian Journal of Botany 54, 2769–2789. Pers. Botanical Magazine, Tokyo 40, 43–47. Gäumann, E. (1959) Die Rostpilze Mitteleuropas. Mayor, E. (1933) Notes mycologiques VIII. Bulletin de Beiträge zur Kryptogamenflora Schweiz 12. la Societe neuchâteloise des Sciences naturelles Buchdruckerei Büchler and Co., Bern, 58, 7–31. Germany. Mayor, E. (1936) Notes mycologiques IX. Bulletin de Hawksworth, D., Kirk, P., Sutton, B. and Pegler, D. la Societe neuchâteloise des Sciences naturelles (1995) Ainsworth’s and Bisby’s Dictionary of the 61, 105–123. Fungi, 8th ed. CAB International, Wallingford, Ogilvie, L (1932) Notes on the rusts of basket willows UK. and their control. Annual Report of the Agri- Helfer, S. (1989) The culture of cereal leaf rusts for cultural and Horticultural Research Station, Long physiological and taxonomical studies. Notes Ashton, Bristol, for 1931, pp. 133–138. from the Royal Botanic Garden, Edinburgh 46, Parmelee, J.A. (1989) The rust (Uredinales) of Arctic 131–139. Canada. Canadian Journal of Botany 67, Helfer, S. (1992) The rust diseases of willows in 3315–3365. Britain. Proceedings of the Royal Society of Pei, M.H. and Ruiz, C. (2000) AFLP evidence of Edinburgh 98B, 119–134. distinct patterns of life-cycle in two forms of Hiratsuka, N. (1927) Japanese species of Melampsora rust on Salix viminalis. Mycological Melampsora (Studies on Melampsoraceae Research 104, 937–942. of Japan IV). Journal of the Society of Agri- Pei, M.H., Royle, D.J. and Hunter, T. (1993) Identity culture and Forestry, Sapporo 19, 180–195 and host alternation of some willow rusts [in Japanese]. (Melampsora spp.) in England. Mycological Hiratsuka, N. (1932) Inoculation experiments Research 97, 845–851. with some heteroecious species of the Pei, M.H., Royle, D.J. and Hunter, T. (1995) A Melampsoraceae in Japan. Japanese Journal of comparative study of stem-infecting and leaf- Botany 6, 1–33. infecting forms of Melampsora rust on Salix 28 Ming Hao Pei

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Gaddam Bagyanarayana Department of Botany, Osmania University, Hyderabad 500 007 (A.P), India

Introduction salicis-albae, M. salicis-cavaleriei, M. salicis-cupularis, M. salicis-warburgii and This chapter describes a morphotoxonomic M. yezoensis are recognized as valid. M. study of the species of Melampsora infect- allii-fragilis f. sp. galanthi-fragilis, M. epitea ing the host genera Salix and Chosenia f. sp. abieti-capraearum, M. epitea f. sp. (Salicaceae). A total of 19 species and ten f. arctica, M. epitea f. sp. lapponum, M. epitea spp. are recognized and described. Out of f. sp. laricis-epitea and M. epitea f. sp. the ten f. spp., six are proposed as new com- ribesii-purpureae are treated as new combi- binations. M. allii-fragilis, M. amygdalinae, nations. A key to the described species, M. capraearum, M. chelidonii-pierotii, detailed morphological characters of the M. choseniae, M. dimorphospora, spore forms, host range and geographical M. epiphylla, M. epitea, M. epitea f. sp. distribution are provided. This study is euonymi, M. epitea f. sp. repentis, M. based on examination of hundreds of epitea f. sp. reticulatae, M. epitea f. sp. Melampsora-infected specimens including tsugae, M. kamikotica, M. kupreviczii, types, original collections and other auth- M. larici-pentandrae, M. larici-urbaniana, entic specimens obtained from several M. paradoxa, M. ribesii-viminalis, M. International Herbaria (Table 3.1).

Telia on Chosenia

Urediniospores evenly thickened M. choseniae Azb. et Kov. Urediniospores: 14–25 × 14–18 mm Teliospores: 28–64 × 10–14 mm Urediniospores apically thickened M. kamikotica Kaneko et Hirats. Urediniospores: 20–33 × 15–18 mm (apically 5–6.5 mm) Teliospores: 25–53 × 64–10 mm

Telia on Salix

Autoecious macrocyclic M. amygdalinae Kleb. Urediniospores: 20–40 × 11–18 mm Teliospores: 18–50 × 7–14 mm ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 29 30 G. Bagyanarayana

Table 3.1. Abbreviations, names and addresses of herbaria.

Abbreviation Name and address of the herbarium

B Botanischer Garten and Museum, Berlin-Dahlem, 1000 Berlin 33, Konigin Luise Strasse 6-8, Berlin C The Botanical Museum and Herbarium, University of Copenhagen, 130 Gothersgade, DK-1123, Copenhagen K, Denmark DAOM National Mycological Herbarium, Research Branch, Biosystematics Research Institute, Wm. Saunders Building, C. E. F., Ottawa, Canada K1A OC6 DAVFP Canadian Forestry Service, Forest Pathology, Forest Research Laboratory, Victoria, BC, Canada FH The Forlow Herbarium of Harvard University, 20 Divinity Avenue, Cambridge, Massachusetts, USA G Conservatoire et Jardin Botanique, Institut de Botanique Systematique de I’Universite, Case Postale 60, CH-1292 Chambésy Suisse, Genève, Switzerland HCIO Herbarium Cryptogamiae Indiae Orientalis, Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, New Delhi-12, India K Royal Botanic Gardens, Kew, Richmond, Surrey, England LEV New Zealand Department of Agriculture Horticultural Research Centre, Levin Mycological Herbarium, New Zealand PAV Instituto Di Botanica Orto Botanico, Laboratoria Cryttogamico, via. S. Epifanio, Case Postale 99, 27100-Pavia, Italy S Swedish Museum of Natural History, Section for Botany, 104 05 Stockholm 50, Sweden WRSL Instytut Botaniczy, Universytetu Wroclawskiego, ul Kanonia 6/8, Wroclaw, Poland IMI IMI Herbarium CABI Bioscience UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY, UK

Heteroecious or the aecial stages not known Telia sub-cuticular Telia sub-epidermal

Telia sub-cuticular

1. Teliospores apically thickened 2 1. Teliospores apically not thickened 3 2. Apical thickening 3–10 mm M. capraearum Thum. Urediniospores: 18–22 × 13–16 mm Teliospores: 30–45 × 7–18 mm 2. Apical thickening 2.5–4.8 mm M. epiphylla Diet. Urediniospores: 12.8–16 × 11.2–15 mm Teliospores: 24–45 × 8–16 mm 3. Urediniospore wall not thickened 4 3. Urediniospore wall thickened M. yezoensis Miyabe et Mats. Urediniospores: 16–28 × 12.5–20 mm Teliospores: 16–33.6 × 6.4–13 mm 4. Teliospores short 5 4. Teliospores long, 64 mm in length M. chelidonii-pierotii Mats. Urediniospores: 16–24 × 13–16 mm Teliospores: 20–64 × 6–8 mm Species of Melampsora on Salix 31

5. Urediniospores small, up to 22 mm M. ribesii-viminalis Kleb. Urediniospores: 15–22 × 14–18 mm Teliospores: 25–40 × 7–15 mm 5. Urediniospores lengthy, more than 32 mm Aecia on Allium M. allii-fragilis Kleb. Urediniospores: 22–33 × 12–15 mm Teliospores: 30–48 × 7–14 mm Aecia on Galanthus M. allii-fragilis f. sp. galanthi-fragilis (Kleb.) comb. nov. Urediniospores: 25–38 × 12–16 mm Teliospores: 25–46 × 8–16 mm

Telia sub-epidermal

1. Urediniospores one type 2 1. Urediniospores two types M. dimorphospora Kaneko & Hirat. 2. Urediniospores apically smooth 3 2. Urediniospores evenly echinulate 4 3. Urediniospores apically thick M. larici-pentandrae Kleb. Urediniospores: 26–44 × 12–16 mm Teliospores: 19–30 × 9–12 mm 3. Urediniospores apically not thickened M. salicis-albae Kleb. Urediniospores: 20–36 × 11–17 mm Teliospores: 25–45 × 7–10 mm 4. Urediniospores apically thickened M. larici-urbaniana Mats. Urediniospores: 15–27 × 13–17 mm Teliospores: 35–70 × 8–16 mm 4. Urediniospores apically not thickened 5 5. Urediniospores more than 15 mm6 5. Urediniospores up to 15 mm M. kupreviczii Zenk. Urediniospores: 10–15 × 10–12 mm Teliospores: 30–37 × 10–15 mm 6. Urediniospores length up to 37 mm M. salicis-cavaleriei Tai. Urediniospores: 18–37 × 11–18 mm Teliospores: 36–69 × 7–11 mm 6. Urediniospores less than 30 mm7 7. Urediniospore wall thickness up to 3 mm8 7. Urediniospore wall thickness less than 2.5 mm M. salicis-warburgii Sawada. Urediniospores: 13–28 × 15–18 mm Teliospores: 34–42 × 8–15 mm 8. Teliospores length up to 40 mm M. paradoxa Diet & Holw. 8A Urediniospores: 15–26 × 15–21 mm M. paradoxa Diet & Holw. 8A Teliospores: 29–43 × 11–14 mm 8B Urediniospores: 21–23 × 17–18 mm[wall 1.5–2 mm] M. salicis-cupularis 8B Teliospores: 28–39 × 11–14 mm 8. Teliospores length up to 50 mm M. epitea (Kunze & Schm.) Thum. 32 G. Bagyanarayana

A collective species having nine formae speciales on different aecial hosts

Urediniospores: 2–25 × 10–19 mm Teliospores: 20–50 × 7–15 mm Aecia on Saxifraga Urediniospore wall 2 mm thick M. epi. f. sp. arctica (Rostr.) comb. nov Urediniospore wall more than 2 mm thick (2.5–3.5) M. epi. f. sp. reticulatae (A. Blytt.) Jorst. Aecia on Abies M. epi. f. sp. abieti-capraearum (Tub.) comb. nov. Aecia on Euonymus M. epi. f. sp. euonymi (Kleb.) Boerema & Verhoev. Aecia on Larix M. epi. f. sp. larici-epitea (Kleb.) comb. nov. Aecia on Orchidaceae M. epi. f. sp. repentis (Plowr.) Boerema & Verhoeven Aecia on Ribes M. epi. f. sp. ribesii-purpureae (Kleb.) comb. nov. Aecia on Tsuga M. epi. f. sp. tsugae Ziller Aecia on Viola M. epi. f. sp. lapponum (Lindf.) comb. nov.

Melampsora allii-fragilis Kleb., Pringsh. carinatum L., A. cepa L., A. fistulosum L., Jahrab. Wiss. Bot. 35, 674; 1901 A. moly L., A. neapolitanum Cyr., A. obligum L., A. ochroleucum W. et K., A. = Uredo allii-fragilis Arth., Result, Scient. odorum Ten., A. oleraceum L., A. pedemon- Congr. Intern. Bot. Vienne p. 338; 1905, tanum Willd., A. porrum L., A. pulchellum 1906. Don., A. sativum L., A. schoenoprasum L., Spermogonia foliicolous, lenticular, A. scenescens L., A. sphaerocephalum L., sub-epidermal, 200 mm wide. Aecia A. ursinum L., A. victorialis L., A. vineale caeomoid, amphigenous and caulicolous, L. Uredinia and telia on Salix fragilis L., aggregated, 0.5–1.0 mm wide, sub- S. pentandra. epidermal, erumpent, pulverulent, orange; Distribution: Europe. aeciospores 18–26 × 12–20 mm, irregularly Specimens examined: FH & S. angular or ellipsoid, rarely globoid, wall Morphologically, Melampsora allii- 1–2 mm thick, verrucose. Uredinia minute, fragilis Kleb. is strikingly similar to M. amphigenous, mainly hypophyllous, salicis-albae Kleb. and both have Allium as 0.5 mm, sub-epidermal, erumpent, pul- their aecial host. The only distinguishing verulent, orange; urediniospores 22–33 × feature is that the telia are sub-cuticular 12–15 mm, ellipsoid, ovate or oblong, wall in the former species whereas they are 3 mm thick, echinulate; paraphyses capitate sub-epidermal in the latter species. Host- or clavate, up to 70 mm long, 15–20 mm alternation and host selection of M. allii- wide, wall 2–5 mm thick. Telia minute, fragilis was studied by Klebahn (1902, 1903) amphigenous, mostly epiphyllous, scattered and Mayor (1934, 1936, 1958). or aggregated, 0.2–0.5 mm wide, sub- cuticular, not erumpent, dark brown; telio- spores 30–48 × 7–14 mm, rounded at both Melampsora allii-fragilis Kleb., f. sp. the ends, wall 1 mm thick, pale brown. galanthi-fragilis (Kleb.) Bagyanarayana Type: On Salix fragilis L., Triglitz in der comb. nov. Prignitz, Brandenburg, Germany. Hosts: Spermogonia and aecia on Allium = Uredo galanthi Kirchner, Lotos 6, 179; ampeloprasum L., A. ascalonicum L., A. 1856. Species of Melampsora on Salix 33

= Caeoma galanthi Schroet., Abhandl. 1 mm, but when in groups up to 3–10 mm, Schles. Ges. Vaterl. Kult. 1869/72. 72; fusing, sub-epidermal, erumpent; aecio- 1869. = Melampsora galanthi-fragilis Kleb., spores 16–24 × 14–20 mm, globose, angulato- Zeitschr. F. Pl. Krankh. 12, 27; 1902. globose or ovoid, wall 2 mm thick, minutely Spermogonia flat, slightly elevated, lens verrucose. Uredinia minute, hypophyllous, shaped, sub-epidermal, 80–100 mm high, rarely epiphyllous and culmicolous, 120–155 mm wide. Aecia caeomoid, scattered, 0.5 mm, orange, sub-epidermal, amphigenous, often hypophyllous, in small erumpent, pulverulent; urediniospores circular groups, often fusing, 1–2 mm long, 20–40 × 12–20 mm, ovate to oblong, wall sub-epidermal, erumpent, pulverulent, 1–5 mm thick, echinulate; paraphyses orange; aeciospores 16–25 × 12–20 mm, capitate, up to 80 mm in length, 10–15 mm globose, sub-globose or angular, wall 1–2 mm in width, wall 2–5 mm thick. Telia minute, thick, verrucose. Uredinia mostly hypo- 0.3–0.5 mm, usually hypophyllous, rarely phyllous, rarely epiphyllous, scattered epiphyllous, aggregated in small groups, or aggregated, 0.5–1 mm in diameter, sub- sub-epidermal, non-erumpent, in the begin- epidermal, erumpent, pulverulent, orange; ning reddish-brown, later blackish-brown; urediniospores 25–38 × 12–16 mm, ovoid, teliospores 18–40 × 7–14 mm, cylindrical, ellipsoid, sometimes pyriform, wall 3 mm often irregular, round at both ends, wall thick, echinulate; paraphyses mostly 1 mm thick, yellowish-brown. capitate, up to 70 mm long, 16–24 mm Type: On Salix amygdalina L., Elsfleth in wide, wall 2–5 mm thick. Telia mostly epi- Oldenburg, Germany. phyllous, rarely hypophyllous, scattered Hosts: On Salix amygdalina L., S. laevigata or aggregated, 0.25–1 mm in diameter, Bebb., S. lucida, S. triandra L., S. pentandra sub-cuticular, not erumpent, dark brown; L., S. pentamira. teliospores 24–46 × 8–16 mm, more or less Distribution: Austria, Canada, Europe and round at both the ends, wall 1.5 mm thick, UK. pale brown. Specimens examined: FH, G, S. Hosts: Spermogonia and aecia on Galanthus This is the only autoecious macrocyclic nivalis L., G. plicatus M.B. Uredinia and species of the genus Melampsora on Salix. telia on Salix fragilis L., S. pentandra L. Morphologically M. amygdalinae is very Distribution: Europe. close to M. larici-pentandrae Kleb., but the Specimens examined: S. urediniospores are not apically thickened as Melamspora allii-fragilis f. sp. galanthi- in the case of M. larici-pentandrae. Raabe fragilis Bagyanarayana very much resembles (1939) found that the mycelium can over- M. allii-fragilis Kleb., except it differs in winter in the sprouts and buds of Salix having Galanthus as its aecial host. Indeed, triandra and can produce urediniospores some of the earlier workers have treated this in the following spring without requiring a as a synonym of M. allii-fragilis. Schroeter fresh infection. (1893) noticed the host alternation of M. allii-fragilis f. sp. galanthi-fragilis, which was later proven by Klebahn (1902, 1903). Melampsora capraearum (D.C.) Thüm., Mith. Forstl. Versuch SW, Oestree 2, 34, 36; 1879 Melampsora amygdalinae Kleb., Pringsh. Jahrb. Wiss. Bot. 34, 352; 1900 = Uredo farinosa var. Uredo salicis-capreae Pers., Syn. Meth. Fung. p. 217; 1801. Spermogonia amphigenous, lenticular, = U. capraearum D.C., Lam & D.C. Syn. Pl. slightly elevated, orange, 100 mm wide, Gall p. 48; 1806. 50 mm high. Aecia caeomoid, on young = Sclerotium salicinum Pers., Moug & leaves, usually hypophyllous, rarely epi- Nestl. Strip Crypt. Veg. Rehn. 386; 1810. phyllous, sometimes on branches up to Pers. Ex DC Fl. Fr. 5, 114; 1815. 34 G. Bagyanarayana

= Lecythea salicinia Lev., Ann. Sc. Nat. Specimens examined: G, S. p. 375; 1847. Tul. Ann Science Natt. VII, According to Klebahn (1914) M. 6–7; 1854. capraearum is one of the most commonly = Melampsora salicinia (D.C.) Lev., Ann. seen rusts of central Europe. This rust is eas- Sci. Nat. Bot. III 8, 375; 1847. ily distinguishable from other melampsoras = Caeoma laricis Hartg, Wichtige Krankh. d. on Salix by having teliospores with heavy Waldaume p. 93; 1874. apical thickening and a conspicuous germ = M. farinosa (Pers.) Schroet., Cohn. Krypt. pore. On the basis of experiments carried out Fl. Schles 1, 360; 1887. by Jacky (1899), Klebahn (1897, 1899, 1900, = M. larici-caprearum Kleb., Frostl. Naturw. 1905, 1908), Mayor (1918) and O. Schneider Zeitschr. 6, 469–470; 1897. Zeistchr. f. Pfl. (1906), Gäumann recognized two formae Krankh 7, 326; 1897. speciales. (i) f. sp. typica Kleb.; and (ii) f. sp. = M. capraearum (D.C.) Thüm f. sp. typica grandifoliae O. Schneid. But this treatment Klebh. does not look justified as both the f. spp. = Uredo larici-caprearum Arth., Result have the same aecial hosts, the only differ- Scient. Congr. Intern. Bot. Vienne p. 305; ence being in the infection types produced 1906. on the telial hosts. Gäumann (1959) himself Spermogonia amphigenous, scattered further commented that ‘on the other hand or in small groups, sub-cuticular, up to the results of Schneider (1905) allow one to 80 mm wide, 45 mm high. Aecia caeomoid, presume the existence of small local races’. minute, 0.25–1 mm, scattered or aggregated, pale orange, sub-epidermal, erumpent, pulverulent; aeciospores 12–25 × 12–17 mm, globose, angulato-globose or ellipsoid, wall Melampsora chelidonii-pierotii Mats., 2 mm thick, finely verrucose. Uredinia hypo- Bot. Mag. Tokyo 40, 46; 1926 phyllous, scattered or aggregated, 1–2 mm diameter. Up to 5 mm on juvenile leaves, Spermogonia epiphyllous, deposited in orange; urediniospores 14–24 × 12–16 mm, circular groups. Aecia caeomoid, ovoid or ellipsoid, wall 2–2.5 mm thick, hypophyllous, occasionally caulicolous, echinulate; paraphyses numerous, capitate, scattered or aggregated 1–2 mm wide, often up to 80 mm in length, 19–25 mm in width, fusing, erumpent, pulverulent, orange-red, wall 5–6 mm thick. Telia epiphyllous, dense, aeciospores 13–20 × 12–15 mm, globoid or aggregated, often fusing, sub-cuticular, non- ovoid, wall 2 mm thick, verrucose. Uredinia erumpent, 0.5–3 mm, dark orange-brown to hypophyllous, scattered, erumpent, pul- dark reddish-brown or blackish-brown; verulent, orange–yellow; urediniospores teliospores (21) 25–45 × 8–16 mm, wall 16–24 × 13–16 mm, ovoid or ellipsoid, wall 1–2 mm, thick at sides and apically up to 2–3 mm, thick, echinulate; paraphyses 10 mm thick, dark cinnamon brown. capitate, up to 65 mm long, 17–22 mm Type: On L., Germany, wide, wall thickened apically up to 12 mm. Bavaria, De Jhumen, March 1896. Telia amphigenous, mostly epiphyllous, Hosts: Spermogonia and aecia on Larix scattered, sub-cuticular, non-erumpent, decidua Mill., L. leptolepis Murr., L. reddish-brown; teliospores 20–64 × 6–8 mm, occidentalis Nutt., L. sibirica Led., Pseudo- cylindrical or wedge-shaped, wall 1 mm larix kaempferi (Fort.) Gond. Uredinia and thick. telia on Salix aurita L., S. bakka Kimura, Type: On Salix pierotii Miq., Morioka, S. caprea L., S. cinerea L., S. daphnoides Japan. Vill, S. dasyclados Wimm., S. elegans Hosts: Spermogonia and aecia on Wall., S. grandifolia Ser., S. hastate L., Chelidonium majus L., Corydalis incisa S. imprimis, S. phlomoidis Bieb., S. phylici- Pers. Uredinia and telia on Salix pierotii folia, S. rosmarinifolia L., S. smithiana Miq. (S. pseudo-korensis Koidz.) and S. Willd., S. tetrasperma Roxb. chaenomeloides Kimura (S. glandulosa Distribution: Eurasia. Seem.). Species of Melampsora on Salix 35

Distribution: Japan and maritime provinces (2) catenulate in short chains, finely and of Russia. densely verrucose, subglobose or broadly Matsumoto (1926) discovered the rela- ellipsoid, 20–24 × 15–22 mm, wall 2–2.5 mm tionship between the Melampsora on Salix thick, colourless, germ pores scattered; pierotii, Chelidonium majus and Corydalis paraphyses numerous, intermixed, capitate incissa. To this rust he gave the name to clavate, 35–103 × 22–30 mm, wall 3–4 mm M. chelidonii-pierotii. The other Salix thick, slightly thickened at apex. Telia rust having sub-cuticular telia, with which amphigenous, subepidermal, usually sur- M. chelidonii-pierotii is comparable, is M. rounding uredinia, rounded, 0.2–0.5 mm ribesii-viminalis Kleb. However, morpho- in diameter, reddish brown in dry state; logically M. chelidonii-pierotii has lengthy teliospores adhering laterally, prismatic in teliospores; in addition it differs in its aecial surface view of telium, oblong and rounded host range. at both ends in side view, rarely acute above, arranged in a single layer, one- celled, occasionally two-celled with a Melampsora choseniae Azb. et Koval, horizontal or oblique septum, 20–50 × m m Novit. Syst. Pl. Vasc. 247–248; 1967 7.5–13.5 m, wall c. 1.5 m thick, yellowish-brown, not thickened at apex. Spermogonia and aecia not found. Uredinia Type: On Salix koriyanagi Kimura, Pref. hypophyllous, 0.25–0.8 mm in diameter, Aichi, Anjyo-cho, 5 Dec. 1938, K. round or oval; urediniospores round to Kuwazuka, HH. 77875. oval, 14–25 × 14–17.8 mm, wall densely Hosts: Salix koriyanagi Kimura. verrucose, 1.5–2 mm thick, hyaline; Distribution: Japan. paraphyses capitate rarely clavate, M. dimorphospora is a unique species 11.6–17.6 mm wide, wall 2–2.5 mm thick. which produces two kinds of spores, e.g. Telia epiphyllous, subepidermal, blackish caeomid uredinia producing catenulate, orange-brown, 0.1–0.5 mm; teliospores verrucose uredio spores and uredinoid uredinia producing catenulate uredinio- prismatic, 28.5–63.9 × 10–14 mm, apically not thickened, wall 1.5–2.5 mm, thick. spores. No other species of Melampsora is Type: On Chosenia arbutifolia (Pall.) A. known to produce this kind of dimorphic Skv., 3–09–1955, coll. By E.Z. Koval, urediniospore. 15–08–1964, Leg M. Azbukina, Inst. Bot. Acad. Sci. URSS (Leningrad) Conservatur. Hosts: Chosenia arbutifolia. Melampsora epiphylla Diet., Englers Bot. Distribution: Maritime provinces of Russia. Jahrb. 32, 50; 1902

= M. larici-opaca Miyabe et Matsumoto, Melampsora dimorphospora Kaneko et Trans. Sapporo Nat. Hist. Soc. 6, 22–37; Hiratsuka, f., Trans. Mycol. Soc. Japan 1915. 23, 201–210; 1982 Spermogonia amphigenous; aecia hypophyllous; aeciospores globose, 15–20 × Spermogonia and aceia unknown. Uredinia 12.5–18 mm; uredinia minute, mostly hypo- hypophyllous, occasionally epiphyllous, phyllous, very rarely epiphyllous, scattered, scattered, rounded, erumpent, 0.2–1 mm in sub-epidermal, erumpent, pulverulent, diameter, brownish-yellow in dry state, circular, pale yellowish-orange, 0.6 mm, sometimes containing one- or two-celled urediniospores 12.8–16 × 11.2–15 mm, usu- free teliospores; urediniospores are of ally globoid, rarely ovate, wall 1–2 mm thick, two types: (1) borne singly on pedicels, finely and prominently echinulate; para- echinulate, subglobose, broadly ellipsoid or physes clavate to capitate, sometimes globose, 19–30 × 17–25 mm, wall 2.5–3 mm curved, 35 mm long, 10–15 mm wide, thick, colourless, germ pores scattered; hyaline. 36 G. Bagyanarayana

Telia epiphyllous, in dense clusters, orange; aeciospores 15–25 × 10–19 mm, very closely aggregated sub-cuticular, not globoid, angulately globoid or ovoid, wall erumpent up to 1 mm, dark cinnamon- 1.5–2.5 mm, finely verrucose. Uredinia brown; teliospores 24–45 × 8–16 mm, cylin- amphigenous, sub-epidermal, in the begin- drical, sessile, single-celled, wall 1 mmthick, ning covered by the rudimentary peridium, brown, apically the corners are thickened, later on erumpent, pulverulent, 0.5–1.5 mm; up to 2.5–4.8 mm, dark cinnamon-brown. urediniospores 12–25 × 10–19 mm, wall Type: On Salix sachalinensis Fr. Schm. 1.5–3 mm thick, echinulate; paraphyses (= Salix shikokiana Makino) Japan: S. numerous, capitate, up to 80 mm long, Kusanoi, November 22, 1899. 15–25 mm wide. Telia minute, amphi- Hosts: Spermogonia and aecia on Larix genous, but mostly hypophyllous, scattered decidua Mill. and L. leptolepis; uredinia or aggregated, often fusing, sub-epidermal, and telia on Salix sachalinensis Fr. Schm. non-erumpent, 0.3–1 mm in diameter, (=Salix shikokiana Makino). blackish-brown; teliospores 20–50 × Distribution: China, Japan, former USSR. 7–15 mm, rounded at both the ends, wall Specimens examined: Type only. 2 mm or slightly thicker at the apex. Melampsora epiphylla Diet., resembles Type: On Salix alba L., Bayreuth, Bavaria, M. capraearum Thuem., in having epiphyl- Germany (Thüm, Hert, Mye, Oec. 388. lous, sub-cuticular telia, but differs from it in sub-nom. M. salicina). having small urediniospores and the apical Hosts: Spermogonia and aecia on species of walls of the teliospores are not thickened to Abies, Euonymus, Larix, Ribes, Saxifraga the same extent as those of M. capraearum. and several genera of Orchidaceae. Uredinia and telia on species of Salix. Distribution: Canada, China, Europe, India, USA. Melampsora epitea (Kunze. & Schm.) Specimens examined: DAVFP, LEV, PAV, Thüm., Mith. Forstl. Versuchsw. Oesterr S, G, WRSL, HCIO, B. 2, 38, 40; 1879 The willow rust, M. epitea has various races which do not differ greatly in their = Uredo epitea Kunze. & Schm. Mycol. morphological characters but differ only Hefte 1, 68; 1817. in their aecial hosts; all these should be con- = M. hartigii Thüm., Mith. Forstl, sidered as formae speciales. Most of the wil- Versuchsw. Oesterr 2, 41; 1879. low rusts are included under this collective = M. coleosporioides Diet., Englers Bot. species. This follows Hylander et al. (1953), Jahrb. 32, 50; 1902. Wilson and Henderson (1966), Boerema and = M. humilis Diet., Englers Bot. Jahrb. 32, Verhoeven (1972) and Ziller (1974). 50; 1902. = M. microsora Diet., Englers Bot. Jahrb. 32, 50; 1902. = M. humboldtiana Speg., Ann. Mus. Mat. Melampsora epitea f. sp. Buenos-Aires 23, 28; 1912. abieti-capraearum (Tub.) Bagyanarayana = M. larici-miyabeana Miyabe et Mats., comb. nov. (Fig. 3.1) Trans. Sapporo Nat. Hist. Soc. 6, 22–35; 1915. = Caeoma abietis-capraearum Tub. = M. kiusiana Hirat. f., Bot. Mag. Tokyo, 57, Centralbl. Bak. II, 241; 1902. 281; 1943. = M. abieti–capraearum Tub. Centralbl. Spermogonia amphigenous, sub- Bak. II, 241; 1902. cuticular, variable in shape and size, = M. americana Arth., Bull. Torrey Club 47, 60–120 mm high, 150–200 mm wide. Aecia 465; 1920. caeomoid, amphigenous, scattered or aggre- Spermogonia amphigenous, pale yellow, gated, sub-epidermal, erumpent, pulveru- 70–115 mm wide, 60–70 mm high. Aecia lent, rarely having a rudimentary peridium, hypophyllous, solitary, parallel to the Species of Melampsora on Salix 37

Fig. 3.1. (A) M. epitea f. sp. abieti-capraearum on Salix nigra (Uredinia & Telia); (B) M. allii-fragilis f. sp. galanthi-fragilis on Salix fragilis (Telia); (C) M. amygdalinae on Salix triandra (Telia); (D) M. epitea f. sp. arctica (Uredinia); (E) M. capraearum on Salix sp. (Uredinia & Telia); (F) M. larici-pentandrae on Salix pentandrae (Uredinia & Telia). midrib, oblong, 0.4–0.6 mm long, often fus- sub-epidermal, non-erumpent, blackish- ing, sub-epidermal, erumpent, pulverulent, brown; teliospores 20–30 × 7–12 mm, orange yellow; aeciospores 15–24 × oblong, wall 1 mm thick, light brown. 12–18 mm, globoid or ellipsoid, sometimes Type: On Salix caprea L., Barnau, near angular, wall 1.5–2.5 mm thick, verrucose. Chiemsee, Bavaria, Germany. Uredinia minute, hypophyllous, scattered, Hosts: Spermogonia and aecia on Abies 0.5 mm, sub-epidermal, erumpent, pul- amabilis (Dougl.) Forb., A. balsamea (L.) verulent, orange; urediniospores 15–20 × Mill., A. cephalonica Loud., A. concolor 10–16 mm, globoid to broadly ellipsoid, wall Lindl. & Gord., A. grandis (Dougl.) Lindl., 1.5–2 mm thick, echinulate; paraphyses A. lasiocarpa (Hook) Nutt., A. nordman- capitate, up to 50 mm long, 15–25 mm wide, niana (Stev.) Spach., A. pectinata D.C., wall 1.5–3.5 mm thick. Telia hypophyllous, A. pinaspo Boiss. Uredinia and telia on scattered, 2 mm wide, more or less fusing, Salix amygdalina L., S. appendiculata Vill., 38 G. Bagyanarayana

S. argyrocarpa Anderss., S. aurita L., S. erumpent, pulverulent; aeciospores 15–26 × bebbiana Sarg., S. caprea L., S. cinerea 15–21 mm, globoid, ovoid, rarely angular, L., S. commutata Bebb., S. cordata Muhl., wall 2–3 mm thick, finely verrucose. S. exigua Nutt., S. humilis Marsh., S. Uredinia minute, amphigenous, chiefly humboldtiana Willd., S. incana Schrank, hypophyllous, scattered or aggregated, S. interior Rowlee, S. longifolia Wall ex 0.5 mm in diameter, sub-epidermal, erum- Anderss, S. mackenzieana Barr. Apud pent, pulverulent, bright orange-yellow; Anderss, S. nigra Marsh, S. nigricans Sm., urediniospores 15–25 × 12–20 mm, globoid, S. pedicellaris Pursh., S. petiolaris Sm., broadly ellipsoid, wall 2 mm thick, echinu- S. purpurea L., S. repens L., S. scouleriana late; paraphyses numerous, capitate, up to Barr., S. sericea Muhl. 65 mm long, 15–24 mm wide, wall 4–6 mm Distribution: Argentina, Canada, Europe, thick. Telia minute, amphigenous, scattered, USA. 0.3–0.5 mm in diameter, often fusing, reddish- Specimens examined: DAVFP, FH, HCIO, brown, sub-epidermal, non-erumpent; telio- S. spores 25–45(50) × 8–17 mm, oblong, wall Tubeuf (1902) created the species 1.5 mm thick, pale golden-brown. Melampsora abieti-capraearum to accom- Lectotype: On Salix groenlandica modate a rust on Salix having Abies as its (Anderss.) Lundst., Christianshaab, aecial host. Arthur (1920) termed this Salix– Greenland. Abies rust as M. americana. Later, Arthur Hosts: Spermogonia and aecia on Anti- (1934) identified this rust as equivalent to phylla oppositifolia (L.) Forr., Leptasea M. abieti-capraearum. However, morpho- flagellaris (Willd.) Small, Saxifraga logically M. abieti-capraearum is similar adscendens L., S. bracteata D. Don., S. to M. epitea, a collective species having cernus L., S. caespitosa L., S. exilis Steph., several formae speciales on different aecial S. groenlandica (Anderss.) Lundst., S. hosts. As such M. abieti-capraearum is hypnoides L., S. oppositifolia L. Uredinia reduced to the rank of a forma specialis of and telia on Salix anglorum Cham., S. M. epitea. aquilonica Kimura, S. arctica Pall., S. Host alternation was established by balsamifera (Hook.) Barratt, S. chamissoni Tubeuf (1902, 1905). Fraser (1912, 1913) Anderss, S. fuscescens Anderss, S. glauca and Weir and Hubert (1918) made cultural L., S. glaucops Anderss., S. groenlandica studies of this rust fungus. (Anderss.) Lundst., S. herbacea L., S. lanata L., S. myrtillifolia Anderss., S. myrsinites L., S. ovalifolia Trautr., S. petrophila Rydb., S. poloris Wah., S. pyrenaica Gou., S. Melampsora epitea (Kunze & Sch.) pyrifolia Anderss., S. reptans Rupr., S. Thüm., f. sp. arctica (Rostr.) rotundifolia Trautr., S. sitchensis Sanson, S. Bagyanarayana, comb. nov. stolonifera Cav., S. subreniformis Kimura, S. yezoalpina Koidz. = M. alpina Juel., Ofvens K. Vetensk. Akad. Distribution: Canada, Europe, Greenland, Forh. 51, 417; 1894. Japan, USA. = Uredo alpina Arth., Res. Sci. Congr. Bot. Specimens examined: DAOM, FH, S. Vienne 338, 1906. Melampsora arctica Rostrup, is mor- = U. rostrupiana Arth., N. Am. Flora 7, 100; phologically very much similar to M. epitea, 1907. a collective species having Salix as its telial = M. arctica Rostr., Medd on Gronl. 3, 535; host. As such it has been reduced to the 1888. level of a forma specialis. The other Salix Spermogonia amphigenous, scattered Melampsora having Saxifraga as its aecial or in groups, sub-epidermal, 150–160 mm host is M. epitea f. sp. reticulatae (Blytt) wide, 90–130 mm high; aecia minute, Jorst., from which M. epitea f. sp. arctica caeomoid, amphigenous, chiefly hypo- differs in having smaller urediniospores phyllous, 0.3–0.5 mm, sub-epidermal, with thinner walls. Species of Melampsora on Salix 39

Gäumann (1959) recognized two bio- Hosts: Spermogonia and aecia on Euony- logical races under the species M. alpina mus europaeus L., E. latifolia Scop., E. Juel. They are M. alpina Juel and M. verrucosa Scop.; uredinia and telia on reticulatae Blytt. However, Arthur (1934) Salix aurita L., S. caprea L., S. cinerea L., considered M. alpina Juel as identical S. incana Schrank., S. viminalis L. with M. arctica and treated M. alpina as a Distribution: Europe. synonym of M. arctica. But Gäumann (1959) Specimens examined: C, G, S. preferred to retain M. alpina and considered Nielsen and Rostrup (1884, see M. arctica as different. The present study Gäumann, 1959) studied the genetic rela- reveals that there is no justification in the tionship between the caeoma developed on retention of the species M. alpina, accord- Euonymus europaeus L. and a Melampsora ingly the author has followed Jørstad (1923) grown on Salix caprea L. and S. cinerea L. and Arthur (1934). Klebahn (1899) extended these studies and created the species M. euonymi- capraearum. However, Boerema and Verhoeven (1972) reduced it to the level of a Melampsora epitea Thüm., f. sp. forma specialis. The author agrees with the euonymi (Kleb.) Boerema et Verhoeven, treatment of Boerema and Verhoeven. Neth. J. Pl. Path. 78, 24; 1972

= M. euonymi-capraearum Kleb., Forstl. Naturw. Zeischr. 6, 469, 1897. Melampsora epitea Thüm., f. sp. = M. euonymi-capraearum Kleb. ex Kleb., lapponum (Lindf.) Bagyanarayana Pringsh. Jahrb. Wiss. 34, 358, 1900. comb. nov. = M. euonymi-incarnae O. Schneid., Central Blatt. f. = Caeoma violae Lindf., Sv. Bot. Tidskr. 4, = Uredo euonymi-capraearum Arth., 193, 1910. Result. Scient.Congr. Intern. Bot. Vienne = M. lapponum Lindf., Sv. Bot. Tidskr. 7, 338, 1905, 1906. 48, 1913. Spermogonia prominent, 200 mm wide, Spermogonia not known. Aecia hypo- 80 mm high. Aecia caeomoid, mostly hypo- phyllous, aggregated in groups, circularly phyllous, occasionally epiphyllous, often deposited, 0.3–1 mm in diameter, sub- aggregated in groups, up to 2 mm, sub- epidermal, erumpent, pulverulent, orange; epidermal, erumpent, pulverulent, bright aeciospores 14–27 × 12–20 mm, globose to orange; aeciospores 18–26 × 12–20 mm, ellipsoid, wall 2–2.5 mm thick, verrucose. sub-globose, ovoid or ellipsoid, slightly Uredinia minute, hypophllous, 0.2–0.4 mm angular, wall up to 5 mm thick, verrucose. in diameter, sub-epidermal, erumpent, Uredinia minute, hypophyllous, scattered pulverulent, orange; urediniospores 14–24 × or aggregated, 0.5 mm, sub-epidermal, 10–20 mm, globose, sub-globose, ovate erumpent, pulverulent, orange; uredinio- or ellipsoid, sometimes even angular, spores 14–20 × 12–17 mm, globose, sub- echinulate; paraphyses clavate to capitate, globose or ovoid, walls unevenly thickened, up to 55 mm in length, 15–30 mm in width, 1.5–4 mm, echinulate; paraphyses capitate, wall 1–4 mm thick. Telia minute, usually up to 70 mm long 18–25 mm wide, wall epiphyllous, 0.2–0.5 mm in diameter, sub- 2.7 mm thick. Telia minute, hypophyllous, epidermal, non-erumpent, orange-brown; deposited in groups, 0.5 mm in diameter, teliospores 30–50 × 6–12 mm, rounded at sub-epidermal, non-erumpent, dark brown; both ends, wall 1 mm thick, brown. teliospores 24–40 × 7–14 mm, rounded at Type: On Salix lapponum L., Njuonjes, Lale both the ends, wall 1 mm, slightly thickened lappomark, Sweden. apically, light brown. Hosts: Aecia on Viola epipsila Ledeb., V. Type: On L., Steinbek, palustris L., uredinia and telia on Salix Hamburg, Germany. lapponum L. 40 G. Bagyanarayana

Distribution: Northern and central Europe. Teliospores prismatic, wedge-shaped or Specimens examined: S. irregular, usually rounded at both ends, According to Lindfors, the supposed 20–50 × 7–14 mm, walls light brown, uni- difference between M. lapponum and typi- formly thin, about 1 mm; pore imperceptible. cal M. epitea is that M. lapponum has wider Hosts: Spermogonia and aecia on Larix. uredo paraphyses. In addition, as the aecial Uredinia and telia on Salix aurita, S. host is Viola, M. lapponum is treated here as caprea, S. cinerea, S. hastate, S. oxycarpa, a forma specialis of M. epitea. S. repens var. argentea, S. viminalis. Distribution: Eurasia, North Africa, India. M. larici-epitea Kleb. is morphologi- cally indistinguishable from M. epitea, Melampsora epitea f. sp. larici-epitea therefore reduced to the level of forma Bagyanarayana comb. nov. specialis. Uredinio- and teliospores on numerous species of Salix (comprising ten = M. larici-epitea Kleb., Ztschr. Pflanzenkr. sections). Heterogeneous species break up IX, 1899. into closely related biological forms adapted = Caeoma laricis Hartig., Wichtige Krankh. to different hosts. d. Waldbaume, 1874, S. 93, pr. P. = Uredo larici-epitea Arth., Result. Scient. Congr. Intern. Bot. Vienne 1905, 1906, p. 338. Melampsora epitea f. sp. repentis Spermogonia subcuticular, round– (Plowr.) Boerema and Verhoeven Neth. conoid, 70–100 mm wide, 30–40 mm high. J. Pl. Path. 78, 24; 1972 Aecia hypophyllous, single or in rows, with corresponding yellow spots on the = Uredo confluens var. orchidis Arth. and upper side of the leaf, round or elongate, Schw., Consp. Fung. Nisk. 122; 1805. 0.5–1.5 mm long, pale orange-coloured; = Caeoma orchidis (Alb. and Schw.) Wint., aecia of the fungus on Salix retusa are sur- Rabh. Krypt. Fl. 1, 256; 1882. rounded by a crown of capitate, thin-walled = Melampsora repentis Plowr., Zeitschr. f. paraphyses. Aeciospores globoid, ellipsoid Pfl. Krankh. 1, 131; 1891. or slightly angular, 15–25 × 10–18 mm; wall = M. orchidi-repentis Kleb., Jahrb. Wiss. about 1.5–3.0 mm thick, with a delicate, Bot., 34, 369; 1900. sparse, very short-ridged structure about Spermogonia lenticular, sub-epider- 0.5 mm thick, at intervals of approximately mal, with flat hymenium, 170 mm wide and 1 mm. 80 mm high. Aecia caeomoid, hypophyllous, Uredinia either amphigenous or con- scattered or aggregated, often circularly fined to the upper or lower side of the leaves, deposited, 1–2 mm in diameter, often fus- on yellow spots, 0.25–1.5 mm, orange- ing, sub-epidermal, erumpent, pulverulent, yellow. Urediospores usually ovoid, globoid orange-yellow; aeciospores 15–23 × or angular, 12–25 × 9–19 mm, slightly vary- 14–27 mm, globoid, angularly globose, ing on different host plants; walls rather ovoid, wall 1–1.5 mm thick, verrucose. thick, 1.5–3.5 mm, echinulate, paraphyses Uredinia minute, hypophyllous, scattered capitate, 35–80 mm long, width 15–24 mm; or aggregated, 0.3–0.8 mm in diameter, wall 3–5 mm thick, apically thickened up to sometimes fusing, sub-epidermal, erum- 10 mm. pent, pulverulent, orange; urediniospores Telia generally hypophyllous, on Salix 12–20 × 12–16 mm, globose, ovate or ellip- retusa, S. miyabeana, S. sachalinensis, also soid, wall 1.5 mm thick, echinulate; para- epiphyllous; sub-epidermal, dark brown, physes capitate, up to 75 mm long, 15–20 mm sometimes tinged with violet, small, wide, wall 2.5 mm thick. Telia minute, 0.25–1.0 mm, often closely packed or hypophyllous, rarely epiphyllous, sub- coalescing in small groups covering portions epidermal, not erumpent, dark brown; of the blade delimited by the small veins. teliospores 17–50 × 7–15 mm, both the ends Species of Melampsora on Salix 41

round, rarely irregular, wall 1 mm thick, light globoid, slightly angular, ovoid, wall 2–3 mm brown. thick, verrucose. Uredinia minute, hypo- Type: On Orchis latifolia L., Germany, Otto phyllous, scattered or aggregated, 0.5 mm in Jaap, Hamburg, June 1, 1905. diameter, orange, sub-epidermal, erumpent, Hosts: Spermogonia and aecia on Gymna- pulverulent; urediniospores 17–30 (35) × denia comopsea (L.) R. Br., Leucoorchis 15–20 (25) mm, globoid, broadly ellipsoid, albida (L.) E. Mey, Listera ovata (L.) R. Br., ovoid, wall 2–3.5 (5) mm thick, echinulate; Orchis angustifolia Lois., O. incarnata L., paraphyses numerous, capitate, up to 95 mm O. insectifera Crantz., O. latifolia L., O. long, 20–30 mm wide, wall thickened up to majalis Rchb., O. mascula L., O. militaris L., 10 mm. Telia minute, amphigenous, dense, O. moris L., O. muscifera Huds., O. scattered, 0.4–0.8 mm in diameter, sub- praetermissa, O. sambucina L., O. epidermal, not erumpent, dark brown; telio- strictifolia Opiz., Platanthera bifolia (L.) spores 30–45 (50) × 7–10 (14) mm, rounded L.C. Rich. Uredinia and telia on Salix aurita at both the ends, wall 1.5 mm thick, slightly L. and S. repens L. thickened above, pale brown. Distribution: Europe and USA. Type: On Salix reticulata L., Finland, Specimens examined: FH, K, S. Finmark, Norland, Alps, Dovre. Plowright (1891), transferred aecio- Hosts: Spermogonia and aecia on Saxifraga spores from Orchis maculata to Salix repens aizoides L., uredenia and telia on Salix and obtained uredinial and telial stages. amygdalina L., S. bebbiana Sarg., S. Similarly, he also succeeded in infecting brachycarpa Nutt., S. breweri Bebb., S. Orchis maculata using teliospores from caprea L., S. coulteri Anderss., S. piperi Salix repens. To this fungus Plowright gave Bebb., S. purpurea L., S. reticulata L., S. the name M. repentis. For the sake of better uva-ursi Pursh. clarity, Klebahn (1900) named this fungus as Distribution: Europe. M. orchidi-repentis (Plowr.). Kleb. Gäumann Specimens examined: FH, HCIO, S, WRSL. (1959) was not in agreement with the Melampsora epitea var. reticulatae views of Klebahn. However, Boerema and (Blytt.) Jorst., differs from M. epitea var. Verhoeven reduced M. repentis Plowr., from arctica (Rostr.) Bagyanarayana in having the status of a species to that of a forma bigger urediniospores with thick walls and specialis. The author has followed Boerema longer uredinial paraphyses. and Verhoeven in treating M. repentis as a From their field observations, Jaap forma specialis. (1908) and Schneider (1910) found Saxifraga aizoides as an alternate host. Henderson (1953) determined the genetic relationship. Scaramella (1932) found the Melampsora epitea f. sp. reticulatae perennation of the mycelium in Salix, (A. Blytt.) Jorst., Skr. Norske. N. vidensk. whereas Henderson (1953) found the Akad. Osla 6, 31; 1940 perennation of the mycelium in the buds of Saxifraga. Dietel (1930) reported the = Uredo saxifragarum D.C., Fl. Franc. 6, 87; presence of two-celled spores in the telia. 1815. = Caeoma saxifragae Wint., Rabh. Krypt. Fl. Ed. 1, 258; 1882. = M. reticulatae A. Blytt., Chria. Vidensk. Melampsora epitea f. sp. Selsk. Forh. 1886 6, 65; 1896. ribesii-purpureae (Kleb.) Bagyanarayana Spermogonia scattered or aggregated, comb. nov. (Fig. 3.2B) 90–125 mm high, 150 mm wide, light orange. Aecia caeomoid, amphigenous, mostly epi- = M. ribesii-purpureae Kleb., Pringsh. phyllous, solitary, 0.5–1 mm in diameter, Jahrb. Wiss. Bot. 35, 667; 1901. sub-epidermal, erumpent, pulverulent, = M. ribesii-salicum Bub., Arch. Naturw. orange; aeciospores 16–25 × 14–20 mm, Landesdurchf. Bohn. 5, 200; 1908. 42 G. Bagyanarayana

= M. ribesii-auritae Kleb., Pringsh. Jahrb. similar to that of M. epitea the collective spe- Wiss. Bot. 35, 670; 1901. cies. The other Melampsora on Salix having = M. ribesii-grandifoliae O. Schneid., Ribes as its aecial host is M. ribesii-viminalis, Central blatt f. Bakteriol. 15, 238; 1905. but the telia in M. ribesii-viminalis are = M. ribesii-epitea Kleb., Krypt. fl. Mark. sub-cuticular whereas in M. epitea f. sp. Brandenb. Va (Pilze III), 708; 1913. ribesii-purpureae they are sub-epidermal. = M. confluens (Pers.) Jackson, Brookl. Bot. The development and specialization Gard. Mem. I, 210; 1918. of M. epitea f. sp. ribesii-purpureae was = M. ribesii-epitea Kleb. f. sp. ribesii-auritae studied by Klebahn (1900, 1902, 1903), Kupr., Kuprevich & Transchel, Fl. Pl. Crypt. Schneider (1906) and Mayor (1924, 1958). URSS 4, Ured. 1, 374; 1957. According to Scaramella (1932), the = M. ribesii-epitea Kleb. f. sp. ribesii- dikaryotic mycelium can overwinter in purpureae Kupr., Kuprevich & Transhel, Fl. the branches of Salix purpurea and some Pl. Crypt. URSS 4, Ured. 1, 374; 1957. other Salix species, so that new infection Spermogonia chiefly epiphyllous, need not take place in every spring. aggregated, sub-epidermal 60–80 mm high, 150–200 mm wide. Aecia often hypo- phyllous, scattered or circularly aggregated, 0.5–1.5 mm in diameter, often fusing, sub- Melampsora epitea f. sp. tsugae Ziller, epidermal, erumpent, pulverulent, orange; Can. J. Bot. 37, 115; 1959 aeciospores 15–26 × 12–20 mm, globose, angularly globose or ellipsoid, wall = Caeoma dubium C.A. Ludwig, Phytopath. 1.5–3 mm thick, minutely verrucose. 5, 281; 1915. Uredinia chiefly hypophyllous, scattered or Spermogonia on current year’s needles, aggregated, 1–3 mm, orange, sub-epidermal, occasionally on cones; minute, lenticular, erumpent, pulverulent; urediniospores originating under the epidermis. Aecia 15–25 × 14–20 mm, globose, sub-globose or caeomoid, on current year’s needles, on ovate, wall 2–3.5 mm, echinulate; para- cones, scattered, sub-epidermal, erumpent, physes clavate to capitate, up to 80 mm long, pulverulent, pale yellow, up to 1 mm; 15–25 mm wide. Telia minute, often hypo- aeciospores 15–24 × 14–21 mm, globose, phyllous, sub-epidermal, non-erumpent, sub-globose, ovoid, wall apically thickened, scattered or aggregated, 0.2–0.5 mm in verrucose. Uredinia minute, hypophyllous, diameter, dark brown; teliospores scattered or aggregated, sub-epidermal, 20–32 × 7–12 mm, round at both ends, wall erumpent, pulverulent, pale yellow, up to 1 mm, light brown. 0.6 mm; urediniospores 14–20 × 12–17 mm, Type: On Salix purpurea L., Trignitz inder globoid to ellipsoid, wall 1–2 mm, uniformly Prignitz, Brandenburg, Germany. thick, echinulate; paraphyses numerous, Host: Spermogonia and aecia on Ribes prismatic, wall 1 mm thick, brown. Telia alpinum L., R. atropurpureum C.A.M., R. hypophyllus, teliospores 6–14 × 16–30 mm. aureum Pursh., R. grossularia L., R. nigrum Host: Spermogonia and aecia on Tsuga L., R. reclinatum (L.) Mill., R. rubrum L., R. heterophylla (Raf.) Sarg., and T. merten- sanguineum Pursh., R. spicatum Robs., R. siana (Bong.) Carr. Uredinia and telia on uva-crispa L., R. vallicola. Uredinia and Salix scouleriana Barr. telia on Salix arbuscula L., S. aurita L., S. Distribution: Canada and USA. bebbiana Sarg., S. bicolor Fries., S. caprea Specimen examined: DAVFP. L., S. daphnoides Vill., S. glauca L., S. gran- Ziller (1959) created M. epitea f. sp. difolia Seringe., S. nigricantis, S. purpurea tsugae as a race of the M. epitea complex. L., S. rubra Huds., S. scouleriana Berr. Ziller (1974) is of the opinion that M. epitea Distribution: Europe and USA. f. sp. tsugae is morphologically indistin- Specimens examined: DAOM, S. guishable from M. abieti-carpraearum. Klebahn’s Melampsora ribesii- However, these two rusts differ in their purpureae is morphologically very much aecial host range, as shown by Ziller (1959). Species of Melampsora on Salix 43

The basidiospores of M. epitea f. sp. tsugae coronate–capitate; 17.5–20 mm, wall 2.5 mm do not infect Abies sp., but infect Tsuga sp., thick. Telia epiphyllous, sub-epidermal, whereas the basidiospores of M. epitea f. prismatic, 30–37 × 10–15 mm, wall very thin sp. abieti-capraearum do not infect Tsuga 0.5–0.7 mm, apically not thickened, germ sp. but infect the Abies species only. pore distinct. Type: Salix brachypoda (Trautv. et May.) Kom. Melampsora kamikotica Kaneko et Hosts: Salix brachypoda. Hiratsuka, f., Trans. Mycol. Soc. Japan Distribution: Amur River, Svobodnensk, 23, 201–210; 1982 1959, B.A. Tomolin, Russia.

Spermogonia and aecia unknown. Uredinia amphigenous, scattered, rounded, erum- Melampsora larici-pentandrae Kleb., pent, 0.3–0.5 mm in diameter, pulverulent, Forstl. Naturw. Zeitscher 6, 470; 1897. yellow; urediniospores obovoid, ellipsoid (nomen provisorium) Zeitschr f. Pfl. × m or broadly ellipsoid, 20–33 15–18 m, Krankh. 7, 330; 1897 (Fig. 3.2A) echinulate, wall 2–2.5 mm thick at sides, 2.5–7.5 mm thick at apex, colourless, germ = Uredo minutissima Opiz., Sezman pores 4–6, scattered, cytoplasm yellow; Rostlin Kvitney Ceske 152, 1852. paraphyses numerous, intermixed, capitate = U. larici-pentandrae Arth., Result. Scient. to clavate, 40–75 × 15–25 mm, wall Congr. Intern. Bot. Vienne 338, 1; 1905, 2–2.5 mm thick at sides, 5–6.5 mm thick at 1906. apex. Telia amphigenous, sub-epidermal, = M. minutissima (Opiz.) Bub., Arch., scattered, rounded, 0.3–0.8 mm in dia- Naturw. Landesdurchf. Bohn 5, 194; 1908. meter, reddish-brown; teliospores adhering Spermogonia amphigenous, sub- laterally, prismatic in surface view of cuticular, 60–100 mm wide, 30–50 mm high. telium, one-celled, oblong and rounded at Aecia caeomoid, minute, mostly hypo- both ends in side view, arranged in a single phyllous, scattered, orange; aeciospores layer, 25–53 × 10–16 mm, wall 1 mm thick, 15–26 × 13–20 mm, ovoid, globoid or slightly yellowish brown, not thickened at apex. angular, wall 2 mm thick, finely verrucose. Hosts: Chosenia arbutifolia. Uredinia mostly hypophyllous, very rarely Distribution: Japan. epiphyllous, scattered or aggregated, often Type: On Chosenia arbutifolia (Pall.) A. fusing, up to 1 mm in diameter, sub- Skvortz. Central Honshu, Pref. Nagano, epidermal, erumpent, pulverulent, orange; Kamikochi, 10 September, 1979, Dr Shigeru urediniospores 25–45 × 17–17 mm, ovoid Kaneko HH – 73060; TMI – 7105. or broadly ellipsoid, wall 2 mm, apically M. kamikotica differs from M. thickened up to 5 mm, echinulate; choseniaei in having longer, apically paraphyses clavate to capitate, up to 70 mm thickened walls. long, 12–17 mm wide. Telia hypophyllous, minute, scattered or aggregated, sub- epidermal, not erumpent, often fusing, Melampsora kuperviczii Zenk., Novit. 0.5 mm, dark brown; teliospores 23–38 × Syst. Pl. Vasc. 2 Non Vasc., 6–12 mm, round at both ends, wall 1.5 mm 197–199; 1964. thick, light brown. Type: On Salix pentandra L., Borsteler Spermogonia and aecia not found. Uredinia Jager, near Hamburg, Germany. hypophyllus, 0.25–0.5 mm in diameter, Hosts: Spermogonia and aecia on Larix round or oval, paraphyses hyaline; decidua L., L. sibirica Ldb. Uredinia and urediniospores globose, ovate, elliptical, telia on Salix fragilis L. and S. pentandra L. angular, 10–15 × 10–12 mm, wall densely Distribution: Europe. verrucose; 2 mm thick, hyaline; paraphyses Specimens examined: S. 44 G. Bagyanarayana

Fig. 3.2. V. S. of Telium (450×). (A) M. larici-pentandrae; (B) M. epitea f. sp. ribesii-purpureae: (C) M. ribesii-viminalis; (D) M. yezoensis.

Klebahn (1897) created the species M. dense, scattered, sub-epidermal, erumpent, larici-pentandrae,aSalix Melampsora pulverulent, orange-yellow; urediniospores having Larix as its aecial host. Later, Bubák 15–26 × 10–17 mm, ovoid, oblong or ellip- (1908) named the Salix–Larix rust as M. soid, wall 2–3 mm thick, echinulate; para- minutissima. However, the name M. physes capitate, up to 70 mm in length, larici-pentandrae Kleb., is preferred over 16–25 mm in width, wall 3–4 mm thick. M. minutissima as it is the earlier, validly Telia hypophyllous, scattered often fusing, published name. sub-epidermal, not erumpent, dark reddish- The characteristic feature of this species brown; teliospores 35–70 × 8–15 mm, pris- is the urediniospore wall, which is conspic- matic, cylindrical, round at both the ends, uously thickened apically, a feature not wall 1.5 mm, light brown. commonly seen in other species of Type: Toisusu urbaniana (Seem.) Kimura Melampsora. Klebahn (1897, 1899, 1902, (= Salix urbaniana Seem.), Hokkaido, 1903) made a detailed study of the Sapporo-shi, 4 October 1901, J. Hanzawa development and host specialization of this SAPA: HH78307. species. Host: Aecia on Larix decidua, L. kaempferi Sarg. Uredinia and telia on Salix arctica × S. cuneata and Toisusu urbaniana (Seem.) Kimura (= S. urbaniana Seem.) Melampsora larici-urbaniana Mats., Ann. Distribution: Japan. Missouri Bot. Gard. 6, 311; 1919 Specimens examined: Herb. Hiratsuka. Matsumoto, in 1916, carried out exten- Spermogonia not known. Aecia caeomoid, sive inoculation experiments using telio- hypophyllous, scattered, pale, orange- spores from Salix urbaniana to inoculate yellow; aeciospores 15–26 × 13–20 mm, Larix decidua, and aeciospores from Larix ovoid, globoid or ellipsoid, wall 3–4 mm decidua to inoculate Salix urbaniana.In thick, echinulate. Uredinia hypophyllous, both ways he got positive results. To this Species of Melampsora on Salix 45

rust, Matsumoto (1919) gave the name M. fusing, 0.5 mm in diameter, sub-epidermal, larici-urbaniana. non-erumpent, orange-brown or reddish- Another Melampsora species on Salix, brown; teliospores 20–50 × 8–15 mm, pris- having Larix as its aecial host and matic or oblong, wall 2.5–3.5 mm thick, resembling M. larici-urbaniana,isM. larici- uniform, cinnamon-brown. pentandrae Kleb. However, M. larici- Type: On Salix sp., in high mountains, urbaniana differs from M. larici-pentandrae Gunnison County, Colorado, North both in its urediniospore and teliospore America, Bartholomew. morphology. Hiratsuka et al. (1992) reported Hosts: Spermogonia and aecia on Larix 4–6 scattered germ pores in the uredinio- americana Michx., L. decidua Mill., L. spores and an apical thickening in the laricina (Du Roi.) K. Koch, L. lyallii Parl., urediniospore walls. However, the present and L. occidentalis Nutt. Uredinia and telia author failed to notice these features. on Salix alba L., S. amygdaloides L., S. arctica Pall., S. babylonica L., S. barclayi, S. bigelowii Torr., S. bonplondiana H.B. & K., S. brachycarpa Nutt., S. candida Flugge., Melampsora paradoxa Diet. et Holw., S. caprea L., S. cinerea L., S. cordata Muhl., Hedw. Beibl. 40, 32; 1901 S. delnortensis C.K. Schneid., S. discolor Muhl., S. exigua Nutt., S. fendleriana = M. bigelowii Thüm., Mitth. Forstl. Vers. Anderss., S. flavescens Nutt., S. fluviatilis Oest. 2, 37; 1879. Nutt., S. geyriana Anderss., S. glaucops = Lecythea macrosora Perck., Bot. Gaz.5, Anderss., S. gracilistyla Miq., S. herbacea 35; 1880. L., S. humilis Marsh., S. irrorata Anderss., = M. larici-retusae Ed. Fisch., Ured. S. jepsoni Benth., S. lutea Nutt., S. Schweiz. 487, 1904. mackenzieana Barr., S. missourensis Bebb., = M. larici-nigricantis O. Schneid., Centrbl. S. monticola, S. nigricans Sm., S. nigra Bakteriol. II, Abt. 13, 233; 1904. Marsh., S. parksiana Ball., S. petrophila = M. larici-purpureae O. Schneid., Centrbl. Rydb., S. phylicifolia L., S. planifolia Bebb., Bakteriol. II, Abt. 13, 233; 1904. S. pseudocoradata (Anders.) Rydb., S. = M. larici-reticulatae O. Schneid., Centrbl. purpurea L., S. repens L., S. reticulata L., Bakteriol. II, Abt. 15, 233; 1905. S. rostrata Richards, S. scouleriana Barr., = Uredo bigelowii Arth., Result. Scenti. S. silesiaca Willd., S. sitchensis Sanson, S. Congr. Intern. Bot. Vienne, 338, 1906. subcaerulea, S. yestita Pursh., S. vitellina Spermogonia minute, amphigenous, L., and S. wardi Bebb. scattered or in small groups, pale yellow, Distribution: North and South America. inconspicuous, conical, sub-cuticular, Specimens examined: DAOM, DAVFP, 60–80 mm in diameter, and 50 mm height. HCIO, FH, S, G, WRSL. Aecia caeomoid, mostly hypophyllous, scat- Gäumann (1959) considers M. paradoxa tered or aggregated in small groups, minute, (= M. bigelowii) as an American form of a 0.1–0.3 mm, pale orange, sub-epidermal, Salix–Larix Melampsora complex, having erumpent, pulverulent, aeciospores 17–28 × many biological races. The three races he 15–23 mm, globose or sub-globose, wall classified as belonging to M. paradoxa from 2–3 mm thick, finely verrucose. Uredinia Europe are M. larici-capraearum, M. larici- mainly hypophyllous, scattered or aggre- epitea and M. larici-pentandrae. However, gated, minute, 0.3–0.6 mm in diameter, in the present study M. capraearum (= M. orange, turning to pale yellow, sub- larici-capraearum) and M. larici-pentandrae epidermal, erumpent, pulverulent; uredinio- are treated as distinct species, whereas M. spores 15–26 × 15–21 mm, globose or larici-epitea is treated as a part of the M. sub-globose, wall 2.5–3.5 mm thick, epitea complex. paraphyses capitate, up to 70 mm long and Through his field observations and 20–25 mm in width, 3.5 mm thick. Telia inoculation experiments, Ziller (1970) amphigenous, scattered or aggregated, often found that M. paradoxa overwinters on 46 G. Bagyanarayana

willow as uredinial mycelia. Weir and relationship of M. ribesii-viminalis. Accord- Hubert (1916), Overholts (1926) and Shope ing to Scaramella (1932) the dikaryotic (1940) have studied host alternation of this mycelium overwinters in the branches of species. Salix viminalis.

Melampsora ribesii-viminalis Kleb., Melampsora salicis-albae Kleb., Pringsh. Pringsh. Jahrb. Wiss. Bot. 34, 363; Jahrb. Wiss. Bot. 35, 679; 1901 1900 (Fig. 3.2C) = Caeoma allii-ursini (D.C.) Wint., Rabh. = Uredo ribesii-viminalis Arth., Result. Krypt. Fl. 1, 255; 1822. Scient. Congr. Intern. Bot. Vienne 338; = Melampsora allii-salicis-albae Kleb., 1905, 1906. Zeitschr. f. Pfl. Krankh 12, 19; 1902. Spermogonia generally epiphyllous, = Uredo allii-salicis-albae Arth., Result. sometimes hypophyllous, sub-epidermal, Scient. Cong. Inter. Bot. Vienne 338; 1905, aggregated, 60–80 mm high, 150–190 mm 1906. wide. Aecia caeomoid, hyphophyllous, Spermogonia prominent, lenticular scattered or mostly aggregated in circular 120 mm high, 150–200 mm wide. Aecia groups, often fusing, up to 1.5 mm, orange, caeomoid, hypophyllous and caulicolous, sub-epidermal, erumpent, pulverulent; aggregated in small groups, 1 mm in diame- aeciospores 16–24 × 14–17 mm, globoid to ter, sub-epidermal, erumpent, pulverulent, ovoid, rarely angular, wall 2–3.5 mm thick, orange; aeciospores 17–26 × 15–20 mm, verrucose. Uredinia minute, hypophyllous, globoid or broadly ellipsoid, sometimes scattered or aggregated, 0.2–0.4 mm, pale irregular or angular, wall 1.5–2 mm thick, orange-yellow, sub-epidermal, erumpent, verrucose. Uredinia foliicolous, on young pulverulent; urediniospores 15–22 × shoots and on the bark of branches, on the 14–18 mm, globoid to ovoid, wall 2 mm long, leaves usually hypophyllous, rarely epi- 18–25 mm wide. Telia minute, epiphyllous, phyllous, densely aggregated, up to 2 mm scattered or aggregated, 0.2–0.5 mm, long, on the bark usually solitary, projecting sub-cuticular, non-erumpent, dark brown; from the cracked periderm, up to 5 mm teliospores 25–40 × 7–15 mm, prismatic or long, sub-epidermal, erumpent, pul- irregular, rounded at both ends, wall 1 mm, verulent; uredinispores 20–36 × 11–17 mm, light brown. oblong, sometimes clavate or pyriform, Type: On Salix viminalis L., Bremen, wall 2–2.5 mm thick, echinulate; para- Germany. physes often capitate, up to 70 mm long, Hosts: Spermogonia and aecia on Ribes 12–20 mm in width, wall 2–3 mm thick. alpinum L., R. aureum Pursh., R. gros- Telia hypophyllous, rarely epiphyllous, sularia L., R. nigrum L., R. reclinatum (L.) scattered or aggregated, sub-epidermal, Mill., R. rubrum L., and R. uva-crispa L. not erumpent, dark-brown; teliospores Uredinia and telia on Salix triandra L., and 24–45 × 7–10 mm, irregularly prismatic, S. viminalis L. rounded at both the ends, wall 1 mm thick, Distribution: Europe. light brown. Specimens examined: S. Type: On Salix alba L., Bremen, Germany. Melampsora ribesii-viminalis Kleb., Hosts: Spermogonia and aecia on Allium differs from M. epitea f. sp. ribesii-purpureae ampeloprasum L., A. ascalonicum L., A. (Kleb.) Bagyanarayana, the other Ribes– carinatum L., A. cepa L., A. cernum Roth., Salix Melampsora, in having sub-cuticular A. coerulescens Boiss., A. gaditanum Per. telia. Lar., A. glaucum Hort., A. kansuense Regel, Klebahn (1900, 1902, 1903) and Mayor A. kartaviense Regel, A. longicuspis Regel, (1927, 1929) studied the biological A. moly L., A. nutans L., A. obliqum L., A. Species of Melampsora on Salix 47

ochroleucum W. et K., A. odorum Ten., A. Host: Salix cavaleriei. oleraceum L., A. pedemontanum Willd., Distribution: Yunnan, China. A. platyspathum Schrenk, A. polyanthum Epiphyllous uredinia and telia, slender Schult., A. pulchellum Don, A. pyrenaicum teliospores, angular urediniospores are Costa, A. sativum L., A. schoenoprasum L., the distinguishing characters of this A. scenescens L., A. sphaerocephalum L., species. A. strictum Schrad., A. suaveolens Jacq., A. tartaricum L., A. ursinum L., A. victorialis L., A. vineale L., A. wallichianum Steud., A. yunanense Diels. Melampsora salicis-cupularis Wang Nat. Uredinia and telia on Salix alba L., S. Acad of Peiping 6(40), 225; 1949 babylonica L., S. fendleriana Anderss., S. geyriana Anderss., S. glandulosa V. Seem., Uredinia hypophyllous, minute, scattered, S. hindsiana Benth., S. humboldtiana brown; urediniospores sub-globose, ovate, Willd., S. lasiandra Benth., S. longifolia oblong to ellipsoid, yellow, 21–23 × Wall ex Anderss., S. melanopsis Nutt., S. 17–18 mm, echinulate, hyaline, 1.5–2 mm, pentandra L., S. pyrifolia Anderss., S. retusa germ pores obscure; paraphyses numerous, L., S. scouleriana Barr., S. sitchensis San., capitate, hyaline, up to 98 mm long, S. tetrasperma Roxb., S. vitellina L. 24–35 mm wide, wall 7 mm, thick. Telia Distribution: Europe, India. hypophyllous, sub-epidermal, minute, Specimens examined: G, HCIO, S, FH. scattered or aggregated, confluent, blackish. The development and host selection Teliospores prismatic, oblong-cylindric, of M. salicis-albae was studied by Klebahn apically not thickened, orange-brown, (1900, 1902, 1903, 1905), Schneider (1910) 28–39 × 11–14 mm. and Mayor (1929, 1934, 1936, 1958). The Type: On Salix cupularis Shanxi: Tai-Pai- aecial host range is exactly the same as that Shan, from Pao-Ma-Liang down to under of M. allii-fragilis. According to Klebahn Wen-Kung Miao, alt. 3300 m. 14 August (1905) the dikaryotic mycelium can 1942. Y.C. Wang 4073. overwinter in the bark of the susceptible Host: Salix cupularis. varieties of Salix. Distribution: Shanxi, China.

M. salicis-cavaleriei Tai., Farlowia Melampsora salicis-warburgii Sawada, 3(1), 102; 1947 Trans. Nat. Hist. Soc. Formosa 21, 227–235; 1931 Uredinia epiphyllous, scattered or slightly aggregated, round, 0.2–0.5 mm diameter, Spermogonia and aceia unknown. Uredinia golden yellow; urediniospores angular– amphigenous, rounded, 0.1–0.3 mm in subglobose, oblong or clavate, verrucose, diameter, but often confluent; uredinio- 18–37 × 11–18 mm, wall 1.5–2 mm thick, spores mostly obovoid, 15–18 × 13–28 mm, paraphysis capitate, hyaline. Telia epiphyl- walls 2–2.5 mm thick, echinulate, but lous, scattered or densely aggregated, smooth at apex, germ pores scattered; round, 0.2–0.5 mm diameter, pale paraphysis wall thickened at apex, up to cinnamon-brown, sub-epidermal, telio- 7 mm thick. Telia mostly hypophyllous, spores cylindrical, prismatic or clavato- rounded; teliospores 34–42 × 8–13 mm, wall cylindric, yellow, apex flat or round, not thickened at apex. 36–69 × 7–11 mm, wall 1–1.5 mm thick. Type: On Salix warburgii Seem, Taiwan. Type: On Salix cavaleriei Yunnan, Hosts: Salix warburgii, S. pierotii, S. Kunming, 10 November 1942, W.F. Chiu subfragilis. 7607, Type 8486. Distribution: Taiwan and Japan. 48 G. Bagyanarayana

Melampsora yezoensis Miyabe et Mats., under whose guidance this work was Matsumoto Trans., Sapporo Nat. Hist. carried out. The kind help of the Curators/ Soc. 6, 29; 1915 (Fig. 3.2D) Directors of the following international her- baria – B, C, DAOM, DAVFP, FH, G, HCIO, Spermogonia minute, epiphyllous, orange- H H, IMI, K, LEV, PAV, WRSL, etc. – is brown, up to 0.2 mm. Aecia caeomoid, gratefully acknowledged. I sincerely thank hypophyllous, just below the spermogonia, the following scientists and researchers for in circular rings, often fusing, 2–3 mm their help and co-operation: the late Dr D. wide, sub-epidermal, erumpent, pale Krishnamurthy, Dr Sesikaran, Mr Rama- yellow; aeciospores 25–20 × 13–17 mm, chandran, National Institute for Nutrition, ovoid or angular, wall 1.5–2.5 mm thick, Hyderabad, India; Dr Uwe Braun, Martin echinulate. Uredinia minute, amphigenous, Luther University, Germany; Dr Halvor B. mostly hypophyllous, sub-epidermal, Gjaerum, Plant Protection Research Insti- erumpent, pulverulent 0.4 mm, pale yellow; tute, Norway; the late Professor Nohide urediniospores 16–28 × 12.5–20 mm, ovoid Hiratsuka, Tottori Mycological Research or ellipsoid, rarely globoid, wall 2–5 mm Institute, Japan; the late Dr D.B.O. Savile thick, echinulate; paraphyses capitate to and Dr Mary Esther Elliott, Biosystematics clavate, up to 50 mm long, 12–20 mm wide. Research Institute, Canada; the late Dr Wolf Telia minute, amphigenous, scattered or G. Ziller and Dr Y. Hiratsuka, Canadian aggregated in small groups, up to 0.4 mm, Forestry, Canada; Miss Ann Ansell, Interna- often fusing, sub-cuticular, non-erumpent, tional Mycological Institute, UK; Dr Ming dark brown; teliospores 16–33.6 × H. Pei, Rothamsted Experimental Station, 6.4–13(16) mm, cylindrical, wedge-shaped, UK. I also thank the University Grants rarely irregular, wall 1 mm thick, light Commission and Council of Scientific and brown. Industrial Research, New Delhi for financial Type: On Salix jessoensis V. Seem, assistance, and officials of Osmania Sapporo, Japan. University for the physical facilities. Hosts: Spermogonia and aecia on Corydalis ambigua Cham. et Schl. Uredinia and telia on Salix breweri Bebb., S. hondoensis Koidz., S. jessoensis V. Seem, S. lasiandra References Benth., S. longiflora Wall ex Anderss., Arthur, J.C. (1920) New species of Uredineae. Bulletin S. scouleriana Barr. of the Torrey Botanical Club 47, 467. Distribution: China, Japan. Arthur, J.C. (1934) Manual of the Rusts in Specimens examined: Herb. Hiratsuka. United States and Canada. Purdue Research Matsumoto (1915) created the species Foundation, Lafayette, Indiana. M. yezoensis to accommodate a Melamp- Boerema, G.H. and Verhoeven, A.A. (1972) Check- sora having its spermogonial and aecial list for scientific names of common parasitic stage on Corydalis and the telial stage on fungi. Series 1a: Fungi on trees and shrubs. Salix. The other Salix rust having Corydalis Netherlands Journal of Plant Pathology 78 as its aecial host, with sub-cuticular telia, (suppl. 1). is M. chelidonii-pierotii Mats., from which Bubák, F.R. (1908) Die Pilze Böhmens. I. Uredinales. Archiv der naturwissenschaflichen M. yezoensis differs in its teliospore Landesdurchforschung von Böhmen 13(5). morphology and in the possession of Dietel, P. (1930) Kleine Beitrage zur Uredineenkunde. thick-walled urediniospores. Jahresbericht des Vereins für Naturkunde Zwickauin Sachsen 1928–30, p. 1–9. Fraser, W.P. (1912) Cultures of heteroecious rusts. Mycologia 4, 188. Acknowledgements Fraser, W.P. (1913) Further cultures of heteroecious rusts. Mycologia 5, 238. The author expresses his deep sense of Gäumann, E. (1959) Die Rostpilze Mitteleuropas. gratitude to the late Professor P. Ramachar, Beiträge zur Kryptogamenflora Schweiz 12. Species of Melampsora on Salix 49

Buchdruckerei Büchler and Co., Bern, Matsumoto, T. (1919) Culture experiments with Germany. Melampsora in Japan. Annales of the Missouri Henderson, D.M. (1953) Some Scottish mountain rust Botanical Garden 6, 309–316. fungi. Transactions of the British Mycological Matsumoto, T. (1926) On the relationship between Society 36, 315–319. Melampsora on Salix pierotii Miq. and Caeoma Hiratsuka, N., Sato, S., Katsuya, K., Kakishima, M., on Chelidonium majus L. and Corydalis incissa Hiratsuka, Y., Kaneko, S., Ono, Y., Sato, T., Pers. Botanical Magazine, Tokyo 40, 43–47. Harada, Y., Hiratsuka, T., and Nakayama, K. Mayor, E. (1918) Contribution a l’etude de la flore (1992) The Rust Flora of Japan. Tsukuba mycologique des environs de Ieysin. Bulletin de Shuppankai, Tsukuba, Ibaraki, Japan. la Murthienne Societe Valaisanne des Sciences Hylander, N., Jørstad, I. and Nannefeldt, J.A. (1953) Naturelles 52, 113–149. Enumeratio Uredinearum Scandinavicarum. Mayor, E. (1924) Notes mycologiques. Bulletin de la Opera Botanica 1, 18–26. Societe Neuchateloise des Sciences Naturelles Jaap, O. (1908) Zweites verzeichnis zu meinem 48, 367–396. exsiccate nwerk Fungiselecti exsiccate Ser. Mayor, E. (1927) Notes mycologiques VI. Bulletin de V–VIII, nebst Beschreibungen never Arten und la Societe Neuchateloise des Sciences Naturelles Bumerkungen. Verhhandlungen des Botani- 51, 53–76. schen Vereins fur die Provinz, Brandenburgund Mayor, E. (1929) Notes mycologiques VII. Bulletin de die angrenzenden Lander 49, 1907, 7–29. la Societe Neuchateloise des Sciences Naturelles Jacky, E. (1899) Untersuchungen über einige 54, 45–59. Schweigerische Rostpilze. Ber. Schweiz. Mayor, E. (1934) Notes mycologiques VIII. Bulletin de Botanical Gazette 9, 49–78. la Societe Neuchateloise des Sciences Naturelles Jørstad, A. (1923) Chytridineae, Ustilagineae and 58, 1933, 7–31. Uredineae from Novaya Zemlya. Report of Mayor, E. (1936) Notes mycologiques X. Bulletin de la the scientific results of the Norwegian Societe Neuchateloise des Sciences Naturelles Expedition of Novaya Zemlya 1921, No. 18, 61, 105–123. pp. 3–12. Mayor, E. (1958) Etude experimentale de quelques Klebahn, H. (1897) Vorläufiger Bericht über Uredinees heteroiques. Uredineana 5, 149–324. Kulturversuche mit heteroecischen Rost- Overholts, L.O. (1926) Mycological notes for 1925. pilizen. Zeitschrift für Pflanzen Krankhieten 7, Mycologia 18, 179–185. 129–130. Plowright, W.B. (1891) Einige Impfversuche mit Klebahn, H. (1899) Kulturversuche mit hetero- Rostpilzen. Zeitschrift für Pflanzen Krankhieten ecischen Rost pilzen. VII. Zeitschrift für Pflanzen 1, 130–131. Krankhieten 9, 14–26, 88–99, 137–160. Raabe, A. (1939) Parasitische Pilze der Umgebung Klebahn, H. (1900) Kulturversuche mit hetero- von Tubingen. Ein Beitrag Zur Kryptogamen ecischen Rost pilzen. VIII. Jahrbucher für flora Subwestdeutschlands. Hedwigia 78, Wissenschaftlichen Botanik 34, 660–710. 1–106. Klebahn, H. (1902) Kulturversuche mit Rost pilzen. X. Scaramella, P. (1932) Contribute aila flora micdogica Zeischrift für Pflanzen Krankhieten 12, 17–44, del piccolo S. Bernardo (Val d’ aostal). Malphigia 132–151. 31, 178–224. Klebahn, H. (1903) Kulturversuche mit hetero- Schneider, O. (1905) Weitere Versuche mit ecischen Rost pilzen. XI. Jahrbuch der Schweizerischen Weidenmel ampsoren. Hamburgischen Wissenschaftlichen Anstalten Vorlaufige Mitteilung. Zentralblatt für Bakterio- 20 (1902), 3, 1–56. logie, Parasitenkunde, Infektionskrankheiten Klebahn, H. (1905) Kulturversuche mit hetero- und Hygiene. 2. Naturwissenschaftliche ecischen Rost pilzen. XII. Zeischrift für Pflanzen Abteilung 15, 232–234. Krankhieten 15, 65–108. Schneider, O. (1906) Experimentelle Untersuch- Klebahn, H. (1908) Kulturversuche mit hetero- ungen über Schweizerischen Weidenrostpilze. ecischen Rost pilzen. XIII. Zeischrift für Pflanzen Zentralblatt für Bakteriologie, Parasitenkunde, Krankhieten 17, 1907, 129–157. Infektionskrankheiten und Hygiene. 2. Natur- Klebahn, H. (1914) Kulturversuche mit hetero- wissenschaftliche Abteilung 16, 74–93, ecischen Rost pilzen. XV. Bericht (1912 and 159–176, 192. 1913). Zeischrift fur Pflanzen Krankhieten 24, Schneider, O. (1910) Beitrag zur Kenntnis 1–32. der Schweizerischen Weidenmelampsoren. Matsumoto, T. (1915) Impfversuche mit Melampsora Zentralblatt für Bakteriologie, Parasitenkunde, auf Japani-schen weiden. Sapporo Natural Infektionskrankheiten und Hygiene. 2. History Society Transactions 6, 22–35. NaturwissenschaftlicheAbteilung 25, 436–439. 50 G. Bagyanarayana

Schroeter, J. (1893) Zur Entwicklungsgechichete Weir, J.R. and Hubert, E.E. (1918) Notes on forest tree der Uredineen. Jahresbericht Schlesischen rusts. Phytopathology 8, 118. Gesellschaft fur Vaterlandische Cultur. II. Wilson, M. and Henderson, D.M. (1966) British Rust Botanische Sektion 71, 31–32. Fungi. Cambridge University Press, Cambridge, Shope, P.F. (1940) Colorado rusts of woody plants. UK. University of Colorado Stud. D. 1, 105–127. Ziller, W.G. (1959) Studies of Western trees rusts. V. Tubeuf, C. Von. (1902) Infections versuche mit The rusts of hemlock and fir caused by uredineen der Weibtanne. Zentralblatt für Melampsora epitea. Canadian Journal of Botany Bakteriologie, Parasitenkunde, Infektionskrank- 37, 109–119. heiten und Hygiene. 2. Naturwissenschaftliche Ziller, W.G. (1970) Studies of Western trees rusts. VIII. Abteilung 9, 241. Inoculation experiments with conifer needle Tubeuf, C. Von. (1905) Infections versuche mit rusts (Melampsoraceae). Canadian Journal of uredineen. Naturwissenschaftliche Zeitschrift Botany 48, 1471–1476. fur Forst.u Landwirtschaft (Stuttgart) 3, 41–46. Ziller, W.G. (1974) The Tree Rusts of Western Weir, J.R. and Hubert, E.E. (1916) Successful inocula- Canada. Publication No. 1329. Canadian tions of Larix occidentalis and Larix europea with Forestry Service, Environmental Canada, Melampsora bigelowii. Phytopathology 6, 37. Ottawa, Canada. 4 A Brief Summary of Melampsora Species on Populus

Ming Hao Pei1 and Yan Zhong Shang2 1Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK; 2College of Forestry, Agricultural University, Huhehot, China

Background the family Salicaceae. Diversity of poplars is centred in East Asia (China and Russia) Rust caused by Melampsora is one of the and Pacific and Atlantic regions of North most important leaf diseases of poplars America. There is no consensus on how (Populus). Some 13 species and two many species should be recognized in hybrids of Melampsora have been described Populus. Taxonomists from the East tend to on Populus. A recent revision of the taxon- recognize many species by adopting the omy of the poplar rusts was presented by narrow species concept, while Western Bagyanarayana (1998). This chapter will workers favour the broad species concept give a brief summary of the species of and recognize fewer species. Thus the Melampsora described on Populus.No number of species in Populus can be from attempt has been made to revise the poplar 30 to 100, according to different authorities. rust taxonomy because more meaningful The genus is divided into the five classification can be achieved when further sections: Aigeiros (European and American information on evolutionary and genetic black poplars), Tacamahaca (balsam relationships among the poplar rusts poplars) and Leuce (aspens and true white becomes available. poplars), Leucoides and Turanga.

The Genus Populus Section Aigeiros (black poplars)

Poplars occur naturally across the northern The majority of cultivated poplars belong to hemisphere between the latitudes 30° and the section Aigeiros. There are two genetic 72°N (Wang and Fang, 1984). Poplars are centres: the Mediterranean region and fast growing, easily propagated and can be North America. The Eurasian species is cultivated under various growing condi- P. nigra (black poplar) with several sub- tions. They are now widely grown in vari- species and with natural stands from the ous parts of the world for the purpose of Mediterranean region extending northward urban amenity, farmland sheltering and for to central Europe and eastward to central timber, pulp and biomass production. The Asia. The most important endemic genus Populus L. is closely related to Salix species in North America is P. deltoides (willow) and they are grouped together in (eastern cottonwood; eastern parts of North

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 51 52 Ming Hao Pei and Yan Zhong Shang

America). Populus × euramericana (syn. Sections Turanga and Leucoides P. × canadensis Moench.) is the name given to cover the vast numbers of hybrid clones Populus euphratica is the main species of derived from P. deltoides and P. nigra. the section Turanga and many other names placed in this section are often considered as local forms. Populus euphratica can Section Tacamahaca (balsam poplars) tolerate poor soils, extreme heat and soil salinity. Its distribution extends from the ° This section is the largest in the genus Altai mountains (45 N) southward to the Populus. Balsam poplars are naturally dis- equator and westward to the Middle East tributed in Asia and in North America. In (FAO, 1979). Except for one species in general, they occur naturally further north North America, the several species grouped than black poplars. Wang and Fang (1984) in the section Leucoides are confined to included 36 species (sensu stricto), native China and the Himalayas. or introduced, in the Chinese flora of Salicaceae. Some examples for balsam poplars from Asia are P. laurifolia, P. Melampsora Species on Poplars maximowiczii, P. koreana, P. simonii and P. szechuanica. Examples for the Taca- Most Melampsora species on poplars were mahaca section from North America are described in the late 19th to early 20th cen- P. trichocarpa (black cottonwood), P. turies. By 1915, ten Melampsora species on balsamifera, (balsam or southern poplar) Populus were complied in the Monographia and P. angustifolia (willow-leafed poplar). Uredinearum III by Sydow and Sydow (1915). As in willow rusts, the rust species on poplars were established mainly based Section Leuce (aspens and true on their morphology, alternate hosts and white poplars) telial host range. The aecial hosts of poplar Melampsora include conifers, dicotyle- donous and monocotyledonous plants This section is further divided into aspens (Table 4.1). None of the Melampsora (sub-section Trepidae) and white poplars species described on poplars is known (sub-section Albidae). Aspens occur in to be autoecious (capable of completing a northern and mountainous regions of full life cycle on the same host). Several Eurasia and in North America. In the sub- species, M. larici-tremulae, M. pinitorqua, section Trepidae, the most important M. magnusiana and M. rostrupii are indis- species are P. tremula (European or tinguishable in their morphology but have trembling aspen; distributed in Europe, different aecial hosts. Hylander et al. (1953) western Asia and North Africa), P. and Wilson and Henderson (1966) adopted tremuloides (American or quaking aspen, M. populnea (Pers.) Karst. to include these North America) and P. tremula var. morphologically indistinguishable species davidiana (David’s aspen, the Far East). In (sensu stricto, to be used in this chapter). the sub-section Albidae, western taxono- mists recognize only one species, P. alba L. (white poplar). This species has many sub-species and varieties (treated as species Morphology of Poplar Rusts by eastern taxonomists) which occur naturally in the Mediterranean, eastern Typically, poplar rusts are macrocyclic, Europe and central Asia. The grey poplar, producing five different spore stages, P. × canescens (Aiton) Sm., which is widely i.e. basidiospores, spermatia, aeciospores, distributed in Europe, is considered to be a urediniospores and teliospores, during natural hybrid of P. tremula and P. alba their life cycle. With M. ciliata, M. (Bean, 1976). pruinosae, M. microspora, M. osmaniensis Melampsora Species on Populus 53 Shang

continued . (2000) . (1992) . (1992)

et al

et al et al Gäumann (1959); and Pei (1984) Shang and Pei (1988) Newcombe Sydow and Sydow (1915); Gäumann (1959); Bagyanarayana (1998) Sydow and Sydow (1915); Gäumann (1959) Hiratsuka Sydow and Sydow (1915); Bagyanarayana (1998) References Sydow and Sydow (1915); Gäumann (1959) Eurasia, N. Africa USA Japan Hiratsuka Distribution Eurasia Eurasia, Australia, New Zealand India, Kashmir Europe, Russia northern Africa southern Africa (Ait.) Sm.

P. simonii, P. Henry, , S. Wats.,

euramericana Ledebour, × (Dode) Guinier Fischer, Schneider canescens P. Hook. P. alba ×

P. Miq, P. balsamifera, Tacamahaca P. fremontii . L., Marsh, ,

P. wilsonii P. maximowiczii P. trichocarpa Sec. euramericana P. suaveolens L. × James,

× P. trichocarpa , P. alba Populus Oliv. Oliv., × Kom.,

P. Rehd., P. sieboldii L., Carr. , P. koreana, P. laurifolia

P. deltoides Wall. P. deltoides sp. USA , L., , ,

Turanga Aigeiros Tacamahaca Aigeiros Tacamahaca Tacamahaca Aigeiros Aigeiros Tacamahaca Leucoides canescens euramericana × ×

P. Sect. Sec. Sec. Sec. Sec. Sec. Telial host P. nigra P. koreana P. simonii P. nigra Sec. P. balsamifera P. ciliata Sec. P. deltoides Populus P. nigra P. Sec. P. angustifolia P. ciliata P. aximowiczii ussuriensis Sec. P. lasiocarpa P. euphratica Sec. Leuce P. tremula Sec. Leuce P. tremula species/forms on ,

Melampsora spp. spp. and spp. spp. spp. spp. Aecial host Abies Allium Arum Larix Larix Chelidonium majus Corydalis (Bub.) Kleb. Kleb. Imai Kleb. Wagner Bagyan. et Ram. ? sp. List of hosts and distribution of Hartig.) Barcl. ?

columbiana × M. laricis M. pulcherrima Maire) Table 4.1. (= Melampsora M. abietis-populi M. allii-populina M. ciliata M. M. cumminsii M. larici-populina M. larici-tremulae (= M. magnusiana 54 Ming Hao Pei and Yan Zhong Shang ) ) ) ) ) . (1986)

et al Ziller (1955, 1974); Shain (1988); Spiers and Hopcroft (1994) Spiers and Hopcroft (1994) Ziller (1974) Sydow and Sydow (1915); Gäumann (1959 Sydow and Sydow (1915); Gäumann (1959 References Azbukina (1974) Bagyanarayana (1998 Shang Sydow and Sydow (1915); Gäumann (1959 Germany Bagyanarayana (1998 North America southern Europe South Africa India Australia New Zealand Distribution New Zealand Australia? USA Canada Europe Europe ex-USSR Iran N.E. China West and Central Asia Mixchx.

euramericana Kom. × canescens canescens × × Tacamahaca

P. P. ‘Robusta’, ‘Serotina’ and ‘I-214’ , , Sec. × P. yunnanensis × Schrenk.

Miq. P. alba P. alba , ,

Aigeiros Tacamahaca Aigeiros Aigeiros Aigeiros Aigeiros Aigeiros Tacamahaca euramericana euramericana × × Sec. Sec. Sec. Sec. Telial host Sec. Sec. Sec. P. nigra, P. deltoides, P. P. maximowiczii, P. simonii, P. trichocarpa Sec. Leuce P. tremuloides, P. grandidentata P. P. deltoides P. nigra, P. usbekistanica Sec. P. P. nigra, P. fremontii P. trichocarpa, P. angustifolia, P. balsamifera Sec. Leuce P. sieboldii Sec. Leuce P. tremula Sec. Turanga P. pruinosa Sec. Leuce P. tremula spp.

. spp. spp. spp. spp. spp. spp. spp Aecial host ? Abies Larix Picea sitkensis Pseudotsuga P. taxifolia Tsuga Pinus Abies concolor Larix Picea sitkensis Pseudotsuga menziesii Pinus ? Pinus Mercurialis perennis Arth. (Farl.) . Spiers .?

deltoidae tremuloidae

et al Bagyan. Jacks. Tranz. Rostr. Tranzsch. ?

Continued sp. f. sp. Wagner

M. albertensis Shang

M. abietis-canadensis et Eremeeva = C.A. Ludwig) f.sp. Table 4.1. Thüm. (= Melampsora M. medusae M. medusae-populina M. microspora M. multa M. occidentalis M. osmaniensis et Ram. M. pinitorqua M. pruinosae M. rostrupii Melampsora Species on Populus 55

and M. multa, only uredinio- and teliospore Telia and teliospores stages have been described. Table 4.2 lists the main morphological characteristics of Teliospores overwinter on fallen leaves of poplar rusts. hosts and, in spring, produce basidiospores which infect aecial hosts. The majority of the Melampsora species on Populus form telia mainly on the lower sides of leaves. In Uredinia and urediniospores contrast, M. larici-populina produces telia on the upper sides of leaves (epiphyllous), The urediniospores are capable of produc- and M. pruinosae and M. multa produce ing many cycles of the same form of spores them on both sides of leaves (amphio- during the season, causing disease epidem- genous). All the Melampsora species on ics on poplars. The majority of poplar rusts poplars are known to form telia beneath the form uredinia mainly on the lower side epidermal cells of the hosts (sub-epidemal). of leaves (hypophyllous), except for M. The teliospore walls in most poplar rusts pruinosae, which produces uredinia on are uniformly thick (< 2.5 mm). In contrast, both sides of leaves (amphigenous). The teliospores walls in M. occidentalis are surface of urediniospores is covered with noticeably thickened (up to 6 mm) at the spines (echinulae). Broadly, there are two apex. Teliospores of M. cumminsii are types of urediniospores, one type having described as having thickened (3–5 mm) relatively small urediniospores with an upper walls, but with prominent apical evenly echinulate surface and the other pores. having relatively large urediniospores often with a smooth patch on the surface. Urediniospores of the Melampsora occur- Basidiospores and spermagonia ring on Sect. Leuce (M. populnea complex), M. ciliata and M. pruinosae are relatively As in willow rusts, basidia and basidio- small (less than 30 mm long) and evenly spores are produced from overwintered echinulate. Others on sections Aigeiros and teliospores in spring. Each basidium pro- Tacamahaka are relatively large (maximum duces four spherical basidiospores which length 35–50 mm) and often have a smooth infect alternate hosts and form sperma- area on the surface of spores. As an excep- gonia. Spermagonia are either sub-cuticular tion, M. microspora has small uredinio- or sub-epidermal, and often form as groups, spores and its urediniospore walls are especially on dicotyledonous plants. mostly smooth and only faintly verrucose. The location of the smooth area on the surface of urediniospores can be characteris- tic of certain species. For example, Aecia and aeciospores urediniospores of M. larici-populina and M. allii-populina have a smooth surface Aeciospores of poplar rusts are produced at the apex, while M. medusae has a on the alternate hosts as the consequence smooth surface on the equatorial zone. of fertilization between spermatia and Melampsora × medusae-populina, a pre- spermogonia. The majority of macrocyclic sumed hybrid between M. larici-populina poplar rusts produce small aecia (a few and M. medusae, has a smooth surface both millimetres or less). However, M. rostrupii at the apex and on the equatorial zone. In M. forms large aecia (up to 3 cm on stems) on larici-tremulae (M. laricis), urediniospores the alternate host Mercurialis perennis are sometimes found to have a smooth area (Wilson and Henderson, 1966). M. pulcher- on the equator (Chapter 8). In some species, rima, which is confined to the Mediterra- such as M. larici-epitea, M. medusae and nean, is usually treated as a synonym of M. M. occidentalis, urediniospore walls are rostrupii (Bagyanarayana, 1998; Cellerino, often thickened at the equator. 1999). This rust is similar to M. rostrupii in 56 Ming Hao Pei and Yan Zhong Shang m) m; m; m m m m; m; m; m; m m m m m m m m mthick mthick m m mthick mthick m m 6–10 7–10 7–13 10–17 10–15 3–11 8–9 7–12 m long m thick, mthick × × × × × × × × m m m 35–60 wall 1–1.5 slightly thickened at the apex (2.5–3 wall 1 wall 1–1.5 40–55 wall 1–2 wall 1 20–45 25–48 25–45 40–60 wall 1–2 40–53 30–40 Size and wall of teliospores mm mm 1 mm Hypophyllous, sub-epidermal; 0.25–1 Hypophyllous, sub-epidermal; 1 Mostly hypophyllous, sub-epidermal Epiphyllous, sub-epidermal Mostly hypophyllous, sub-epidermal; up to 0.5 mm Hypophyllous, sub-epidermal; Hypophyllous; minute Position and size of telia c. m; m m; m; m; m; m m m m m; m 23–51 m m m m m m 13–25 mthick mthick mthick mthick × m m m m 14–22 13–22 7–15 8–17 12–18 12–18 × × × × × × m m wall evenly thick, 2–3 50–60 wall 3–5 40–54 thick at the apex wall up to 10 thick at the apex Up to 60 wall 3–6 34–65 40–45 wall 3–5 Wall up to 10 45–85 35–50 wall 3–6 Size and wall of paraphyses ; . m; m m; m; m; m; m; m; m m m m m m m m m m mthick mthick mthick mthick m m m m Populus 11–18 15–23 12–19 13–20 14–22 10–15 15–20 13–18 m long; m thick, × × × × × × × × m m m m m at the equator m smooth at the apex; wall evenly thick, 2–4 24–38 23–35 evenly echinulate; wall 2.5–3 thickened up to 10 17–26 evenly echinulate evenly echinulate; wall 1.5–2 30–50 wall 2 smooth at the apex; oftenupto7 thick at the equator often smooth at the equator Size and wall of urediniospores 15–22 evenly echinulate; wall 1.5–2 19–38 wall 1.5–2 mm 0.5 Mostly hypophyllous, usually less than1mm hypophyllous Hypophyllous; 0.3–0.5 mm 0.3–0.5 mm Mainly hypophyllous Position and size of uredinia c. species/forms described on ; m Hypophyllous 16–29 m, m m Hypophyllous, m Hypophyllous; m Mainly m Hypophyllous 21–35 m m m m m m m 13–22 14–19 16–21 14–19 12–16 12–20 17–27

Melampsora mthick × × × × × × × m wall 2 wall thickened at the equator 17–23 14–17 14–23 19–26 17–22 Size of aeciospores 20–33 mm mm mm 1mm 0.5–1 0.75 Up to 4 0.3–1.5 mm Length of aecia Morphological characteristics of Barcl. Hypophyllous 18–26

columbiana × (Far Eastern) 14–27 Table 4.2. Species/form M. abietis-populi Imai M. alli-populina Kleb. M. ciliata M. Newc. M. larici-populina Kleb. M. larici-tremulae Kleb. (European) M. magnusiana Wagner M. medusae Thüm. Melampsora Species on Populus 57 ; m; m m m m m m m m; m; m; m m m mthick mthick m m m thick at m 10–20 7–11 10–13 10–13 6–16 7–12 6.5–13 mthick mthick × × × × × × × m m m at the apex m sides, thickened up to 6 wall 1–2 40–66 40–52 wall 1–1.5 40–50 30–68 20–50 wall 1–2.5 22–45 Teliospores in 1 or 2, rarely 3, layers, 30–45 wall 1 wall 1 mm mm mm mm 0.5 Hypophyllous, sub-epidermal, up to 0.6 Hypophyllous, sub-epidermal; Hypophyllous; 0.5–1 Mostly hypophyllous, sub-epidermal Mainly epiphyllous Amphigenous, sub-epidermal Amphigenous, sub-epidermal; 0.3–0.8 c. m; m m; m; m; m; m m m m m; m; m m m m mthick 15–25 m m thick at mthick mthick m × m m 20–25 15–23 8–16 16–20 16–25 mthick × × × × × m up to 15 m at the apex × m wall 3–7 the apex 40–60 Up to 75 up to 6.5 wall 3–6 46–55 wall 1.5–4 60 wall 5 18–50 thickened up to 12 45–60 42–62 wall up to 16 thick at the apex m; m; m; m; m; m; m; m m m m m m m m thick, mthick m m 17–13 12–16 12–25 14–18 11–13 16–18 16–25 morthick mthick × × × × × × × m m mthick m m at the equator m m at the equator m in the equator m m evenly echinulate, wall 2–3 thickened up to 8 or thickened up to 6 30–50 evenly echinulate; wall 2 14–23 smooth at the apex and the equator, thickened up to 10 faintly verrucose, almost smooth; up to 4 25–50 18–25 wall 3 11–17 wall 3–4 smooth at the apex; often thickened at the equator 26–44 mm mm mm 1 Mainly hypophyllous; 0.5–1.5 mm 0.3–0.5 Mainly hypophyllous; sub-epidermal Mostly hypophyllous Amphigenous 20–28 Hypophyllous; 0.2–0.4 c. m Hypophyllous, m; m Hypophyllous, m m m 11–17 22–27 11–17 × × × wall thickened at the equator 15–22 26–35 13–24 on mm mm on stems mm 0.3–1 mm Up to 20 Up to 5 leaves, up to 30 Shang, Spiers Tranz. et Eremeeva Jacks. Rostr. Tranzsch. Wagner M. medusae- populina M. microspora M. multa Pei et Yuan M. occidentalis M. pinitorqua M. pruinosae M. rostrupii 58 Ming Hao Pei and Yan Zhong Shang

its host range (M. pulcherrima occurs on principis-rupprechtii Mayer, Picea wilsonii P. alba and alternates on Mecurialis Mast, Picea meyeri Rehd. et Wils., Pinus annosum). According to the literature tabuliformis Carr and Sabina chinensis (L.) (Gäumann, 1959), aeciospores of M. rost- Antoine (Shang et al., 1986). However, the rupii are ovoid to oblong, rarely spherical possibility of M. multa having alternate and almost always polygonal, while aecio- host(s) may not be ruled out as teliospores spores of M. pulcherrima are spherical to of the rust germinated well and produced ellipsoid, rarely oblong, and polygonal only abundant basidiospores in the inoculation when immature. M. pinitorqua also forms experiments. aecia extended (up to 2 cm) along the For both M. occidentalis and M. pine needles. The size of aeciospores in medusae, host alternation appears to be European populations of M. larici-tremulae obligatory for their existence in western appears to be smaller than those in the Far Canada (Ziller, 1974). It appears that M. East (Table 4.2). larici-populina and M. larici-tremulae also require the alternate host larch to continue their existence in north-eastern China (M.H. Pei and Y.Z. Shang, north-eastern China, Host Alternation and Overwintering 1981, unpublished observations). On the other hand, M. pinitorqua was found to The two rust species native to North produce uredinia on young shoots of aspen America, M. occidentalis and M. medusae, before signs of infection appeared on the have a wide range of conifers as their alter- aecial host, Pinus, in costal Italy (Longo nate hosts. M. occidentalis infects Abies, et al., 1975). M. magnusiana and M. rostrupii Larix, Picea, Pseudotsuga and Pinus at the have also been reported to overwinter as aecial stage. In addition to these conifers, mycelia in infected buds of poplars in China M. medusae is also capable of infecting (Xu, 1998). Tsuga. Such wide aecial host range is rather unusual among the rust fungi, considering that the majority of rust genera alternating on conifers have only one host genus as the Distribution and Spread aecial host. Some poplar rust species are separated Current distribution maps of poplar rusts solely on the basis of having different alter- provide some classic examples of the nate hosts. For example, M. larici-tremulae, spread of plant pathogens between conti- M. pinitorqua, M. rostrupii and M. magnusi- nents. M. larici-populina, the most wide- ana, all occur on aspens or white poplars spread of all poplar rust species, is native to and share a similar morphology. However, Eurasia but has now spread to Australasia, M. larici-tremulae alternates on Larix, M. North and South Americas. Both M. pinitorqua on two-needled species of Pinus, occidentalis and M. medusae are native M. rostrupii on Mercurialis and M. magnusi- to North America. While M. occidentalis ana on Chelidonium and Corydalis. Of the is still confined to North America, M. poplar rusts native to Eurasia, M. abietis- medusae now occurs in Europe, Australasia populi is the only species having Abies as and southern Africa. In Europe, M. the alternate host. In M. allii-populina, two medusae was first found near Barcelona, formae speciales, f. sp. allii-populina having Spain (Fragoso, 1925) and then in southern Allium and Arum as aecial hosts and f. sp. France (Dupias, 1943). Later, it was found muscaridis-populina having Muscari as in Portugal and further French locations aecial host, were recognized (Gäumann, (J. Pinon, Nancy, 2003, personal 1959). communications). With M. multa, several inoculation Members of the Salicaceae are not attempts using basidiospores failed to native to Australasia, and all the poplars produce aecia on the tested Larix grown in Australia and New Zealand have Melampsora Species on Populus 59

been introduced. M. medusae was first verrucose urediniospores, was recorded in detected in Australia in January 1972 on the former USSR and Iran (Bagyanarayana, poplars near Sydney (Walker and Hartigan, 1998). In the former USSR, it was found on P. 1972). Within 2 months this rust had spread nigra in Uzbekistan and on P. usbekistanica over a large area from Melbourne to south (= P. nigra var. afghanica Ait. et Heml.) Queensland. The Eurasian M. larici- in Tadjikistan (Z. Azbukina, Vladivostok, populina was found near Sydney 13 months 2003, personal communications). M. multa, later, and within 2 months had spread to which forms teliospores in 1–3 layers, most of New South Wales. It is not yet clear was found on P. × euramericana clones how these pathogens were introduced into in Liaoning Province, north-eastern China Australia, but infected propagating material (Shang et al., 1986). that had been imported illegally was the most likely avenue (Wilkinson and Spiers, 1976). Both rusts were found in New Zealand in 1973. The introduction of these Interspecific Hybridization rusts to New Zealand most likely occurred via high-trajectory wind currents from Two interspecific hybrids, M. medusae- Australia (Wilkinson and Spiers 1976; populina and M. × columbiana have been Close et al., 1978). described on poplars. M. larici-populina is now well estab- M. medusae-populina caused an out- lished in New Zealand, unlike M. medusae, break of rust on some previously resistant which occurs rarely. The widespread occur- clones of P. × euramericana and P. rence of M. larici-populina is mainly due to deltoides × P. yunanensis in New Zealand in the strong wind currents generally experi- March 1991 (Spiers and Hopcroft, 1994). enced within New Zealand, and successful This rust exhibited various morphological overwintering on semi-evergreen Lombardy and physiological characteristics of both M. poplars widely planted in the North larici-populina and M. medusae. For exam- Island and Nelson (Viljanson-Rollinson ple, urediniospores of M. medusae-populina and Cromey, 2002). Alternate hosts, such had smooth areas both at the apex and the as larch, may also play an important role equator. This rust appeared only in 1991 and in providing inoculum throughout the year died out (Spiers and Hopcroft, 1994; Spiers, (Spiers, 1990). 1998). It is probable that M. medusae- In North America, Newcombe and populina may not be fit enough to complete Chastagner (1993) reported that in 1991 two a full sexual life cycle. There are many poplar leaf rusts (M. larici-populina and M. examples in nature where hybrids between medusae f. sp. deltoidae) were detected for species or forms are often unfit to produce the first time in the Pacific Northwest. Pinon the next generation. In the willow rust M. et al. (1994) reported that M. larici-populina larici-epitea, it was found that F1 hybrids was found in 18 counties of California. between f. sp. larici-epitea typica and f. sp. They collected uredinia from two separate larici-retusae were predominantly sterile locations in California and one location in (Pei et al., 1999). Washington, and compared their virulence Newcombe et al. (2000) reported the patterns with European races (coded as E1, occurrence of natural hybrids between M. E2, . . .) using a set of poplar differentials medusae and M. occidentalis in the Pacific (see Chapter 12, this volume). It was shown Northwest, USA, and named them as that that race E1 was predominant in both M. × columbiana. This hybrid taxon showed California and Washington, but that three intermediate characters between M. med- other un-named races existed at a low usae and M. occidentalis in uredinial and frequency. Races E2 and E3 were not found telial morphology. The hybrids frequently in any of the locations. contained ribosomal ITS sequences from M. microspora, which differs from other both M. medusae and M. occidentalis. Some poplar rusts by having small and faintly also contained homozygous ITS sequences 60 Ming Hao Pei and Yan Zhong Shang

of either M. medusae or M. occidentalis, relationships among poplar rusts. It is indicating that the primary F1 hybrids may expected that applications of other have produced further generations through molecular techniques, such as amplified the sexual life cycle. Examination of the fragment length polymorphism (AFLP) and herbarium specimens, which were nearly a microsatellites, will benefit research on pop- century old, suggested that this hybrid group ulation genetics and disease epidemiology was widely distributed in North America. of poplar rusts.

Acknowledgements Concluding Remarks This study was funded by the Department As there are fewer species in Populus for Environment, Food and Rural Affairs (30–100 species) than in Salix (300–500 (DEFRA), UK, and the European Union. species), the taxonomy of poplar rust is Rothamsted Research receives grant-aided expected to be less complicated than that of support from the Biotechnology and Biolog- willow rusts. Yet, current knowledge does ical Sciences Research Council of the UK. not allow satisfactory revision of the taxon- omy of Melamspora on poplars. It should be pointed out that existing records of the host range of some poplar rusts need to be References treated with caution. For example, accord- ing to the records (Bagyanarayana, 1998), Azbukina, Z.M. (1974) Rust Fungi of the Soviet Far P. alba has been included in the host range East [in Russian]. Nauka Publishers, Moscow, of M. latici-populina. However, mycologists pp. 120–140. with experience of working on this rust Bagyanarayana, G. (1998) The species of Melampsora would feel that such records may be on Populus (Salicaceae). In: Jalkanen, R., Crane, P.E., Walla, J.A. and Aalto, T. (eds) Proceedings incorrect, since there is no confirmed of the First IUFRO Rusts of Forest Trees W.P. evidence indicating that M. larici-populina Conference, 2–7 August, Saariselkä. Finnish causes infections on aspens or white Forest Research Institute, Rovaniemi, pp. 37–51. poplars. A similar case may be found Bean, W.J. (1976) Trees and Shrubs Hardy in the with M. occidentalis, which has also been British Isles, Vol. IV, 8th edn. John Murray, recorded on P. alba. London, pp. 293–328. Several species of poplar rusts, M. Cellerino, G.P. (1999) Review of poplar diseases: 3. microspora, M. cumminsii, M. osmaniensis Diseases caused by fungi. Available at website and M. multa, were described on the basis http://www.efor.ucl.ac.be/ipc/pub/celle01/ that they are morphologically different from celle01.htm (accessed 27 September 2004). Close, R.C., Moar, N.T., Tomlinson, A.I. and Lowe, other species of poplar rusts. Apart from A.D. (1978) Aerial dispersal of biological mate- the descriptions of the uredinial and telial rial from Australia to New Zealand. International morphology, little is known of their occur- Journal of Biometeorology 22, 1–19. rence, life cycle and genetic relationships Dupias, G. (1943) Contribution à l’étude des with other poplar rusts. It is not yet certain Urédinées de la Haute-Garonne. Bulletin de la whether they are genetically distinct species Societe d’Histoire Naturelle de Toulouse 78, or occasional variants which may occur 32–52. in nature. Further surveys of rusts from FAO (1979) Poplars and Willows in Wood Production their original localities will help to under- and Land Use. FAO Forestry series 10. Food and stand whether they exist as established Agriculture Organisation of the United Nations, Rome. populations. Fragoso, R.G. (1925) Flora Iberica. II. Uredinales. Recent studies of ribosomal DNA Museo Nacional de Ciencias Naturales, Madrid, sequences of poplar rusts (Tian et al., 2004; p. 424. Chapter 8) have given revealing insights Gäumann, E. (1959) Die Rostpilze Mitteleuropas. into the genetic identity and evolutionary Beiträge zur Kryptogamenflora Schweiz 12. Melampsora Species on Populus 61

Buchdruckerei Büchler and Co., Bern, Spiers, A.G. (1998) Melampsora and Marssonina Germany, pp. 130–144. pathogens of poplars and willows in New Hiratsuka, N., Sato, T., Katsuya, K., Kakishima, M., Zealand. European Journal of Forest Pathology Hiratsuka, Y., Kaneko, S., Ono, Y., Sato, S., 28, 233–240. Harada, Y., Hiratsuka, T. and Nakayama, K. Spiers, A.G. and Hopcroft, D.H. (1994) Comparative (1992) The Rust Flora of Japan. Tsukuba studies of the poplar rusts Melampsora medusae, Shuppankai, Ibaraki, Japan, pp. 271–300. M. larici-populina and their interspecific hybrid Hylander, N., Jørstad, I. and Nanfeldt, J.A. (1953) M. medusae-populina. Mycological Research Enumeratio Uredinearum Scandinavicarum. 98, 889–903. Opera Botanica 1, 1–102. Sydow, P. and Sydow, H. (1915) Monographia Longo, N., Moriondo, F. and Naldini Longo, B. (1975) Uredinearum III. Bornträger, Leipzig, The status of Melampsora pinitorqua Rostr. in pp. 334–350. Italy. European Journal of Forest Pathology 5, Tian, C.M., Shang, Y.Z., Zhuang, J.W., Wang, Q. 147–152. and Kakishima, M. (2004) Morphological and Newcombe, G. and Chastagner, G.A. (1993) First molecular phylogenetic analysis of Melampsora report of the Eurasian poplar leaf rust fungus, species on poplars in China. Mycoscience 45, Melampsora larici-populina, in North America. 56–66. Plant Disease 77, 532–535. Viljanson-Rollinson, S.L.H. and Cromey, M.G. (2002) Newcombe, G., Stirling, B., McDonald, S. and Pathways of entry and spread of rust pathogens: Bradshaw, H.D. (2000) Melampsora × Implications for New Zealand’s biosecurity. columbiana, a natural hybrid of M. medusae New Zealand Plant Protection 55, 42–48. and M. occidentalis. Mycological Research 104, Walker, J. and Hartigan, D. (1972) Poplar rust in 261–274. Australia. Australasian Plant Pathology Society Pei, M.H., Royle, D.J. and Hunter, T. (1999) Hybridi- Newsletter 1, 3. sation in larch-alternating Melampsora epitea Wang, C. and Fang, C.F. (1984) Salicaceae. Flora of (M. larici-epitea). Mycological Research 103, the People’s Republic of China, Vol. 20 (2) [in 1440–1446. Chinese]. Science Press, Beijing, pp. 2–78. Pinon, J., Newcombe, G., and Chastagner, G. (1994) Wilkinson, A.G. and Spiers, A.G. (1976) Introduction Identification of races of Melampsora larici- of the poplar rusts Melampsora larici-populina populina, the Eurasian poplar leaf rust, on and M. medusae to New Zealand and their Populus species in California and Washington. subsequent distribution. New Zealand Journal of Plant Disease 78, 1011. Science 19, 195–198. Shain, L. (1988) Evidence for formae speciales in the Wilson, M. and Henderson, D.M. (1966) The British poplar leaf rust fungus, Melampsora medusae. Rust Fungi. Cambridge University Press, Mycologia 80, 729–732. Cambridge, UK. Shang, Y.Z. and Pei, M.H. (1984) Study on the leaf rust Xu, M.Q. (1998) Leaf rust of Populus section Leuce in of Davids European Aspen caused by Melam- China. In: Jalkanen, R., Crane, P.E., Walla, J.A. psora larici-tremulae Kleb. [in Chinese]. Journal and Aalto, T. (eds) Proceedings of the First of North-easternForestry Institute 12 (1), 47–55. IUFRO Rusts of Forest Trees W.P. Conference, Shang, Y.Z. and Pei, M.H. (1988) Different resistance 2–7 August, Saariselkä. Finnish Forest Research to Melamspora laricis Hart. and Melamspora Institute, Rovaniemi, pp. 53–56. larici-populina Kleb. among three sections and Ziller, W.G. (1955) Studies of western tree rusts: II. hybrids of poplar [in Chinese]. Journal of Melampsora occidentalis and M. albertiensis, Northeast Forestry University 16(4), 12–20. two needle rusts of Douglas fir. Canadian Journal Shang, Y.Z., Pei, M.H. and Yuan Z.W. (1986) A new of Botany 33, 177–188. rust fungus on poplars [in Chinese]. Acta Ziller, W.G. (1974) The Tree Rusts of Western Mycologia Sinica. Supplement 1, 180–184. Canada. Publication No. 1329. Canadian Spiers, A.G. (1990) Melampsora leaf rusts of poplar. Forestry Service, Environmental Canada, Forest Pathology in New Zealand 20, 1–6. Ottawa, Canada. This page intentionally left blank 5 Variability and Population Biology of Melampsora Rusts on Poplars

Pascal Frey1, Pierre Gérard2, Nicolas Feau3, Claude Husson1 and Jean Pinon1 1UR Pathologie Forestière, INRA, F-54280 Champenoux, France; 2Laboratoire Ecologie, Systématique et Evolution, UMR ENGREF-UPXI-CNRS 8079, Université Paris-Sud, 91405 Orsay, France; 3Centre de Recherche en Biologie Forestière, Université Laval, Sainte-Foy (QC), G1K 7P4, Canada

Background collective species to include four species which are difficult to distinguish in their There are eight Melampsora species morphology, M. pinitorqua, M. larici- infecting poplars in Europe (Pinon, 1973; tremulae, M. rostrupii, and M. magnusiana Cellerino, 1999). Three of them, M. larici- (Chapter 1, this volume). Since aspens and populina, M. allii-populina, and M. white poplars are not widely planted for medusae are pathogenic on the poplars of commercial cultivation and exist only as the sections Aigeiros and Tacamahaca, i.e. wild and ornamental trees in Europe, the P. nigra, P. deltoides, P. trichocarpa, and incidence of rust on these species has a their interspecific hybrids. Most of the much lower economic impact. commercial poplar cultivation in Europe is In this review, we will focus mainly made up of P. × euramericana (P. deltoides × on the three Melampsora species attacking P. nigra) and P. × interamericana (P. tricho- poplars of the Aigeiros and Tacamahaca carpa × P. deltoides) hybrids. Rust was the sections. Among these three species, M. most damaging disease of poplars in the larici-populina is responsible for most of past decade (Chapter 12, this volume). Five the economic losses, causing the breakdown other Melampsora species, M. pinitorqua, of several important resistance genes which M. larici-tremulae, M. rostrupii, M. mag- had been released (Chapter 12, this volume). nusiana, and M. pulcherrima, are patho- At present, M. allii-populina is much less genic on species of the Populus (formerly frequent and its economic impact is neg- Leuce) section, i.e. P. alba, P. tremula, and ligible (Frey and Pinon, 1997). The North their hybrids. Although these five species American species, M. medusae, present are difficult to distinguish, since they have in Europe since the early 20th century, is in common evenly echinulated uredinio- very rare and restricted to south-western spores, Pinon (1973) proposed a diagnosis Europe (south-western France, Spain and key based on the morphology of the Portugal; Pinon, 1986, 1991). Although urediniospores and the paraphyses. Some registered as a quarantine pathogen in the authors follow the proposal of Wilson and EU, this species causes no economic losses Henderson (1966) to adopt M. populnea as a in Europe.

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 63 64 P. Frey et al.

Interspecific Variability (Zambino and Szabo, 1993; Nakamura et al., 1998; Vogler and Bruns, 1998; Hantula The advances of molecular biology have et al., 2002; Maier et al., 2003). In order brought new approaches to investigations to determine the phylogenetic relationships of rust fungi, and molecular tools are among the poplar Melampsora, we studied increasingly being used in phylogenetic the ITS sequence of nine isolates of M. studies, DNA-based diagnostics and the larici-populina, M. allii-populina and M. study of genes involved in pathogenicity. medusae. Additional sequence information available from the GenBank database (http://www.ncbi.nlm.nih.gov/), including a sequence from M. occidentalis, was added Comparison of internal transcribed to this study (Table 5.1). The full-length ITS spacer sequences sequences (ITS1, 5.8S, ITS2) were aligned using MULTALIN software (http://www. The nuclear ribosomal DNA, and especially toulouse.inra.fr/multalin.html) (Corpet, the internal transcribed spacer (ITS) 1988) and a phylogenetic tree was con- regions, has widely been used to assess structed using parsimony method in PAUP genetic variability at the species level 4.0, using a heuristic search with the in many fungal genera, including rusts default settings (Swofford, 2002). The tree

Table 5.1. ITS sequences of Melampsora spp. used in this study.

Year of GenBank Species Isolate code Original host collection Collection site accession Sourcea

M. larici- 95XA1 P. × interamericana 1995 N France AY375267 (1) populina ‘Beaupré’ 93ID6 P. × euramericana 1993 NE France AY375268 (1) ‘I 45-51’ 97A1 P. × euramericana 1997 Morocco AY375269 (1) 97J10 P. × euramericana 1997 South Africa AY375270 (1) ‘I 488’ 9014-A P. maximowiczi × 2002 Québec, AY429656 (2) P. balsamifera Canada GHOYAH00-2 P. × euramericana 2000 SW England AY444774 (3) ‘Ghoy’ TSH-R16975 P. nigra ‘Italica’ 2001 Jilin, China AB116836 (4) M. allii- 96B6-1 Allium vineale 1996 NE France AY375271 (1) populina 96M24-1 Muscari comosum 1996 NE France AY375272 (1) BEAUPMK95 P. × interamericana 1995 N England AY444773 (3) ‘Beaupré’ TSH-R04141 P. laurifolia 1986 Xinjiang, China AB116875 (4) M. medusae 88MM1 P. × euramericana 1988 SW France AY375273 (1) ‘Gaver’ 88MM2 P. × interamericana 1988 SW France AY375274 (1) ‘Beaupré’ 97CN1 P. × interamericana 1997 SW France AY375275 (1) ‘Boelare’ 97-4 P. deltoides 1997 South Carolina, AF087711 (5) USA M. occidentalis 97-13 P. trichocarpa 1997 Washington AF087710 (5) State, USA aSource: (1) This study, (2) Innes et al. (2004), (3) Pei et al. (2004), (4) Tian et al. (2004), (5) Newcombe et al. (2000). Variability and Population Biology of Melampsora on Poplars 65

obtained (Fig. 5.1) shows three clearly 2004). Exactly the same ITS sequence was distinct clades: M. larici-populina, M. found in one isolate from France, two allii-populina, and one clade containing isolates from Canada (Innes et al., 2004) and M. medusae and M. occidentalis. The inter- 16 isolates from China (Tian et al., 2004), specific polymorphism is relatively high whereas a few single nucleotide poly- (average identity 87–90%), whereas the morphisms (SNPs) were found in other intraspecific polymorphism within isolates isolates from France, Morocco, South Africa of the same species is limited to a few point and England (Fig. 5.1). One surprising mutations (average identity 99–100%). feature is the position of M. occidentalis in Recently, M. larici-populina was detec- this tree: the ITS sequence of M. occidentalis ted in north-eastern Canada (Innes et al., differs from that of M. medusae only by one

Fig. 5.1. Phylogenetic tree obtained with the aligned internal transcribed spacer (ITS) sequences of Melampsora spp. listed in Table 5.1. The numbers above the branches are the bootstrap values from 1000 replicates. GenBank accession numbers are indicated. 66 P. Frey et al.

TT→CG substitution at position 78–79 absence of M. medusae in the producer’s (Newcombe et al., 2000), although the two nursery. Therefore, since 1993, all the species differ clearly for several characteris- commercial nurseries producing poplar tics, such as the morphology of uredinio- saplings or cuttings in France have been spores and the host range. Clearly, the ITS inspected annually by the French Plant region does not discriminate between these Protection Service (Laboratoire National de two species sufficiently. la Protection des Végétaux, LNPV) for the presence of M. medusae. The official detec- tion method is based on observation of the morphological characters of the uredinio- Development of species-specific polymerase spores. No M. medusae has been detected chain reaction primers and application to since 1998, even in south-western France. the detection of M. medusae In collaboration with LNPV, we assessed the polymerase chain reaction (PCR)-based Although M. larici-populina, M. allii- detection method, along with the official populina and M. medusae are easily method. In 2002, 333 rust samples were distinguishable under a light microscope at collected in 154 commercial nurseries the uredinial stage, species-specific primers throughout France. All the samples were were developed in order to: (i) distinguish tested by PCR with the three primer pairs. M. larici-populina and M. medusae at The results of the molecular detection the aecial stage on larch; (ii) distinguish method were consistent with those of the between the three species at the telial stage morphological diagnosis: M. larici-populina on fallen poplar leaves; and (iii) detect M. was detected in 87% of the samples, M. medusae when present at a low frequency allii-populina in 16% of the samples, and mixed with M. larici-populina and/or M. medusae was never detected. In 2003, M. allii-populina. From the alignment of although the number of samples collected the ITS sequences, three primer pairs was reduced greatly due to the exceptional were designed (unpublished data). The drought that year, the results were similar three primer pairs were tested on a world- to those in 2002, without any occurrence of wide collection of 110 isolates belonging M. medusae. In the future, the molecular to the three species. All the isolates detection method will be proposed as the resulted in a positive amplification with official method for M. medusae detection, the corresponding primer pair. No cross- since the detection threshold is reduced by reaction was observed between the three 10,000-fold, compared to the morphological species. method. We also determined the detection threshold of the M. medusae-specific primers. By adding serial dilutions of M. medusae DNA into M. larici-populina Interspecific Hybrids in Poplar Rusts DNA, the detection threshold was found to be 10−5 to 10−6, which corresponds to less In the 1990s, two interspecific hybrids of than 10 urediniospores of M. medusae Melampsora spp. were discovered in two within a sample containing 2 mg (i.e. different geographic areas. M. medusae- 800,000) urediniospores. The same sensitiv- populina, a hybrid of M. larici-populina ity was found by adding serial dilutions and M. medusae, was first discovered in of M. medusae urediniospores into M. New Zealand in 1991 (Spiers and Hopcroft, larici-populina urediniospores. 1994) and was subsequently found in Since M. medusae is registered as a South Africa. M. × columbiana, a hybrid quarantine pathogen in the EU (Anon., of M. occidentalis and M. medusae, was 2000), the trade of poplar plants between discovered in 1995 in the Pacific North- and within EU countries is subjected to west region of the USA (Newcombe et al., a phytosanitary passport which states the 2000). Variability and Population Biology of Melampsora on Poplars 67

Melampsora medusae-populina Melampsora ¥ columbiana

M. medusae-populina was first discovered Melampsora × columbiana, a hybrid of M. in New Zealand in 1991 (Spiers and occidentalis and M. medusae, was discov- Hopcroft, 1994), where both M. larici- ered in 1995 in the Pacific Northwest region populina and M. medusae were introduced of the USA by Newcombe et al. (2000). in 1973 (Van Kraayenoord et al., 1974). This This new rust taxon was discovered after new rust taxon presents several morpho- many P. × interamericana cultivars became logical characters which are intermediate infected by rust. This hybrid arose after between the two supposed parental species, M. medusae was introduced to the the most evident being the presence of both Pacific Northwest in 1991, where only M. apical and equatorial smooth patches on the occidentalis was present (Newcombe, surface of the urediniospores. The rust was 1998). Although less obvious than in discovered after it attacked several ‘rust- the case of M. medusae-populina, the resistant’ poplar cultivars (Spiers and Hop- morphology of the urediniospores of croft, 1994). These authors hypothesized M. × columbiana is intermediate between that this hybrid arose in Australia, where that of the parental species. The hybrid both supposed parental species are present, status also is evident from the host range: and that it may have been transported to whereas M. medusae is mostly pathogenic New Zealand by wind currents. The mecha- to P. deltoides clones and M. occidentalis nism of hybridization still remains an to P. trichocarpa clones, M. × columbiana enigma. Spiers and Hopcroft (1994) seemed combines both host ranges and is highly to favour the hypothesis of anastomosis at pathogenic on P. × interamericana hybrids. the uredinial, i.e. dicaryotic, stage on pop- Thus the hybrid rust is particularly adapted lar, instead of a heterologous plasmogamy to the hybrid poplars (Newcombe et al., on larch, which is the common alternate 2001). host for both parental species. Newcombe et al. (2000) also studied Independently, we found M. medusae- the ITS region of M. medusae, M. populina in 1997 in samples originating occidentalis and M. × columbiana. The from South Africa (unpublished data). Since ITS sequences of M. medusae and M. both M. larici-populina and M. medusae occidentalis were found to be 99.7% identi- have been described in South Africa since cal, as they only differ by two base pairs 1967 and 1985, respectively, M. medusae- at one position (Fig. 5.1). Of six isolates populina in this country may have origi- of M. × columbiana, two were found con- nated from: (i) transportation from New taining ITS sequences from both parental Zealand or Australia; or (ii) a new hybridiza- species, whereas three presented only the tion event. In order to confirm the hybrid ITS sequence of M. medusae, and one that of status of M. medusae-populina, we studied M. occidentalis. Therefore, the authors sug- the ITS region of four mono-uredinial iso- gested that the hybrids may have produced lates of this taxon. After amplification, the advanced generations such as F2 and/or amplicons were cloned and several clones backcrosses. were sequenced. Within each M. medusae- These two hybrids illustrate perfectly populina isolate, two types of ITS sequences the risk of interspecific hybridization were found, one matching exactly that of between fungal species after a geographical M. larici-populina and the other matching transposition of one (or both) species into that of M. medusae (Frey et al., 1999). Fur- a new region (Brasier, 1995; Newcombe ther confirmation of the hybrid status of M. and Frey, 2002). Phylogenetically close, but medusae-populina was obtained with ran- geographically distant, fungal species may dom amplified polymorphic DNA (RAPD) hybridize when they come into contact in fingerprinting. For most of the RAPD prim- the same niche, and the resulting hybrids ers tested, the hybrid showed DNA bands may become a ‘superpathogen’ in some from M. larici-populina and M. medusae. cases (Brasier et al., 1999). 68 P. Frey et al.

Population biology of Melampsora defined as the mean number of virulences larici-populina per isolate (Andrivon and de Vallavieille- Pope, 1995). Objectives Along with these virulence markers, we also developed molecular markers. RAPD Since M. larici-populina is responsible for markers were used to compare the genetic most of the economic losses in poplar culti- structure of M. larici-populina populations. vation in Europe, our efforts in population Microsatellites (or single-sequence repeat, biology have focused mainly on this spe- SSR) markers are currently being developed. cies. The populations of M. larici-populina These co-dominant markers, suitable for have shown very rapid changes during the automation, will be very useful tools for the past two decades, through the rise of new study of migration, selection and genetic virulence that overcame several resistance drift within and between populations of genes (Chapter 12, this volume). After the M. larici-populina. occurrence of a new virulence, a very rapid spread of the new virulent rust was observed, associated with the appearance of Genetic structure of populations from wild numerous new pathotypes through recom- versus cultivated poplar stands bination (Pinon et al., 1998). In the mean- In order to assess the effect of the nature time, no obvious changes were noticed on of the host population, we compared the the impact of rust on wild stands of P. nigra. structure of M. larici-populina populations Taking advantage of the presence in France from wild and cultivated poplar stands. We of both wild stands of P. nigra and culti- selected wild riparian P. nigra stands along vated stands planted with exotic hybrids two rivers in the French Alps (Durance and × × such as P. euramericana and P. inter- Drac rivers), and cultivated stands in two americana, our studies were aimed mainly main areas for poplar cultivation (Picardie, at comparison of the structure of M. larici- northern France, and Franche-Comté, populina populations in both pathosystems north-eastern France). For each region, sites (Pinon and Frey, 1997; unpublished data). with and without larch (Larix decidua) Furthermore, some aspects of the biology in their vicinity (< 1 km) were selected in and the epidemiology of this pathogen order to take the role of sexual reproduction remained unknown. For instance, the on the structure of M. larici-populina popu- relative role of sexual reproduction versus lations into account. Pathotype analysis asexual survival remained unclear. There- clearly distinguished the populations from fore, we examined more closely the effect wild and cultivated stands. Populations of the presence of larch on the structure of from cultivated stands comprised a M. larici-populina populations. significantly higher (P = 0.0001) number of pathotypes (Np = 13.1 ± 4.0) than those ± Markers used from wild stands (Np = 5.3 2.2). Both the richness and the complexity of the popula- We have described a universal differential tions were significantly higher (P = 0.0001) set of poplar clones which discriminates in cultivated than in wild stands (Fig. 5.2). eight virulences of M. larici-populina Populations from cultivated stands (Chapter 12). All the isolates studied were were composed of numerous pathotypes made from single uredinia and then combining many virulences, such as patho- inoculated on to leaf discs of the poplar type 1–3–4–5–7, which has been the most differential set. Parameters studied were the frequent since 1994 (Pinon et al., 1998; Miot frequency of each virulence, the frequency et al., 1999). In contrast, populations from of each pathotype, the richness of the popu- the wild stands were composed of a few lations assessed with the Alpha, Shannon simple pathotypes, such as pathotypes 0, 2 and Gleason indices (Pinon and Frey, 1997), and 4. These findings are consistent with and the complexity of the populations, those of previous studies (Pinon and Frey, Variability and Population Biology of Melampsora on Poplars 69

Fig. 5.2. Richness and complexity of 51 populations of Melampsora larici-populina collected from wild and cultivated poplar stands in 2001–2003. Circles, wild stands; squares, cultivated stands; black symbols, stands with larches in their vicinity (< 1 km); open symbols, stands without larches in their vicinity.

1997). The high complexity of M. larici- pathotypes (P = 0.001) either for cultivated populina populations in cultivated stands or for wild stands. It also increased the raises the question of the cost of unnecessary richness of the populations (P = 0.001), virulence (Miot et al., 1999; Chapter 12). but did not increase the complexity RAPD markers did not distinguish (P = 0.9). Surprisingly, we did not observe clearly populations of M. larici-populina a reduction of the linkage disequilibria in from wild versus cultivated stands. The the presence of larch, neither for virulence overall genetic distances between popula- markers nor for RAPD markers. tions were low. Nei’s unbiased genetic Furthermore, a more detailed study distance (Nei, 1978) between paired popula- of the space–time dynamics of M. larici- tions ranged from 0.01 to 0.07, suggesting a populina populations along the Durance high gene flow at the country scale. Genetic River gave us a better view of the natural distances were very low between pairs pathosystem. Every year, rust epidemics of populations from the same region. seem to begin in the area where both poplar Similarly, F-statistics showed low values and larch occur, in the upstream of the (FST ranging from 0.02 to 0.09), suggesting river. During summer, the epidemics spread a low degree of differentiation between downstream following the continuous ripar- populations. ian forest of P. nigra. The dates of infection of P. nigra in the downstream part of the river Effect of the presence of larch clearly suggest that there is no asexual survival for urediniospores of M. larici- The presence of larch in the vicinity populina. Thus the only primary inoculum (< 1 km) of the selected poplar stands source under this climate is larch. These significantly increased the number of findings are consistent with previous 70 P. Frey et al.

observations in the cultivated stands (Frey only for about two centuries, whereas the et al., 1998). fungus and its wild host, P. nigra, have Comparison between regions with and coevolved for millions of years. Therefore, without larch plants was also made in order an in-depth study of the population genet- to assess the role of sexual reproduction ics and population dynamics in the wild on the structure of populations of other stands is useful, to have a better view of the larch-alternating Melampsora species. M. cultivated pathosystem. medusae populations from the areas in Canada where both poplar and larch occur, exhibited a higher genetic diversity com- pared to those from the areas in southern Acknowledgements Canada and in the USA where only poplars occur (Bourassa et al., 1998). Similar results This research was partly funded by grants were obtained by comparing sexual popu- from GIP Ecofor and from INRA and DGAL lations of M. larici-epitea on willow from (Ministry of Agriculture). Sweden and asexual populations from Northern Ireland (Samils et al., 2001; Chapter 7). References

Andrivon, D. and de Vallavieille-Pope, C. (1995) Race diversity and complexity in selected populations Conclusions of fungal biotrophic pathogens of cereals. Phytopathology 85, 897–905. Although M. allii-populina and M. medusae Anon. (2000) Council Directive 2000/29/EC of 8 are present in some parts of Europe and May 2000 on protective measures against the can infect cultivated poplar stands (mainly introduction into the Community of organisms P. × euramericana and P. × interamericana harmful to plants or plant products and against their spread within the Community. Official cultivars), most of the damage is caused by Journal L 169, 10/07/2000, 1–112. M. larici-populina. With its ability to over- Bourassa, M., Bernier, L., Milligan, B.G. and Hamelin, come several pathotype-specific resistance R.C. (1998) Sympatry between alternate hosts genes, this species is presently the main affects population structure of poplar rust. In: concern for poplar breeders (Lefèvre et al., Jalkanen, R., Crane, P.E., Walla, J.A. and Aalto, 1998; Dowkiw et al., 2003). There were T. (eds) Proceedings of the First IUFRO Rusts great differences in the structure of M. of Forest Trees Working Party Conference, larici-populina populations from cultivated 2–7 August 1998, Saariselkä. Finnish Forest and from wild poplar stands. The low com- Research Institute, Research papers 712, plexity of M. larici-populina populations pp. 65–69. Brasier, C.M. (1995) Episodic selection as a force in from wild stands may suggest a cost for fungal microevolution, with special reference unnecessary virulence, whereas the high to clonal speciation and hybrid introgression. complexity of populations from cultivated Canadian Journal of Botany 73, S1213–S1221. stands may suggest the opposite. This Brasier, C.M., Cooke, D.E. and Duncan, J.M. (1999) apparent contradiction may result from the Origin of a new Phytophthora pathogen through difference in the rate of evolution of viru- interspecific hybridization. Proceedings of the lence markers versus molecular markers. National Academy of Sciences of the USA 96, Virulence markers have been shown to 5878–5883. evolve very rapidly in the cultivated stands Cellerino, G.P. (1999) Review of poplar diseases. during the past 30 years. However, the International Poplar Commission, FAO. Available at Website www.efor.ucl.ac.be/ genetic distances between populations from ipc/pub/celle01/celle01.htm (accessed 28 cultivated and wild stands were low, sug- September 2004). gesting it is unlikely that there is a strong Corpet, F. (1988) Multiple sequence alignment with barrier between cultivated and wild stands. hierarchical clustering. Nucleic Acids Research Anyway, the cultivated stands have existed 16, 10881–10890. Variability and Population Biology of Melampsora on Poplars 71

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Mirko Liesebach1 and Irmtraut Zaspel2 1Federal Office and Research Centre for Forests, Department of Forest Genetics, Hauptstrasse 7, A-1140 Vienna, Austria; 2Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Eberswalder Chaussee 3A, D-15377 Waldsieversdorf, Germany

Background More than 20 species of the genus Melampsora have been recognized on Over 300 species are described in the genus willows worldwide, and most of them have Salix and hybridization between Salix a complex life cycle and alternate on very species occurs commonly (Lautenschlager- different herbaceous and woody plants Fleury, 1994; Zander, 2000; Newsholme, (Pei et al., 1997). To different species and 2002). This provides a rich genetic resource populations of rust pathogens, species and with which to combat the threat of pest varieties of willows have different degrees and disease attacks through breeding of resistance and tolerance (Hunter et al., (Ahman and Larsson, 1994). Willows are 1996). hosts to a wide range of phytophagous Three species of rusts occur on shrub insect species and fungal pathogens. Leaf willows for biomass. One of them is the rusts caused by Melampsora spp. are highly heterogeneous collective species important fungal pathogens on willows in M. epitea (Kunze et Schm.) Thüm complex. central Europe. Pei et al. (1999a) have defined eight putative Willow rusts can cause severe damage pathotypes within this M. epitea complex. to the leaves and shoots of their host plants Five of these pathotypes belong to the forma and lead to leaf senescence and premature specialis (f. sp.) larici-epitea typica (LET), defoliation, thus debilitating the plant two to f. sp. larici-retusae (LR) and one to f. (Helfer, 1992; Pei et al., 1999a). With the sp. larici-daphnoides (LD). The groupings of introduction of willow short-rotation cop- willow rust by Gäumann (1959) and Butin pice (SRC) plantations in northern and (1960) were similar. Butin (1960) divided north-western Europe, rusts have become M. epitea into five subspecies, including major pathogens in energy forestry, resulting M. larici-epitea. The M. larici-epitea in in reduced growth rates and decreased central Europe and Switzerland was yields by as much as 40% (Parker et al., further divided into six formae speciales 1993; Tucker and Sage, 1999). Severe rust (Gäumann, 1959), including LET, LR and can predispose plants to infections by LD. Much of the pioneering work on the secondary pathogens, which often kill the species identity, host alternation and host plants. range in willow rusts in central Europe

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 73 74 M. Liesebach and I. Zaspel

was carried out in the late 19th to early conservation in central Europe, especially 20th century (reviewed by Gäumann, 1959). in Germany. Since then, little work has been done on willow rusts in central Europe. Klenke (1998) discussed the taxonomy of willow Melampsora Species on Willow in rusts but his work was restricted to Saxonia Germany and their Life Cycles only. There has been no recent investigation The majority of Melampsora species have a on the range of pathotypes of the different macrocyclic life cycle, during which they formae speciales in Germany. These differ- develop five spore stages and alternate on ent types cannot be distinguished by their very different host species (alternate hosts). morphology. They can only be identified The most important alternate host of through standardized pathotyping using Melampsora species infecting willows and a range of willow clones as differentials. poplars in SRC plantations is larch. The life Willow clones used for differentials may cycle of M. larici-epitea is illustrated in Fig. belong to several sections and species. 6.1. During summer, rust can be observed An understanding of genetic variability in mostly on the lower sides of willow leaves pathogens is thought to be highly important as yellow to orange pustules (uredinia). The for the management of diseases of fast- urediniospores are responsible for many growing tree species like willows. At the cycles of asexual reproduction and hence beginning of the 21st century, we conducted the spread of rust disease in a growing sea- a new study to identify and characterize son. Probably as a reaction to low tempera- different rust species and forms posing tures and senescence of leaves, uredinia threats to willows for renewable energy and are transformed into telia which serve as

Fig. 6.1. Life cycle of Melampsora epitea on Salix viminalis and Larix decidua. Genetic Diversity of Melampsora Willow Rusts in Germany 75

the overwintering form of the rusts after leaf Variation in Size and Appearance fall. In spring, teliospores germinate to pro- of Uredinia duce basidiospores which infect alternate hosts. Consequently, fertilization between Recently an investigation (Liesebach and spermatia and receptive hyphae results in Zaspel, unpublished data) was conducted formation of aecia. Inoculations with aecio- to evaluate the occurrence of Melampsora spores from the larch hosts Larix decidua, collected from 28 locations in Germany L. kaempferii and L. × eurolepis on to S. (Fig. 6.2). There were three different types viminalis, S. daphnoides, S. caprea and of locations: willow collections (C, n = 8), hybrid willows showed that all three SRC plantations (P, n = 9) and willow sites species/hybrids of the genus Larix are in the landscape (N, n = 11). For the pur- capable of serving as alternative hosts of pose of comparison, samples from Sweden willow rusts. (3 sites), Northern Ireland (1), Belgium (1), According to the literature, important France (1) and Georgia (1), and from rust species and sub-species in Germany and Populus (6) were also investigated. In all, their main willow hosts and alternate hosts, leaves bearing well-developed uredinia can be compiled as in Table 6.1. In contrast were collected from the 35 locations to Butin (1960), Klenke (1998) described ten (Fig. 6.2). A large proportion of the assessed Melampsora species on Salix and classified willows were S. viminalis and its hybrids. M. repentis as a distinct species, renamed as Altogether, more than 500 willow samples M. arctica Rostr. em. Braun. This rust occurs were studied and the specific phenotypic very rarely on S. aurita. He also placed M. traits of all rusts, as well as the host clones, ribesii-epitea Kleb. in the M. epitea complex. were recorded. During the period of study, Further work is needed to clarify the the incidence and severity of rust changed discrepancies between the systems of Butin from site to site in each year and between (1960) and Klenke (1998) and the taxonomy the years. The rust severity was assessed of the willow rusts. using a 5-step scale, separately for the

Table 6.1. Rust species and formae speciales described by Butin (1960) (B) and Klenke (1998) (K) in Germany.

Rust Willow species Alternate host species Source

Melampsora allii-fragilis Kleb. S. fragilis Allium B, K Melampsora ari-sacina Raabe S. fragilis Arum K Melampsora galanthi-fragilis Kleb. S. fragilis Galanthus K Melampsora amygdalinae Kleb. S. amygdalina, S. triandra, Autecious B, K S. pentandra Melampsora caprearum (DC.) Thün. S. aurita, S. caprea, Larix (not obligate on B, K S. cinerea alternate host species) Melampsora epitea (Kunze et Schm.) Thüm. (a) M. abieti-caprearum Tub. S. caprea Abies B, K (b) M. euonymi-caprearum Kleb. S. cinerea Euonymus B, K (c) M. larici-epitea Kleb. S. viminalis Larix B, K (d) M. repentis Plowr. S. repens Orchidaceae B (e) M. ribesii-purpureae Kleb. S. purpurea Ribes B, K (f) M. ribesii-epitea Kleb. S. caprea, S. cinerea Ribes K Melampsora larici-pentandrae Kleb. S. pentandra Larix B, K Melampsora ribesii-viminalis Kleb. S. viminalis Ribes B, K Melampsora salicis-albae Kleb. S. alba Allium (not obligate on B, K alternate host species) Melampsora arctica Rostr. em. Braun S. repens, S. aurita Orchidaceae K 76 M. Liesebach and I. Zaspel

Fig. 6.2. Locations of sites where Melampsora rusts were assessed and collected. C, willow collections; P, short-rotation coppice (SRC) plantations; N, willow sites in the landscape. expanded leaves and for the younger, disease on the expanded leaves (Liesebach partially expanded leaves (modified after et al., 2002). With some willow species, such McCracken and Dawson, 1992; Ahman, as S. alba hybrids, S. cinerea hybrids, 1998; Hunter and Pei, unpublished) (Table S. daphnoides hybrids, S. nigricans hybrids, 6.2). S. purpurea hybrids, S. schwerinii, and S. × On 55% of the samples, rust pustules undulata, the disease was only observed occurred only on the expanded leaves, while on the expanded leaves. It was noted that, on the other 45%, the younger, partially with the exception of S. schwerinii, only expanded leaves were infected. In many hybrids showed this type of infection. cases, the infection of the younger leaves In some samples of the willow species S. occurred following the appearance of acutifolia, S. adenophylla, S. humboltiana, Genetic Diversity of Melampsora Willow Rusts in Germany 77

S. kazbekensis, S. magnifica, S. megeriana into five groups (Table 6.3). In one of these and S. × sericans, infections were observed groups uredinial pustules were found only only on younger leaves. on the lower side of the leaves, and in a few There were differences on which side of cases on both sides. The most important the leaf surface the pustules developed, i.e. biomass species S. viminalis and its hybrids, whether the pustules were only on the lower S. × dasyclados and S. × aquatica, were or the upper sides, or on both sides, of the included in this group. Salix caprea, S. leaves. In some cases stem infections were daphnoides and their hybrids were placed also found. Table 6.3 lists rust samples into another group which was characterized grouped by willow genotype and the by having pustules only on the lower side in position of the leaves where the uredinia about 50% of the samples and on both sides occurred. About 67% of the infections were of the leaves in the other 50% of the samples. observed only on the lower side of the The third main group included the species leaves. In 31% of samples, the pustules were S. alba, S. aurita, S. fragilis, S. nigra, on both sides of the leaves. Very seldom S. repens, S. triandra and S. × sericans,on (2%) did the pustules appear only on the which pustules were predominantly on both upper side of the leaves. sides of the leaves. In the two remaining According to the results from the assess- groups, the sample size was too small to give ment, the host clones could be grouped a general description.

Table 6.2. Rust disease assessment key for willow clones (Liesebach et al., 2002).

Numerical Category for the Category for the younger, scale expanded leaves partially expanded leaves Description

0 None No rust can be detected 1 Very slight – Only one pustule or a single leaf is infected 2 Slight Slight Leaves bear a few conspicuous uredinia or more, but barely recognizable rust pustules 3 Moderate Moderate Leaves frequently bear rust pustules, up to an average of 1–5% leaf area covered by uredinia 4 Severe Severe Most leaves bear numerous pustules

Table 6.3. List of willow species/hybrids and the frequency of leaf sides on which rust pustules appeared.

Frequency of leaf sides on which rust pustules appeared, and willow hosts

Lower side: 100% About 2/3 50% About 1/3 – Both sides: – About 1/3 50% About 2/3 100%

Willow species/ S. schwerinii S. viminalis S. alba hybrids S. triandra S. humboltiana hybrid S. × undulata S. viminalis S. aurita hybrids S. alba S. kazbekensis S. cineria hybrids hybrids S. caprea S. aurita S. magnifica S. purpurea S. × aquatica S. caprea hybrids S. repens S. megeriana hybrids S. × dasyclados S. daphnoides S. fragilis S. adenophylla S. triandra S. cinerea S. daphnoides S. × sericans S. acutifolia hybrids S. pentandra hybrids S. nigra S. cordata S. fragilis hybrids S. purpurea S. nigricans hybrids S. repens hybrids S. × hirtei 78 M. Liesebach and I. Zaspel

In addition, 60 willow clones (10.9%) S. daphnoides and S. aurita and their showed stem infections. The stem infections hybrids. Furthermore, the size and colour of were frequently found on the clones of S. × the pustules were estimated using a 5-step sericans and S. nigra. There were similar scale (Table 6.4). It was noted that small degrees of infection on leaves and stems in pustules were associated with the light- the clones of S. purpurea and S. caprea yellow colour, while larger pustules were and their hybrids, and S. × smithiana, from associated with colours towards orange. which only small-size samples were col- The results from the assessments of lected. Stem infection was found only rarely the pustule colours were not as clear-cut as on the clones of S. × dasyclados, S. × aqua- those from the assessment of the pustule tica, S. viminalis and its hybrids, S. triandra, sizes. The colour of the uredinia varied between different willow species and species hybrids. Most clones of S. viminalis Table 6.4. Scoring system for the size and the had pustules with light yellow or yellow colour of the rust pustules. colour (Fig. 6.3A). On S. daphnoides clones, Size of the rust pustules Colour of rust pustules pustules were predominantly orange, and light-yellow pustules were missing (Fig. Small Light-yellow 6.3B). The colour step ‘(yellow-) brown’ was Small–medium Yellow rarely found. This colour category might be Medium Yellow-orange associated with old pustules. Medium–large Orange For clones of S. viminalis (grown mostly Large (Yellow-) brown in SRC plantations) and S. daphnoides

Fig. 6.3. The frequencies of pustule colours and sizes in Melampsora on (A) Salix viminalis and its hybrids (n = 191), and (B) Salix daphnoides and its hybrids (n = 52). Genetic Diversity of Melampsora Willow Rusts in Germany 79

(clones with high salicylic acid content), the young shoots of clones appeared 10 or more frequency of the colour and the size of days earlier than those on the clones having the pustules are shown in Fig. 6.3. On both infections only on expanded leaves. species, pustules were assessed from small The hybrid clone S. caprea × S. to large. However, small pustules were viminalis ‘77666’ was found to be highly predominant on S. viminalis (Fig. 6.3A) and susceptible. Disease started very early on the large ones on S. daphnoides (Fig. 6.3B). younger, partially expanded leaves (Fig. 6.4, With selected rust samples, uredinio- bottom) and increased continuously to a spores were measured using a light micro- high level. In the third year, two further scope. Most samples had spores that were clones, S. caprea × S. viminalis ‘Coles’ and more or less round, with a diameter of S. nigra ‘SN2’, were clustered in this group. 15–20 mm. Some samples from S. fragilis had The remaining two groups were represented a different spore size (35–40 mm long and by one clone each. On these clones, rust 12.5–15 mm wide). All the S. fragilis grew in development was different: the infection meadows. According to its morphology and started earlier but was only temporary, and collection sites, the rust was identified as was concentrated on the expanded leaves. M. allii-fragilis. In the newly established field trial at Hann. Münden and the clone collection Waldsieversdorf, S. daphnoides clones had hardly any rust in the year of establishment. Development of Rust Infection Rust infection started during the following years and these clones were severely The development of rust infection was observed on a set of 35 clones and clones grown in a SRC trial over three consecutive years (Liesebach et al., 2002; unpublished data). Rust severity was assessed using a 5-step scale (0–4 numerical scales) (Table 6.2). The assessment was carried out sepa- rately for the expanded leaves and for the younger, partially expanded leaves. Differences were observed in the development of the rust disease between the years. The first appearance of disease varied by about 2 weeks, probably due to the weather conditions. According to the onset and the severity of rust disease, the clones assessed could be assigned to six groups. Between the years there were slight differ- ences in the clones assigned to different groups. A few resistant clones, such as S. schwerinii ‘77077’, S. fragilis × S. alba ‘77802’ and a few S. viminalis clones, did not have any rust pustules, or had only occa- sional pustules. Most clones had moderate rust infections on the expanded leaves (Fig. Fig. 6.4. Development of the Melampsora rust infection assessed on two clones: S. viminalis 6.4, top). The infection started between the ‘79046’ (top) and S. caprea × S. viminalis ‘77666’ 160th and 235th Julian day. A few clones (bottom) in 2002. Closed circles indicate the had moderate rust infections on the partially infection on expanded leaves; open circles indicate expanded leaves and young shoots. The the infection on partially expanded leaves. (From infections on partially expanded leaves on Liesebach et al., 2002.) 80 M. Liesebach and I. Zaspel

affected in July/August and defoliated at the unique to the rust samples from S. caprea. end of August. At the beginning of Septem- The rust samples derived from S. purpurea, ber, the plants flushed again and the tips of S. × sericans, S. nigra, S. aurita, S. acuti- young shoots were immediately infected. folia, S. cordata, S. pentandra, S. repens, S. Soon after, the plants showed dieback triandra and S. × hirtei only caused slight symptoms. infections on some of the tested clones. Most of the rust samples produced visi- ble symptoms 8–10 days after inoculation on the leaf discs. But some developed faster: Pathogenicity Tests the first uredinia occurred 6 days after inoculation and produced a large number of Virulence patterns of the rusts from central pustules within several days. Those ‘fast- Europe in relation to the known pathotypes infecting’ samples were from S. viminalis were examined using the leaf disc method clones as well as several hybrid clones. described by Pei et al. (1993, 1999a). After To evaluate these results, a correspon- inoculation, the leaf discs were incubated dence analysis was conducted using SAS at 16°C with 16 h day−1 illumination at an (Rasch et al., 1998). The result is shown in intensity of about 80 mE m−2 s−1. The num- Fig. 6.5. The first two dimensions explain ber of rust pustules on each leaf disc was 61.3% of total inertia (Dimension 1: 33.5% observed from the 6th day after inoculation and Dimension 2: 27.8%). In this analysis and counted until 2 weeks after inocula- only the samples which had more or less tion. From these observations, ‘infection round spores with a diameter between 15 types’ were assigned as: 0 = no pustules; and 20 mm were included. Samples of M. 1 = up to 5 pustules per leaf disc; 2 = 6–10 allii-fragilis from S. fragilis, which could be pustules per leaf disc; 3 = more than 10 recognized easily by their large spore size pustules per leaf disc. In total, 76 rust were not included. In the plot, several samples were tested on 28 willow clones groups can be distinguished. In one group all (13 S. viminalis,7S. × aquatica and S. × the tested rust samples from S. × aquatica dasyclados,2S. daphnoides,1S. alba,1S. and S. × dasyclados and the willow clones nigra,1S. × stipularis,1S. caprea hybrid, 1 belonging to S. × aquatica and S. × dasy- S. × hirtei,1S. fragilis hybrid). Altogether, clados were clustered. They belonged to at least 43 virulence patterns were found. the forma specialis larici-retusae. A second The virulence patterns were deter- group combined most samples collected mined mainly by the origin of the rust from S. daphnoides. Rust samples of the samples. Samples derived from S. viminalis forma specialis laricii-daphnoides were caused infections mostly on the tested S. more or less clustered on S. daphnoides. viminalis clones. Hence these 22 patterns However, some samples from S. daphnoides showed only a slight variation. The samples could not be placed into this forma specialis, derived from S. × stipularis also infected indicating that there may be further genetic mostly S. viminalis clones. Those from differentiation. A third group, which S. × aquatica infected the tested clones of included all the rust samples from S. S. × aquatica and S. × dasyclados. Samples viminalis clones, was assigned to f. sp. larici- collected from S. fragilis caused infections epitea typica. This forma specialis group only on S. fragilis or did not cause any also contained rust samples from different infections. The infections observed on S. species of willows. daphnoides were mostly caused by rusts The samples from S. caprea could not collected from S. daphnoides. However, be assigned to one of the known formae four more patterns were recorded from rusts speciales of M. epitea. Possibly they collected from S. daphnoides. Also, only a might belong to another rust species (M. slight infection was observed on S. caprea caprearum) or to another form or species in the test using the rust collected from S. (sensu stricto)ofM. epitea that alternates on caprea in the field. Six infection types were aecial hosts other than larch (such as M. Genetic Diversity of Melampsora Willow Rusts in Germany 81

1

M. epitea

0 M. larici-retusa

−1 S.viminalis S. daphnoides S. caprea −2 S. x aquatica Dim. 2 (27.8%) Dim. S. fragilis further S. viminalis S. daphnoides −3 M. larici-daphnoides S. caprea hyb. S. x aquatica S. fragilis hyb. further −4 −10 1 2 3 4 5 Dim.1 (33.5%) Fig. 6.5. Two-dimensional correspondence analysis for 28 willow clones (grey symbols) and for 75 rust samples coming from corresponding willow hosts (black symbols). abieti-caprearum, M. euonymi-caprearum The ITS regions are a convenient target or M. ribesii-epitea). All other rust samples region for molecular identification of fungi, derived from willow species, which were because in fungi the ITS regions can be only represented by a single or few samples, readily amplified with universal primers were not differentiated in Fig. 6.5 (they are that are complementary to sequences within indicated as −). the rRNA genes. Furthermore, the multi- copy nature of the rDNA repeat makes it easy to amplify from small, or diluted DNA samples. Several studies have demonstrated Differentiation Using ITS-RFLP and that the ITS regions are often highly variable Sequencing of ITS unit and LSU among morphologically distinct fungal species, but intraspecific variation is low. The ribosomal DNA (rDNA) of fungi Taxon-selective primers for the ITS regions contains one transcriptional unit with a in the nuclear ribosomal repeat unit are cluster of genes coding for 18S, 5.8S and available for rusts (Gards and Bruns, 1993; 28S rRNAs and two internal transcribed Vogler, 1995). spacers, ITS1 and ITS2 (White et al., 1990). The use of polymerase chain reaction The complex, non-coding and variable ITS (PCR)-based techniques, such as ITS- regions and the coding and conserved 5.8S restriction fragment length polymorphism rRNA gene are useful in measuring close (ITS-RFLP) and sequencing of DNA frag- genealogical relationships, because they ments, made it possible to study genetic exhibit greater interspecies differences than diversity at the molecular level and the the 18S and 28S genes. The coding regions phylogenetic relationships of isolates of within rDNA repeat units are known to be Melampsora spp. In our study, DNA was highly conserved in the non-transcribed extracted from a collection of 205 isolates spacer regions, both between and within (Liesebach and Zaspel, unpublished data), species (Long and Dawid, 1980). of which willow rust isolates accounted 82 M. Liesebach and I. Zaspel

for 199. For comparison, six Melampsora original DNA or RNA target region) using isolates from poplar were also included. the primer pair ITS1 and ITS4, as well as the Protocols for PCR amplification of ITS numbers of the fragments generated by regions and RFLP analysis are described by restriction digestion with TaqI, EcoRI, MboI Liesebach and Zaspel (2004). The primer and SspI. The restriction sites are shown in pair ITS1 (5′ TCC GTA GGT GAA CCT GCG Fig. 6.6. The 5.8S-ITS region of willow rusts G3′) and ITS4 (5′ TCC TCC GCT TAT TGA was in a range from 625 bp to 628 bp. The TAT GC 3′), permitted the amplification of sequences of all the tested willow rusts a single DNA fragment of Melampsora could be aligned in 630 nucleotide containing ITS1, ITS2, 5.8S and partial positions. The uncut amplicon of M. sequences of 18S and 25S rDNA genes, of a larici-populina had a length of 644 bp. total size between 625 and 628 bp. RFLPs The analysis of the sequences of ITS1/ of the 5.8S-ITS region were generated from ITS4 units confirmed most of the patterns the amplified products by restrictions with obtained from the RFLP analyses. Based TaqI, EcoRI, MboIorSspI. Using the four on the sequence information, some main restriction enzymes in combination, a total groups can be distinguished. The nucleotide of 12 RFLP patterns could be distinguished differences between the patterns of the clearly. Table 6.5 shows the frequency of the Melampsora samples from Salix (38) and multi-locus patterns. those from Populus (2) were from 6.7 to 8.5% A set of 40 DNA samples, representing (Table 6.7). The variation between the three most of the patterns detected by RFLP and main groups from Salix was lower (≤ 4.1%). the resulting subgroups, were sequenced. There were two patterns (I, II) within the Table 6.6 gives the length of the uncut first main group. The nucleotide differences amplicon (a PCR product that is a copy of the between the two patterns were 0.3 and 2.2%.

Table 6.5. RFLP patterns of ITS region, frequency and host species of isolates, and information on the collection.

RFLP Melampsora rusts from species/hybrids Isolated from collection sites pattern Frequency (number of isolates) (Fig. 6.2)

I 14 S. alba (2); S. fragilis (8); S. purpurea (1); C6, C8, N1, N2, N3 S. triandra (3) II 3 S. purpurea (3) C4 III 40 S. adenophylla (1); S. alba (2); S. aurita (3 + 1 C1, C3, C4, C5, C6, N1, N3, hybrid); S. caprea (14 + 9 hybrids); S. × sericans N4, N5, N6, N8, N11, (9); S. × stipularis (1) N12, N13, N15, P1, P7 IV 97 S. acutifolia (3); S. daphnoides (9); S. fragilis C1, C2, C3, C4, C5, C6, C8, (2 + 1 hybrid); S. magnifica (1); S. pentandra N1, N2, N3, N5, P1, P2, (1); S. purpurea (2); S. triandra (5); S. repens P3, P4, P5, P6, P7, P11 (2 hybrid); S. viminalis (50 + 1 hybrid); S. × aquatica (7); S. × dasyclados (5); S. × hirtei (2); S. × stipularis (5); S. × calodendron (1) V 1 S. triandra (1) P11 VI 1 S. aurita (1) N5 VII 1 S. daphnoides (1) C4 VIII 20 S. daphnoides (20) C1, C3, C4, C5, P3 IX 3 S. daphnoides (3) C4, C5 X 6 S. nigra (6) C1 XI 12 S. alba (1 + 1 hybrid); S. aurita (1 + 1 hybrids); C1, C3, C4, N1, N5 S. caprea (3 + 2 hybrids); S. cinerea (2 + 1 hybrid) XII 1 S. caprea (1) N10 Po 6 Populus spp. (6) C1, C8, N1, N8, P2, P10

RFLP, restriction fragment length polymorphism; ITS, internal transcribed spacer. Genetic Diversity of Melampsora Willow Rusts in Germany 83

Table 6.6. Lengths of the uncut amplicons of ITS region and number of the fragments generated by restriction digestion with TaqI, EcoRI, MboI and SspI.

Fragments Amplicon Pattern Frequency (bp) TaqI EcoRI MboI* SspI

I 6 625 4 2 4 1 II 2 626 4 2 3 1 III 4 626 3 2 3 2 IV 17 626 3 1 3 2 V 1 626 5 1 3 1 VI 1 627 3 1 3 2 VII 1 628 3 1 3 1 VIII 4 626 3 1 3 1 IX 1 627 3 1 3 1 X 1 626 2 1 3 2 Po 2 644 3 2 3 1

ITS, internal transcribed spacer. *Including one fragment of 3 bp in size, which was not visible on the gel.

Fig. 6.6. Map of restriction sites along the rDNA-ITS region for the Melampsora collection (Mel. = Melampsora sp. from Salix; M. p. = Melampsora larici-populina). Solid arrows indicate invariable sites, dotted arrows indicate variable sites; arrows from below belong to M. larici-populina.

The variation within pattern I was 1% or The ITS1/ITS4 sequences of Melamp- less. The second main group comprised sora isolates were used to infer phylogenetic seven patterns (III, IV, VI, VII, VIII, IX, X) relationships among these isolates. The which differed in less than 1.8% of their sequence data were aligned using the nucleotide positions. Between these sam- CLUSTALX program (Thompson et al., 1997). ples the differences were very small (< 2%). For calculating the tree, the neighbour- Sample 5.1 (pattern V) represented a distinct joining (N-J) method was used (Saitou and group with a 3% nucleotide difference to Nei, 1987). Bootstrap sampling was carried other Melampsora samples from willow. out using 1000 replications to assess the 84 M. Liesebach and I. Zaspel

Table 6.7. Percentage divergence between 18 Melampsora isolates. (Each sequence is only listed once. The sample numbers are a combination of the RFLP patterns and the different sequence types.)

Sample 1.1 1.2 1.3 1.4 2.1 3.1 3.2 3.3 10.1 8.1 8.2 7.1 9.1 8.3 4.1 6.1 5.1 Po 1.2 0.3 I 1.3 0.5 0.8 1.4 0.6 1.9 1.9 2.1 1.9 2.2 2.2 1.6 II 3.1 2.9 3.2 2.9 2.9 2.9 III, X 3.2 3.9 3.4 3.9 3.9 3.9 0.2 3.3 2.7 3.9 2.9 3.9 3.9 0.2 0.3 10.1 3.4 3.7 3.4 2.7 3.1 0.5 0.6 0.6 8.1 3.4 3.7 3.4 3.9 3.9 1.1 1.3 1.3 1.1 IV, VI, VII, VIII, IX 8.2 3.5 3.8 3.5 3.2 3.2 1.3 1.4 1.4 1.3 0.2 7.1 3.7 4.1 3.7 3.1 3.4 1.6 1.8 1.8 1.3 0.5 0.7 9.1 3.5 3.9 3.5 2.9 3.2 1.3 1.4 1.4 1.9 0.2 0.3 0.3 8.3 3.5 3.8 3.5 3.2 3.2 1.3 1.4 1.4 1.3 0.2 0.3 0.7 0.3 4.1 3.7 4.9 3.7 3.9 3.4 1.1 1.3 1.3 0.8 0.6 0.8 0.8 0.5 0.5 6.1 3.7 4.9 3.7 3.9 3.4 1.1 1.3 1.3 0.8 0.6 0.8 0.8 0.5 0.5 > 0.7 5.1 3.9 3.4 3.9 2.7 3.4 2.9 3.9 3.9 2.9 2.4 2.6 2.9 2.6 2.6 > 2.7 2.7 V Po 7.2 7.5 7.5 7.2 7.4 6.7 6.9 6.9 7.2 6.9 6.7 7.3 7.2 6.7 > 6.9 6.9 8.5 Po confidence limits of the branches (Felsen- nigra, S. × sericans and S. × stipularis. stein, 1985). The program TREEVIEW (Page, Four subgroups can be distinguished. One 1996) was used to construct the phylo- subgroup contained only the samples from genetic tree, which was rooted using S. daphnoides (patterns VII, VIII, IX). The the sequences ITS1/ITS4 units of Melamp- sequences of 18 samples, mostly from sora larici-populina (GenBank accession: S. viminalis, S. × aquatica, S. × dasyclados, AY375267) as the outgroup. S. × stipularis, S. fragilis, S. daphnoides The phylogenetic tree constructed and S. triandra, were closely related to a based on the results is shown in Fig. 6.7. The sample from S. aurita (pattern VI). The next two Melampsora samples from Populus (Po) branch was formed by the samples from formed a distinct group. The sequences of S. × sericans and S. aurita (pattern III), and these samples (M. larici-populina) were con- one sample from S. nigra (pattern X). firmed by that from the database (GenBank The large subunit (LSU) ribosomal DNA accession: AY375267). The analysis showed sequence data were also used to infer phylo- two clear lineages in willow rusts. The genetic relationships of Uredinales (Maier willow rust samples differed between these et al., 2003). We used the primer pair NL1 (5′ lineages in about 4% of their nucleotides. GCA TAT CAA TAA GCG GAG GAA AGG There was a close phylogenetic relation- 3′) and NL4 (5′ GGT CCG TGT TTC AAG ship among the Melampsora isolated from ACG G 3′) to amplify the 28S rDNA (nuclear the rusts on S. fragilis, S. triandra and S. large subunit rDNA) (Guadet et al., 1989). A purpurea (RFLP patterns I, II, V). This main set of 32 samples, including three rust iso- group can be divided into three subgroups. lates from Populus sp., were sequenced. The Two samples from S. purpurea were placed identified 28S region of Melampsora isolates in one of these subgroups (pattern II). The was used to infer phylogenetic relationships samples in pattern I were mostly derived among these isolates. The sequence data from S. fragilis. were aligned using the CLUSTALX program The second main group comprised the (Thompson et al., 1997). The primer pair samples from S. viminalis, S. daphnoides, NL1 and NL4 amplified a single DNA frag- S. × dasyclados, S. × aquatica, S. aurita, S. ment of the 28S rDNA genes of Melampsora, Genetic Diversity of Melampsora Willow Rusts in Germany 85

Fig. 6.7. Neighbour-joining tree based on nucleotide divergences. The numbers on the nodes are the frequencies (in per cent) with which a cluster appears in a bootstrap test of 1000 runs. The tree is rooted with M. larici-populina (Po) as outgroup. For an explanation of sample numbers see Table 6.7. of a total size between 597 and 600 bp. The M. larici-populina samples. Another branch maximum nucleotide difference between contained rust samples from S. purpurea the 29 samples from Salix and the three from and assigned to M. ribesii-purpurea.In Populus was only 3%. A neighbour-joining the bottom left of the tree the samples from (N-J) tree was calculated according to the S. fragilis and S. triandra were grouped methods mentioned above (Fig. 6.8). together and they were assigned to M. The tree was clearly composed of five amygdalinae. Melampsora allii-fragilis was branches. One branch was formed by the placed in a separate branch. The branches in 86 M. Liesebach and I. Zaspel

Fig. 6.8. Neighbour-joining tree based on nucleotide divergences of the large subunit 28S region. Bootstrap values for 1000 re-samplings are indicated on each branch. Willow hosts and putative Melampsora species are shown. the upper part of the tree, which appeared to Melampsora willow rusts are highly be predominated by M. larici-epitea, did not variable in their species and pathotype reveal the different formae speciales, i.e. composition. The results are broadly in line larici-epitea typica, larici-retusae, larici- with the taxonomic systems by Gäumann daphnoides and others. Overall, the results (1959) and Butin (1960). Klenke (1998) showed that there is a close phylogenetic indicated a higher degree of variation in relationship between Melampsora spp. iso- the willow rust complex, but his work was lates from rusts on Salix spp. and Populus confined to a restricted region of Germany. spp. (M. larici-populina). Previous work referred to rusts occurring in natural willow stands, and those in SRC plantations were not implicated. M. epitea has been treated as a species Concluding Remarks complex because of different spermagonial and aecial hosts and/or uredinial and telial The present study has demonstrated that, hosts in Europe (Hylander et al., 1953; in central Europe, especially in Germany, Gäumann, 1959; Wilson and Henderson, Genetic Diversity of Melampsora Willow Rusts in Germany 87

1966) and North America (Ziller, 1974). be seen by the different reactions of willow Under existing conditions in Germany, clones – from resistant to highly susceptible the genetic structure of the M. larici-epitea to rust infection. S. viminalis and some population, one of the subspecies of M. S. daphnoides clones were infected but epitea, is of special interest. This rust plays without serious damage. Other clones, espe- an important role in rust disease epidemics cially those belonging to S. × dasyclados on biomass willow clones in countries or S. × aquatica, suffered severe attacks with SRC plantations, such as Sweden and and showed dieback symptoms. the UK. It can be concluded that the three The pathotype composition of an SRC major formae speciales, out of the six listed plantation depends on the species and the by Gäumann (1959), occurred on biomass number of willow clones planted, and is clones in trial plantations and clone further influenced by their age. The develop- collections in Germany. ment of aggressive pathotypes endangering The willow rust population comprises a the sustainability of the whole plantation is number of pathotypes which vary between more likely when only one, or a few, suscep- sites and geographical regions. Using leaf tible host clones have been grown. There- disc pathogenicity tests, Pei et al. (1999b) fore, the concept of multi-clonal plantations identified 12 pathotypes within M. larici- is creating a higher diversity of rust popula- epitea in the UK. Using the same method, tions, resulting in a lower infection level Ramstedt (1999) found 13 types and pre- and expected higher stability of yields dicted about 20 types in Sweden. Our inves- (McCracken et al., 2000). Information on tigation revealed a distinctly higher number the resistance of willow species/clones and of rust types among the samples we tested, genetics of the rust populations is important but the samples included, in addition to the in the management of willow leaf rust in M. epitea complex from cultivated clones, SRC coppice plantations, and in defining several other Melampsora species from strategies in resistance breeding. natural stands. The stem-infecting rusts were recorded frequently but cannot yet be assigned to a distinct pathotype. Acknowledgements The application of molecular tools using PCR-based methods of ITS-RFLP, ran- This research was funded by the EC and dom amplified polymorphic DNA (RAPD) is part of the joint project ‘Integrated, non- and amplification fragment length poly- fungicidal control of Melampsora rusts morphism (AFLP) markers revealed a high in renewable energy willow plantations’ degree of genetic diversity among willow (QLK5-1999-01585). We wish to thank Melampsora (Nakamura et al., 1998; Ms H. Mattauch and E. Ewald for technical Liesebach et al., 2001; Samils et al., 2001; Pei assistance. et al., 2002). Because our molecular method was not exactly the same as that of the oth- ers, our results cannot be compared directly to the results by the cited authors. However, References the extent of variation in willow rusts shows the potential of the rusts to act as aggressive Ahman, I. (1998) Rust scorings in a plantation of Salix pathogens under suitable conditions. viminalis clones during ten consecutive years. The clonal nature of willows as short- European Journal of Forest Pathology 28, rotation coppice crops makes them espe- 251–258. Ahman, I. and Larsson, S. (1994) Genetic improve- cially vulnerable to the build-up of pests ment of willows (Salix) as a source of bioenergy. and diseases when grown in monocultures. Norwegian Journal Agricultural Sciences Also, the intensity of pest damage and 18(Suppl.), 47–56. disease outbreaks can differ markedly Butin, H. (1960) Die Krankheiten der Weide und between Salix species, clones and geograph- deren Erreger. Mitteilungen aus der Biologischen ical locations (Hunter et al., 1996). This can Bundesanstalt für Land- und Forstwirtschaft 98. 88 M. Liesebach and I. Zaspel

Felsenstein, J. (1985) Confidence limits on DNA sequences. Canadian Journal of Botany 81, phylogenies: an approach using the bootstrap. 12–23. Evolution 39, 783–791. McCracken, A.R. and Dawson, M. (1992) Clonal Gards, M. and Bruns, T.D. (1993) ITS primers response in Salix to Melampsora rusts in short with enhanced specificity for basidiomycetes – rotation coppice plantations. European Journal application to the identification of mycorrhizae of Forest Pathology 22, 19–28. and rusts. Molecular Ecology 2, 113–118. McCracken, A.R., Dawson, W.M., Watson, S. and Gäumann, E. (1959) Die Rostpilze Mitteleuropas. Allen, C.Y. (2000) Pathotype composition in Beiträge zur Kryptogamenflora Schweiz 12. Melampsora epitea populations occurring on Buchdruckerei Büchler and Co, Bern, Germany, willow (Salix) grown in mixed and monoculture pp. 144–177. plantations. European Journal of Plant Pathology Guadet, J., Julien, J., Lafay, J.F. and Brygoo, Y. (1989) 106, 879–886. Phylogeny of some Fusarium species, as Nakamura, H., Kaneko, S., Yamaoka, Y. and determined by large-subunit rRNA sequence Kakishima, M. (1998) Differentiation of comparison. Molecular Biology and Evolution 6, Melampsora rust species on willows in Japan 227–242. by using PCR-RFLP analysis of ITS regions of Helfer, S. (1992) The rust diseases of willows in ribosomal DNA. Mycoscience 39, 105–113. Britain. Proceedings of the Royal Society of Newsholme, C. (2002) Willows of the Genus Salix. Edinburgh 98B, 119–134. Batsford Ltd, London. Hunter, T., Royle, D.J. and Arnold, G.M. (1996) Page, R.D.M. (1996) TREEVIEW: An application to Variation in the occurrence of rust (Melampsora display phylogenetic trees on personal comput- spp.) and other diseases and pests, in ers. Computer Applications in the Biosciences short-rotation coppice plantations of Salix in the 12, 357–358. British Isles. Annals of Applied Biology 129, Parker, S.R., Royle, D.J. and Hunter, T. (1993) Impact 1–12. of Melampsora rust on yield of biomass willows. Hylander, N., Jorstad, I. and Nannfeldt, J.A. (1953) Sixth International Congress of Plant Pathology, Enumeratio uredinearum scandinavicarum. Montreal, Canada, 28 July–6 August. National Opera Botanica 1, 1–102. Research Council Canada, Ottawa, p. 117 Klenke, F. (1998) Sammel- und Bestimmungshilfen [abstract]. für phytoparasitische Kleinpilze in Sachsen. Pei, M.H., Hunter, T. and Royle, D.J. (1993) Variation Berichte der Arbeitsgemeinschaft sächsischer of Melampsora rusts occurring on biomass wil- Botaniker 16. lows in the UK. Proceedings IEA Task VIII Joint Lautenschlager-Fleury, D. (1994) Die Weiden von Meeting, Northern Ireland, September 1992. Mittel- und Nordeuropa. Birkhäuser Verlag, Pei, M.H., Parker, S.R., Hunter, T. and Royle, D.J. Basel. (1997) Variation in populations of Melampsora Liesebach, M. and Zaspel, I. (2004) Genetic diversity willow rust and the implications for design of of the mycoparasite Sphaerellopsis filum on short rotation coppice plantations. In: Bullard, Melampsora willow rusts. Forest Pathology 34, M.J., Ellis, R.G., Heath, M.C., Knight, J.D.D. and 292–305. Parker, S.R. (eds) Aspects of Applied Biology Liesebach, M., Zaspel, I. and Stauber, T (2001) Vol. 49, Biomass and Energy Crops. The Associ- Monitoring of Melampsora rusts in Salix. Journal ation of Applied Biologists, Wellesbourne, UK, of Forest Sciences 47 (Special Issue No. 2), pp. 91– 96. 119–122. Pei, M.H., Royle, D.J. and Hunter, T. (1999a) Liesebach, M., Zaspel, I. and Stauber, T. (2002) Hybridization in larch-alternating Melampsora Growth of willows and the influence of epitea (M. larici-epitea). Mycological Research Melampsora infection on the establishment of 103, 1440–1446. biomass plantations. In: Ercan, M., Diner, A., Pei, M.H., Hunter, T. and Ruiz, C. (1999b) Occur- Birler, A.S., Goulding, C. and Zoralioglu, T. (eds) rence of Melampsora rusts in biomass willow Management of Fast Growing Plantations. Inter- plantations for renewable energy in the United national IUFRO Meeting, 11–13 September, Kingdom. Biomass and Bioenergy 17, 153–163. Izmit/Turkey, pp. 150–160. Pei, M.H., Bayon, C., Ruiz, C., Yuan, Z.W. and Long, E.O. and Dawid, I.B. (1980) Repeated genes in Hunter, T. (2002) Genetic variation in Melamp- eukaryotes. Annual Review of Biochemistry 49, sora larici-epitea on biomass willows assessed 727–764. using AFLP. European Journal of Forest Maier, W., Begerow, D., Weiß, M. and Oberwinkler, Pathology 108, 229–236. F. (2003) Phylogeny of the rust fungi: an Ramstedt, M. (1999) Rust disease on willows – approach using nuclear large subunit ribosomal virulence variation and resistance breeding Genetic Diversity of Melampsora Willow Rusts in Germany 89

strategies. Forest Ecology and Managment 121, Vogler, D.R. (1995) Use of molecular techniques in 101–111. rust systematics. In: Kaneko, S., Katsuya, K., Rasch, D., Herrendörfer, G., Bock, J., Victor, N. and Kakishima., M. and Ono, Y. (eds) Proceedings Guiard, V. (eds) (1998) Verfahrensbibliothek. of 4th IUFRO Rusts of Pines Working Party Versuchsplanung und -auswertung, Vol. II. R. Conference, Tsukuba, pp. 9–15. Oldenbourg Verlag, Munich, pp. 208–216. White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Saitou, N. and Nei, M. (1987) The neighbor-joining Amplification and direct sequencing of fungal method: a new method for reconstructing phylo- ribosomal RNA genes for phylogenetics. In: genetic trees. Molecular Biology and Evolution 4, Innis, M.A., Gelfand, D.H., Sninsky, J.J. and 406–425. White, T.J. (eds) PCR Products: a Guide to Samils, B., Lagercrantz,U., Lascoux, M. and Gullberg, Methods and Applications. Academic Press, U. (2001) Genetic structure of Melampsora San Diego, California, pp. 315–322. epitea populations in Swedish Salix viminalis Wilson, M. and Henderson, D.M. (1966) British Rust plantations. European Journal of Plant Pathology Fungi. Cambridge University Press, Cambridge, 107, 399–409. UK. Thompson, J.D., Gibson, T.J., Plewniak, F., Zander, M. (2000) Untersuchungen zur Identifizie- Jeanmougin, F. and Higgins, D.G. (1997) The rung ausgewählter Vertreter der Gattung Salix L. ClustalX windows interface: flexible strategies im NO-deutschen Tiefland, unter besonderer for multiple sequence alignment aided by Berücksichtigung des Salix repens-Komplexes. quality analysis tools. Nucleic Acids Research Mitteilungen und floristische Karten Sachsen- 24, 4876–4882. Anhalt (Halle) 5, 3–137. Tucker, K. and Sage, R. (1999) Integrated Pest Ziller, W.G. (1974) The Tree Rusts of Western Management in Short Rotation Coppice for Canada. Publication No. 1329. Canadian Energy – a Grower’s Guide. Game Conservancy Forestry Service, Environmental Canada, Ltd, Fordingbridge, UK. Ottawa, Canada. This page intentionally left blank 7 Genetic Structure of Melampsora larici-epitea Populations in North-western Europe

Berit Samils Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden

Introduction studies. AFLP is a polymerase chain reaction (PCR)-based DNA fingerprint The population genetic structure of a patho- technique (Vos et al., 1995) that has gen reflects its evolutionary history and its produced highly polymorphic and robust potential to evolve. By making inferences markers, which makes it a powerful tool from the population structure we can get an in genotyping and examination of genetic understanding of the evolutionary mecha- variation (Majer et al., 1996). nisms that lead to adaptations of the patho- This chapter reviews the studies of gen, and this knowledge can contribute to a genetic variation in populations of M. larici- more durable resistance through improved epitea in biomass willow plantations in breeding programmes and strategies for the north-western Europe (mostly in Sweden). deployment of resistance (Leung et al., From these studies some inferences on the 1993; Wolfe and McDermott, 1994; McDon- evolutionary potential of the pathogen will ald and Linde, 2002). Many mechanisms be made. interact in the genetic changes and evolu- tion of populations. The important ones are mutation, reproduction mode, gene flow, selection and random genetic drift. Biology of M. larici-epitea The recent development of molecular markers for Melampsora larici-epitea has M. larici-epitea has a complex life history. provided new opportunities to investigate The fungus alternates between two hosts genetic variation of the fungus. RAPD (ran- (willow and larch) and has five distinct dom amplified polymorphic DNA) markers kinds of spores (Fig. 7.1). The uredinio- were used to distinguish between stem- spores start to form on the lower sides of and leaf-infecting forms of Melampsora willow leaves in the beginning of the on willow (Pei et al., 1997). More recently, summer. These spores are windspread and AFLP (amplified fragment length polymor- serve as the inoculum in repeated asexual phism) was applied to willow rust (Pei and infection cycles throughout the growing Ruiz, 2000; Samils et al., 2001a) and has season. In autumn, telia develop on since been used in a number of population infected leaves and the fungus overwinters

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 91 92 B. Samils

in this form on fallen leaves. In the spring, form species (formae speciales)ofM. larici- basidiospores are produced and they infect epitea on biomass willow are f. sp. larici- larch (Larix), the alternate host, where epitea typica (LET; infecting, for example, sexual reproduction takes place (i.e. mating S. viminalis), f. sp. larici-retusae (LR; infect- between spermatia and receptive hyphae). ing, for example, S. dasyclados/S. burjatica) The sexual phase results in the formation of and f. sp. larici-daphnoides (LD; infecting, recombined aeciospores, which infect new for example, S. daphnoides). These formae willow leaves early in the summer. There- speciales probably constitute genetically after, the asexual uredinial phase follows separated populations, since low fertility in and the cycle is completed. The sexual crossing experiments suggests that they do phase on larch is presumed to be essential not normally interbreed (Pei et al., 1999b). for winter survival of M. larici-epitea, but it This has also been confirmed in extensive has been discussed whether overwintering field surveys where no genotypes in rust in the uredinial form might also occur. So samples expressed virulence to both of the far, however, evidence is lacking. two host species S. viminalis (infected by LET) and S. dasyclados (infected by LR) (Pei et al., 1999a; Ramstedt, 1999). Further Spore dispersal evidence of the formae speciales being genetically separate groups has been pro- vided by DNA marker analyses (Pei et al., In rust fungi, wind-transported uredinio- 2002; Samils et al., 2002). spores (and possibly also aeciospores) are The population studies of M. larici- generally believed to be capable of long- epitea reviewed in this chapter relate mainly distance migration (Roelfs, 1986; Aylor, to f. sp. larici-epitea typica (LET), causing 1990; Nagarajan and Singh, 1990). This leaf rust on S. viminalis, one of the most capability has also been demonstrated important biomass willow species. All for some Melampsora species on poplars studies were conducted in biomass willow and willows (Latch, 1980). The dispersal plantations or experimental fields. range of the drought- and ultraviolet (UV)- sensitive basidiospores is much more limited, probably to around a few hundred metres (Roelfs, 1985), although under Genetic Diversity Within Populations favourable conditions basidiospores have of M. larici-epitea been reported to move as far as 8 km (van Arsdel, 1967). This implies that, in the case Genetic structure relates to the amount and of M. larici-epitea, the distance between distribution of genetic diversity within and willow plantations and larch stands may be among populations. It is useful to distin- important for the rate of winter survival of guish between the two components of the fungus. Only those basidiospores that in genetic diversity, namely gene diversity spring manage to get to a larch plant and and genotypic diversity. Gene diversity find a mating partner will contribute to the refers to the number of alleles at a locus in following season’s initial inoculum in the the population, while genotypic diversity form of recombined aeciospores. refers to the number of genetically distinct individuals, or genotypes, in a population. It should be kept in mind that the absolute Host specificity number of individuals of the rust fungus in the form of uredinia in the epidemic phase Like other rust fungi, M. larici-epitea is can be incredibly large. Even a moderately highly specialized to its host, and geneti- infected willow stand can harbour millions cally differentiated forms have been defined of individual pustules per hectare. But based on the host range (Gäumann, 1959; since M. larici-epitea reproduces asexually Pei et al., 1996). The three most common in repeated infection cycles during the Genetic Structure of Melampsora larici-epitea Populations 93

Fig. 7.1. The life cycle of M. larici-epitea (from Samils et al., 2001). The nuclear condition of the fungus in each of the five spore stages is indicated.

summer, many of the uredinia can belong to Variation Among Willow Clones the same genotype (i.e. they belong to the same clone). There is little or no evidence of race- Studies of Swedish M. larici-epitea specific resistance towards M. larici-epitea populations show high levels of genetic within species of Salix, probably because diversity, both with regard to gene and breeding of biomass willow is still at an genotypic diversity. Samils et al. (2001a) early stage in this relatively new crop. investigated three M. larici-epitea popula- However, some degree of specificity has tions in biomass willow field trials in been indicated. For example, a study by middle and southern Sweden. The three McCracken et al. (2000) showed differences fields were located in Uppsala, Bränna and in pathotype composition between two S. Svalöv, 300–600 km apart (Fig. 7.2). A high viminalis varieties in a mixture experiment. genotypic diversity was demonstrated in To evaluate whether the genetic composi- all populations by AFLP fingerprinting. tion of M. larici-epitea populations differs Most urediniospore isolates (191 out of between clones of S. viminalis, a compari- 197 investigated) were unique multilocus son of rust populations on three willow phenotypes, and only a few clonemates were clones (S. viminalis ‘78112’, ‘78183’ and detected. This means that the effective ‘Rapp’) was made in three Swedish population size is very large. A high gene locations (Samils et al., 2001a) (Fig. 7.2). In diversity was also indicated in these popu- general, M. larici-epitea populations on the lations, with 96% of the AFLP loci being three S. viminalis clones were genetically polymorphic. similar. An exceptional finding was the 94 B. Samils

occurrence of five contrasting isolates from viminalis clones (‘78118’ and ‘78183’) in a S. viminalis ‘Rapp’ in Svalöv in southern mixture experiment in Castlearchdale in Sweden. Although these isolates could not Northern Ireland (Samils et al., 2003) be separated by morphology, they had a (Fig. 7.2). An analysis of molecular variance markedly different AFLP pattern compared based on AFLP showed a relatively large to the prevalent type of the rust. Since these differentiation (19.8%, fST = 0.198; Table isolates might belong to another species, 7.1) between the two willow varieties, and they were excluded from the genetic analy- differences in genotypic composition were ses in the paper. This odd type (designated indicated by the non-random distribution of the ‘B-type’) has also been found on other clonal rust isolates between the varieties. It S. viminalis clones in southern Sweden and seemed that the pathogen population was its genetic relationship to other Melam- composed both of genotypes that are more psora on Salix, as revealed by AFLP mark- adapted to one or other host clone and those ers, has been described (Samils et al., 2002). that are well adapted to both clones. The In contrast to the results from Sweden, a finding of differences between willow clear genetic difference was found between clones in Castlearchdale, but not in Sweden, M. larici-epitea populations on two S. could simply be because different sets of

Fig. 7.2. Map showing the location of Melampsora larici-epitea populations in studies by Samils et al.: 1, Sätuna; 2, Uppsala; 3, Djurby; 4, Bränna; 5, Svalöv; and 6, Castlearchdale.

Table 7.1. Summary of results from analysis of molecular variance (AMOVA) for Melampsora larici- epitea isolates in studies by Samils et al. (The publication where the complete result is presented is given.)

f a Source of variation Populations considered ST

Between Sweden and N. Ireland Djurby and Sätuna (Sw), and Castlearchdale (NI)b −0.089*** Among provinces in Sweden Uppsala, Bränna and Svalövc −0.025*** Between populations within a province Djurby and Sätunab −0.003*** Among willow clones in Sweden S. viminalis ‘78112’, ‘78183’ and ‘Rapp’c −0.001*** Between willow clones in S. viminalis ‘78183’ and ‘78118’d −0.198*** Castlearchdale (N. Ireland)

***P < 0.001 (determined by 1000 randomizations of the data set). af ST (analogous to FST) is an estimate of population subdivision (Excoffier et al., 1992). bSamils et al. (2001b), cSamils et al. (2001a), dSamils et al. (2003) Genetic Structure of Melampsora larici-epitea Populations 95

willow clones were investigated in the within-field clustering of clonal isolates. A two countries (e.g. ‘78118’ was not included single leaf was normally infected by more in Sweden). Another explanation could be than one M. larici-epitea genotype. In fact, that detection of virulence differences with when two uredinia from each of 56 leaves molecular markers is less likely in a popula- were investigated, it turned out that the tion with frequent sexual reproduction, as two uredinia had different genotypes in all seems to be the case in Sweden (Samils et al., cases. The occurrence of sexual reproduc- 2001a), than in a population where asexual tion in the Swedish populations was sup- reproduction is predominant, as might be ported by tests of non-random association the case in Castlearchdale in Northern among loci, which revealed no significant Ireland (Samils et al., 2001b). departure from random mating in either of the two populations. According to weekly records of disease severity in the two fields, disease development in the beginning of Influence of Larch – the Alternate Host the summer started a few weeks earlier in the plantation close to larch. The infection Since larch is the host for sexual reproduc- level was also significantly higher than in tion of M. larici-epitea, it is also the place the non-larch field during a large part of the where new, genetically recombined geno- growth period. types are created in each spring–early For the purpose of the study, the leaf summer. Recombination is an important rust present within a willow field was feature for the evolution of fungal geno- regarded as a separate population. In reality, types. With new combinations of virulence populations might extend over larger areas, genes, recombinants are better adapted to and the two Swedish populations (located the resistances of the host. To investigate 45 km apart) might perhaps better be consid- whether the presence of larch will increase ered as belonging to the same population, as the genetic diversity of willow rust popula- they were shown to be genetically similar. A tions, Samils et al. (2001b) studied the plausible scenario would be that uredinio- genetic structure of M. larici-epitea popula- spores will spread from field to field during tions in two plantations of S. viminalis in the repeated cycles of asexual production the province of Uppland in central Sweden. during the epidemics. The sexual reproduc- On one of the plantations, a larch stand tion in spring might occur only at those loca- was less than 100 m from the willow field, tions where willow and larch grow close whereas no larch plants were present enough to allow basidiospore transmission within 1.5 km of the other plantation. A (i.e. probably less than a few hundred metres comparison was also made with a popula- apart). From these larch plants the recom- tion in an experimental trial in Northern bined aeciospores can spread to nearby wil- Ireland, where larch grew less than 500 m low fields, and the subsequently produced from the willow field. In order to analyse urediniospores can be dispersed throughout the spatial distribution of genetic variation the population range. within the fields, rust samples were col- The M. larici-epitea population in lected in a hierarchical pattern, and the rust Castlearchdale in Northern Ireland showed isolates were analysed with AFLP markers. a different structure compared to the two Comparing the two Swedish populations, Swedish populations. As in the Swedish it turned out that the proximity of the populations, genetic variation was ran- alternate host had no apparent effect on the domly distributed within the field, and no genetic structure, as there was no genetic spatial clustering of clonal isolates was indi- differentiation between the two sites and cated. However, the level of clonality was the level of genotypic diversity was simi- much higher, with the three most frequent larly high for both populations. Most of clones representing 70% of the total sample the molecular variation was present on a (16, 11 and 7 isolates, respectively, out of very fine spatial scale, with no detectable 47). The genotypic diversity calculated by a 96 B. Samils

normalized Shannon’s index was 0.54 in located 300–600 km apart (Samils et al., this population, while it was 0.95 and 1.0, 2001a); and, as already mentioned, no respectively, in the two Swedish popula- genetic differentiation was found between tions. Further, the occurrence of multilocus two populations located 45 km apart in the associations (linkage disequilibrium) indi- province of Uppland in Sweden (Samils cated non-random mating, which is a et al., 2001b) (Table 7.1). When these two characteristic of a predominantly asexual populations were compared to a population population. in Northern Ireland, analysis of molecular The overall results suggest that while variance showed a differentiation of 8.9% sexual reproduction seems to be frequent (fST = 0.089; Table 7.1) between the two and regular in Swedish M. larici-epitea countries. populations, it may be less frequent or less In contrast to the low levels of geo- regular in Castlearchdale. This could be due graphic differentiation in the above studies, to a seasonal bottleneck during winter a case with a high degree of subdivision survival and sexual reproduction, resulting among M. larici-epitea populations was in a limited amount of source inoculum observed in Great Britain. Pei et al. (2000) for the next season and thus leading to investigated M. larici-epitea populations limited genotypic diversity. Alternatively, on Salix × mollisima (S. triandra × S. the milder climate in Northern Ireland might viminalis) during the first year of rust out- allow for winter survival of urediniospores break (in 1992) on this previously highly and sexual reproduction may thus not be resistant clone. The investigation included obligatory, which would also result in lower three plantations in the British Isles: one in genotypic diversity. Long Ashton and one in Markington (both in England, roughly 300 km apart) and the third in Loughall in Northern Ireland. In an analysis of molecular variance based on Genetic Variation Among Populations AFLP, as much as 86% of the variation was of M. larici-epitea due to differences among populations. The authors suggested that the high degree of The amount of genetic variation among differentiation could be due to inbreeding populations can give us indirect measures in small, newly established populations. of the amount of gene flow, since little vari- They further suggested that the previously ation among populations typically implies unknown pathotype was not spread from high rates of gene flow and vice versa. a common source but was spread from Migration of wind-transported uredinio- different sources. spores and aeciospores will be the primary Even though isolated diverged popula- way of gene flow in M. larici-epitea. The tions may temporarily occur in M. rate of gene flow is of importance from a larici-epitea, the overall results, with small breeding point of view since it will give an to moderate differentiation, suggest rela- indication on how fast virulence genes will tively high levels of gene flow between spread among regions. It should be noted populations. that other factors, such as selection and genetic drift, especially in small popula- tions, also affect genetic variation among populations. Conclusions Generally there seems to be relatively little variation among geographically The genetic structure of M. larici-epitea separated willow rust populations in north- populations, with large amounts of both western Europe. An analysis of molecular gene and genotypic variation present variance (AMOVA) based on AFLP showed within most populations, and little geo- only a few per cent differentiation among graphic differentiation, suggests high rates three Swedish M. larici-epitea populations, of genetic recombination through sexual Genetic Structure of Melampsora larici-epitea Populations 97

reproduction and high rates of gene flow. Excoffier, L., Smouse, P.E. and Quattro, J.M. (1992) Such qualities are likely to allow the patho- Analysis of molecular variance inferred from gen populations to readily adapt to a chang- metric distances among DNA haplotypes: appli- ing environment, such as new resistances cation to human mitocondrial DNA restriction sites. Genetics 131, 479–491. in the host population. In other words, M. Gäumann, E. (1959) Die Rostpilze Mitteleuropas. larici-epitea has a high evolutionary poten- Beiträge zur Kryptogamenflora Schweiz 12. tial and can be rated as a ‘high-risk’ patho- Buchdruckerei Büchler and Co., Bern, gen (McDonald and Linde, 2002). This Germany. means that when resistant Salix clones are Lascoux, M., Ramstedt, M., Åström, B. and Gullberg, introduced and cultivated over large areas, U. (1996) Components of resistance of leaf rust they run the risk of losing resistance as (Melampsora laricii-epitea Kleb./Melampsora M. larici-epitea can rapidly acquire and ribesii-viminalis Kleb.) in Salix viminalis L. Theo- spread adaptive genotypes with matching retical and Applied Genetics 93, 1310–1318. virulences. Such losses of resistance have Latch, B.J. (1980) Weeping willow rust in New Zealand. New Zealand Journal of Agricultural already been experienced in the Salix/ Research 23, 535–538. Melampsora pathosystem, where resis- Leung, H., Nelson, R.J. and Leach, J.E. (1993) Popula- tances of the willow clones S. burjatica tion structure of plant pathogenic fungi and bac- ‘Korso’ and S. × mollisima ‘Q83’ were teria. Advances in Plant Pathology 10, 157–205. broken after 8–10 years of cultivation Majer, D., Mithen, R., Lewis, B.G., Vos, P. and (McCracken and Dawson, 1998; Pei et al., Oliver, P. (1996) The use of AFLP fingerprinting 2000). Similar breakdowns have also for the detection of genetic variation in fungi. occurred in the closely related Populus/ Mycological Research 100, 1107–1111. Melampsora pathosystem (Pinon and Frey, McCracken, A.R. and Dawson, W.M. (1998) Short 1997). Therefore, finding ways to slow host rotation coppice willow in Northern Ireland since 1973: development of the use of mixtures adaptation in M. larici-epitea populations is in the control of foliar rust (Melampsora spp.). a major concern. One promising approach European Journal of Forest Pathology 28, is the cultivation of host mixtures, which 241–250. are treated in detail in Part 4 of this book. McCracken, A.R., Dawson, W.M., Watson, S. and Another option is breeding for partial Allen, C.Y. (2000) Pathotype composition in resistance (race-non-specific resistance) Melampsora epitea populations occurring on (Lascoux et al., 1996; Ramstedt, 1999), willow (Salix) grown in mixed and monoculture which is believed to be more durable than plantations. European Journal of Plant Pathology complete resistance (race-specific resis- 106, 879–886. tance). Partial resistance is thought to be the McDonald, B.A. and Linde, C. (2002) Pathogen population genetics, evolutionary potential, combined effect of a number of resistance and durable resistance. Annual Review of components and may be characterized by a Phytopathology 40, 349–379. long latent period, low spore production Nagarajan, S. and Singh, D.V. (1990) Long-distance and low infection frequency (Parlevliet, dispersion of rust pathogens. Annual Review of 1993). A large amount of heritable variation Phytopathology 28, 139–153. for resistance components has been Parlevliet, J.E. (1993) What is durable resistance, a detected among S. viminalis clones general outline. In: Jacob, T. and Parlevliet, J.E. (Lascoux et al., 1996), which raises the (eds) Durability of Disease Resistance. prospects for breeding for partial resistance Kluwer Academic Publishers, Dordrecht, in biomass willows. The Netherlands, pp. 23–39. Pei, M.H. and Ruiz, C. (2000) AFLP evidence of the distinctive patterns of life-cycle in two forms of Melampsora rust on Salix viminalis. Mycological Research 104, 937–942. References Pei, M.H., Royle, D.J. and Hunter, T. (1996) Patho- genic specialization in Melampsora epitea var. Aylor, D.E. (1990) The role of intermittent wind in the epitea on Salix. Plant Pathology 45, 679–690. dispersal of fungal pathogens. Annual Review of Pei, M.H., Parker, S.R., Hunter, T. and Royle, D.J. Phytopathology 28, 73–92. (1997) Variation in populations of Melampsora 98 B. Samils

willow rust and the implications for design of Samils, B. (2001) Population genetic structure of short rotation coppice plantation. Aspects of Melampsora larici-epitea, a willow leaf rust Applied Biology 49, 91–96. fungus. Doctoral thesis. Acta Universitatis Pei, M.H., Hunter, T. and Ruiz, C. (1999a) Agriculturae Sueciae, Agraria 292. SLU Servive/ Occurrence of Melampsora rusts in biomass Repro, Uppsala, Sweden. ISSN 1405-6249. willow plantations for renewable energy in the Samils, B., Lagercrantz,U., Lascoux, M. and Gullberg, United Kingdom. Biomass and Bioenergy 17, U. (2001a) Genetic structure of Melampsora 153–163. epitea populations in Swedish Salix viminalis Pei, M.H., Royle, D.J. and Hunter, T. (1999b) Hybrid- plantations. European Journal of Plant Pathology ization in larch-alternating Melampsora epitea 107, 399–409. (M. larici-epitea). Mycological Research 103, Samils, B., Stepien, V., Lagercrantz, U., Lascoux, M. 1440–1446. and Gullberg, U. (2001b) Genetic diversity in Pei, M.H., Yuan, Z.W., Hunter, T. and Ruiz, C. (2000) relation to sexual and asexual reproduction in Heterogeneous nature of a ‘new’ pathotype of populations of Melampsora larici-epitea. Euro- Melampsora rust on Salix revealed by AFLP. pean Journal of Plant Pathology 107, 871–881. European Journal of Plant Pathology 106, Samils, B., Lagercrantz, U. and Gullberg, U. (2002) 771–779. Genetic relationships among genetically distinct Pei, M.H., Bayon, C., Ruiz, C., Yuan, Z.W. and forms of Melampsora larici-epitea and related Hunter, T. (2002) Genetic variation in species based on AFLP data. Forest Pathology Melampsora larici-epitea on biomass willows 32, 379–386. using AFLP. European Journal of Plant Pathology Samils, B., McCracken, A.R., Dawson, W.M. and 108, 229–236. Gullberg, U. (2003) Host specific genetic Pinon, J. and Frey, P. (1997) Structure of Melampsora composition of Melampsora larici-epitea larici-populina populations on wild and populations on two Salix viminalis varieties cultivated poplar. European Journal of Plant in a mixture trial. European Journal of Plant Pathology 103, 159–173. Pathology 109, 183–190. Ramstedt, M. (1999) Rust disease on willows – Van Arsdel, E.P. (1967) The nocturnal diffusion virulence variation and resistance breeding and transport of spores. Phytopathology 57, strategies. Forest Ecology and Management 1221–1229. 121, 101–111. Vos, P., Hogers, M.B., Reijans, M., van de Lee, T., Roelfs, A.P. (1985) Wheat and rye stem rust. In: Roelfs, Hornes, M., Frijters, A., Pot, J., Peleman, J., A.P. and Bushnell, W.R. (eds) The Cereal Rusts, Kuiper, M. and Zabeau, M. (1995) AFLP: a new Vol. II, Diseases, Distribution, Epidemiology, technique for DNA fingerprinting. Nucleic Acids and Control. Academic Press, Orlando, Florida. Research 23, 4407–4414. Roelfs, A.P. (1986) Development and impact of Wolfe, M.S. and McDermott, J.M. (1994) Population regional cereal rust epidemics. In: Leonard, K.J. genetics of plant pathogen interactions: The and Fry, W.E. (eds) Plant Disease Epidemiology: example of the Erysiphe graminis–Hordeum Population Dynamics and Management. vulgare pathosystem. Annual Review of Macmillan, New York, pp. 129–150. Phytopathology 32, 89–113. 8 Current Taxonomic Status of Melampsora Species on Poplars in China

Cheng-Ming Tian1 and Makoto Kakishima2 1College of Resource and Environment, Beijing Forestry University, 35 Tsinghua Eastern Road, Beijing 100083, China; 2Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Melampsora Rusts on Populus five formae speciales under M. populnea. Cellerino (1999) listed 14 species of The genus Melampsora was established by Melampsora on poplars. Castagne in 1843 based upon M. euphorbiae The classification of Melampsora (Schub.) Cast. About 90 species of Melamp- species on poplars is based mainly on sora, showing either an autoecious or a the morphology of urediniospores and heteroecious life-cycle pattern, have been teliospores, and their host range. However, described worldwide (Kirk et al., 2001). some criteria often overlap and the taxon- Most Melampsora species occur on poplars omy of Melampsora on various poplar and willows, and not all are morphologi- species is not entirely clear at the present cally distinct (Cummins and Hiratsuka, time. With many poplar rusts, identification 2003). of species can be very difficult for several A worldwide literature survey by reasons. First, almost all the morphological Van Kraayenoord et al. (1974) showed that characteristics of teliospores are very simi- 32 Melampsora species names had been lar, and teliospores of some species do described on Populus. Of these, 11 species not appear during the growing season. could be recognized and others treated Secondly, most species infecting poplars as synonyms. Shang et al. (1986b) reviewed are heteroecious, having Abies, Allium, 34 species of poplar rusts reported world- Arum, Chelidonium, Corydalis, Fumaria, wide and recognized 12 species based on Larix, Mercurialis, Papaver, Picea, Pinus the host range and the characteristics of and Tsuga as alternate hosts, but informa- uredinia and telia. In a numerical taxonomi- tion on the alternate hosts is often unavail- cal study, Dai (1989) examined Melampsora able. Some species are also capable of species on poplars using 24 characters overwintering as mycelia in dormant buds derived from urediniospores and telio- of Populus and continue their existence spores, and aecial and telial hosts. As a without infecting alternate hosts. Thirdly, result, 14 species could be recognized. the same species of Populus can be infected Bagyanarayana (1998) studied the morphol- by different Melampsora species. Further- ogy of Melampsora species on Populus more, non-host poplars can sometimes be species and recognized nine species and infected in artificial inoculations.

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 99 100 C.-M. Tian and M. Kakishima

Record of Poplar Rusts in China Melampsora abietis-populi Imai, in Ito et Murayama, Trans. Sapporo Nat. Hist. The earliest report on Melampsora Soc. 17, 164; 1943 species on poplars in China was made by Miyake (1914), based on the specimens = Caeoma abietis-mayrianae S. Imai, 1942. collected in north-eastern China. In the Host plant: II, III on Populus cathayana Rehd. following years, the species of poplar (Shaanxi: Cao and Li, 1999) and P. wilsonii rusts were complied in publications by Schneid. (Hubei: Guo, 1989; Shaanxi: Cao other authors, such as Miura (1928), Huo and Li, 1999; Zhang et al., 1997). and Wang (1934) and Liu and Wang Distribution: China, Japan, Russia. (1935). Tai (1979) reviewed earlier studies This species resembles M. magnusiana of Melampsora spp. on poplars in China and M. laricis morphologically (Fig. 8.1A), and listed five species, M. larici-populina but can be distinguished from the two Kleb., M. laricis Hart., M. magnusiana species by the following features: (a) its Wagn., M. rostrupii Wagn. and M. urediniospores have thin walls (Fig. 8.1B); pruinosae Tranz. Yuan (1984) examined (b) the distance between spinules is less the morphology of Melampsora species compared with that in M. magnusiana and on poplars and recorded three more species M. laricis (Fig. 8.1C); and (c) it alternates on in China, i.e. M. abietis-canadensis (Farl.) Abies. Ludw., M. allii-populina Kleb. and M. Zhuang and Wei (1994) identified some occidentalis Jacks. Shang et al. (1986a) specimens (HMAS 67387, 67388) occurring described a new species, M. multa on P. pseudoglauca Wang et Fu collections Shang, Pei and Yuan on P. × euramericana from Tibet as M. populnea (Per.) Karst. We Moench. collected in Liaoning, China. also saw these specimens and found that, Melampsora magnusiana and M. rostrupii morphologically, they differed from M. were treated by Shang et al. (1986b, 1990) as populnea in having urediniospores with synonyms of M. aecidioides Plowr. thin walls (Fig. 8.1B). In our phylogenetic Recently, M. abietis-populi Imai was analyses, these specimens were clearly reported on Populus wilsonii Schneid. separated from other groups such as M. in Shaanxi and Hubei provinces (Guo, laricis Hartig. or M. populnea (Tian et al., 1989; Zhang et al., 1997; Cao and Li, 2004). Thus, we consider that these speci- 1999). Previously, M. abietis-populi had mens belong to M. abietis-populi. We also only been recorded in Japan (Hiratsuka recorded this species for the first time on et al., 1992; Bagyanarayana, 1998). Zhuang P. yunnanensis Dode collected in Yunnan and Wei (1994) reported M. populnea Province (Tian et al., 2004). (Pers. ex Pers.) Karst., for the first time This species was established by Imai in China, on P. pseudoglauca Wang and (1942) based on a rust fungus on P. nigra var. Fu., and P. szechuanica Schneid. var. italica (Moench) Koehne in Hokkaido, tibetica Schneid. In their report, M. laricis Japan, and the alternation of M. abietis- was treated as a synonym of M. populnea. populi between Populus and Abies was Subsequently, some specimens from dif- proven by Imai (1942) and Hiratsuka et al. ferent regions were also recognized as (1992). In China, no specimens of spermo- M. populnea (Cao and Li, 1999). In total, gonial and aecial stages have been collected 11 Melampsora species names have and no inoculation experiments conducted. been recorded on poplars in China We consider that the thin wall is a stable (Table 8.1). In this chapter, we present feature in distinguishing this species from a review of the current taxonomic status other Melampsora species, based on our of Melampsora species on Populus in morphological and molecular phylogenetic China. studies (Tian et al., 2004). Taxonomic Status of Melampsora Species on Poplars in China 101

Alternate hosts (genus) Larix Larix

Aigeiros Aigeiros , ,

Tacamahaca ,

Host section Tacamahaca Tacamahaca Leuce Leuce Leuce Leuce Turanga Leuce Tacamahaca Leucoides Aigeiros 6–13 5–10 9–12 8–12 9–13 8–14 8–13 7–12 9–13 9–13 8–12 × × × × × × × × × × × m) m ( 30–45 15–32 30–48 27–48 38–59 27–44 27–48 30–60 37–51 38–56 37–60 Teliospore size ., 1986b; Guo, 1989; Zhuang and Wei 1994; Cao and Li, 1999).

et al m) m Smooth parts ( Apical parts Apical parts No No No No No Equatorial parts No No Apical parts m) m 2–3 2–3 2–3 ( Walls 2–2.8 2.8–3.5 2.5–3.5 2.7–3.5 1.7–2.7 2–3 (5.1–6.8) 2–3 (5.1–6.8) Urediniospores 16–25 17–20 16–21 14–17 14–18 13–20 17–20 13–20 20–27 11–22 16–25 m) × × × × × × × × × × × m Size ( 23–46 26–38 19–26 16–28 15–28 17–27 18–27 17–25 27–41 17–32 26–44 spp. reported in China (Tai, 1979; Yuan, 1984; Shang ) )

Melampsora

M. aecidioides M. larici-tremulae ( (

Table 8.1. Species M. larici-populina M. allii-populina M. magnusiana M. populnea M. rostrupii M. laricis M. pruinosae M. abietis-canadensis M. occidentalis M. abietis-populi M. multa 102 C.-M. Tian and M. Kakishima

Fig. 8.1. Urediniospores and teliospores of Melampsora spp. on Populus observed by light microscopy and scanning electron microscopy. (A)–(C) M. abietis-populi on P. wilsonii. (A) Hypophyllous telia; (B) globose or ellipsoid urediniospores with thin walls; (C) urediniospores with echinulate surface. (D)–(E) M. allii-populina on poplars. (D) Amphigenous telia on P. laurifolia; (E) oblong urediniospores with uniformly thickened wall on P. laurifolia; (F) urediniospores with a smooth apex on P. talassica. Bars: (A) 30 mm; (B) 15 mm; (C) 20 mm; (D) 55 mm; (E) 35 mm; (F) 20 mm.

Melampsora abietis-canadensis (Farl.) Host plant: II, III on P. davidiana Dode Ludw. Phytop. 5, 279; 1915 (Shanxi and Jilin: Yuan, 1984), P. cathayana Rehd. (Shanxi: Yuan, 1984). = Caeoma abietis-canadensis Farl., Proc. Distribution: China, Canada, USA. Am. Acad. 20, 323; 1885. Yuan (1984) described M. abietis- = Melampsora populi-tsugae Havis, Trans. canadensis for the first time in China based Wis. Acad. 19, 676; 1919. on the specimens on P. davidiana Dode and Taxonomic Status of Melampsora Species on Poplars in China 103

P. cathayana Rehd. He considered that ‘a perpetuate infection on poplar as the few urediniospores with a smooth area at the uredinial stage. Bagyanarayana (1998) equator’ is an important feature in distin- placed M. allii-populina together with guishing this species from others. Although other four species, as formae speciales of we were not able to see the same specimens M. populnea (Pers. ex Pers.) Karst., based on that Yuan examined, we noticed a smooth different aecial hosts. However, M. allii- area at the equator of urediniospores in some populina can be distinguished from M. other specimens, such as the rust on P. populnea by the urediniospores with a davidiana (Fig. 8.2D). These specimens smooth apex (Fig. 8.1F), and its amphi- were grouped together with other specimens genous telia. Furthermore, it differs from of M. laricis in our phylogenetic analyses other Melampsora species in phylogeny (Tian et al., 2004). In Japan, Ito (1938) and (Tian et al., 2004). Hiratsuka and Karube (1969) also described Previously, M. allii-populina was some specimens of M. laricis that had known in China by the only specimen urediniospores with slightly thickened side collected from P. talassica Komarov from walls, and some urediniospores that had Xinjiang Province (Yuan, 1984). In this a smooth area at the equator. Thus, we chapter we record this species for the first consider that the previously reported M. time from Inner Mongolia on two new poplar abietis-canadensis (Farl.) Ludwig in China hosts, P. opera of section Tacamahaca, and is a consequence of misidentification. Mel- P. davidiana Dode of section Leuce, and also ampsora abietis-canadensis is regarded as a from Xinjiang on P. laurifolia Ledebour. synonym of M. medusae (Cellerino, 1999).

Melampsora laricis Hartig, Allg. Melampsora allii-populina Kleb. Zeit. Forest-Jagdzeit. 61, 326; 1885 Pfl. – Krankh. 12, 25; 1902 = Uredo laricis Arth. Result. Scient. Congr. = Caeoma alliorum Link, sp. Pl. 2, 7; 1825. Intern. Bot. Vienne 338; 1905, 1906. = Caeoma allii-ursini Winter, Pilze = M. larici-tremulae Kleb., Forestl. Nat. Deutschl. I, 255; 1881. Zeits. 468; 1897. = Uredo allii-populina Arth., Rèsult Scient. = M. tremulae f. sp. laricis Hartig, Lehrb. Congr. Intern. Bot. Vienne 338; 1905, 1906. Baumkrankh. Ed. 2, 14; 1889. = Melampsora populnea f. sp. allii- = M. populnea f. sp. laricis (Hart.) Boer. and populina (Kleb.) Bagyanarayana, Proc. 1st Verh., Neth. J. Pl. Path. 78 (1), 82–201; 1972. IUFRO Fore. Trees Working Party Conf. 47; Host plants: 0, I on Larix olgensis Henry, L. 1998. gmelinii (Rupr.) Rupr., L. kaempferi (Sieb. Host plants: II, III on P. talassica Komarov et Zucc.) Gord., L. principis-ruppechtii Mayr. (Xinjiang: Yuan, 1984). and L. sibirica Ledeb. (Heilongjiang: Shang Distribution: China, southern Europe, and Pei, 1984). II, III on Populus alba L. × P. Russia. davidiana Dode (Heilongjiang: Yuan, 1984), Morphological characteristics of P. adenopoda Maxim. (Guangxi: Tai, 1979; urediniospores and teliospores of M. allii- Hubei: Guo, 1989; Shaanxi: Cao and Li, populina are somewhat similar to those of 1999), P. davidiana Dode (Heilongjiang and M. larici-populina, but it is distinguishable Shaanxi: Tai, 1979; Shanxi: Yuan, 1984; by having urediniospores with uniformly Inner Mongolia: Shang et al., 1990; Shaanxi: thickened (2–4 mm) walls (Fig. 8.1E), para- Cao and Li, 1999; Inner Mongolia: Zhuang physes with uniformly thin walls, and and Wei, 2002), P. pseudoglauca Wang et amphigenous telia (Fig. 8.1D). Although M. Fu (Tibet: Zhuang, 1986), P. tomentosa Carr. allii-populina completes its full life cycle (Shaanxi: Tai, 1979), P. tremula L. (Tibet: by infecting herbaceous plants belonging Zhuang, 1986), P. sp. (Henan: Tai, 1979), to Allium, Arum and Muscari, it can P. wilsonii Schneid (Hubei: Guo, 1989). 104 C.-M. Tian and M. Kakishima

Distribution: China, Japan, Europe, Russia, Melampsora larici-populina Kleb., Zeit. Africa, USA. Pfl. –Krankh. 12, 43; 1902 Shang and Pei (1984) were the first to establish the full life history of M. laricis = Caeoma laricis Hart. Wichtige Krankh. (= M. larici-tremulae Kleb.) in China, when Wäldbaume 93; 1847. they produced uredinia and telia on Populus = Uredo larici-populina Arth., Rèsult. spp. in artificial inoculations with aecidio- Scient. Congr. Intern. Bot. Vienne 338; spores from Larix spp. It was proven that 1905, 1906. basidiospores of M. laricis can infect L. = Melampsora populina (Jacq.) Lev., Ann. olgensis, L. gmelinii, L. kaempferi, L. Sci. Nat., 375; 1847. principis-rupprechtii and L. sibirica, and = Melampsora populina Auct. pr. P that its aeciospores can infect the species of Host plants: 0, I on Larix gmelinii (Rupr.) section Leuce. It also infected the hybrids Kuz. (Heilongjiang: Tai, 1979; Pei and having section Leuce as female parents, but Shang, 1984; Zhuang and Wei, 2002), L. not the species of sections Aigeios and kaempferi (Sieb. et Zucc.) Gord. (north-east Tacamahaca, and the hybrids between these China: Tai, 1979; Heilongjiang: Pei and two sections. Shang, 1984), L. olgensis Henry (Heilong- Wilson and Henderson (1966) adopted jiang: Pei and Shang, 1984; Shaanxi: Cao M. populnea as a complex species to and Li, 1999), L. principis-ruppechtii Mayr. include ‘various races’ which are similar in (Heilongjiang: Pei and Shang, 1984; morphology, but different in aecial hosts. Shaanxi: Cao and Li, 1999), L. sibirica Boerema and Verhoeven (1972) also treated Ledeb. (Heilongjiang: Pei and Shang, 1984), M. laricis as a forma specialis (f. sp. laricis) L. sp. (Qinghai and Heilongjiang: Tai, 1979). within M. populnea, based on the aecial II, III on Populus adenopoda Maxim. host. In China, M. laricis has recently been (Shaanxi: Wei and Zhuang, 1997), P. × regarded as synonymous with M. populnea berolinensis Dippel (Inner Mongolia, by Zhuang and Wei (1994) and others (Cao Liaoning, Jilin and Heilongjiang: Tai, 1979; and Li, 1999), but these workers also have Shang et al., 1990), P. × beijingensis Hsu accepted M. magnusiana as a distinct taxon. (Beijing, Inner Mongolia and north-east Although M. laricis is similar to M. mag- China: Tai, 1979; Yuan, 1984; Shang et al., nusiana in the shape and size of uredinio- 1990), P. cathayana Rehd. (Hebei, Liaoning: spores, it differs from M. magnusiana by Tai, 1979; Beijing: Yuan, 1984; Inner having urediniospores with slightly thick- Mongolia: Shang et al., 1990; Shaanxi: ened lateral walls (Fig. 8.2B) and sometimes Wei and Zhuang, 1997; Cao and Li, 1999), with a smooth area at the equator (Fig. 8.2D). P. × euramericana (Dode) Guinier (Inner Furthermore, the uredinia of M. laricis are Mongolia, Liaoning and north-east China: hypophyllous (Fig. 8.2A), scattered and Tai, 1979; Beijing, and Jilin: Yuan, 1984; pale-yellow, while M. magnusiana often Inner Mongolia: Shang et al., 1990), P. × produces uredinia on leafstalks and young euramericana var. regenerata (Henry) Rehd. buds. The uredinia of M. magnusiana are (Inner Mongolia and north-east China: Tai, epiphyllous, often aggregated in groups and 1979), P. davidiana Dode (Hebei: Tai, 1979; yellow (Shang et al., 1990; Y.Z. Shang, 2002, Tibet: Zhuang, 1986), P. deltoides Marsh personal communication). Recently, our (Beijing: Yuan, 1984; Inner Mongolia: phylogenetic analyses placed the M. laricis Shang et al., 1990), P. euphratica Oliv. specimens on P. adenopoda Maxim., P. (Liaoning: Tai, 1979; Inner Mongolia: Yuan, davidiana Dode, and P. tomentosa Carr. 1984), P. harbinensis Wang et Skv. (Inner collected from China into one group, and Mongolia and north-east China: Tai, 1979; showed that this rust has a distant relation- Beijing: Yuan, 1984), P. × gansuensis Wang ship with other Melampsora species occur- et Yang (Inner Mongolia: Shang et al., ring on section Leuce (Tian et al., 2004). 1990), P. koreana Rehd. (Liaoning and Therefore, we consider that M. laricis is a Heilongjiang: Tai, 1979; Inner Mongolia: distinct taxon. Shang et al., 1990), P. laurifolia Ledebour Taxonomic Status of Melampsora Species on Poplars in China 105

Fig. 8.2. Urediniospores and teliospores of Melampsora spp. on Populus observed by light microscopy and scanning electron microscopy. (A)–(D) Melampsora laricis on Populus davidiana. (A) Teliospores; (B) ovoid or ellipsoid urediniospores with walls uniformly thickened or slightly thickened laterally; (C) urediniospores with echinulate surface; (D) occasionally, urediniospores have a smooth area at the equator. (E)–(F) M. multa on P. × euramericana. (E) Telia; (F) ellipsoid or oblong urediniospores with walls uniformly thickened or slightly thickened laterally. Bars: (A) 35 mm; (B) 20 mm; (C) 12 mm; (D) 20 mm; (E) 30 mm; (F) 30 mm.

(Inner Mongolia and north-east China: Tai, (Moench) Koehne (Hebei, Inner Mongolia, 1979; Yuan, 1984; Shang et al., 1990; Heilongjiang and north-east China: Tai, Zhuang, 1999), P. lasiocarpa Oliv. 1979; Shang et al., 1990; Liaoning: Yuan, (Shaanxi: Wei and Zhuang, 1997), P. 1984; Shaanxi: Wei and Zhuang, 1997; Cao manshurica Nakai (Liaoning: Tai, 1979), and Li, 1999), P. nigra var. thevestina P. maximowiczii Henry (north-east China: (Dode) Bean (Inner Mongolia: Yuan, 1984; Tai, 1979; Jilin: Yuan, 1984), P. nigra L. Shang et al., 1990; Shaanxi: Wei and (Inner Mongolia, Liaoning and north-east Zhuang, 1997), P. nigra L. × P. hopeiensis China: Tai, 1979; Shang et al., 1990; Beijing Hu et Chow (Shaanxi: Wei and Zhuang, and Jilin: Yuan, 1984) P. nigra var. italica 1997), P. opera Hsu (Beijing: Yuan, 1984; 106 C.-M. Tian and M. Kakishima

Inner Mongolia: Shang et al., 1990), P. and most of the species of section Aigeiros, pioneer (Heilongjiang: Yuan, 1984), P. and the hybrids within or between these two purdomii Rehd. (Shaanxi: Wei and Zhuang, sections. M. larici-populina did not infect 1997; Cao and Li, 1999), P. pseudoglauca the species of section Leuce or section Wang et Fu (Tibet: Zhuang, 1986; Zhuang Turanga under natural conditions. Uredinio- and Wei, 1994), P. pseudo-simonii Kitag. spores of M. larici-populina infected P. (Beijing, Inner Mongolia, Liaoning and euphratica in inoculation experiments north-east China: Tai, 1979; Yuan, 1984; when urediniospores collected from Shang et al., 1990), P. pseudo-simonii × P. Populus × gansuensis Wang et Yang were deltoides (Beijing: Yuan, 1984), P. × used as an inoculum (Yuan, 1984). However, euramericana ‘Robusta’ (Inner Mongolia the urediniospores produced had uniformly and north-east China: Tai, 1979), P. simonii thickened walls, and only a few uredinio- (Beijing: Liu and Wang, 1939; Hebei, spores had slightly laterally thickened Shanxi, Inner Mongolia, Liaoning, Jilin, walls, differing from the spores used for Heilongjiang and Henan: Tai, 1979; Yuan, inoculation. 1984; Inner Mongolia: Shang et al. 1990; Shaanxi: Wei and Zhuang, 1997; Cao and Li, 1999), P. simonii var. shomliflia (Beijing: Yuan, 1984), P. simonii × P. davidiana Melampsora magnusiana Wagner, Zeit. (Inner Mongolia: Shang et al., 1990), P. sp. Pfl. –Krankh. 7, 340; 1897 (Tibet: Zhuang, 1986), P. szechuanica, P. trichocarpa (Beijing and Inner Mongolia: = M. aecidioides Plowr., Brit. Ured. Ustil. Yuan, 1984), P. ussuriensis Kom. (Inner 241; 1889. Mongolia, Heilongjiang and north-east = M. aecidioides (DC) Schroet., Cohn, China: Tai, 1979; Yuan, 1984; Shang et al., Krypt. Fl. Schles. 3 (1), 362; 1989. 1990), P. × xiaohei Hwang et Liang (Inner = M. rostrupii Wagner, Oest. Bot. Zeit. 46, Mongolia: Shang et al., 1990), P. × 274; 1896 (nomen nudum). xiaozuanica Hsu et Liang (Inner Mongolia: = M. pulcherrima Maire, Myc. Bor. Afr. Shang et al., 1990), P. yunnanensis Dode Fass. 5, 108; 1914. (Yunnan: Tai, 1979), and most cultivars = M. populnea (Pers.) Karst., Brdr. Kanned. of Argeiros and Tacamahaca and some Finl. Nat. Folk. 31, 53; 1879. hybrids between Argeiros and Tacamahaca = M. tremulae Tulasne, Ann. Sci. Nat. 4 (2), (Tai, 1979; Yuan, 1984; Shang et al., 1990). 95; 1854. Distribution: China, Europe, Japan, India, = M. populnea f. sp. magnusiana Bagyan- Korea, Mongolia, New Zealand, Australia, arayana, Proc. IUFRO Rust Forest Tree South America, USA and Russia. Working Party Conf. 48; 1998. M. larici-populina is similar to M. allii- = M. populnea f. sp. rostrupii Bagyan- populina in morphology (Figs 8.1F, 8.3C), arayana, Proc. IUFRO Rust Forest Tree but readily distinguishable from other Working Party Conf. 50, 1998. poplar rusts, such as M. allii-populina,by Host plants: 0, I on Corydalis sp. (Hebei and having epiphyllous telia (Fig. 8.3A), elon- north-east China: Tai, 1979). II, III on gate urediniospores with distinct laterally Populus alba L. (Liaoning and Jiangsu: Tai, thickened walls (Fig. 8.3B), and paraphyses 1979; Shaanxi: Ge et al., 1964; Beijing: with apical walls thickened up to 25 mm. In Yuan, 1984; Inner Mongolia: Shang et al., China, this species is the most prevalent and 1990), P. alba L. var. pyramidalis Bge. widely distributed of all the poplar patho- (Xinjiang: Yuan, 1984; Inner Mongolia: gens. It also has the greatest range of telial Yuan, 1984; Shang et al., 1990), P. hosts and is one of the most damaging forest davidiana Dode (Hebei, Xinjiang and tree diseases in man-made forests in north- north-east China, Tai, 1979; Guizhou: Yuan, ern China. In inoculation experiments (Pei 1984; Shaanxi: Zhang et al., 1997), P. and Shang, 1984), its aeciospores infected hopeiensis Hu et Chow (Inner Mongolia: all the tested species of section Tacamahaca Shang et al., 1990), P. shanxiensis Wang Taxonomic Status of Melampsora Species on Poplars in China 107

Fig. 8.3. Urediniospores and teliospores of Melampsora on poplars observed by light microscopy and scanning electron microscopy. (A)–(C) Melampsora larici-populina on Populus spp. (A) Hypophyllous uredinia and epiphyllous telia on Populus opera; (B) ellipsoid or oblong urediniospores with equatorially thickened walls on P. laurifolia; (C) echinulate urediniospores with a smooth apex on P. simonii × P. nigra var. italica. (D)–(F) Melampsora magnusiana Wagn. on Populus spp. (D) Teliospores on P. alba var. pyramidalis; (E) globose or ovate urediniospores with uniformly thickened walls on P. tomentosa; (F) urediniospores with echinulate surface on P. tomentosa. Bars: (A) 120 mm; (B) 35 mm; (C) 21 mm; (D) 35 mm; (E) 14 mm; (F) 20 mm. et Tung (Inner Mongolia: Shang et al., Distribution: China, eastern Europe, Russia, 1990), P. tomentosa Carr. (Shandong, USA. Shaanxi, north-western China and Shanxi: According to Wilson and Henderson Tai, 1979; Shaanxi: Wei and Zhuang, 1997; (1966), M. magnusiana is morphologically Cao and Li, 1999; Inner Mongolia: Shang very similar to M. rostrupii and both have et al., 1990), and Populus spp. of section the uredinial and telial stages on the same Leuce. species of Populus. The only difference is in 108 C.-M. Tian and M. Kakishima

their aecial hosts. M. rostrupii was a uredinia of this species are epiphyllous nomen nudum of M. aecidioides and is (Fig. 8.3D), often found on leafstalks and placed under M. populnea. Boerema and young buds, aggregated in groups and Verhoeven (1972) reduced M. rostrupii to a yellow. Urediniospores of M. magnusiana forma specialis of the species M. populnea. have uniformly thickened walls (Fig. 8.3E) On a similar basis, Bagyanarayana (1998) and the distance between spinules is 2–3 mm considered five species as formae speciales (Fig. 8.3F). of M. populnea and treated two as formae Zhuang (1999) reported M. aecidioides speciales within M. populnea, i.e. f. sp. Plowr. (on Populus collected from Xinjiang magnusiana and f. sp. rostrupii.He province) as well as M. magnusiana in described the length of urediniospores of China. But many authors considered that M. populnea as over 30 mm. However, these two species names refer to the same urediniospores of M. populnea or M. species and M. magnusiana was a nomen magnusiana occurring on the section Leuce nudum of M. aecidioides (Shang et al., are less than 30 mm long (Wilson and 1990). Recently, morphological and molecu- Henderson, 1966). lar studies suggested that there are no obvi- Three rust species have been reported ous differences among specimens collected on Populus tomentosa in China: M. in China which had been identified as magnusiana (Wang, 1949; Zhou et al., 1979), M. rostrupii and M. magnusiana, and M. M. rostrupii (Ge et al., 1964) and Uredo aecidioides (Tian et al., 2004). tholopsora (Cummins, 1951). The former two species are common in China and differ from each other only in their aecial hosts. M. magnusiana alternates on Corydalis and Melampsora multa Shang, Pei et Yuan, Chelidonium whereas M. rostrupii alter- Acta Mycol. Sin. suppl. I, 180–184; 1986 nates on Mercurialis. In Japan, Hiratsuka (1927, 1932) established that M. magnusiana Host plant: II, III on P. × euramericana cv. alternates between Populus sieboldii and ‘Robusta’, P. × euramericana cv. ‘Serotina’, Chelidonium majus. According to the P. × euramericana cv. ‘I-214’ (Liaoning: records, spermogonia and aecia of M. Shang et al., 1986a). magnusiana have been found on Corydalis Distribution: China (Liaoning). in Hebei province, China (Tai, 1979). This This species was established by Shang record is doubtful as Miura (1928) reported a et al. (1986a) based on a rust fungus on P. × rust species, Caeoma fumariae on Corydalis euramericana which occurred in Liaoning spp. in Caohekou of Liaoning Province, province. The same authors reported that but he did not establish the relationship this species did not infect Larix principis- between M. magnusiana and Caeoma fuma- ruppechtii Mayr., Picea wilsonii Mast., Picea riae. M. yezoensis Miyebe et Matsumoto meyeri Rehd., Pinus tabulaefomis Carr. and also forms aecia on Corydalis. At present, Sabina chinensis (L.) Antonine in artificial host alternation in M. magnusiana and M. inoculation experiments. Morphologically, rostrupii in China is not clear. Field observa- this species resembles M. larici-populina, tions and results from inoculation experi- but differs from other poplar rusts by having ments have shown that M. magnusiana single or 2–3-layered telia and being overwinters on poplar as uredinial myce- amphigenous (Shang et al., 1986a). Our lium. Thus, it can survive and propagate examination of the isotype of M. multa itself in many areas where its alternate host suggested that the morphology of this is absent. In fact, Corydalis and Chelidonium species is almost the same as that of are rarely infected and the teliospores of M. larici-populina, although M. multa M. magnusiana may not be important in the occasionally has two- or three-layered life cycle. telia. It is possible that some specimens of Although M. magnusiana is very similar M. larici-populina Kleb. occasionally pro- to M. laricis in urediniospore shape and size, duce two-layered telia. Further studies are Taxonomic Status of Melampsora Species on Poplars in China 109

needed to clarify the taxonomic status of Distribution: China, Japan, Europe. these closely related rust fungi. Recently, rust species occurring on Populus adenopoda and P. davidiana were treated as M. populnea (Pers. ex Pers.) Melampsora occidentalis Jacks., Karst. in China. Many authors adopted Phytopath. 7, 354; 1917 M. populnea as a complex species and some poplar rusts, such as M. laricis, = Caeoma occidentalis Arth., Bull. Torrey M. magnusiana, M. allii-populina were Bot. Club 34, 591; 1907. included under this species (Wilson and Host plant: On P. cathayana Rehd. Henderson, 1966; Boerema and Verhoeven, (Heilongjiang: Yuan, 1984), P. koreana 1972; Bagyanarayana, 1998). Rehd. (Heilongjiang: Yuan, 1984), P. Zhuang (1986) identified some speci- yunnanensis Dode (Yunnan: Yuan, 1984) mens occurring on Populus pseudoglauca and P. × euramericana cv. ‘Marilandica’ collections from Tibet as M. laricis, and the (Jilin: Yuan, 1984). same author also treated some other speci- Distribution: Canada and USA. mens on P. pseudoglauca collected from This species was reported for the first Tibet as M. populnea (Zhuang and Wei, time in China by Yuan (1984) based on three 1994). We were not able to clarify whether specimens on Populus. The fungus is these two species on P. pseudoglauca were characterized by having spinules at the apex different or the same in their papers. But of urediniospores. Usually, the smooth area these specimens on P. pseudoglauca were at the apex of urediniospores is observed identified as M. abietis-populi based our using a light microscope. However, scan- studies (Tian et al., 2004). ning electron microscopy revealed that in Zhang et al. (1997) reported M. mag- some specimens of M. larici-populina Kleb., nusiana on P. davidiana, but this species such areas are either smooth or have some cannot be distinguished from M. populnea spinules varying in size (Gao, 1982; Shang in reality, and specimens on P. davidiana et al., 1990). We also found that, in some may be M. laricis. specimens of M. larici-populina, many urediniospores have spinules at the apex and only a few urediniospores have a Melampsora pruinosae Tranz., smooth area at the apex. In our phylogenetic Tranzxchel et Serebriannikov, analyses, these specimens were grouped Mycotheca Rossica No. 265; 1912 together with other specimens of M. larici-populina (Tian et al., 2004). Thus, Host plant: II, III on P. euphratica Oliv. we consider that the distribution of M. (Inner Mongolia: Shang et al., 1990; occidentalis in China is doubtful. Xinjiang and Inner Mongolia: Yuan, 1984), P. nigra var. italica (Moench) Koehne (Shaanxi: Wei and Zhuang, 1997), Populus Melampsora populnea (Pers.) Karst., sp. (Xinjiang: Tai, 1979), P. tomentosa Carr. Bidr. Kammed. Finl. Nat. Folk 31, (Shaanxi: Wei and Zhuang, 1997), P. × 53; 1879 xiaozuanica Hsu et Liang (Inner Mongolia: Shang et al., 1990). Host plant: Populus adenopoda Maxim Distribution: China (Xinjiang, Ningxia, (Shaanxi: Cao and Li, 1999), P. davidiana Inner Mongolia), Russia. Dode (Shaanxi: Cao and Li, 1999), P. This species is similar to M. pseudoglauca Wang et Fu (Tibet: Zhuang magnusiana in morphology, but easily dis- and Wei, 1994), P. szechuanica Schneid. tinguishable by its urediniospores, which var. tibetica Schneid. (Tibet: Zhuang and have very small spinules, a short distance Wei, 1994), Populus sp. (Tibet: Zhuang and between spinules (< 2 mm; Fig. 8.4C) and Wei, 1994). thick walls (Fig. 8.4B). Geographically, this 110 C.-M. Tian and M. Kakishima

rust is distributed in China’s north-western Melampsora rostrupii Wagn., Oest. Bot. desert areas, i.e. the provinces of Xinjiang, Zeit. 46, 274; 1896 (nomen nudum) Ningxia and Inner Mongolia. In nature, it has only been recorded on Populus euphratica Host plant: Populus alba L. (Liaoning and and is non-pathogenic to poplars of the Jiangsu: Tai, 1979), P. candicans Ait section Leuce and their hybrids. This rust (Liaoning: Tai, 1979), P. rotundifolia Griff. can cause slight infection on P. tomentosa (Yunnan: Tai, 1979), P. tomentosa Carr. Carr. in artificial inoculations in nurseries, (Shaanxi: Ge et al., 1964; Henan: Tai, 1979), but not under natural conditions (Yuan, P. tremula L. (Hebei: Tai, 1979), P. tremula 1984). No alternate hosts have been found L. var. villosa Weam (Hebei: Tai, 1979), with M. pruinosae. Populus sp. (Yunnan: Tai, 1979). Wei and Zhuang (1997) identified sev- Distribution: China, Europe, Russia. eral specimens on P. tomentosa Carr., and Melampsora rostrupii has been P. nigra var. italica (Moench) Koehne from recorded together with M. magnusiana on Shaanxi province as M. pruinosae. However, the same Populus species in China, such as our work suggested that these specimens P. tomentosa. This species is morphologi- should be identified as M. magnusiana as cally similar to M. magnusiana; the only dif- their urediniospores have larger spinules, ference being in the aecidial host. However, the distance between spinules is longer nobody can distinguish these two species by m (> 2 m) and both uredinia and telia are their morphology. Zhou et al. (1979) investi- amphigenous (Fig. 8.4A, C). gated the distribution of alternate hosts, i.e.

Fig. 8.4. Urediniospores and teliospores of Melampsora pruinosae on Populus euphratica observed by light microscopy and scanning electron microscopy. (A) Amphigenous uredinia and telia. (B) Globose or ovate urediniospores with uniformly thickened wall. (C) Urediniospore surface with small spinules. (D) Amphigenous telia. Bars: (A) 100 mm; (B) 20 mm; (C) 8.4 mm; (D) 65 mm. Taxonomic Status of Melampsora Species on Poplars in China 111

Mercurialis; he considered that the rust on Dai, Y.C. (1989) Study on poplar rusts (Melampsora P. tomentosa in China is M. magnusiana (see spp.) by numerical taxonomic method [in the entry on Melampsora magnusiana). Chinese]. Scientia Silvae Sinicae 25, 87–91. Gao, Y. (1982) Scanning of the urediniospore of poplar leaf rust Melampsora larici-populina [in Chinese]. Acta Microbiologica Sinica 22, 26–30. Acknowledgements Ge, G.P., Jing, Y., Shen, M.M. and Shi, Q. (1964) Observations on the development and We thank Professor Y.Z. Shang (College morphology of Melampsora rostrupii Wagner of Forestry, Inner Mongolia Agricultural on Populus tomentosa. Scientia Silvae Sinicae 9, University, China) for constructive review 221–232. comments and providing many herbarium Guo, L. (1989) Uredinales of Shennongjia, China. In: specimens. We are also grateful to Dr J.Y. Mycological and Lichenological Expedition to Zhuang (Institute of Microbiology, Chinese Shennongjia, Academia Sinica (eds) Fungi and Academy of Sciences), Dr Z.M. Cao and Lichens of Shennongjia [in Chinese]. Beijing World Publishing Co, Beijing, p. 113. Y.M. Liang (Northwest Sci-Tech University Hiratsuka, N. (1927) Notes on Japanese species of of Agriculture and Forestry, China), Q. Melampsora parasitic on species of Larix [in Wang (College of Chinese Medicinal Mate- Japanese]. Journal Society of Agricultural and rials, Jilin Agricultural University, China), Forestry, Sapporo 19, 180–194. Professor Z.Y. Yue (Xinjiang Academic Hiratsuka, N. (1932) Inoculation experiments with of Forestry, China) and Professor Y.D. Mo some heteroecious species of the Melamp- (University of Qinghai, China) for providing soraceae in Japan. Japanese Journal of Botany 6, herbarium specimens. This work was 1–33. supported by the Japan Society for the Hiratsuka, N. and Karube, Y. (1969) Studies on poplar Promotion of Science (JSPS). leaf rusts in Japan [in Japanese]. Bulletin Sugino Women’s College 6, 31–56. Hiratsuka, N., Sato, S., Katsuya, K., Kakishima, M., Hiratsuka, Y., Kaneko, S., Ono, Y., Sato, T. and References Harada, Y. (1992) The Rust Flora of Japan. Tsukuba Shuppankai, Tsukuba, Ibaraki, Japan, Bagyanarayana, G. (1998) The species of Melampsora pp. 271–272. on Populus (Salicaceae). In: Jalkanen, R., Crane, Huo, J.F. and Wang, M.D. (1934) A preliminary list of P.E., Walla, J.A. and Aalto, T. (eds) Proceedings fungi in north China. Annals of the Research of the First IUFRO Rusts of Forest Trees Working Council National University of Peking 1, 1–22. Party Conference, 2–7 August, Saariselkä. Finish Imai, S. (1942) Damage caused by Caeoma Forest Research Institute, Rovaniemi, Finland, abietis-mayrianae on todo-fir seedlings and pp. 37–51. the life-cycle of the pathogen. Annals of the Boerema, C.H. and Verhoeven, A.A. (1972) Phytopathological Society of Japan 12, 68–69. Check-list for scientific names of common Ito, S. (1938) Uredinales-Melampsoraceae. In: Myco- parasitic fungi. Series la: Fungi on trees and logical Flora of Japan. Vol. 2. Basidiomycetes. 2 shrubs. Netherlands Journal of Plant Pathology [in Japanese]. Yokendo, Tokyo, pp. 113–117. 78, 24–25. Kirk, P.M., Cannon, P.F., David, J.C. and Stalpers, J.A. Cao, Z.M. and Li, Z.Q. (1999) Rust Fungi of Qinling (2001) Ainsworth and Bisby’s Dictionary of the Mountains [in Chinese]. China Forestry Publish- Fungi, 9th edn. CAB International, Wallingford, ing House, Beijing, pp. 29–41. UK, pp. 311. Cellerino, G.P. (1999) Review of poplar diseases. (3. Liu, T.N. and Wang, Y.Z. (1935) Materials for study on Disease by fungi. 3.4.1. Rusts caused by Melam- rusts of China (III). Contributions from the Insti- psora spp.) Grugliasco. Available at website tute of Botany, National Academy of Peking 3, http://www.efor.ucl.ac.be/ipc/pub/celle01/ 353. celle01.htm (accessed 27 September 2004). Miura, M. (1928) Flora of Manchuria and East Mongo- Cummins, G.B. (1951) Uredinales of continental lia. Part. III, Cryptogams, Fungi [in Japanese]. China collected by S.Y. Cheo II. Mycologia 43, South Manchuria Railway Co, pp. 230, 393. 78–98. Miyake, I. (1914) Über Chinensiche Pilze. Botanical Cummins, G.B. and Hiratsuka, Y. (2003) Illustrated Magazine 28, 37–56. Genera of Rust Fungi, 3th edn. APS Press, Pei, M.H. and Shang, Y.Z. (1984) Study on the leaf Minnesota, p. 74. rust of cathay poplar caused by Melampsora 112 C.-M. Tian and M. Kakishima

larici-tremulae Kleb [in Chinese]. Journal of Wei, S.X. and Zhuang, J.Y. (1997) Species checklist Northeastern Forestry Institute 12 (4), 40–49. of uredinales of the Qinling Mountains [in Shang, Y.Z. and Pei, M.H. (1984) Study on the leaf Chinese]. In: Mao, X.L. and Zhuang, J.Y. (ed.) rust of Davids European Aspen caused by Fungi of the Qinling Mountains. China Agri- Melampsora larici-tremulae Kleb. Journal of cultural Science and Technology Publishing Northeastern Forestry Institute 12 (3), 47–55. House, Beijing, pp. 37–38. Shang, Y.Z., Pei, M.H. and Yuan, Z.W. (1986a) A Wilson, M. and Henderson, D.M. (1966) The British new rust fungus on poplars [in Chinese]. Acta Rust Fungi. Cambridge University Press, Mycologica Sinica Supplement 1, 180–184. London, pp. 64–93. Shang, Y.Z., Yuan, X.Y. and Hao, J.Z. (1986b) Yuan, Y. (1984) On identification of some species Taxonomy of Melampsora on Populus [in of Melampsora infecting poplar trees in China Chinese]. Journal of Inner Mongolia Forestry and a test of host range of M. pruinosae [in College 8, 126–133. Chinese]. Journal of Beijing Forestry College 6, Shang, Y.Z., Hao, J.Z. and Yuan, X.Y. (1990) Melamp- 48–82. sora on poplars in Nei Monggol [in Chinese]. Zhang, N., Zhuang, J.Y. and Wei, S.X. (1997) Acta Agriculturae Boreali-Sinica 5, 86–92. Fungal flora of the Daba Mountains: Tai, F.L. (1979) Sylloge Fungorum Sinicorum [in Uredinales. Mycotaxon 61, 59–61. Chinese]. Science Press, Academia Sinica, Zhou, Z.M., Shen, R.X., Huo, Z. and Lei, Z.P. (1979) Beijing, pp. 537–539. Study on the tolerance of Populus tomentosa to Tian, C.M., Shang, Y.Z., Zhuang, J.Y., Wang, Q. Melampsora magnusiana [in Chinese]. Journal of and Kakishima, M. (2004) Morphological and Beijing Forestry College 1, 61–66. molecular phylogenetic analysis of Melampsora Zhuang, J.Y. (1986) Uredinales from East Himalaya. species on poplars in China. Mycoscience 45, Acta Microbiologica Sinica 5, 77. 56–66. Zhuang, J.Y. (1999) Rust fungi from the Altai. Journal Van Kraayenoord, C.W.S., Laundon, G.F. and Spiers, of Anhui Agricultural University 26, 261–265. A.G. (1974) Poplar rusts invade New Zealand. Zhuang, J.Y. and Wei, S.X. (1994) An annotated Plant Disease Reporter 58, 423–427. checklist of rust fungi from the Mt. Qomo- Wang, Y.C. (1949) Uredinales of Shensi. Contri- langma region (Tibetan Everest Himalaya). butions from the Institute of Botany, National Mycosystema 7, 45. Academy of Peking 6, 224–225. Zhuang, J.Y. and Wei, S.X. (2002) A preliminary Wang, Y.C., Zang, M., Ma, Q.M. et al. (ed.) Fungi checklist of rust fungi in the Greater Khingan of Xizang (Tibet) [in Chinese]. Science Press, Mountains. Journal of Jilin Agricultural Academia Sinica, Beijing, pp. 39. University 24, 6. 9 Current Status of Poplar Leaf Rust in India

R.C. Sharma1, S. Sharma1 and K.R. Sharma2 1Department of Mycology and Plant Pathology, 2Department of Forest Products, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan-173 230 Himachal Pradesh, India

Poplars and Poplar Rusts in India (DC) Schroet, M. rostrupii Wagner) on P. alba was first reported in 1943 from the The genus Populus represents a significant north-west Himalayas (Cummins, 1943). M. component of the world’s potential renew- larici-populina Kleb. was first recorded on able resources for the 21st century. In their exotic poplars in 1992 from Uttar Pradesh natural range, poplars occur interspersed (Pandey, 1992). Recently, M. populina throughout the forests of temperate regions. (Jacq.) Lev. in its uredinial stage was Six species of Populus are indigenous to recorded on six poplar species from India, of which P. ciliata Wall. ex Royle is Himachal Pradesh and Jammu and Kashmir, fast growing and offers a potential to meet although this still needs to be confirmed the increasing demand of wood for the ply- (Vattiprollu and Agarwal, 2002). In India, wood industry, pulp industry, packaging major work has been done on M. ciliata, and other industrial uses. This species is which will be reviewed in this chapter. well distributed in temperate and sub- temperate regions of the Himalayas between 1200 and 3500 m above mean sea level. Distribution and Hosts Pinus roxburghii, Quercus spp., Cedrus deodara, Pinus wallichiana, Abies pindrow, M. ciliata is a microcyclic rust species Picea smithiana, Pinus gerardiana, Salix recorded from the Indian and the Nepal spp. and Hippophae spp. are the other Himalayas only (Khan, 1994; Vannini et al., important forest tree species in this zone. 1995; Sharma and Sharma, 2000). This rust In addition to indigenous poplar species, a species is widely distributed in the north- number of exotic hybrids/clones developed west Himalayas and causes moderate to from P. deltoides, P. alba, P. nigra, P. severe damage (Bakshi and Singh, 1961; tremula, P. × euramericana, P. yunnanensis Singh et al., 1983). In the north-west and P. maximowiczii are also grown in the Himalayas, M. ciliata is prevalent from an Himalayas and northern Indian plains. altitude of 700 m to 3130 m above mean sea The most predominant Melampsora level. The rust has not been recorded below rust species causing poplar leaf rust in 700 m or above 3130 m. The climate at this India is M. ciliata Barclay, which was first altitude varies from sub-temperate with reported from the vicinity of Shimla in 600–1000 mm annual rainfall to dry, cold 1891 (Barclay, 1891). Another species, M. desert, where 3–5 m snow is experienced populnea (Pers.) Karst. (syn. M. aecididoides during winters. Defoliation usually occurs ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 113 114 R.C. Sharma et al.

when 50% or more of leaf area is covered by was recorded on P. yunnanensis, whereas the rust pustules. In nurseries, leaf shed- the maximum was recorded on P. × eur- ding due to rust infection is 1–2 months ear- americana ‘Rubra Poiret’ (59.5%). The lier than the normal leaf shedding period apparent infection rate per unit area per day (Sharma and Sharma, 2000). Only two was highest in P. nigra × P. trichocarpa stages of the pathogen (uredinial and telial) (9.7 × 10−2), whereas the minimum occurred are known. M. ciliata has been recorded on in P. maximowiczii × P. berolinensis P. ciliata, P. deltoides, P. alba, P. yunnanen- ‘Oxford’ (3.3 × 10−2). The area under the sis, P. × euramericana, P. nigra, P. tremula disease progress curve was maximum in and P. trichocarpa, and hybrids developed P. × euramericana ‘Rubra Poiret’ (26.7) and from P. maximowiczii × P. berolinensis minimum in P. yunnanensis (1.6). Inoculum and P. nigra × P. trichocarpa (Sharma production was highest in P. nigra × P. and Sharma, 2000, 2002; Sharma et al., trichocarpa and lowest in P. maximo- 2001a). This rust occurs in native forests wiczii × P. berolinensis ‘Oxford’ (Sharma of P. ciliata, nurseries and plantations of et al., 2001a). P. ciliata clones, which indigenous and exotic poplars. remained disease free during July, became Another poplar rust recorded on P. alba infected in the month of August. Hybrids of in India is caused by M. populnea (Pers.) P. ciliata × P. maximowiczii also differed in Karst (Bakshi and Singh, 1966). Pandey their susceptibility to M. ciliata (Sharma (1992) recorded the occurrence of M. et al., 2002). larici-populina Kleb. on P. deltoides clones. Between six temperature ranges and The uredinial stage of M. populina (Jacq.) five levels of relative humidity, a tempera- Lev. has been reported on P. capsica, P. ture of 20°C and 100% relative humidity casale, P. ciliata, P. eugenei, P. oxfordii and were best for the germination of uredinio- P. robusta from Jammu and Kashmir and spores of M. ciliata. Low (5°C) and high Himachal Pradesh. (30°C) temperature and relative humidity of 90% did not favour the germination of urediniospores. Inoculum production (urediniospore/uredinium and uredinio- Epidemiology spores/unit leaf area) was affected adversely by maximum and minimum temperature but At 1300 m above mean sea level, at Nauni, favourably by higher relative humidity and rust was first recorded on fully grown trees rainfall. Viability of urediniospores stored of P. deltoides ‘Lux’ in April. However, at at 20°C or higher was lost within 10 the same site in the nursery, rust was first days. Temperature and relative humidity observed on P. ciliata ‘Theog’. At 1500 m adversely affected urediniospore viability above mean sea level in Shilly nursery, rust (Sharma and Sharma, 2001). was recorded in July on P. ciliata. The ini- The intensity of leaf rust is influenced tial appearance of the rust disease varied by the stomatal density in poplar leaves. A according to the altitude and plants grown study on the relationship between stomatal (Khan, 1994; Sharma and Khan, 2000). density/pore size and the incidence of leaf The disease remains static during the rust in 12 P. ciliata hybrids and clones with summer months (May and June) and spreads varying degrees of susceptibility revealed rapidly during August, September and Octo- that stomatal density was positively and ber, when the relative humidity is high and significantly correlated with disease inci- the temperature starts decreasing. Develop- dence, whereas pore size was not (Sharma ment of leaf rust (M. ciliata) in different spe- et al., 2001b). Variation in the timing of initi- cies, hybrids and cultivars of poplar in the ation of leaf rust (M. ciliata) was correlated nursery revealed that the disease index of with variation in the leaf flushing time 9.4% on 10 August increased to 70.0% on 10 of poplar genotypes (Sharma and Sharma, October. The lowest disease index (5.5%) 2004). Status of Poplar Leaf Rust in India 115

Variability in Melampsora ciliata more uredinia per leaf disc. Variation in the dimensions of urediniospores of various It has been observed that there is variation isolates was also observed. The smallest in the dimensions of urediniospores of M. urediniospores were recorded in isolate ciliata collected from various poplar spe- I4, while the largest were in I2 (Sharma and cies grown under different environmental Sharma, 2003). conditions. The smallest urediniospores (20.88 × 15.43 mm) produced on P. ciliata were recorded from a site of wet, temperate, Resistance high hills, while the largest (36.30 × m 19.97 m) were from sub-temperate, sub- Sixty-nine clones and 84 hybrids of P. humid, mid hills. Urediniospores produced ciliata (UHF Selections), 141 clones, 7 on P. deltoides and P. nigra were smaller in hybrids and 48 families of P. deltoides, a sub-temperate, sub-humid, mid hill zone 6 clones of P. × euramericana, 11 cultivars than in a wet, temperate, high hill zone. and 6 species of Populus were screened Variations have also been recorded in the under natural epiphytotic conditions at two dimensions of urediniospores produced on locations. In P. ciliata all the clones were P. alba in a dry, temperate zone (Sharma susceptible, while 14 hybrids remained et al., 2001c). disease-free. One hundred and fifteen clones, all hybrids and families of P. deltoides and all clones of P. × eurameri- Influence of Host Genotype and cana remained rust-free. Eight cultivars, × Pathogen Isolate on Disease P. trichocarpa and P. nigra P. trichocarpa Development also remained disease-free (Sharma, 2000).

In vitro inoculation tests using field collec- tions of M. ciliata revealed that there are Biological Control significant interactions between host geno- type and pathogen isolate in determining Control of plant diseases by the use of the infection type and disease level. Shorter biocontrol agents, rather than with chemi- incubation period for flecking (IPF), latent cal sprays alone, is a supplementary period for eruption of first uredinium (LPU) measure to reduce impacts of disease. and maximum uredinia per leaf disc (ULD) With a forest crop, a long rotation and low were associated with P. deltoides ‘Lux’ and economic returns, biological control is P. deltoides ‘19’, while longer IPF and LPU a particularly attractive option. Fungal and minimum ULD were associated with antagonists, Alternaria alternata and Clado- P. ciliata × P. maximowiczii ‘CMZ-26’. sporium oxysporum were isolated from the Flecking was not observed on P.alba, while phylloplane of poplar leaves infected with on P. yunnanensis, only flecking appeared M. ciliata. Pre-inoculation with these antag- but no uredinia erupted. IPF was positively onists resulted in minimum germination correlated (0.866) with the time periods of urediniospores, minimum eruption of required for eruption of first uredinium uredinia per unit leaf area and maximum and eruption of 50% uredinia (LPU50) parasitization of urediniospores and ure- while it was negatively correlated with dinia. A. alternata was found to be more ULD. Rust isolates I1 and I2 produced only parasitic than C. oxysporum. Reduction in flecking, while I3 and I4 could produce viability of urediniospores was more obvi- well-developed pustules. Time taken to ous with A. alternata than C. oxysporum, show flecking, eruption of first and 50% and this potential may be exploited in the uredinia by isolate I4 was shorter when biocontrol of this disease (Sharma et al., compared to isolate I3, which produced 2002). 116 R.C. Sharma et al.

Chemical Control References

Successful control of leaf rust (M. ciliata)of Bakshi, B.K. and Singh, S. (1961) New and noteworthy P. ciliata in nursery seedlings was achieved records of some mildews and rusts on Indian by spraying carbendazim and mancozeb trees. Indian Forester 87, 547. (Khan et al., 1988). Post-symptom sprays Bakshi, B.K. and Singh, S. (1966) Rusts on Indian forest trees. Indian Forest Record (New Series) 2, of difenoconazole (0.02%), penconazole 139–204. (0.06%) and carbendazim (0.05%) exhib- Barclay, A. (1891) Additional urediniae from the ited excellent eradicant activity and neighbourhood of Simla. Journal of Asiatic resulted in minimum production of Society of Bengal 60, 211–230. urediniospores per uredinium and per unit Cummins, G.B. (1943) Uredinales from north west leaf area when applied on nursery-grown Himalayas. Mycologia 35, 446–458. poplar seedlings. When fungicides were Khan, S.N., Rehill, P.S., Tiwari, R.K., Rawat, D.S. and tested as preventive and post-symptom Misra, B.M. (1988) Control of poplar rust, sprays at fortnightly intervals, difeno- Melampsora ciliata in nurseries. Indian Journal conazole, penconazole and hexaconazole of Forestry 11, 253–255. Khan, Y. (1994) Studies on Melampsora leaf rust resulted in the least disease incidence and of Populus species. M.Sc. thesis, University of uredinia pustules per leaf, and minimum Horticulture and Forestry, Solan, India. rate of spread of disease and area under Pandey, P.C. (1992) Occurrence of destructive rust disease progress curve (Sharma, 2000). parasite on exotic poplars in India. Indian Forester 118, 168. Sharma, R.C. and Khan, Y. (2000) Incidence and Conclusions severity of Melampsora leaf rust of poplar. In: Proceedings of Indian Phytopathological Society – Golden Jubilee International Conference on Leaf rust caused by an autoecious, Integrated Plant Disease Management for microcylic M. ciliata is an important dis- Sustainable Agriculture, IPS, New Delhi, India, ease of Populus species, especially on the pp. 970–971. Himalayan poplars (P. ciliata in particular) Sharma, R.C. and Sharma, S. (2000) Status and distri- in India. In the north-west Himalayas, this bution of foliar diseases of poplar in Himachal rust species is prevalent from an altitude Pradesh. Indian Phytopathology 53, 57–60. of 700 m to 3130 m above mean sea level, Sharma, R.C., Khan, Y., Sharma, S. and Malhotra, R. where the climate varies from sub-tropical (2001a) Development of Melampsora ciliata to temperate, cold desert. This rust species rust on nursery-grown poplars in north-western Himalayas. Forest Pathology 31, 313–319. also infects a number of other exotic Sharma, R.C., Sharma, S. and Gupta, A.K. (2001b) hybrids and clones of poplar. The disease Stomatal characters of Populus ciliata in initiation and incidence is influenced by relation to leaf rust and growth parameters. the altitude and type of poplar species/ Phytomorphology 51, 199–205. hybrids/clones raised. A temperature of Sharma, S. (2000) Studies on epidemiology and man- 20°C and 100% relative humidity is most agement of poplar leaf rust. Ph.D. thesis, Univer- favourable for urediniospore germination. sity of Horticulture and Forestry, Solan, India. The extent of disease is also influenced by Sharma, S. and Sharma, R.C. (2001) Epidemiology the stomatal density. Differential interac- of Melampsora ciliata leaf rust of poplars in tion between host genotypes and pathogen India. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 108, 337–344. isolates reveals the existence of races in Sharma, S. and Sharma, R.C. (2002) Prevalence of M. ciliata. Difenoconazole, penconazole poplar leaf rust in Himachal Pradesh. Indian and hexaconazole fungicides are most Phytopathology 55, 81–83. effective in checking the disease in Sharma, S. and Sharma, R.C. (2003) Influence of nursery-grown poplars. host genotype and pathogen isolate in the Status of Poplar Leaf Rust in India 117

development of poplar leaf rust. Zeitschrift für and Cladosporium oxysporum on germination, Pflanzenkrankheiten und Pflanzenschutz 110, parasitism and viability of Melampsora ciliata 359–365. urediniospores. Zeitschrift für Pflanzenkrank- Sharma, S. and Sharma, R.C. (2004) Relationship heiten und Pflanzenschutz 109, 291–300. between leaf flushing time and initiation of Singh, S., Khan, S.N. and Misra, B.M. (1983) Status poplar leaf rust. Journal of Tropical Forest of Melampsora rusts of poplars in India. Indian Science 16, 369–372. Forester 109, 743–747. Sharma, S., Sharma, R.C. and Sharma, J.N. (2001c) Vannini, A., Cecco, D., Monaci, N. and Anselmi, N. Morphological variability in Melampsora ciliata (1995) Main tree pathogens of western Himala- – The incitant of poplar leaf rust. Indian Forester yan forests in Nepal: Description and risk of 127, 242–248. introduction. Bulletin OEPP 25, 455–461. Sharma, S., Sharma, R.C. and Malhotra, R. (2002) Vattiprollu, P.K. and Agarwal, D.K. (2002) Melamp- Effect of saprophytic fungi Alternaria alternata sora. Indian Phytopathology 55, 248. This page intentionally left blank 10 Melampsora Willow Rust in Chile and Northern Europe: Part of a Metapopulation?

Mauritz Ramstedt and Sergio Hurtado Plant Pathology and Biocontrol Unit, Swedish University of Agricultural Sciences, PO Box 7035, S-75007 Uppsala, Sweden

Background needed. Molecular markers are used increas- ingly to characterize the genetics of fungal As the cultivation of willow grows globally, plant–pathogen populations and to evaluate worldwide monitoring of pathotypes for the levels of genetic diversity and phylo- Melampsora rust, the most serious disease genetic relationships within and between of willow, becomes increasingly important. species (Majer et al., 1996). They have also For safe introduction of promising new been used in attempts to identify particular plant material from breeding work, the races or pathotypes, but this has proven to be possible exchange of inocula for the rust somewhat more difficult. between continents has to be taken into Samils et al. (2001, 2002) have des- account. New clones exhibiting good cribed the structure and variation of willow resistance properties might have a rapid rust from different locations in Sweden breakdown if new unexpected pathotypes and Northern Ireland. Complementing and virulence factors appear. their study, Hurtado and Ramstedt (2002) In Chile, although small local willow compared the Melampsora rust populations plantations have long been used for basket at four distant locations by amplification making, only recently has there been fragment length polymorphism (AFLP) increasing interest in growing willows fingerprinting, as well as pathogenicity for commercial production. This naturally testing (Hurtado and Ramstedt, 2003). Such leads to much larger monoclonal planta- comparisons will facilitate our understand- tions, compared to earlier more diversified ing of pathotype dynamics and the possible populations of willow hosts for the rust. impact of long-distance migration of spores Each growing season in Chile, rust infects on those dynamics. Such studies are also both natural and cultivated stands of required for improvement of management of willows heavily (Hurtado, unpublished). willow rust and safer introduction of new Variability in virulence and aggressiveness cultivars of willows from breeding. of Melampsora rust is one of the main problems for diagnostics in rust control, especially for Salix viminalis, the species The Problems with Rust most widely grown in willow plantations. To study the genetics underlying patho- For low-input tree crops such as willow, type dynamics, molecular genetic tools are disease control by chemical means is

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 119 120 M. Ramstedt and S. Hurtado

difficult and economically and environ- virulence factors possible in the rust mentally unrealistic. The use of resistant or population. tolerant clones is therefore the priority, and breeding and development of a desirable planting design become important issues. One problem encountered is that it is Pathogenicity/Aggressiveness/Diversity assumed that a willow will be in the ground for 15–25 years, while its enemy, the rust, A range of rust pathotypes, divided into develops very quickly. With recombination formae speciales, have been reported from every year, with countless numbers of the UK and Sweden (Pei et al., 1996; spores, the rust can always be one step Ramstedt et al., 2002). Although the dis- ahead in selection and adaptation to hosts criminating resistance mechanisms are not present. Rusts are also known for their fully understood, different stages of inter- great heterogeneity, with a wide range of actions during germination and penetration virulence spectra, even in relatively small have been described by Hurtado and populations. Different pathotypes attack Ramstedt (2000) and shown to be different willow clones/species. Pathotypes non-specific in several aspects. can have a narrow or broad host range, even An important question to be answered though the European rusts on short-rotation is what role the local adaptation of the forest (SRF) willows are mainly restricted pathogen and the long-distance dispersion within the host range of their respective play in development of new pathotypes, and formae speciales (Pei et al., 1996) with a possibly new virulence factors. Hubbes limited diversity of potential hosts (Tables (1983) reported that approximately 20 10.1 and 10.2). Chilean rusts have not been possible species of Melampsora rust on found to follow the same pattern and seem willows existed worldwide, each with its to have much higher multi-virulent proper- own host preference and virulence pattern. ties, the same isolate being compatible on M. epitea Thüm, the main Melampsora several species of willows (Table 10.3). The species infecting willow plantations in nature of this virulence difference is not Europe (Royle and Ostry, 1995), is consid- known, reliable markers to separate the ered a collective species that was supposed specificity for different clones or groups of to be divided into varieties of races, sub- clones have not yet been found. Whether species or formae speciales (Hylander et al., there exists a genotype–pathotype correlation 1953; Wilson and Henderson, 1966; Royle for M. epitea, and therefore a correlation and Hubbes, 1992). between genetic diversity and pathotype In SRF plantations in Europe, M. epitea diversity, remains to be investigated. comprises a number of pathotypes which In Sweden, where breeding for willow differ in virulence and aggressiveness on resistance has gone on since 1986 (Gullberg, Salix (Ramstedt, 1989, 1999; Pei et al. 1996; 1989), the high variability of genotype Ramstedt et al., 2002). In most cases, these virulence in rust populations also makes pathotypes are morphologically indistin- evaluation of new willow clones in field guishable from each other and have the same trials troublesome. Field clone trials will not alternate host. A pathotype is considered always be exposed to the rust of interest, to be a heterogeneous group of genotypes, since it might not be present in high enough only having in common the same pattern number at the time or site of the trial. When of clone-specific virulence for a given set of the compatible willow clone appears in clones. Pathotyping is therefore a means of greater numbers/larger plantations, this new monitoring the current state of interaction pathotype might be able to multiply rapidly, between pathogen virulence and plant resulting in unexpected problems for the resistance for a pathogen population and new clone. To have a sustainable breeding host-plant population. system, therefore, rust testing must also When discussing pathotypes, it is impor- be conducted in the lab using all relevant tant to remember that this classification is Melampsora Willow Rust in Chile and Northern Europe 121

Table 10.1. Pathotypes found in Sweden 1991–96.

Test clones

Pathotype 1 2 3 4 5 6 7 8 9 f. sp. No. Code Mull Q83 Stip B.H. 149 Calo Kors Him 139

LD 1 0.0.0 2 0.0.4 +

LR 3 0.3.0 + + 4 0.3.2 + + + 5 0.3.4 + + + 6 0.7.0 + + + 7 0.7.2 + + + + 8 0.7.4 + + + + 9 1.7.0 + + + + 10 4.1.6 + + + + 11 4.3.0 + + + 12 4.7.0 + + + + 13 4.7.2 + + + + + 14 4.7.4 + + + + +

LET 15 5.0.0 + + 16 5.0.1 + + + 17 5.0.2 + + + 18 5.0.3 + + + + 19 5.0.7 + + + + + 20 5.1.2 + + + + 21 5.1.3 + + + + + 22 5.1.6 + + + + + 23 5.1.7 + + + + + + 24 5.4.3 + + + + 25 6.1.0 + + + 26 6.1.6 + + + + + 27 7.0.2 + + + + 28 7.0.3 + + + + + 29 7.0.7 + + + + + + 30 7.1.1 + + + + + 31 7.1.3 + + + + + + 32 7.1.5 + + + + + + 33 7.1.6 + + + + + + 34 7.1.7 + + + + + + + 35 7.4.2 + + + + + 36 7.4.3 + + + + + + 37 7.5.3 + + + + + + +

Virulence factor 123456789

+, the pathotype shows virulence on the test clone. +, indicate recombinations between the main pathotype, groups (formae speciales) LR (M. larici-retusue) and LET (M. larici-epitea typica). LD, M. larici-daphnoides. not a measure for genetic relatedness, since genotypes. The members of the same virulence pattern (pathotypes) is only one pathotype may have different origin and, in minor part of the fungus genome. This some cases, may not be closely related. means that similar pathotypes not only Although those classifications sometimes might be, but most probably are, different do crudely reflect the relatedness, and 122 M. Ramstedt and S. Hurtado

Table 10.2. Clones used for the pathotype coding.

No. Clone Species Virulence valuesa Formae specialesb

European willow differential 1 Mullatin S. viminalis 1 LET 2 Q 83 S. triandra × viminalis 2 LET 3 Stipularis S. aurita × viminalis × caprea 4 LET (LR)

4 Calodendron S. caprea × cinerea × viminalis 1 LR/LET 5 Korso S. burjatica 2 LR 6 Himalayas S. disperma 4 LR (LET)

7 Bowles Hybrid S. viminalis 1 LET 8 78149 S. viminalis × caprea 2 LET 9 78139 S. daphnoides 4 LD/LR/LET

Chilean willow differential 10 Chimbarongo S. viminalis 1 11 Chuyaca S. triandra × viminalis 2 12 Valdivia S. aurita × viminalis × caprea 4

13 L. de Aculeo S. caprea × cinerea × viminalis 1 14 Cascadas S. burjatica 2 15 Pte. Chanco S. disperma 4

16 Santiago S. viminalis 1 17 Llanquihue S. viminalis × caprea 2 18 San Javier S. daphnoides 4 aThe virulence code values for a positive reaction are added together within each subgroup of test clones to give a three-digit code for virulence, e.g. 7.5.3 (1 + 2 + 4 = 7, 1 + 4 = 5, 1 + 2 = 3) bLET, larici-epitea typica; LR, larici retusae; LD, larici-daphnoides. amounts of genetic diversity (but only not only to develop more slowly during the concerning virulence), they do not represent season but also to have more difficulties in amounts of general diversity. To describe selection for more aggressive ‘take-all’ the genetic diversity accurately, molecular pathotypes, such as those that can be found tools are required (Samils et al., 2001; in monoclonal plantations. Multi-virulent Hurtado and Ramstedt, 2002). The types, which are required for a successful specificity found in European willow rust attack of all clones in a mixture, are sup- is, to a large extent, expressed by grouping posed to be less aggressive than pathotypes them into different formae speciales (f. sp.). specialized for one or a few hosts. The host range of each forma specialis is However, in our studies we have found limited to certain species of willow. Within that the level of multi-virulence of Melamp- a forma specialis, the rust genotypes differ sora rust does not seem to affect the level of mainly quantitatively but not so much aggressiveness (unpublished) in a negative qualitatively in virulence properties. way, thereby making the defence somewhat To overcome the problem due to the more vulnerable. Rust isolates, which, in rapid selection and adaptation of rust viru- laboratory tests, were compatible with a lence to previously tolerant or resistant range of different clones or species (S. vimi- clones, mixture plantations have been nalis, S. dasyclados, S. daphnoides, S. established using different clones, instead of disperma and S. caprea), did not give a planting traditional monoclonal plantation lower disease index than the isolates with a (McCracken and Dawson, 1997, 1998). In more narrow host spectrum. such mixtures, as the hosts have different Nevertheless, the clonal mixtures have, susceptibility patterns, the rust is expected over a number of years, performed well, Melampsora Willow Rust in Chile and Northern Europe 123

Table 10.3. Pathotypes found in Chile 1997–98 according to Swedish willow differential.

Pathotype Test clones f. sp. No. Code Mull Q83 Stip B.H. 149 Calo Kors Him 139

1 0.0.0 s 2 0.0.2 +

LR 3 0.2.0 + 4 0.3.0 + + s 5 0.3.2 + + + s 6 0.4.0 +

X 7 1.2.0 + +

LET 8 1.0.0 + 9 1.0.2 + + 10 2.0.2 + + 11 1.0.4 + + 12 2.2.4 + + + 13 4.3.3 + + + + + 14 5.0.3 + + + + s 15 5.1.2 + + + + s 16 5.1.3 + + + + + s 17 5.2.1 + + + + 18 7.0.0 + + + 19 7.0.4 + + + + 20 7.0.7 + + + + + + s 21 7.1.3 + + + + + + s 22 7.4.2 + + + + + s

! 23 7.7.7 + + + + + + + + +

Virulence factor 123784569

+, the pathotype shows virulence against the test clone. s, pathotypes also found in Sweden. LR, M. larici-retusae; X, atypical formae speciales, pathogenic to one type-species from each f. sp. group; LET, M. larici-epitae typica; !, unexpected multivirulent f. sp., pathogenic to all species and clones of all f. sp. groups tested.

giving higher harvest and lower rust inci- Population Structure dence (McCracken and Dawson, 1997, 1998). This indicates that the rust might Uncontrolled long-distance exchange of have difficulties in adapting to the mixtures, fungal inocula between countries may or in competing with other rust pathotypes occur, since spores can be transported long in the population. Another possibility is distances by nature (wind; water; animal that, hitherto, the trials have been too small, carriers, such as migrating birds; etc.) and as compared to all commercial monoclonal humans (e.g. transportation of willow mate- plantations, to affect the total rust popula- rial). Within countries, long-distance tion. Therefore, an important task in plan- inoculum exchange by wind-borne rust ning commercial mixtures would be to have spores almost certainly plays a role in the a variation of mixture set-ups that would local population structure of M. epitea, and minimize the risk of adaptation of suitable in local as well as global evolutionary rust pathotypes to the set-up of hosts. dynamics of pathotype structure. 124 M. Ramstedt and S. Hurtado

From Chile’s rust population, 64 out of The proposed formae speciales for 89 isolates studied, constituting 23 different European rust were, however, less pro- pathotypes, were compatible on the nounced in Chile (Table 10.3) than in European test clones (Table 10.4). Non- Sweden (Table 10.1) (Ramstedt et al., 2002), compatible isolates were from willow even if a few pathotypes seemed to dominate species expected to harbour other formae the Chilean rust collection. An important speciales of the rust, and neither were these difference is that we found several isolates compatible on several other Chilean clones. in Chile that were compatible on all This was the case particularly with rust from clones in the Swedish differential: a multi- the endogenous S. humboltiana, which was virulence that has never been found in not infected by the rust from other willows Europe’s rust population (Ramstedt, 1998, (Table 10.5). All Chilean S. viminalis iso- 1999; Pei et al., 1996, 1997). lates (43), as well as rust from S. caprea and Each virulence component detected S. cinerea, however, were compatible on the earlier in Sweden’s rust population was European differentials (Table 10.4). present in Chile’s rust population. Isoenzyme analysis showed that the genetic Table 10.4. The 23 pathotypes found among 89 variation of Salix viminalis (an introduced isolates in Chile, according to the European willow willow species) in Chile was low, compared differentials. to the endemic species S. humboldtiana. Comparison of rust populations for the two No. of isolates Pathotype countries supports our hypothesis that inter- Species tested code continental inoculum exchange can be a Salix viminalis 2 1.0.4 s significant determinant of local pathotype 2 2.2.4 s structure, and consequently can be important 3 4.3.3 s for willow-resistance breeding. In Chile, 7 5.0.3 s larch is an introduced species and its pres- 2 5.1.2 s ence is rare and dependent on human plant- 12 5.1.3 s ing. Hence, larch plants in Chile can hardly 2 7.0.0 s be considered a natural alternate host for M. 1 7.0.7 s epitea. In fact, whether M. epitea has a natu- 3 7.0.4 s ral alternate host in Chile is unknown, and 4 7.1.3 s 1 7.4.2 s therefore it is not known whether the rust 4 7.7.7 s has the opportunity to reproduce sexually. If S. humboldtiana 12 0.0.0 s sexual recombination is rare in Chile, the 1 0.3.0 s nature of successful strategies in gaining 1 0.3.2 s superior fitness in the pathogen might be 1 0.4.0 s different for the two countries. For instance, 5 1.0.2 s if reproduction is essentially asexual in S. caprea 1 0.2.0 s Chile, then pathotype competition becomes 2 1.0.0 s important, as opposed to genotype competi- 1 1.2.0 s tion. Hence, maintaining common patho- 2 5.2.1 s S. cinerea 1 1.0.0 s types might require active intercontinental 2 1.2.0 s exchange between the two countries, at least S. fragilis 2 0.0.0 s sufficient for establishing a common basic 1 0.0.2 s strategy that is locally adaptable in both 2 2.0.2 s countries. S. purpurea 3 0.0.0 s From this study we conclude that there S. tortuosa 3 0.0.0 s is a striking difference between the Chilean S. alba 4 0.0.0 s and Swedish rust isolates. The Chilean iso- S. babylonica 1 0.0.0 s lates had greater multi-species virulence 1 0.2.0 s capacities than that found in the Swedish s, pathotypes also found in Sweden. isolates. For example, certain Chilean Melampsora Willow Rust in Chile and Northern Europe 125

Table 10.5. Compatibility pattern of Melampsora rust isolates from different willow clones and species.

Compatibility on willow test clones

Rust isolated from humb. alba caprea cinerea fragilis purpurea tortuosa vim. vim. hybr.

Chilean willows S. humboldtiana + − (+) (+) (+) S. fragilis − − + S. tortuosa − − + − − S. babylonica − − + S. alba − + S. caprea − − + + S. purpurea − − + S. cinerea − − + + S. viminalis − (+) + + (+) (+) + + Swedish willows S. viminalis − − + + − (+) (+) + + S. dayclados − − + + − − − − S. daphnoides − − (+) + − − − − + humb., humboldtiana; vim., viminalis; hybr., hybrid. isolates were able to attack both S. viminalis trial near Lectour in southern France during clones and pure S. dasycladus types the summer of 1999. All isolates were taken (‘Korso’) (Table 10.3, Fig. 10.1), the patho- from S. viminalis clones at one single types 1.2.0 and 7.7.7 being good examples location. The isolates from Northern Ireland (Table 10.3). were collected during the autumn of 1998 in Perhaps Chile’s rust population has a mixture trial at Castlearchdale with 20 simply made use of genetic heterogeneity in Salix clones of 10 different species. a different form from that of the Swedish Unweighted pair group method with population, that of more multi-species arithmetic mean (UPGMA) analysis of virulence. Thus Chilean rust pathotypes are the data obtained from two-primer combina- less confined to specific species of willows tions (Fig. 10.2) did not group the isolates (Table 10.3). Also, Chile’s Melampsora rust according to the geographical locations population has yet to adapt to a markedly of their collection. Note, though, that the heterogeneous willow host population of Jaccard coefficient is a conservative measure natural willow stands. In Europe, however, of similarity, compared to the simple match- large monoclonal willow plantations have ing coefficient, which can be as much as 0.4 encouraged Melampsora rust genotypes times greater. These results are consistent to become more clone-specific. However, with those of Samils et al. (2001, 2002) who increase of commercial plantations in Chile found that, when Swedish populations might change this, making a new screening 700 km apart were compared, no genetic of rust worthwhile in the near future. differentiation was found between the geo- In a comparison of rust from several graphically separated locations. Samils and countries, 10 French, 24 Swedish, 24 North- co-workers also noted that genetic diversity ern Irish and 35 Chilean rust isolates were was high within a population. analysed using AFLP. The Swedish isolates were collected from Skåne in the south to Uppland in central Sweden. Eleven of the isolates were collected from the willow Gene Flow: Long-distance Migration clone S. viminalis ‘78183’, which is the most widely used S. viminalis clone in short- When geographical distance among patho- rotation forest plantations in the country. gen populations correlates poorly with The French isolates were collected in a clone genetic distance, as appears to be the case 126 M. Ramstedt and S. Hurtado on willow collected from Chile and Sweden. Melampsora Pathotype frequencies of Fig. 10.1. Melampsora Willow Rust in Chile and Northern Europe 127

Fig. 10.2. Dendrogram generated after UPGMA using amplification fragment length polymorphism (AFLP)-based genetic distance. Numbers on the branches are bootstrap values (%) obtained from 500 replicate analyses. 128 M. Ramstedt and S. Hurtado

for Melampsora rust on willows, any sexually reproducing species capable of population sub-division that does exist can long-distance dispersal. It is also consistent reflect changes due to the different selec- with weak within-population selection. Our tion pressure existing in the geographical results affirm that M. epitea has high levels areas (McDonald and McDermott, 1993). It of gene and genotypic variation within pop- would be difficult to say whether the rust ulations. However, we also found variation isolates in our study could be grouped between our four distant populations, sug- according to the willow hosts, because of gesting that adaptation by natural selection the small numbers of samples for the spe- to local hosts and local environmental cies other than S. viminalis. It is, however, conditions was strong enough to maintain likely that the willow species present will the same degree of variation, despite long- determine the fitness for the Melampsora distance dispersal. The long-distance dis- rust and affect the structure of local persal, however, can explain the finding of populations. unusual genotypes within each location, Gene flow between populations may be which were more similar to genotypes in sufficient to keep populations from diverg- a distant location. Although high rates of ing due to random processes such as genetic long-distance migration reduce variation drift. Thus, although the number of isolates between populations, it facilitates local used in this study was too limited to draw adaptation by input of genetic variation. To definitive conclusions, the results do sug- estimate the potential for sustainable dis- gest that country-defined, geographically ease resistance, new resistance genes should distant populations have not developed be tested at many locations. This would not separately from each other, but instead they only serve as a guard against unexpected represent part of a common population, that epidemic outbreaks, but would also provide is, part of the same cohesive species meta- clues for pathogenic variation in different population. Long-distance migration of rust pathogen populations at several locations. spores can be a powerful means of maintain- Almost 40% of the 23 pathotypes found ing such stability, and wind transportation in Chile were also found in Sweden, and of those spores can be an effective means of 25% of the 36 pathotypes found in Sweden long-distance migration. Wind transporta- were also found in Chile. Also, all virulence tion has been reported earlier as the cause of components found in Sweden were also introduction in 1978 of M. coleosporioides detected in Chile, suggesting that active Diet., first in Asia and then Australia, and intercontinental exchange cannot be ruled further to New Zealand the same year, by out. From the well-documented cases of riding the trans-Tasman air current (Latch, long-distance migration of rust (Wilkinson 1980). The same wind current may have and Spiers, 1976; Latch, 1980; Aylor, 1990; been responsible for the introduction in Nagarajan and Singh, 1990) and especially New Zealand from Australia of the poplar of wind-borne rust spores, the possibility rusts M. larici-populina and M. medusae. of active intercontinental exchange, at least The weather conditions in eastern Australia one-way exchange, seems likely. indicate that spores were transported at high In addition to wind-borne spores, trans- altitudes, probably above 3000 m. Spores fer of Melampsora inoculum by humans carried at lower levels are likely to have (cuttings, clothes, aeroplanes, boats) is also a been washed-out by the rain (Wilkinson and probable, and very likely, way for Melamp- Spiers, 1976). sora rust to spread both from Europe to Chile Studying the genetic structure of the M. and from Chile to Europe. While the growing epitea populations in Swedish S. viminalis season in the southern hemisphere differs plantations, Samils et al. (2001) concluded from that in the northern hemisphere, there that the genetic structure observed in M. is still a certain period when the climate epitea (i.e. high levels of both gene and conditions favourable for Melampsora genotypic variation, most of which is found overlap in both hemispheres (March–April within populations) is consistent with a or September–October). This will give the Melampsora Willow Rust in Chile and Northern Europe 129

Melampsora inoculum plenty of time to Hylander, N., Jørstad, I. and Nannfeldt, J.A. (1953) be transported by the winds present at that Enumeratio Uredinearum Scandinavicarum. time of the year. According to Nagarajan and Opera Botanica 1, 1–102. Singh (1990) the probability of viable spores Latch, B.J. (1980) Weeping willow rust in New Zealand. New Zealand Journal of Agricultural reaching a target and causing infection after Research 23, 535–538. a ‘long journey’ is very low, but very real. Majer, D., Mitchen, R., Lewis, B.G., Vos, P. From these studies, we conclude that and Oliver, R.P. (1996) The use of AFLP the M. epitea populations in Chile and fingerprinting for the detection of genetic Sweden may well be influenced by active variation in fungi. Mycological Research 100, intercontinental exchange of inocula, as 1107–1111. is well documented for other pathogens McCracken, A.R. and Dawson, W.M., (1997) (Pedgley, 1982). The importance of possible Growing clonal mixtures of willow to reduce long-distance transport of rust spores effect of Melampsora epitea var. epitea. deserves consideration by Salix breeding European Journal of Forest Pathology 27, 319–329. programmes. In addition, the consequence McCracken, A.R. and Dawson, W.M. (1998) Short of unknown, unmonitored and unstudied rotation coppice willow in Northern Ireland new patterns of clone-specific virulence since 1973: development of the use of mixtures arriving from another region, country or in the control of foliar rust (Melampsora spp.). continent can be devastating. European Journal of Forest Pathology 28, Thus, as willow cultivation becomes 241–250. increasingly global and important, world- McDonald, B.A. and McDermott, J.M. (1993) wide monitoring of the dynamics of patho- Population genetics of plant pathogenic fungi. type structure in Melampsora populations BioScience 43, 311–319. becomes increasingly important in Nagarajan, S. and Singh, D.V. (1990) Long-distance dispersion of rust pathogens. Annual Review of understanding of long-distance inoculum Phytopathology 28, 139–153. exchange, controlled and uncontrolled, and Pedgley, D. (1982) Windborne Pests and Diseases. the effects of such exchange. Metereology of Airborne Organisms. Ellis Horwood, Chichester, UK. Pei, M.H., Royle, D.J. and Hunter, T. (1996) Pathogenic specialization in Melampsora epitea References var. epitea on Salix. Plant Pathology 45, 697–690. Aylor, D.E. (1990) The role of intermittent wind in the Pei, M.H., Parker, S.R., Hunter, T. and Royle, D.J. dispersal of fungal pathogens. Annual Review of (1997) Variations in populations of Melampsora Phytopathology 28, 73–92. willow rust and the implications for design of Gullberg, U. (1989) Växtförädling av Salix, short rotation coppice plantations. Aspects of 1986–1989. Research Notes 42, Swedish Applied Biology 49, 91–96. University of Agricultural Sciences, Sweden. Ramstedt, M. (1989) Diseases of Salix energy Hubbes, M. (1983) A Review of the Potential plantations in Sweden, 1988. Proceedings of the Diseases of Alnus and Salix in Energy International Energy Agency Task II Joint Plantations. Ontario Tree Improvement and Workshop, Bristol, UK, pp. 76–81. Forest Biomass, Toronto. Ramstedt, M. (1998) Diversity of the Melampsora Hurtado, S. and Ramstedt, M. (2000) Compatible and rust on willows – consequences for SRF incompatible reaction of Melampsora rust on planting strategies. Proceedings of the First willow leaves. Scandinavian Journal of Forestry IUFRO Rusts of Forest Trees WP Conference, Research 15, 405–409. 2–7 August, Saariselkä, Finland. Finnish Forest Hurtado, S. and Ramstedt, M. (2002) AFLP Research Institute, Research papers 712, comparision of distant Melampsora epitea 123–130. (willow rust) populations. Mycological Research Ramstedt, M. (1999) Rust disease on willows – 106, 1400–1407. virulence variation and resistance breeding Hurtado, S. and Ramstedt, M. (2003) Comparing strategies. Forest Ecology and Management 121, Chilean and Swedish rust populations: possible 101–111. impact of long-distance transport of Melampsora Ramstedt, M., Hurtado, S. and Åström, B. (2002) rust. Forest Pathology 33, 69–80 Pathotypes of Melampsora rust on Salix in 130 M. Ramstedt and S. Hurtado

short-rotation forestry plantations. Plant Samils, B., Lagercranz, U. and Gullberg, U. (2002) Pathology 51, 185–190. Genetic relationships among genetically distinct Royle, D.J. and Hubbes, M. (1992) Diseases and pests forms of Melampsora larici-epitea and related in energy crop plantations. Biomass and species based on AFLP data. Forest Pathology Bioenergy 2, 45–54. 32, 379–386. Royle, D.J. and Ostry, M.E. (1995) Disease and pest Wilkinson A.G. and Spiers, A.G. (1976) Introduction control in the bioenergy crops poplar and of the poplar rusts Melampsora larici-populini willow. Biomass and Bioenergy 9 (1–5), 69–79. and M. medusae to New Zealand and their Samils, B., Lagercrantz,U., Lascoux, M. and Gullberg, subsequent distribution. New Zealand Journal of U. (2001) Genetic structure of Melampsora Science 19, 195–198. epitea populations in Swedish Salix viminalis Wilson, M. and Henderson, D.M. (1966) The British plantations. European Journal of Plant Pathology Rust Fungi. Cambridge University Press, 107, 399–409. Cambridge, UK. 11 Disease Scoring by Taking Inoculum Densities into Consideration in Leaf Disc Inoculations with Poplar and Willow Rusts

Ming Hao Pei1 and Tom Hunter2 1Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK; 24 Wally Court Road, Chew Stoke, Bristol BS40 8XL, UK

Disease Scoring Methods used for diameter as small and > 0.4 mm as large; and Poplar and Willow Rusts in the number of uredinia: 3–6 as few and ≥ 7as Leaf Disc Inoculations numerous. Their inoculation experiments appeared to have used very high doses of Assessment of disease is an essential part inoculum, i.e. c. 7500 spores being spread of inoculation experiments designed to over 16 mm diameter leaf discs (2.01 cm2) characterize disease resistance/pathogen using cotton wool. Newcombe et al. (2000, virulence. In the studies of Melampsora 2001) assessed resistance/susceptibility rusts on poplar and willow, the leaf according to infection types based on the disc inoculation technique has been used size and the presence of chlorotic or necrotic widely to test host resistance/pathogen flecking, in inoculation experiments with virulence, because of its simplicity and M. × columbiana, a possible hybrid between efficiency. Various methods have been M. medusae and M. occidentalis. used to score the disease in leaf disc In studies of pathogenic variation in M. inoculation experiments. epitea, Pei et al. (1996) inoculated willow With the poplar rust M. medusae, leaf discs at the inoculum densities 70–300 Prakash and Thielges (1987) used a 0–7 spores per 11 mm diameter (0.95 cm2) disease rating, i.e. 0, no macroscopic symp- leaf disc and, based on the number and toms; 1, necrotic flecks only; 2, necrotic estimated size of uredinia, scored infection flecks and some with minute uredinia; types using a 0–4 scale. 3, necrotic flecks with few medium-sized In the above-mentioned cases, inocu- uredinia; 4, few, small uredinia; 5, few, large lum pressure was not clearly quantified and uredinia; 6, numerous, small uredinia; the criteria for disease scores were some- 7, numerous, large uredinia. In a study what ambiguous. To date, disease scoring of the North American poplar rust, M. in leaf disc or detached leaf inoculation occidentalis, Hsiang and Castagner (1993) experiments with rust pathogens is still modified Prakash and Thiegle’s ratings by based largely on the assessors’ experience defining the size of uredinia: < 0.4 mm and skills.

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 131 132 Ming Hao Pei and T. Hunter

Inoculum Pressure and Disease Inoculum Density and Uredinial Severity in Rust Fungi Number/Area in Poplar and Willow Rusts In rust fungi, it is known that inoculum densities (viable spores applied on to a unit Host clones and rust isolates tested area of host tissue) can markedly influence the number of pustules/lesions produced. For poplar rust, four poplar clones – P. In wheat leaf rust Puccinia graminis, nigra ‘Vereecken’, P. nigra × P. deltoides inoculation densities up to 2000 spores/cm2 ‘Spijk’, P. trichocarpa ‘Trichobel’ and P. were linearly correlated with resulting trichocarpa × P. deltoides × P. deltoides numbers of uredinia on wheat seedlings ‘75028’ – and four M. larici-populina iso- (Peterson, 1959). In bean rust Uromyces lates having different degrees of virulence appendiculatus, a linear relationship was to the tested clones were used. found between the amount of inocu- For willow rust, six willow clones – lum (200–900 spores/cm2) and resulting S. burjatica ‘Korso’, S. × calodendron, S. × uredinia, with approximately 1 out of 10 mollissima ‘Q83’, S. × stipularis, S. vimi- spores producing uredinia (Schein, 1964). nalis ‘Mullatin’ and S. viminalis ‘78183’ – Recently, quantitative relationships and four M. larici-epitea isolates were used. between inoculum density and disease Pathogenicity of the isolates corresponded variables were investigated in willow rust, to LET1, LET3, LET4 and LR1 (see Pei et al., M. larici-epitea (Pei et al., 2002), and poplar 1996). rust, M. larici-populina (Pei et al., 2003). The results from both studies suggested that rust resistance/susceptibility was expressed by the differences in both the number and the Inoculation experiments size of uredinia, and that there were close correlations between uredinial pustule area Leaf discs, 1.6 cm diameter for poplars and and spore production. 1.1 cm diameter for willows, were placed A practical problem in testing disease on blotting-paper bridges soaked in tap resistance/pathogen virulence involving a water in 25 compartments of 10 × 10 cm number of different isolates is that it is square Petri dishes. Poplar rust spore almost impossible to achieve deposition suspensions were adjusted to seven levels of identical numbers of spores on the (4, 8, 16, 32, 64, 128 and 256 × leaf surface for each isolate, because 103 spores/ml) and willow rust spore each isolate has to be inoculated separately. suspensions to five levels (1.5, 3, 12, 25 and Furthermore, viability of spores may vary 50 × 104 spores/ml). The spore suspensions considerably between isolates. Results from were sprayed on to the target area (1 ml per different inoculations would be more 10 × 10 cm area) using a Humbrol air brush comparable if the inoculum densities (Humbrol Ltd., England). Ten leaf discs could be accounted for in inoculation were used as replicates in poplar and five experiments. discs in the willow for each host clone/ In this chapter, we further examine the rust isolate/inoculum density combination. relationships between inoculum density Inoculum density for each rust isolate was and uredinial number/area in poplar and determined by examining the number of willow rusts, using the extensive data germinating spores on the water agar which obtained from the quantitative inoculation had been placed in the target area and experiments with M. larici-epitea (Pei et al., then incubated at 16°C for 24 h. After 2002) and M. larici-populina (Pei et al., inoculation, the leaf discs were incubated 2003). in a growth chamber at 16°C with 16 h per Disease Scoring for Poplar and Willow Rust 133

day illumination at an intensity of 80 mE/ A series of models were fitted, i.e. a m2/s1. single overall line, parallel lines (a common Thirteen days after inoculation, reac- slope but different intercepts for the dif- tions on leaf discs were recorded using a dig- ferent clone/isolate combinations), separate ital camera (Olympus C-2500L). Then, for lines with different slopes but with a each leaf disc, the spores were counted using common intercept (intercept = 0) and a haemocytometer. Image analysis software finally separate lines with different slopes as (SIGMASCAN Pro 5.0; SPSS Inc.) was used to well as intercepts. The analyses were carried count uredinial numbers and to measure out using GENSTAT 5 Release 4.2 (Genstat the diameters of erumpent uredinia. Further Committee, 2000). details of the inoculations with the poplar rust were described in Pei et al. (2003) and that with the willow rust in Pei et al. (2002). Poplar rust

Inoculum densities of M. larici-populina Data analyses were estimated up to 680 viable spores/cm2. Compared to a single overall line, the Uredinial pustule area was calculated using relationship between inoculum density and the total number and average diameter of uredinial number was better expressed by the uredinia on the leaf disc. A square root the model for parallel lines for different transformation was used to transform the clone/isolate combinations (%VAF data. A simple linear regression model increased from 48.3 to 73.1, P < 0.001) (y = ax + b) was used to examine the (Table 11.1). Allowing the slopes and relationships between inoculum density (x) intercepts to change independently for and uredinial number/area (y). In the equa- each combination further improved the tion, a is the slope factor and b the inter- fit (P < 0.001; %VAF = 79.1) compared to cept. A measure of the strength of the linear the parallel lines model. The model with relationship was given by the percentage a common intercept (intercept = 0) but variance accounted for, calculated as: individual slopes gave significant increase % variance accounted for (VAF) = in fit compared to a single overall line 100 × (total mean square − residual having intercept = 0 (%VAF increased from mean square)/total mean square. 48.3 to 77.8, P < 0.001).

Table 11.1. Percentage variance accounted for (%VAF) for different models fitted to the plots between inoculum density and uredinial number/area of M. larici-populina and M. larici-epitea.

Willow (inoculum densities Willow (inoculum densities Poplar up to 740 spore/cm2) up to 400 spore/cm2)

Uredinial Uredinial Uredinial Uredinial Uredinial Uredinial Model number area number area number area

Single overall line 48.3 43.6 55.6 36.1 41.3 36.1 Parallel lines 73.1 69.4 66.8 58.6 65.3 63.1 Lines with independent 79.1 75.2 68.4 61.9 70.2 70.2 slopes and intercepts Single overall line having 48.3 43.7 43.9 20.3 33.0 33.4 intercept = 0 Individual lines with 77.8 73.6 58.3 48.7 52.4 64.3 common intercepts (= 0) 134 Ming Hao Pei and T. Hunter

Between inoculum density and %VAF increased from 41.3 to 65.3) between uredinial pustule area, parallel lines gave inoculum density and uredinial number. a better correlation than the single overall Allowing the slopes and intercepts to line (%VAF increased from 43.6 to 69.4, change independently for each combination P < 0.001). The model having individual improved the correlation (%VAF = 70.2, slopes and intercepts gave slight but signifi- P < 0.001) compared to the parallel lines cant improvement compared to the parallel model. The model with a common intercept lines model (%VAF = 75.2, P < 0.001). The (intercept = 0) gave significant increase in fit model with a common intercept (inter- compared to a single overall line having cept = 0) but having individual slopes gave intercept = 0 (%VAF increased from 33.0 to significant increase in fit compared to a 52.4, P < 0.001). single overall line (%VAF increased from For the data obtained at the inoculum 43.7 to 73.6, P < 0.001). densities < 400 spores/cm2, parallel lines gave a better fit than single overall line (%VAF increased from 36.1 to 63.1, P < 0.001) between inoculum density and Willow rust uredinial pustule area. The model having individual slopes and intercepts gave Inoculum densities of M. larici-epitea were significant improvement compared to the estimated up to 740 viable spores/cm2. parallel lines model (%VAF = 70.2, Between inoculum density and uredinial P < 0.001). The model with a common number, the model for parallel lines for dif- intercept (intercept = 0) gave significant ferent clone/isolate combinations showed increase in fit compared to a single overall a significant increase in fit compared to line having intercept = 0 (%VAF increased a single overall line (P < 0.001; %VAF from 33.4 to 64.3, P < 0.001). increased from 55.6 to 66.8). Allowing the slopes and intercepts to change independently for each combination also improved the fit (%VAF = 68.4, P < 0.001) Disease Scoring by Taking Inoculum compared to the parallel lines model. Densities into Consideration The model with a common intercept (intercept = 0) gave significant increase in Based on the present results, we propose fit compared to a single overall line (%VAF that inoculum densities should be taken increased from 43.9 to 58.3, P < 0.001). into account when disease is scored in leaf Between inoculum density and disc or detached leaf inoculations. This can uredinial pustule area, parallel lines gave be done conveniently by using the linear better fit than a single overall line (%VAF relationships between inoculum density increased from 36.1 to 58.6, P < 0.001). The and uredinial number/area. In our analyses, model having individual slopes and inter- parallel lines and individual lines having cepts gave slight but significant improve- different slopes and intercepts gave some- ment compared to the parallel lines model what better correlations between inoculum (%VAF = 61.9, P < 0.001). The model with a density and uredinial number/area com- common intercept (intercept = 0) gave sig- pared with the individual lines with a nificant increase in fit compared to a single common intercept (intercept = 0). However, overall line having intercept = 0 (%VAF because of its simplicity and the biological increased from 20.3 to 48.7, P < 0.001). implications that no uredinia develop with- When analyses were done with the out inoculum, individual lines with a com- data restricted to inoculum densities mon intercept may be reasonably adequate < 400 spores/cm2, the model for parallel to weigh the differences in disease due to lines for different clone/isolate combina- the differences in the amount of inoculum tions showed a significant increase in fit applied. As rust resistance/susceptibility compared to a single overall line (P < 0.001; was expressed by the differences in both the Disease Scoring for Poplar and Willow Rust 135

number and size of uredinia (Pei et al., are used, given the assumption that 2002, 2003), the use of uredinial pustule y = 0.175x represents the mid-point for the area data would be more meaningful in dis- scale 4 rating, then the mid-point for scale 3 ease scoring because the uredinial pustule is 0.125, scale 2 is 0.75 and scale 1 is 0.25. area is calculated based on both the number Lines (dashed in Fig. 11.1) can be drawn and the size of uredinia. arbitrarily as y = 0.15x to indicate the border A possible way of scoring disease is to between the scales 3 and 4, y = 0.10x use a highly susceptible clone/isolate com- between 2 and 3 and y = 0.05x between 1 bination as a reference and then calculate and 2. Then disease scores for various clone/ the slope based on its inoculum density and isolate combinations can be obtained by uredinial pustule area. With other combina- plotting their uredinial pustule area against tions, slopes can also be calculated accord- respective inoculum densities. It should be ing to respective inoculum densities and noted that, at very low inoculum densities, uredinial pustule area measurements. Then, small variations in uredinial pustule area the slopes can be categorized into different data may give very different disease scores. scales and the disease scores can be obtained Therefore, care should be taken to avoid accordingly. Otherwise, percentage figures applying extremely low or excessively high may be given to indicate the disease severity number of spores in inoculation experi- in relation to the highly susceptible ments. It appears that the proposed method reference clone. of disease scoring may be well suited for As an example, the data for the inoculum densities between 100 and uredinial pustule area and inoculum density 400 spores/cm2 for both poplar and willow of four M. larici-populina isolates on two rusts. poplar clones were plotted in Fig. 11.1. Apart from inoculum density, disease Populus nigra ‘Vereecken’ was infected by scores can be influenced by temperature, all the four isolates, P. nigra × P. deltoides light, the age and growing conditions of ‘Spijk’ by isolates BE and BO. For the model plants. Therefore, these conditions should having individual lines with a common be standardized in the preparation of inocu- intercept, the slope factor for the most lation experiments. Consideration should susceptible clone/isolate combination BE/ also be given to the fact that viability of ‘Spijk’ was 0.175. If 0–4 scale disease ratings spores may vary and spore concentration

Fig. 11.1. Disease scoring on poplar clones ‘Vereecken’ and ‘Spijk’ based on pustule area and inoculum density of M. larici-populina. Dotted lines are arbitrarily drawn to indicate the boundaries between scores when 0–4 scale rating is used. 136 Ming Hao Pei and T. Hunter

may not necessarily reflect the number of of uredinia (42.7% variance). Secondly, the viable propagules deposited on a host sur- size of host leaf area would not affect the face. It is desirable to use, wherever possible, slope factor. For example, the slope factor fresh spores for inoculation and to quantify obtained based on uredinial pustule area inoculum pressure according to both the and inoculum density on a 2 cm2 leaf disc density and viability of the spores. would be the same as that obtained on a 10 cm2 leaf segment. Thirdly, the data obtained between different inoculation experiments can be made more comparable Concluding Remarks by including known host clone/rust isolate combinations in both inoculation experi- We examined the relationships between ments. The proposed method would also be inoculum density and uredinial number/ useful in studies of disease resistance/ area using the data obtained from quantita- pathogen virulence in other plant/rust tive inoculations with poplar rust M. larici- systems. populina and willow rust M. larici-epitea, using leaf discs. Simple regression models having parallel lines, individual lines with different slopes and intercepts, and individ- Acknowledgements ual lines with a common intercept were fitted for different host clone/rust isolate This study was funded by the Department combinations. For poplar rust, all the for Environment, Food and Rural Affairs models fitted well, % variance accounted (DEFRA), UK, and the European Union. for being 69–79. In willow rust, reasonable Rothamsted Research receives grant-aided linear relationships between inoculum support from the Biotechnology and Biolog- density and uredinial number/area were ical Sciences Research Council of the UK. maintained at the inoculum densities < 400 spores/cm2 (% variance accounted for being 52–65). A method was proposed to score References disease in leaf disc inoculations using slope factors which were calculated based on the Genstat Committee (2000) GenStat 5 for Windows, uredinial pustule area and inoculum Release 4.2, 5th edition. Numerical Algorithms density. Group, Oxford, UK. The proposed method has several Hsiang, T. and Castagner, G.A. (1993) Variation in attractive attributes. First, disease scores Melampsora occidentalis rust on poplars in the based on uredinial pustule area can be Pacific Northwest. Canadian Journal of Plant indicative of the extent of spore production Pathology 15, 175–181. Johnson, R. and Taylor, A.J. (1976) Spore yield because uredinial pustule area is closely cor- of pathogens in investigation of the race- related to the number of spores produced. specificity of host resistance. Annual Review Spore production is one of the most impor- of Phytopathology 14, 97–119. tant driving variables in the development of Newcombe, G., Stirling, B., McDonald, S. and disease epidemics and is also one of the most Bradshaw, H.D. (2000) Melampsora × accurate and least subjective ways of assess- columbiana, a natural hybrid of M. medusae ing the growth of pathogens and the suscep- and M. occidentalis. Mycological Research 104, tibility of hosts (Johnson and Taylor, 1976). 261–274. In M. larici-populina, there was a close cor- Newcombe, G., Stirling, B. and Bradshaw, H.D. relation between the number of spores pro- (2001) Abundant pathogenic variation in the new hybrid rust Melampsora × columbiana on duced and uredinial pustule area (Spearman hybrid poplar. Phytopathology 91, 981–985. rank correlation 0.94) (Pei et al., 2003). In M. Pei, M.H., Royle, D.J. and Hunter, T. (1996) Patho- larici-epitea, spore yield was better corre- genic specialization of Melampsora epitea lated with uredinial pustule area (account- var. epitea on Salix. Plant Pathology 45, ing for 61.2% variance) than to the number 679–690. Disease Scoring for Poplar and Willow Rust 137

Pei, M.H., Ruiz, C., Hunter, T., Arnold, G.M. and Peterson, L.J. (1959) Reactions between inoculum Bayon, C. (2002) Quantitative relationships density and infection of wheat by uredospores of between inoculum of Melampsora larici-epitea Puccinia graminis var. tritici. Phytopathology 49, and corresponding disease on Salix. Plant 607–614. Pathology 51, 443–454. Prakash, C.S. and Thielges, B.A. (1987) Pathogenic Pei, M.H., Ruiz, C., Harris, J. and Hunter, T. variation in Melampsora medusae leaf rust of (2003) Quantitative inoculations of poplars. Euphytica 36, 563–570. poplars with Melampsora larici-populina. Schein, R.D. (1964) Design, performance, and use European Journal of Plant Pathology 109, of a quantitative inoculater. Phytopathology 54, 269–276. 509–513. This page intentionally left blank 12 Interactions Between Poplar Clones and Melampsora Populations and their Implications for Breeding for Durable Resistance

Jean Pinon and Pascal Frey UR Pathologie Forestière, INRA, F-54280 Champenoux, France

Background ‘Beaupré’ becoming severely infected by new pathotypes of M. larici-populina. This In the 1950s, bacterial canker (Xantho- latter species is now a major concern all monas populi (Ridé) Ridé and Ridé) was over Europe. a major concern in the northern part of Variability within a given Melampsora Europe in the area used for poplar cultiva- species is mainly defined by the presence tion. During the 1960s, the major foliar dis- of pathotypes (or physiological races), ease on poplar in Europe was Marssonina this second level of variability becoming brunnea (Ell. and Ev.) Magn., following its increasingly important. The first evidence introduction from North America. In the of pathotypes was mentioned for M. larici- past, rust caused problems sporadically, populina by Van Vloten (1949), although only in favourable weather conditions the occurrence of different pathotypes in and following infections by Discosporium the field did not become obvious until populeum (Sacc.) Sutton, which resulted in the 1980s. At present, eight virulences are decline. Three Melampsora species are known within M. larici-populina, with pathogenic on cultivated poplars in Europe: potentially 256 pathotypes (Table 12.1). M. larici-populina Kleb., M. allii-populina During the past 20 years, many cultivars Kleb. and M. medusae Thuem. The latter have lost their complete resistance to M. is confined to south-western France, Spain larici-populina, due to new virulences and and Portugal. To date, no significant development of new pathotypes. Virulences damage caused by M. medusae has been were also described within M. allii-populina observed, even though susceptible cultivars (Frey and Pinon, 1997). To date, no major are widely planted (Pinon, 1986). M. allii- change in clone behaviour has been populina was frequent in western France observed in relation to the pathotypes of (Pinon, 1991) and its frequency increased in M. allii-populina. This is probably due to the Belgium (M. Steenackers, personal commu- fact that no breeder took susceptibility to nication) due to the cultivation of ‘Beaupré’ this species into account and therefore no which is relatively susceptible to M. allii- selection was made for complete resistance. populina. Since 1997 the frequency of this It has been demonstrated that popu- species has decreased significantly with lations of M. larici-populina are not ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 139 140 J. Pinon and P. Frey

Table 12.1. Virulences known within M. larici-populina.

Virulence number Year of description Initial location Clone used to detect virulence

1 1982 Belgium, France ‘Ogy’ 2 1986 France ‘Aurora’ 3 1949 The Netherlands ‘Brabantica’ 4 1974 France ‘Unal’ 5 1982 France, Belgium? ‘Rap’ 6 1994 France ‘87B12’ 7 1994 Belgium, France ‘Beaupré’ 8 1997 Belgium, France ‘Hoogvorst’

homogeneous and can evolve very quickly pathotype. This was confirmed in France in a given location. During the same growing after experimental inoculations in the season, populations are diverse, according laboratory (Pinon and Bachacou, 1984). to sites, and a new virulence can spread Studying pathotype populations, Pinon and easily, with recombinations giving rise to Bachacou (1984) found that most isolates new pathotypes. Host population is the pathogenic to ‘Unal’ were also pathogenic main selection force exerted on rust popula- to ‘Rap’, but with some exceptions, and tions (Pinon and Frey, 1997) and the fungus they therefore proposed the existence of can adapt efficiently to the host. Climatic two distinctly different virulences. In 1986, aspects do not seem to play a major role in some completely resistant cultivars from population evolution. Italy (‘Cima’, ‘Luisa Avanzo’) and the Neth- The main challenge for poplar breeding erlands (‘Hees’, ‘Ellert’) became infected and cultivation, therefore, is incorporation at INRA, Nancy in France, and artificial of durable resistance to rusts that are com- inoculations confirmed the existence of a posed of diverse and changing populations. new virulence (Pinon and Peulon, 1989). Some Populus deltoides ‘Marsh’ clones studied at INRA, Orléans were completely Virulences and Pathotypes within resistant to the above-mentioned virulences Melampsora on Poplar but showed some infections in the nursery. Isolates collected on these clones (87B11, Melampsora larici-populina 87B12, 87B29 and 87B37) expressed a new virulence (Pinon and Lefèvre, 1994). In Bel- Variability within M. larici-populina gium in 1994 (Steenackers et al., 1994), and in France shortly afterwards (Pinon, 1995), The variability of pathogenicity within another breakdown of complete resistance M. larici-populina is detected at two levels: was detected and subsequent rust out- virulence and aggressiveness. Recognition breaks brought about a major economical by Van Vloten of the first pathotypes in impact. Many Belgian (‘Beaupré’, ‘Boelare’, 1949 was not considered by breeders for ‘Gibecq’, ‘Ghoy’, ‘Primo’) and Dutch (‘Barn’, several decades. In 1974 rust infections ‘Donk’, ‘Dorskamp’, ‘Flevo’) clones lost were detected on poplar cultivars such their complete resistance. According to as ‘Unal’ and ‘Hunnegem’, which had the extent of symptoms seen in Belgium on been selected for complete resistance adult trees in 1994, it is probable that this (V. Steenackers, personal communication), new virulence had appeared 1 or 2 years indicating a new virulence in the fungus. previously. The eighth virulence was Steenackers (1982) observed breakdown of found, again in Belgium and in France complete resistance on ‘Isières’, ‘Ogy’, in 1997, when the complete resistance of ‘Spijk’ and ‘Rap’ in Belgium in 1982 and newly released cultivars ‘Hoogvorst’ and suggested that this was caused by a new ‘Hazendans’ was broken down. Interactions Between Poplar Clones and Melampsora Populations 141

For simplicity, pathotypes were gath- Europe but was detected in New Zealand ered into groups, with ‘E’ meaning Europe before its description in France. (Table 12.2): E1 pathotypes are older Complete (or pathotype-specific) resis- pathotypes which have never exhibited tance is known in cultivars of P. × eura- virulences 1, 2, 5, 6, 7 or 8. Most frequently mericana Guinier (syn. P. × canadensis they have one virulence (4) or two (3, 4). E2 Moench) and P. × interamericana Brockh. pathotypes have at least virulence 1, E3 viru- Both hybrids have P. deltoides as a parent, lence 2, E4 virulence 7 and E5 pathotypes which is the probable source of pathotype- virulence 8. For scientific studies in pathol- specific resistance (Dowkiw, 2003). As ogy and tree breeding, it is recommended pointed out by Newcombe (1998), P. that isolates are described by their composi- deltoides has not evolved with M. larici- tion in virulences (pathotypes) according to populina but is the source of complete the number allocated in Table 12.1. (exapted) resistance to this rust agent. It After the introduction of M. larici- seems that breeders have, in the past, mainly populina to Australia and its migration used this source of resistance for their to New Zealand, pathotypes were also hybrids. Frey and Pinon inoculated a few described in these countries. It was possible P. trichocarpa Torr. and Gray clones with to compare European pathotypes with the different pathotypes, but none exhibited ones from New Zealand where European pathotype-specific resistance. In 1975, cultivars were included in pathological we assessed many P. trichocarpa clones in studies. Pathotypes (NZ-1 and NZ-2) Orléans and all those assessed were found to described in New Zealand (Latch and be infected. Among P. nigra L., 46 clones of Wilkinson, 1980) have the same host range French origin (‘Gard’, ‘Durance’ and ‘Drôme as pathotypes described in Europe as E1 rivers’) were inoculated with 36 isolates of and E3. NZ-2 was probably introduced from M. larici-populina from the same origin.

Table 12.2. Groups of pathotypes of M. larici-populina responsible for infection on the most common cultivars.

Groups of Corresponding pathotypes needed virulence Type of cultivars

Differential (pathotype-specific resistance) E2 1 ‘Isières’, ‘Ogy’, ‘Rap’, ‘Spijk’ E3 2 ‘Altichiero’, ‘Bellotto’, ‘Luisa Avanzo’, ‘Aurora’, ‘Carpaccio’, ‘Cima’, ‘Enza’, ‘Ellert’, ‘Hees’, ‘Grimminge’, ‘Olona’, ‘Orba’, ‘Tiepolo’, ‘Veronese’ E4 7 ‘A73’, ‘Barn’, ‘Beaupré’, ‘Boelare’, ‘Donk’, ‘Dorskamp’, ‘Flevo’, ‘Gaver’, ‘Ghoy’, ‘Gibecq’, ‘H-402’, ‘H-490’, ‘Kopecky’, ‘Muur’, ‘Oglio’, ‘Oudenberg’, ‘Pannonia’, ‘Pegaso’, ‘Primo’, ‘Soligo’, ‘Tasman’, ‘Vesten’ E5 8 ‘Hazendans’, ‘Helix’, ‘Hoogvorst’

Universal cultivars (no pathotype-specific resistance) All No virulence ‘Adda’, ‘Adige’, ‘Agathe F’, ‘Alcinde’, ‘Arno’, ‘Bellini’, ‘Bietigheim’, needed among ‘BL Costanzo’, ‘Blanc du Poitou’, ‘Blom’, ‘Boccalari’, ‘Branagesi’, 1, 2, 6, 7, 8 ‘Brenta’, ‘Büchig’, ‘Cappa Bigliona’, ‘Carolin’, ‘Columbia river’, ‘Culasso’, ‘Dora’, ‘Dvina’, ‘Eridano’, ‘Eugenei’, ‘Faux-Gaver’, ‘Florence Biondi’, ‘Fritzi Pauley’, ‘Gattoni’, ‘Gelrica’, ‘Gomol’, ‘Guardi’, ‘Guariento’, ‘Heimburger’, ‘Hunnegem’, ‘I-45/51’, ‘I-154’, ‘I-214’, ‘I-455’, ‘I 74–76’, ‘Isonzo’, ‘Italica’, ‘Koster’, ‘Lambro’, ‘Lena’, ‘Lima’, ‘Lux’, ‘Mella’, ‘Mellone Carlo’ (= MC), ‘Muhle Larsen’, ‘Neva’, ‘Onda’, ‘Ongina’, ‘Panaro’, ‘Pourtet’, ‘Raspalje’, ‘Rintheim’, ‘Robusta’, ‘Rochester’, ‘Scott Pauley’, ‘Serchio’, ‘Sesia’, ‘Sile’, ‘Stura’, ‘Tannenhoft’, ‘Tardif de Champagne’, ‘Taro’, ‘Ticino’, ‘Timavo’, ‘Trebbia’, ‘Trichobel’, ‘Triplo’, ‘Unal’, ‘Veneziano’ 142 J. Pinon and P. Frey

These isolates were all pathogenic to all the bearing virulence 7 have been detected clones, although various levels of general each year. Without taking virulence 8 into resistance were observed on the same clones account, 20 pathotypes were found and, following natural infections in the nursery. when virulence 8 is included, 25 are known Pathotype-specific resistance in P. tricho- altogether. Some are less complex, combin- carpa and P. nigra cannot be ruled out, ing two virulences (4, 7), while others are although it seems infrequent. The frequency more complex (7 virulences, 1, 2, 3, 4, 5, 6 of pathotype-specific resistance in P. del- and 7 and 1, 2, 3, 4, 5, 7 and 8). Miot (1999) toides is unknown, but it appears quite easy conducted several laboratory experiments to to find and it is likely to have been favoured determine the effect of the accumulation of by breeders. unnecessary virulences on fungal fitness. In The second level of variability is aggres- each experiment, one simple and one com- siveness. The first evidence for variability in plex isolate were mixed and inoculated on aggressiveness between isolates belonging compatible cultivars. Many cycles were to the same pathotype was found among 1, 3, successively performed and the proportion 4, 5 and 7 pathotypes collected in 1998 on of the two pathotypes was determined. As a ‘Beaupré’ at Moy-de-l’Aisne (Aisne, France). result, a significant change in the proportion On ‘Beaupré’, under constant temperature of the isolates was found, but there was no (20°C) in the laboratory, the latent period clear tendency suggesting that unnecessary of these isolates was 7 days, compared to virulences had a cost for the fungus. The 8 days required by the older isolates of results were typically dependent on the the same pathotype. Variation in infection choice of the isolates. This is in agreement (described as number of uredinia per unit of with previous observations that the original leaf area) was also recorded, although this pathotypes, with five virulences, were still characteristic was most sensitive to experi- in the majority, even 10 years after their first mental conditions and was less reliable appearances. It was concluded that this fun- in distinguishing isolates. Dowkiw (2003) gus is able to accumulate virulences, thus considered sporulation. While most isolates increasing its host range, without losing produced small uredinia on selected clones, aggressiveness or fitness. he found that some isolates were able to develop significantly larger uredinia on the Interactions between poplar clones and same clones. Finally, all three quantitative pathotypes of M. larici-populina parameters used to quantify isolate aggres- siveness seemed to vary between isolates, Clone susceptibility to Melampsora species including those within the same pathotype. has been reviewed previously (Lemoine Probably aggressiveness is favoured each and Pinon, 1978; Pinon, 1992; Pinon and year through sexual reproduction of the Valadon, 1997). Furthermore, many poplar fungus on larch and is then selected as an clones have been inoculated in the labora- advantage. tory to establish their susceptibility to A frequently asked question in plant M. larici-populina pathotypes. Only those pathology is, ‘What is the cost for the fungus clones that are commercially available will of unnecessary virulences?’ In order to des- be cited, with no reference to experimental cribe rust populations, rust samples have clones (Table 12.2). A new case was found been collected from ‘Robusta’, which is, up with clone ‘A4A’. This clone was initially to the present, susceptible to all known free of rust when introduced in France. pathotypes (i.e. absence of pathotype- From 1998 some infection was detected on specific resistance). Since the detection of this clone and the level of infection has the first isolates with virulence 7, 10 years increased year by year. Pathotypes infecting ago, the most frequent isolates are still 1, 3, 4, this clone belong to the E4 group but were 5 and 7, despite the fact that only virulence 7 of a new type, combining virulences 2 and is necessary to infect the common cultivar 7. This new combination is likely to have ‘Beaupré’. Since 1994, new pathotypes appeared during sexual recombination on Interactions Between Poplar Clones and Melampsora Populations 143

larch. In addition, a few experimental However, when inoculated in the laboratory clones were also infected, only by such separately with E1 and E4 isolates, it pathotypes. Of the clones tested, only ‘A79’’ appeared to be less susceptible to E4 than to has remained completely resistant. E1 (longer latency and less infection). The Some cultivars showed quantitative same response was observed with the clone interactions with pathotypes. For example, ‘Unal’ (Fig. 12.1). Consequently, ‘Raspalje’ ‘Blanc du Poitou’ was almost resistant to E3 has become more popular, but there is a risk isolates, but somewhat more susceptible to that its increasing cultivation could exert a E4 (without damage). During the epidemic selection pressure on the rust populations, on ‘Beaupré’, it became evident that another with an increase in the proportion of the interamerican cultivar, ‘Raspalje’’, per- more pathogenic E1 pathotypes. The risk formed better against rust than had been will also increase with the concomitant previously recorded. This clone was decrease of ‘Beaupré’ cultivation leading to compatible with all known pathotypes. fewer E4 pathotypes.

RELATIVE LATENCY 100 90 80 70 60 50 40 30

Relative latency (%) Relative 20 10 0

UNAL

ROBUSTA RASPALJE BEAUPRE

E1 E4

RELATIVE INFECTION 100 90 80 70 60 50 40 30 20 Relative linfection (%) linfection Relative 10 0

UNAL

ROBUSTA RASPALJE BEAUPRE Fig. 12.1. Quantitative interactions between clones and M. larici-populina isolates of E1 (pathotype 3–4) and E4 (pathotype 3–4–7) groups. Data (latency and infection, i.e. number of uredinia) expressed as relative values from the most pathogenic isolate on each cultivar. 144 J. Pinon and P. Frey

Cultivars in which complete resistance others were infected only by some isolates was broken, were still able to exhibit various (i.e. differential clones) (Table 12.3). levels of general resistance. For instance, According to the pathogenicity of with E4 infection, ‘Beaupré’ may have been isolates originating from poplars and from badly damaged, while ‘Soligo’ remained alternate hosts, a first list of differential quite healthy. The general resistance was clones is proposed (Table 12.3). ‘Altichiero’ usually poor within P. × interamericana may be added to this list, although further but better (and often sufficient in practice) pathotype studies are needed to confirm that within P. × euramericana. Some exceptions the virulence necessary to infect this clone may exist, e.g. the level of susceptibility of is distinct from the present list of eight ‘Ghoy’ is relatively high and this clone must virulences. This list is not necessarily com- only be used where the risk of rust is low. plete, because a selected set of clones was used to inoculate the isolates and, also, our collection of isolates may not represent all the variability of the pathogen. Melampsora allii-populina As isolates from different alternate hosts were tested, their pathogenicity to Formae speciales were described within poplar clones was compared, in relation to M. allii-populina according to the host their original alternate host. No link was range of alternate hosts. Viennot-Bourgin found, and it is hypothesized that genes for (1937) described one forma specialis (f. sp. pathogenicity to poplar and to alternate allii-populina) which infected Arum spp. hosts are completely different (Frey and and Allium spp., while another (f. sp. Pinon, 1997). The differential cultivars for muscaridis populina) was pathogenic M. allii-populina belong to either pure to Muscari comosum and Allium species (i.e. P. trichocarpa for ‘Fritzi sphaerocephalum. On the latter, he Pauley’) or hybrids (P. × euramericana and described (under a light microscope) thin P. × interamericana). This is the first time spinules at the apex of the urediniospores that a balsam poplar was found as a source of that were not observed on recent samples complete resistance towards a rust agent. using a scanning microscope (Frey and Only one clone of P. nigra was included Pinon, 1997). Dupias (1965) described a (‘Italica’) and, therefore, it cannot be con- third forma specialis (f. sp. typica) patho- cluded that there is an absence of complete genic on all known alternate hosts. No resistance in this species. In the future, it experimental data were collected to confirm will be necessary to collect more M. allii- the validity of these formae speciales and populina isolates on P. nigra and to inocu- it would be necessary to conduct genetic late them on to a wider range of P. nigra analysis involving isolates from single clones to determine whether complete pustules. resistance exists in this host species that Magnani (1966) reported a possible vari- co-evolved with the rust pathogen. ability in pathogenicity to poplars within M. allii-populina, and indicated the presence of Table 12.3. Reaction of poplar clones to isolates pathotypes. However, his experimental data of M. allii-populina. could also be interpreted as being due to differences in aggressiveness, potentially Universal clones Differential clones attributed to inoculum quality. Fourteen iso- ‘Aurora’, ‘Beaupré’, ‘Bellini’, ‘Altichiero’, lates from poplar (on various clones and ‘Bellotto’, ‘Boelare’, ‘Büchig’, ‘Carpaccio’, locations) and 28 on alternate hosts (Allium ‘Carpaccio’, ‘Fritzi Pauley’, ‘Cima’, ‘Fritzi spp., Arum italicum and Muscari comosum) ‘Ghoy’, ‘I-154’, ‘I-214’, Pauley’, ‘I-154’, were collected and inoculated on to a wide ‘Isières’, ‘Italica’, ‘Primo’, ‘Isières’, ‘Luisa range of poplar clones (Frey and Pinon, ‘Raspalje’, ‘Rintheim’, Avanzo’, ‘NL 1997). Many clones were infected by all ‘Robusta’, ‘Tiepolo’, ‘Unal’, 2842’, ‘Rap’, ‘Véronèse’ ‘Spijk’ of the isolates (i.e. universal clones) while Interactions Between Poplar Clones and Melampsora Populations 145

Work was carried out on M. allii- between poplar clones and isolates or patho- populina populations. For example, in 1993 types of the rust. No information is yet populations were collected in south-eastern available on the variability of the fungus France (where wild P. nigra is predominant) for aggressiveness. Laboratory inoculations and in a nursery of poplar cultivars indicated that the infection efficiency of (Guémené-Penfao, Loire-Atlantique) in M. allii-populina was lower than that of M. western France (Fig. 12.2). Seven virulences larici-populina, with approximately twice were examined and it appeared that the fre- the number of urediospores needed to get quencies of the virulences varied according the same level of infection. to sites. To the present time 42 different pathotypes of M. allii-populina have been detected in France. Some isolates were sim- ple, being pathogenic on cultivars such as Melampsora medusae ‘Beaupré’ but without any of the virulences listed above. Others were more complex, In Europe, M. medusae was introduced a combining up to 7 virulences (all except long time ago and first described in Spain virulence 1). The most frequent pathotype by Fragoso (1925) and then in south- in 1990 in Fontainebleau and Nancy was western France by Dupias (1965). It was 2–4–5–8, which was collected from Muscari identified on ‘I-214’ leaves received from comosum and Allium niveale. Portugal in the 1980s. Little attention was Unlike M. larici-populina, no cultivar given to M. medusae, for two probable rea- with defeated complete resistance to M. sons. First, no real damage or epidemic was allii-populina has been selected up to the observed (for unclear reasons), despite the present. The effect of poplar populations on fact that some cultivars popular in France pathogen populations is therefore not docu- were found to be susceptible to M. medusae mented. In addition, no studies were con- in New Zealand (Pinon, 1986). Secondly, its ducted to examine quantitative interactions distribution was restricted to south-western

Fig. 12.2. Frequency of seven virulences in five populations of M. allii-populina in 1993. South-eastern France: Mézel, Gréoux, Sisteron, Manosque; western France: Guémené-Penfao. In brackets: number of isolates studied. 146 J. Pinon and P. Frey

Europe. Evidence from surveys carried out in also infected by M. allii-populina and France, showed that the infected area may M. larici-populina. Van Kraayenoord (1984) be defined as a triangle between Bordeaux, published information on the level of Toulouse and Bayonne. The rust studied susceptibility of many clones. fitted the description of M. medusae f. sp. Some variability in pathogenicity was deltoidae defined by Shain (1988). described within M. medusae in North During the 1980s, there was also one America (Prakash and Thielges, 1987; case of infection in the western part of Newcombe et al., 2000) and Australasia France (Guémené-Penfao) on ‘Rap’. In 1999 (Singh and Heather, 1982). Interactions infections were found in Nancy (C. Husson, between poplar clones and rust isolates 1999, personal communication) that were often quantitative and sometimes allowed some recent clones to be added to clearly qualitative. Temperature may the host list. In the following year, both in affect the expression of poplar clone × Guémené-Penfao and in Nancy, M. medusae M. medusae isolate interactions (Singh and was no longer detectable. It seemed that this Heather, 1982; Prakash and Thielges, 1989). fungus might sometimes move from south- Although some clones were described as western France over long distances, during possessing complete resistance and isolates the summer, and initiate very small and as having matching virulence, no official list discrete infections far from its established of differential clones was proposed. Also, locations. However, in both cases, it seemed poplar clones used in each study were differ- unable to overwinter. This fungus is a quar- ent and they were often not widely grown antine pathogen in the EU and the French cultivars (experimental clones). Comparing Plant Protection Service inspects poplar data from these two geographical areas, it plants in nurseries. Between 1993 and 1997, was found that some clones could act as M. medusae was detected in some commer- differential clones, possibly in the case of cial nurseries in south-western France. ‘Eugenei’, ‘I-154’ and ‘I-455’ (Pinon, 1992). Since 1998, it has not been detected in France (J. Tournut, Nancy, 2003, personal communication). Therefore, only a list of clones which have been found to be infected Impact of the Breakdown of with M. medusae may be drawn up. Slight Complete Resistance infections were found on some cultivars in Bordeaux between 1988 and 1991 (Table Until the present, only the breakdown of 12.4). In most cases, these clones were resistance by M. larici-populina affected

Table 12.4. Clones found infected by M. medusae in France.

Natural infection in nursery

Bordeaux (1988–1991) Nancy (1999) Laboratory inoculation

‘Alabama’, ‘Aladin’, ‘Albane’, ‘Altichiero’, ‘Angulata ‘Hazendans’, 72501a, 041.3.402a, 71077–308a, de Chautagne’, ‘Barn’, ‘Batard de Haute Rive’, ‘Hoogvorst’, 71093R1a, 71097R2a, ‘Beaupré’, ‘Blanc du Poitou’, ‘Carolin’, ‘Carpaccio’, ‘NL 2233’, 72142R4a, 72156R5a, ‘Cima’, ‘Culasso’, ‘Donk’, ‘Estoup’, ‘Florence ‘NL 3261’, 92519R9a, 95545R1a, Biondi’, ‘Gaver’, ‘Ghoy’, ‘Gibecq’, ‘Guariento’, ‘NL 4040’ 96546R1a, 87B12b, ‘Aurora’, ‘Hees’, ‘Heidemij’, ‘Hoogvorst’, ‘I-214’, ‘I 45–51’, ‘Beaupré’, ‘Brabantica’, ‘Flevo’, ‘I-455’, ‘I-488’, ‘Isières’, ‘Luisa Avanzo’, ‘Lux’, ‘Hoogvorst’, ‘I 45-51’, ‘Italica’, ‘Olin’, ‘Rap’, ‘Raspalje’, ‘Robusta’, ‘Spijk’, ‘Unal’, ‘Ogy’, ‘Pourtet’, ‘Rap’, ‘Veneziano’, ‘Veronese’, ‘Virginiana’, ‘Virginie de ‘Robusta’, ‘Sarrazin’, ‘Simonii’, Frignicourt’ ‘Unal’, ‘Vereecken’ aNative Populus nigra clones. bP. deltoides from INRA, Orléans. Interactions Between Poplar Clones and Melampsora Populations 147

poplar cultivation in Europe. In France, at in some short-rotation coppices. Virulence least, the breakdown of complete resistance 7 was detected in Nancy on larch in by pathotypes from group E2 (virulence 1 spring 1996. Infection increased steadily on and 5) had no significant effect. The reason ‘Beaupré’ in the nursery, while on ‘Robusta’ for this was that the corresponding clones it fluctuated, according to the climate or were not cultivated commercially. Patho- assessment date (Fig. 12.4). The frequency of types with virulence 2 (E3 group) induced virulence 7 (E4 group) increased during the significant damage on ‘Luisa Avanzo’, same period. In 2002, 68% of the isolates on grown mainly in south-western France. In ‘Robusta’ had virulence 7 and some cases of this area, although larch is infrequent, mortality were observed in relatively young infections were serious and some stands stands of ‘Beaupré’. Damage due to M. larici- had to be sprayed with fungicide. Damage populina is important. According to Gastine due to rust was sometimes severe, although et al. (2003) the current annual increment of the overall effect was limited because this ‘Beaupré’ was reduced by 20–30% in 1998 clone was grown in a relatively small area. and by 50–60% in 2000. Some clones susceptible to virulence 2 More recently, virulence 8 was discov- were assessed each year for rust infection ered (E5 group). This virulence is necessary in Nancy. More than 15 years after the to infect some recent cultivars such as outbreak of this group of pathotypes, ‘Hoogvorst’ and ‘Hazendans’. Isolates with infection is still heavy in the nursery virulence 8 were found just at the time when (Fig. 12.3). these cultivars were registered in Belgium, The most important damage was that and so laboratory tests could be conducted due to the outbreak of virulence 7 (E4 patho- in France in order to forecast their level of types) because this overcame the complete susceptibility. ‘Hoogvorst’ was found to be resistance of a popular cultivar, ‘Beaupré’. as susceptible to E5 pathotypes as ‘Beaupré’ Virulence 7 was detected for the first time in was to E4 pathotypes (Fig. 12.5). Even if northern France in 1994. In 1997 a serious infection was somewhat less on ‘Hazen- outbreak occurred in this region. Some dans’, poplar growers were advised against stands planted close to larch were defoliated planting these two clones. E5 pathotypes early in July. Before the occurrence of E4 therefore did not have these clones to build pathotypes, ‘Beaupré’ was infected by up their population and had difficulty M. allii-populina without damage, except spreading in France where their frequency

90 80 70 60 50 % 40 30 20 10 0 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Years

Virulence 2 frequency Infection

Fig. 12.3. Relationship between the infection level of cultivars susceptible to virulence 2 and virulence 2 frequency in Nancy. 148 J. Pinon and P. Frey

Fig. 12.4. Relationship between infection and virulence 7 frequency in Nancy.

Fig. 12.5. Comparison of the aggressiveness of E4 and E5 pathotypes on five cultivars. Aggressiveness is expressed as a relative disease index (number of uredinia divided by latency) adjusted to 100 for ‘Robusta’ inoculated with E5. remains low. The area planted with these Adaptation of Poplar Cultivation on the two clones in France is low and their health Evolution of Pathotype Populations remains good, in contrast to recent mortali- ties described in Belgium on ‘Hoogvorst’. E3 pathotypes affected mainly two clones, Comparing clone populations and frequen- planted generally in south-western France, cies of the relevant virulences, it became ‘Luisa Avanzo’ and ‘Cima’. These two clones obvious that poplar rust fits perfectly with were a minority in poplar plantations in Van der Plank’s theory on the effect of clonal France. Their maximum nursery plant pro- popularity on infection (Van der Plank, duction was around 4–6% between 1987 1968). and 1992 (Fig. 12.6). Later their proportion Interactions Between Poplar Clones and Melampsora Populations 149

Fig. 12.6. Changes in poplar plant production in France according to the Ministère de l’Agriculture, de l’Alimentation, de la Pêche et des Affaires Rurales (1987–2001). in new plantations decreased to as low as of ‘I-214’ and ‘’I-45/51’ against rust was not 0.08% during the winter of 2001–2002. This modified by the new virulences of M. larici- decrease in popularity was due to rust populina. ‘Dorskamp’ and ‘Flevo’ had origi- susceptibility, as well as to declines relating nally been completely resistant and became to flooding during the growing season. infected with the emergence of virulence 7. Virulence 7 was first detected, at very Nevertheless, they possessed a good level of low frequency, in 1994. Advice was given general resistance and some new clones such to growers to reduce the proportion of as ‘Triplo’ and ‘Koster’ have also been pro- ‘Beaupré’ and ‘Boelare’ in new plantations. duced. There has been renewed interest in During the winter of 1996–97, ‘Beaupré’ one clone of P. × interamericana (‘Raspalje’). represented 57% of the total number of In the past, it had been relatively susceptible poplar plants purchased (Fig. 12.6). In to rust, and so ‘Beaupré’ was preferred addition, this clone was especially popular because of its health and fast growth. When in the northern half of France, where 80% virulence 7 developed, ‘Beaupré’ became of new plantations were planted with badly infected while ‘Raspalje’ appeared ‘Beaupré’. Damage became evident in north- healthier, especially when planted close to ern France in 1997 and, from the winter of ‘Beaupré’. ‘Raspalje’ was less susceptible to 1996–97 until the winter of 2001–2002, the E4 pathotypes than to E1 pathotypes (Fig. general demand for this clone decreased 12.1). Because ‘Beaupré’ is still common, sharply, and in winter 2001–2002, ‘Beaupré’ in many places E4 pathotypes are the most contributed only 2.75% of the plantations frequent, especially in northern France. (Fig. 12.6). Such a boom and bust situation had been described previously by Van der Plank (1968) for agricultural crops. Testing Clones Considering Fungal In the meantime, new interest emerged Diversity in older cultivars, most of which (‘I 214’, ‘Dorskamp’, ‘I-45/51’, ‘Flevo’) belonged to Methods used for rust resistance testing P. × euramericana. Hence the performance must be reliable. One risk is to declare a 150 J. Pinon and P. Frey

clone as resistant when it is not, either mid-July. For the first time it was possible because the pathotypes capable of causing to compare poplar clones for their suscepti- infection were not present in the test or bility to M. allii-populina. The reactions of present only at a very low frequency. Three clones ranged from free of rust to severely levels of variability must be taken into infected. In the future, those clones with account: species, virulences and aggressive- defeated resistance need to be tested under ness. Although complete resistance was sufficient inoculum pressure of the relevant easily defeated, susceptible clones became virulence so that they fully express their acceptable if it was demonstrated that there susceptibility. was a high level of tolerance, i.e. obvious infection without serious suppressive effect on growth. Melampsora larici-populina

Because of the high risk of infection and Melampsora allii-populina damage posed by M. larici-populina,a sophisticated strategy has been developed In the past, M. allii-populina occurred (Pinon and Berthelot, 2004). Most of the mixed with M. larici-populina, especially time, clones are evaluated in the nursery in western France. Rust was assessed in a with a controlled inoculum, so as to ensure nursery located in this area (Guémené- that all virulences are present. Larch plants Penfao) and samples were taken from plants are grown in the trial and in spring poplar for further species identification under the leaves bearing telia are placed on the microscope. This was not completely satis- ground under the larch. These infected factory because the proportion of infection leaves had been collected during the previ- due to each Melampsora species differed ous autumn from differential clones (Table according to clone, and it would have been 12.1). Samples were also collected from too time-consuming to determine the pre- ‘Robusta’ at the time of each assessment, in cise proportions. In 1999 a clonal trial was order to quantify the prevalent pathotype established in the nursery with an alternate population. For most clones, laboratory host (leek). Leaves bearing telia of M. allii- tests were conducted to determine which populina, collected in the previous year, virulence(s) was (were) required to infect were placed near to the leeks. However, clones being tested. Symptoms are also host alternation was largely unsuccessful. assessed on control clones (including those In 2000, the clonal trial was inoculated of Table 12.1) of known susceptibility. in early June in a different way. All larch Results were interpreted by taking the fol- plants present in the nursery were sprayed lowing into account: the level of symptoms with a fungicide in order to prevent their on each clone, the pathotype population infection by M. larici-populina. Therefore and the virulences required for infection of the nursery could only be infected by clones. This strategy has proved successful, M. larici-populina later in the season but a complementary approach has also by inocula coming from outside. Plants of been developed for clones with inter- ‘’Beaupré’ (susceptible to M. allii-populina) mediate reactions to the rust, as it is were grown in a glasshouse at INRA, Nancy, difficult to define the acceptable threshold inoculated in the laboratory with several for such clones. Recently, shoot growth on isolates of M. allii-populina (covering the some clones was compared between fungi- whole range of known virulences) and cide-sprayed and unsprayed control plants. transported to Guémené-Penfao nursery on It appeared that the rust impact was much 6 June, just before sporulation. On 19 June greater on P. × interamericana than on all clones were observed and around 60% P. × euramericana. Within a hybrid type, showed first symptoms. Epidemics started clones with the same level of infection did and infection was well established by not suffer from rust to the same extent. Interactions Between Poplar Clones and Melampsora Populations 151

Therefore, not only clones with fewer rust Sporulation was less intense on ‘Robusta’ symptoms, but also those exhibiting a fair and this is one of the reasons why this clone level of tolerance, may be selected. After is less damaged by rust than ‘Beaupré’. a genetic analysis, Dowkiw (2003) showed Nursery tests are preferred because they that the link between resistance (expressed are less time-consuming and can take into by symptoms) and tolerance (growth under account the effect of plant physiology and infection) is weak and consequently, these phenology on rust development. Neverthe- two characters may have to be selected less, when a new virulence does appear, an independently. He suggested that a major early prognosis can be obtained on a limited gene for partial resistance is inherited from number of clones in laboratory tests. the P. trichocarpa parent. When a new virulence appears, break- ing down the complete resistance of some Mixing Clones and M. larici-populina clones, nursery tests do not allow a pertinent Populations evaluation of such clones, because the frequency of the new virulence is too Poplar cultivation is generally based on low. Several years are needed to increase the monoclonal stands. Mixing cultivars is a new virulence frequency to such a level that potential approach to rust control. This allows the clones to express their true level approach is well documented for willow of resistance. This was evident in the rust (Chapters 16 and 17, this volume). popular clones such as ‘Beaupré’ when E4 These authors obtained positive effects on appeared, and in the recently released culti- health and growth from willow mixtures. vars such as ‘Hoogvorst’ and ‘Hazendans’ Miot et al. (1999) studied a mixture of three when E5 was detected. Poplar nursery staff, poplar clones whose complete resistance poplar growers and officers in charge of was defeated by different virulences of forest policy need to be able to make an early M. larici-populina (P. × jackii ‘Aurora’, P. × prognosis and, through laboratory tests, the interamericana ‘Boelare’ and ‘Rap’). Each future of such cultivars can be forecast. Tests clone was inoculated with an isolate were conducted on discs cut from leaves avirulent on the two other clones. Over 3 taken from poplar cuttings grown in the years, the benefit of the mixture was small greenhouse. ‘Robusta’, which is susceptible on infection and often none on growth. The to all pathotypes, was used as a control experiment was located in a glade in a and latency and infection (the number of beech and oak forest with little poplar uredinia per disk) were measured. Figure cultivation in the surrounding areas. 12.5 shows the high level of susceptibility of Nevertheless, natural infection occurred ‘Beaupré’, ‘Boelare’ and ‘Ogy’ to E4 and an during the second and third years due even more serious infection by E5. With to 1–3–4–5–7 isolates. Such isolates were such early tests it was possible to forecast virulent on 2 out of 3 mixture components. possible heavy infections on ‘Hazendans’ As discussed previously, M. larici-populina and ‘Hoogvorst’ by E5. In 2003 those is able to cumulate virulences and maintain cultivars were badly damaged in Belgium a fair level of aggressiveness and fitness. (J. Van Slycken, Belgium, 2003, personal This feature may reduce the interest of pop- communication). A method, based on a par- lar clonal mixtures and, even more, such ticle counter, was also developed in cooper- mixtures may favour complex and aggres- ation with Husson (Husson, unpublished sive isolates. data) to estimate the level of sporulation. Sporulation depends on the clone being tested and, to some extent, on the isolate or pathotype. ‘Beaupré’ and ‘Boelare’ sup- Discussion ported a high level of sporulation of the pathogen and the level increased progres- The breakdown of complete resistance to sively after the appearance of uredinia. M. larici-populina, the low level of general 152 J. Pinon and P. Frey

resistance in many P. × interamericana quality and plasticity) was incomplete or cultivars and the popularity of these missing. cultivars had a major impact on poplar cul- The breakdown of resistance also had a tivation in Europe. The population of the marked influence on poplar breeders, who pathogen changed rapidly, with new viru- were obliged to select new clones and lent isolates spreading easily on the poplar change their strategy, choosing alternative clones with defeated resistance. In any parents and initiating new crosses. Numer- given location, the frequency of such iso- ous vigorous cultivars were discarded. Inter- lates increased quickly. In addition, higher est in complete resistance (not necessarily aggressiveness (shorter latency and higher durable) was questioned. Dowkiw et al. sporulation) was later detected. When a (2003) demonstrated recently that there new virulence (virulence 7) was detected in is a positive side-effect of complete (but complex isolates, the incubation period was defeated) resistance. This resistance is longer on clones with defeated resistance linked with general resistance towards some (e.g. ‘Beaupré’) than on universal cultivars virulences. They suggested that this general such as ‘Robusta’. Later, isolates with resistance implicates a major gene from reduced incubation periods were detected. P. trichocarpa. Dowkiw (2003) also proved At present, the pathogen population is that progenies with a given P. trichocarpa much more diverse in cultivation areas, father can exhibit various level of resistance made up of complex isolates (combining according to the P. deltoides mother parent. many virulences) with a high level of It means that some associations of parents aggressiveness. Dowkiw (2003) also found are more interesting than others. Also some longer incubation periods with complex backcrosses of P. × interamericana towards isolates having the recent virulence 8. P. deltoides carried out by the Instituut voor It is too early to know if cultivating more Bosbouw en Wildbeheer (IBW) in Geraards- cultivars with general resistance will reverse bergen (Belgium) produced hybrids with a the present situation towards populations sufficient level of resistance. Thus it seems with fewer and simpler pathotypes. Until too early to discard definitively, complete now, population studies were mainly resistance. Recently it was found, among devoted to virulences and more recently to P. deltoides clones under study at INRA fungal genotypes. A key question to be asked Orléans, that some individuals were without is about the variability in aggressiveness, for this complete resistance but had a good level which some evidence is available. Again, it of general resistance. These clones may be is not known if the cultivation of cultivars used as parents for creating new P. × inter- with higher levels of general resistance will americana clones. New hybrid combina- exert some selection pressure on the fungus tions can also be made. For instance, for more aggressiveness. IBW recently submitted hybrids between The high level of adaptability of this P. trichocarpa and P. maximowiczii. These pathogen to its host is clearly demonstrated clones proved to exhibit only general and the choice of clones offering diversity is resistance in laboratory tests and the a prerequisite for sustainable poplar cultiva- first symptom assessments in the nursery tion. Poplar growers were quite often obliged suggested a good level of resistance. to spray in order to control the disease, and Basic knowledge is still needed in new plantations were made up of clones defining clone management in space and with more general resistance, but less vig- time. This is essential because host popula- our. Increase of diversity was advocated, but tions exert an effective and rapid selection this was sometimes difficult to achieve as pressure on the pathogen population, which many clones were genetically related. Some affects the health of poplars, under a new clones with more durable resistance boomerang effect. The ability of the fungus were proposed for cultivation, but often to develop complex isolates with high information on their characters at adult age aggressiveness and fitness makes things (adaptation to ecological conditions, wood more complicated. The diversification of Interactions Between Poplar Clones and Melampsora Populations 153

resistance sources and the deployment of et de la structure de la population hôte rust tolerance are important areas for future sur l’évolution des populations de l’agent investigation. pathogène. PhD thesis, The University Henri Poincaré, Nancy, France. Miot, S., Frey, P. and Pinon, J. (1999) Varietal mixture of poplar clones: effects on infection by Melamp- Acknowledgements sora larici-populina and on plant growth. European Journal of Forest Pathology 29, The authors thank Claude Husson for 411–423. his collaboration, especially in the Newcombe, G. (1998) A review of exapted resistance development of a method for sporulation to disease of Populus. European Journal of Forest assessment. Pathology 28, 209–216. Newcombe, G., Stirling, B., McDonald, S. and Bradshaw, H.D. Jr (2000) Melampsora columbiana, a natural hybrid of M. medusae References and M. occidentalis. Mycological Research 104, 261–274. Dowkiw, A. (2003) Analyse génétique de la résistance Pinon, J. (1986) Situation de Melampsora medusae en et de la tolérance de peupliers hybrides Populus Europe. Bulletin OEPP 16, 547–551. deltoides × Populus trichocarpa aux rouilles Pinon, J. (1991) Eléments de répartition des rouilles foliaires à Melampsora larici-populina. PhD des peupliers cultivés en France. Comptes- thesis, The University of Orléans, France. rendus de l’Académie d’agriculture de France Dowkiw, A., Husson, C., Frey, P., Pinon, J. and 77, 109–115. Bastien, C. (2003) Partial resistance to Melamp- Pinon, J. (1992) Variability in the genus Populus in sora larici-populina leaf rust in hybrid poplars: sensitivity to Melampsora rusts. Silvae Genetica genetic variability in inoculated excised leaf 41, 25–34. disk bioassay and relationship with complete Pinon, J. (1995) Présence en France d’une nouvelle resistance. Phytopathology 93, 421–427. race de Melampsora larici-populina, agent de Dupias, G. (1965) Les rouilles des peupliers dans les la rouille foliaire des peupliers cultivés. Revue Pyrénées et le Bassin sous-pyrénéen. Bulletin de Forestière Française 47, 230–234. la Société Mycologique de France 81, 188–196. Pinon, J. and Bachacou, J. (1984) Existence de deux Fragoso, R.G. (1925) Flora Iberica. II. Uredinales. groupes d’isolats différant par leur pouvoir Museo Nacional de Ciencias Naturales, Madrid, pathogène chez Melampsora larici-populina pp. 198–199. Kleb. Comptes-rendus de l’Académie Frey, P. and Pinon, J. (1997) Variability in d’agriculture de France 70, 114–122. pathogenicity in Melampsora allii-populina Pinon, J. and Berthelot, A. (2004) La prise en compte expressed on poplar cutivars. European Journal des problèmes sanitaires par le GIS Peuplier. Les of Forest Pathology 27, 397–407. Cahiers du DSF, 1–2003/2004 (La santé des Gastine, F., Berthelot, A., Bouvet, A., Servant, H. and Forêts [France] en 2002). Min. Agri. Alim. Pêche Roy, B. (2003) La protection phytosanitaire du Aff. Rur. (DGFAR), Paris, pp. 84–87. cultivar ‘Beaupré’ est-elle efficace? Informations Pinon, J. and Frey, P. (1997) Structure of Melampsora Forêt 2, 6 pp. larici-populina populations on wild and Latch, B.J. and Wilkinson, A.G. (1980) New poplar cultivated poplar. European Journal of Plant clones help distinguish races of Melampsora Pathology 103, 159–173. larici-populina Kleb. in New Zealand. Pinon, J. and Lefèvre, F. (1994) A new virulence found Australasian Plant Pathology 9, 112–113. among isolates of Melampsora larici-populina. Lemoine, M. and Pinon, J. (1978) Différences clonales Réunion de la Commission Internationale du de sensibilité des peupliers aux rouilles à Peuplier, Izmit (Turkey), 3–7 October. Melampsora larici-populina et M. allii-populina. Pinon, J. and Peulon, V. (1989) Mise en évidence Revue Forestière Française 30, 181–185. d’une troisième race physiologique de Magnani, G. (1966) Investigations on the possibility Melampsora larici-populina Kleb en Europe. of physiological specialization in Melampsora Cryptogamie Mycologie 10, 95–106. allii-populina. Pubblicazioni del Centro di Pinon, J. and Valadon, A. (1997) Comportement des Sperimentazione Agricola e Forestale Roma 8, cultivars de peupliers commercialisables dans 127–133. l’Union européenne vis-à-vis de quelques Miot, S. (1999) Rôle de la variabilité de Melampsora parasites majeurs. Annales des Sciences larici-populina agent de la rouille des peupliers Forestières 54, 19–38. 154 J. Pinon and P. Frey

Prakash, C.S. and Thielges, B.A. (1987) Pathogenic Steenackers, M.; Steenackers, V. and Delporte, T. variation in Melampsora medusae leaf rust of (1994) A new physiological race of Melampsora poplars. Euphytica 36, 563–570. larici-populina in Belgium. FAO–CIP, Réunion Prakash, C.S. and Thielges, B.A. (1989) Interaction du groupe de travail sur les maladies, Izmit of geographic isolates of Melampsora medusae (Turkey), 3–7 October. and Populus: effect of temperature. Canadian Van der Plank, J.E. (1968) Disease Resistance in Plants. Journal of Botany 67, 486–490. Academic Press, New York. Shain, L. (1988) Evidence for formae speciales in Van Kraayenoord, C.W.S. (1984) National poplar leaf rust fungus Melampsora medusae. Report on Activities Related to Poplar and Mycologia 80, 729–732. Willow. International Poplar Commission, Singh, S. and Heather, W.A. (1982) Temperature Ottawa. sensitivity of race–cultivar interactions in Van Vloten, H. (1949) Kruisingsproeven met Melampsora medusae Thum and Populus rassen van Melampsora larici-populina spp. European Journal of Forest Pathology 12, Klebahn. Tijdschrift over Plantenziekten 55, 123–127. 196–209. Steenackers, V. (1982) Nouvelle race physiologique Viennot-Bourgin, G. (1937) Contribution à l’étude de de Melampsora larici-populina en Belgique la flore cryptogamique du Bassin de la Seine (provisional communication). FAO–CIP 22nd (11ième note). Deux urédinées nouvelles. Revue session du groupe de travail maladies de Pathologie et d’Entomologie Agricole de Casale–Monferrato (Italy), September 1982. France 24, 78–85. 13 Transgenic Hybrid Aspen with Altered Defensive Chemistry: a Model System to Study the Chemical Basis of Resistance?

Johanna Witzell1, Marlene Karlsson1, Marisa Rodriguez-Buey2, Mikaela Torp1 and Gunnar Wingsle1 1Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden; 2Umeå Plant Science Center, Department of Plant Physiology, Umeå University, S-901 87 Umeå, Sweden

Phenolic Compounds of Salicaceae Thus, their possible importance for the Plants resistance of willows and poplars to patho- gens has remained poorly understood. In Plants of the Salicaceae family (poplars and willows, Melampsora rust infection has willows) are rich in phenolic secondary been shown to induce the accumulation metabolites, including low molecular of certain phenolic metabolites (Hakulinen, weight phenolic glycosides (salicylates) 1998; Hakulinen et al., 1999; Hakulinen and and high molecular weight polyphenols Julkunen-Tiitto, 2000), but the molecular (flavonoids and condensed tannins) mechanisms behind this induction, or its (Lindroth et al., 1987, 2001; Nichols-Orians possible consequences to the invading et al., 1993; Ruuhola, 2001). During recent fungi, have not been studied. decades, there has been considerable accu- mulation of evidence corroborating their versatile role as mediators of interactions MYB Genes Regulate Phenylpropanoid between Salicaceae plants and their insect Metabolism in Plants and mammalian herbivores (Pasteels et al., 1983; Tahvanainen et al., 1985; Koleh- The biosynthesis of phenolic compounds mainen et al., 1994; Ruuhola et al., 2001). involves different metabolic pathways. Aro- Phenolic metabolites are also recognized as matic amino acids, such as phenylalanine, antimicrobial agents and signal compounds which serve as precursors in the synthesis in many plant–pathogen interactions (for of phenolics, are products of the shikimic reviews see Nicholson and Hammer- acid pathway. Enzymatic deamination of schmidt, 1992; Dixon and Paiva, 1995). phenylalanine yields trans-cinnamic acid, However, there are only a few reports con- which can be further metabolized to sidering their functions in the interactions para-coumaric acid. The subsequent between pathogens and Salicaceae plants additions of hydroxyl groups and other (Scaysbrook et al., 1992; Hakulinen, 1998). substituents yield a variety of derivatives, ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 155 156 J. Witzell et al.

phenylpropanoids, which are precursors of the gene of interest and regenerated as more complex phenolic substances, such as described previously (Nilsson et al., 1992). lignin (Ruuhola, 2001). The regulation of Several lines carrying the antisense con- the enzymes of phenylpropanoid metabo- struct of PttMYB46 or PttMYB76 were lism is likely to be complex, given the high recovered, multiplied as cuttings and structural diversity of phenolics, as well as rooted. Wild type (WT) plants (clone T89) the great variation in phenolic levels due to were multiplied in the same way. The plant inherent factors (genotype, ontogeny) plants were grown in a climate chamber and environmental factors (Coleman et al., illuminated by Osram HQI-TS 400 W lamps 1992; Herms and Mattson, 1992). At the giving a minimum quantum flux density of transcriptional level, different branches of 150 mE m−2 s−1, with 16 h photoperiod and the phenylpropanoid metabolism have been 20°C/16°C day/night temperature cycles. shown to be regulated by DNA-binding pro- The transformed lines showed altered teins encoded by the MYB-domain genes phenotypes with a stunted appearance. (Grotewold et al., 1994; Sablowski et al., More detailed studies confirmed that the 1994; Moyano et al., 1996; Tamagnone transgenic lines had reduced number and et al., 1998). Members of the MYB family length of internodes. In addition, some lines have been found in nearly all eukaryotes, showed increased trichome frequency and and in plants they comprise the largest fam- anatomical alterations in the stem tissues. ily of transcription factors. In addition to Such notable morphological and anatomical regulation of the phenylpropanoid path- deviations from the WT plants suggested way, they have been shown to influence underlying metabolic and chemical differ- hormone responses (Urao et al., 1993), cell ences (Karlsson, 2003). The lines 46 III, 46 shape (Noda et al., 1994), trichome differen- IV, 76 III and 76 V, which showed a clearly tiation (Oppenheimer et al., 1991) and plant deviating phenotype from the WT, were cho- defence responses (Yang and Klessig, 1996). sen for chemical analyses. The bark of the transgenic 76 V plants was found to have a markedly decreased carbohydrate/lignin ratio, suggesting enhanced allocation of car- Hybrid Aspen Carrying Antisense bon to phenylpropanoid metabolism in the Constructs of MYB Genes Show Altered bark of these plants (Karlsson, 2003). This Phenotype and Chemical Deviations inspired us to compare the levels of individ- ual phenolic compounds in WT and trans- Recently, two MYB genes, PttMYB46 and genic plants. We used liquid chromatogra- PttMYB76, were isolated and cloned from phy (HPLC) to measure the concentrations of hybrid aspen (Karlsson, 2003). These genes methanol-extractable phenolics in the leaf were selected from hybrid aspen expressed tissues of the WT and transgenic plants. sequence tag (EST) library from the Swed- From 6-week-old plants (n = 5 for WT and ish Centre for Tree Functional Genomics each line), the first full-grown leaf was col- project database (PopulusDB) and their lected, air-dried at room temperature and cDNA cloned. A polymerase chain reaction stored in a desiccator until extracted and (PCR) fragment carrying HindIII and BamHI analysed as described by Karlsson (2003). restriction sites was generated from the 3′ The analyses showed that the phenolic non-conserved part of the genes and cloned pool of aspen leaves was dominated by in antisense orientation in a pJIT60 vector salicin (2-O-b-D-glucoside of salicyl alcohol) between the double cauliflower mosaic and its derivatives (salicortin and tremu- virus (CaMV) 35S promoter and the CaMV lacin). In addition, chlorogenic acid and 35S terminator (for more details, see cinnamic acid derivatives and some Karlsson, 2003). Cuttings of hybrid aspen flavonoids (quercetin and kaempferol deriv- (Populus tremula L. × P. tremuloides atives) were detected. Clear-cut qualitative Michx., clone T89) were transformed with differences in phenolic profiles were not Agrobacterium tumefaciens strains carrying detected between the three genotypes, Transgenic Hybrid Aspen with Altered Defensive Chemistry 157

whereas quantitative differences were lines with altered phenolic profiles provide apparent. Compared to the WT plants, trans- a much narrower genetic background for genic lines had elevated levels of phenolic variation in resistance. This should acids and reduced levels of total salicylates facilitate identification of the chemical in their full-grown leaves (Fig. 13.1). characters that are critical for resistance or susceptibility. As a first attempt to explore this field, we performed experiments where we inocu- Transgenic Plants with Altered Phenolic lated the detached leaves of the WT and dif- Levels: a Model System to Study the ferent transgenic lines with conidia from Chemical Basis of Resistance? Pollacia radiosa, the anamorph of Venturia tremulae (Ascomycotina). This fungus is the Phenolic compounds are frequently impli- causal agent of aspen and poplar leaf and cated as potential defensive chemicals twig blight, with the characteristic ‘shep- against pathogens in Salicaceae plants herd’s crook’ symptoms. The leaves were (Bradshaw et al., 1991; Scaysbrook et al., detached from young trees grown in the 1992; Nicolescu et al., 1996). So far, how- same conditions as above, surface-sterilized ever, the possible links between the pheno- and maintained on MS/agar medium. lic metabolism and pathogen resistance in Conidia suspension or water (control) was Salicaceae plants have remained unclear. spread on the leaves. During a 14-day In several cases, the analyses of different incubation period, the development of the aspects of phenolic metabolism did not disease was monitored by measurements of include testing of the actual resistance. In the necrotic area on leaves (Fig. 13.2). The most of the studies addressing the question, results of the inoculation experiments indi- the degree of resistance/susceptibility has cated varying responses to infection among been correlated with the chemical composi- the studied aspen genotypes. Compared to tion or concentrations of a clone or a selec- WT, the line 76 III did not show marked tion of clones. However, this approach is differences in sensitivity to infection. How- not without drawbacks and may not lead ever, on leaves of the line 76 V infection to profound understanding of the role seemed to spread faster than on WT leaves of secondary chemicals in plant–pathogen (Fig. 13.2A). The lines carrying the antisense interactions, or to identification of reliable construct of PttMYB46 seemed to be sensi- biochemical markers for disease resistance/ tive to the infection. We found that in the susceptibility in Salicaceae plants. As com- line 46 IV the first symptoms appeared 2 pared with traditional studies with differ- days earlier than in WT. Moreover, although ent clones, the transformed hybrid aspen the infection on leaves of line 46 III started at

Fig. 13.1. Relative concentrations of (A) salicylates (sum amount of salicin, salicortin and tremulacin) and (B) phenolic acids (sum amount of three chlorogenic acid and two cinnamic acid derivatives) in hybrid aspen (clone T89 = wild type) and transgenic plants. 158 J. Witzell et al.

Fig. 13.2. Infected necrotic area expressed as the percentage of the total leaf area from (A) hybrid aspen (s, n) and its transgenic lines 76 III (p) and 76 V (o), carrying an antisense construct of PttMYB76 gene; and (B) hybrid aspen (u) and its transgenic lines 46 III (p) and 46 IV (|), carrying an antisense construct of PttMYB46 gene. Shown are the means of two different experiments using at least three different leaves from two individuals for every line in every experiment. Vertical bars represent standard error of the mean. the same time as on WT leaves (i.e. 5 days Conclusions after the inoculation) it spread faster than on WT leaves (Fig. 13.2B). The results suggest The phenylpropanoid pathway produces a that in our study system the analysed variety of phenolic secondary metabolites, phenolic compounds did not participate which may function as signal compounds in the defence against the pathogen and defensive chemicals in plant–pathogen infection, or the observed changes in the interactions. Members of the MYB family of phenolic pool did not compromise the plant transcription factors have been recognized response to the pathogen. It is also possible as regulators of different branches of that the pathogen was able to use the higher phenylpropanoid metabolism. Recently, amount of phenolic compounds as a carbon two MYB genes, PttMYB46 and PttMYB76, source. were isolated and cloned from hybrid At present, we are testing the sensitivity aspen. The morphological and anatomical of the transgenic plants against infections by deviations of transgenic plants from the other fungal pathogens that use different wild-type plants suggested underlying strategies for infection than the studied chemical differences, which could be ascomycete fungus. Among these are leaf linked to phenylpropanoid metabolism. We rusts (Melampsora sp., Basidiomycotina) studied the levels of methanol-soluble phe- and stem-infecting Neofabraea populi nolic compounds in four transgenic hybrid (Ascomycotina). Detailed studies on the aspen lines, each carrying an antisense con- resistance of the hybrid aspen plants struct of one of these genes. Marked differ- carrying antisense constructs of MYB ences in the levels of individual phenolic genes against pathogens and other natural compounds were found between wild-type enemies, such as insect herbivores, should and transgenic lines. In general, the level of provide us with novel insights into the phenolic acids was raised and the level molecular and chemical basis of resistance of salicylates (salicin and its derivatives, in plants. The amenability of hybrid aspen salicortin and tremulacin) was reduced to modern molecular techniques (e.g. in the transgenic plants. These transgenic microarrays) and the increasing knowl- lines may provide a novel model system for edge of its genetics open completely investigations of the role of phenolics in new possibilities for this type of interactions between poplars and their investigation. natural enemies, such as Melampsora rusts. Transgenic Hybrid Aspen with Altered Defensive Chemistry 159

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Alessandro Ragazzi1, Nicola Longo2, Biancamaria Naldini2, Salvatore Moricca2 and Irene Dellavalle3 1Dipartimento di Biotecnologie Agrarie, Sezione di Patologia Vegetale, Universita di Firenze, Piazzale delle Cascine 28, 50144 Frienze, Italy; 2Dipartimento di Biologia Vegetale, Universita di Firenze, Via la Pira 4, 50121 Firenze, Italy; 3CNR, Istituto per la Protezione delle Piante, Area della Ricerca del CNR di Firenze, Via Madonna del Piano, 50019, Sesto Fiorentino, Italy

Background 1996; Epstein and Nicholson, 1997; Mendgen, 1997). Penetration into host tissue is the very first Patton and Johnson (1970) reported that step of infection by fungal pathogens. penetration on the needles of Pinus strobus Research conducted so far with rust fungi L. by the basidiospores of Cronartium has shown that there are essentially two ribicola J.C. Fisher ex Rabenh. was initiated types of penetration: (i) direct penetration, indirectly through the stomatal openings when the rust germ tube invades directly and followed by the formation of a sub- through an epidermal cell wall; and (ii) stomatal vesicle. However, later studies indirect penetration, when it first enters a with many rust fungi confirmed the direct stoma and then invades the cell (Fig. 14.1). penetration by the basidiospores (Miller The most generally accepted theory et al., 1980; Gray et al., 1983; Gold and regarding penetration is that direct penetra- Mendgen, 1984, 1991; Hopkin et al., 1988; tion is associated with the germ tubes of Longo et al., 1988, 1991, 1994, 1997; Morin basidiospores (monokaryotic stage), while et al., 1992) and the researchers considered indirect penetration is associated with the the indirect penetration of P. strobus by germ tubes of aeciospores and uredinio- C. ribicola (Patton and Johnson, 1970) and spores (dikaryotic stage). With direct of Pinus banksiana Lamb. by Cronartium penetration the (basidiospore) germ tube comandrae Pk. (Bergdhal and French, 1985) develops an intra-epidermal infection as anomalies to be explained. structure (vesicle and primary infection Gold and Mendgen (1984) reported two hypha), while with indirect penetration the other cases of indirect penetration from (aeciospore/urediniospore) germ tube forms basidiospores: Crysomyxa abietis on Picea a substomatal infection structure (Littlefield abies (Grill et al., 1978) and Coleosporium and Heath, 1979; Bushnell and Roelfs, 1984; spp. on Pinus spp. (Bauer, 1983). They Hoch and Staples, 1991; Mendgen et al., considered that direct penetration from

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 161 162 A. Ragazzi et al.

Fig. 14.1. Diagrammatic representation of the monokaryotic (A) and dikaryotic (B) infection process of a rust fungus. (A) Direct penetration; (B) indirect penetration. (Modified from Mendgen, 1997.) basidiospores was typical of rusts on angio- results were obtained with Cronartium sperms, while indirect penetration from quercuum (Berk.) Miyabe ex Shirai f. sp. basidiospores was typical on gymnosperms. fusiforme (Cumm.) Burds. and Snow on Penetration of gymnosperm leaves was indi- Pinus elliottii Engelm. var. elliottii (Miller rect because of the high level of cutinization et al., 1980) and with Endocronartium and the thick epidermal cell wall. On the harknessii (J.P. Moore) Y. Hiratsuka on other hand, Longo et al. (1988, 1991, 1997) Pinus contorta Dougl. var. latifolia Engelm. found that basidiospore germ tubes of (Hopkin et al., 1988). Melampsora pinitorqua (Br.) Rostr. and M. From these findings it can be concluded larici-tremulae Kleb. penetrate into Pinus that the same organ of a gymnosperm can be spp. and Larix decidua Mill. directly, penetrated either directly or indirectly by regardless of any organ they infect. Similar a basidiospore germ tube. The manner of Basidiospore-derived Penetration by Cronartium and Melampsora 163

penetration depends on the rust species and size observed, only a few entered stomata. In probably on the tree organ each rust usually some cases a germ tube appeared initially to infects in nature. Thus M. pinitorqua, C. lodge in a stomatal antechamber, but then quercuum f. sp. fusiforme and E. harknessii grew out of it again, continuing its growth penetrate directly into growing shoots, without penetrating the stomatal opening. where there are few or no stomata, while C. Several germ tubes may enter the same ribicola and C. comandrae penetrate indi- chamber, but usually only one continues to rectly into the needles, which have many grow, remaining more or less attached to the stomata. walls of the subsidiary cells that are located above the guard cells, until it crosses the stomatal opening (Fig. 14.5). C. flaccidum penetrates into the Mode of Penetration of Cronartium spp. stomatal opening by the apex of the germ tube, without forming appressoria. At Cronartium flaccidum (Alb. et Schw.) Wint. the stomatal opening, germ tube diameter is a macrocyclic, heteroecious rust that decreases by about half; it becomes flattened forms spermogonia and aecia on some and ribbon-like, and takes up only a small, species of pine, and uredia and telia on and sometimes peripheral, part of the open- some angiosperms such as Vincetoxicum ing (Figs 14.5 and 14.6). Within the stoma hirundinaria Medicus, Peonia spp. and the germ tube is more flattened when closer Gentiana spp. Basidiospores of C. flac- to the side of the stomatal opening, less so cidum infect the primary and secondary when near centre. Preferential points of needles of pine (Wilson and Henderson, germ tube passage through the stomatal 1966). Its haploid mycelium, as it colonizes opening were not noted. The ultrastructure the mesophyll, forms stomata in the tissue of the germ tube at the stomatal opening it invades, giving rise to yellowish spots is typical of a vegetative hypha, with its that are the first symptom of rust infection cytoplasm rich in organelles and granules of (Raddi, 1976; Ragazzi and Moriondo, 1979, glycogen. Once a germ tube passes through 1980). the stomatal opening and enters the Ragazzi et al. (1987) and Ragazzi and substomatal chamber, the hypha derived Dellavalle Fedi (1992) examined the germ directly from this germ tube becomes tubes of C. flaccidum on the needles of Pinus an infection structure. The term ‘infection pinaster Ait. and Pinus nigra Arn. subsp. structure’ is used to refer to the hypha that laricio (Poiret) and concluded that, like forms inside the substomatal chamber before C. ribicola (Patton and Johnson, 1970), its it initiates intracellular colonization (Longo basidiospore germ tubes entered indirectly et al., 2000) (Figs 14.5 and 14.6). through the stoma, and not directly through The lack of an appressorium-like struc- the epidermal cell wall. However, these ture had already been noted in C. flaccidum authors also stressed that the behaviour by Ragazzi and Dellavalle Fedi (1992), in of the germ tube after it had invaded the C. ribicola by Patton and Johnson (1970) and substomatal antechamber was still unclear in C. comandrae by Bergdahl and French (Fig. 14.2). (1985). The mode of penetration of the three Longo et al. (2000) examined the pene- species is similar at their monokaryotic tration and early colonization of P. pinea stage. With C. flaccidum the germ tube needles by C. flaccidum and found that, on narrows at the point where it passes through the needle surface, the germ tubes were gen- the stomatal opening and remains distinctly erally single, not branched, fairly uniform in flattened thereafter. Similar observations diameter, and grew in length without direct- were made with C. ribicola (Patton and ing themselves towards a stoma (Figs 14.3, Johnson, 1970) and with C. comandrae 14.4). Penetration into a stomatal ante- (Bergdahl and French, 1985). chamber was therefore a random event, and In E. harknessii on P. contorta (Hopkin indeed, of the many germ tubes of varying et al., 1988) and C. quercuum f. sp. fusiforme 164 A. Ragazzi et al.

on P. elliottii var. elliottii (Miller et al., anticlinal wall of one of the subsidiary cells 1980), basidiospore germ tubes in some surrounding the stoma. There it formed an cases invaded the stomatal antechamber intracellular structure like that produced by apparently in a manner of indirect penetra- direct penetration through the epidermal tion, but then immediately penetrated the cell wall.

Figs 14.2–6. Basidiospore-derived Penetration by Cronartium and Melampsora 165

The appressorium of the Uredinales French, 1985). In contrast, with E. harknessii (Littlefield and Heath, 1979; Harder and (Hopkin et al., 1988) or C. quercuum f. sp. Chong, 1984; Hoch and Staples, 1991) is fusiforme (Miller et al., 1980), penetration in highly differentiated at the dikaryotic infec- some cases is initiated indirectly into the tion stage and is separated from the germ stomatal antechamber, but then it becomes tube by a septum. At the monokaryotic stage, direct, through the anticlinal walls of the the appressorium is generally present but subsidiary cells of the stoma. not well differentiated, being merely a swelling of the apex of the germ tube and not normally separated from it by a septum (Miller et al., 1980; Gray et al., 1983; Gold Mode of Penetration of Melampsora spp. and Mendgen, 1984; Hopkin et al., 1988; Longo et al., 1991, 1994; Morin et al., 1992). Melampsora pulcherrima (Bub.) Maire, the At the dikaryotic stage the penetration causal agent of Mediterranean white poplar peg is described as a thin protuberance (Populus alba L.) rust, forms its spermo- growing from the innermost layer of the gonia and aecia on Mercurialis annua L. appressorium wall, which advances, (Magnani, 1961; Moriondo et al., 1989). growing apically into the stomatal opening Naldini et al. (1993) showed that hap- (Littlefield and Heath, 1979). loid infection M. pulcherrima begins on the The penetration peg of the mono- host leaves and only thereafter spreads to karyotic stage also grows directly out of the the stem and lateral branches. Its infection appressorium but it does not differentiate process has been described by Longo et al. like that of the dikaryotic stage (Gold and (1994). The germ tubes generally measure Mendgen, 1984; Mendgen, 1997). The apex 5–6 mm in length, but may go up to 20 mmin which penetrates into the cuticle of the some cases, making them long enough to epidermal cell is very thin. It enlarges in the reach any point of the periclinal wall of an cell wall and, as it grows into the host cell, is epidermal cell, or the anticlinal walls of two sheathed by the cell’s plasma membrane. contiguous cells. However, the penetration As far as the basidiospore-derived pre- is always directly through the wall of an epi- penetration is concerned, C. flaccidum dif- dermal cell. Direct penetration also occurs fers from most other observed rusts in that its through the walls of the stomatal subsidiary penetration is always indirect (Longo et al., cells, and sometimes through the wall of the 2000). The same type of penetration is also guard cells (Fig. 14.7). The fully developed, seen with C. ribicola (Patton and Johnson, intracellular infection structure is a more or 1970) and with C. comandrae (Bergdahl and less round vesicle which is attached to the

Fig. 14.2. Scanning electron micrograph of Cronartium flaccidum on a needle of Pinus pinaster. The germ tube (GT) of a basidiospore (B) is going into the stomatal antechamber (SA). ×1000. (Ragazzi and Dellavalle Fedi, 1992). Fig. 14.3. Epifluorescence optics of Cronartium flaccidum on a cotyledon of Pinus pinea. The basidiospore germ tubes (GT) show a random behaviour without moving directly towards the stomata (S). B, Basidiospore; VB, vascular bundle. ×220. (Longo et al., 2000.) Fig. 14.4. Epifluorescence optics of Cronartium flaccidum on a secondary needle of Pinus pinea. The basidiospore germ tubes (GT) show a random behaviour without moving directly towards the stomata (S). ×180. (Longo et al., 2000.) Fig. 14.5. Light microscopy (thin transverse section) of Cronartium flaccidum on a cotyledon of Pinus pinea. Stomatal indirect penetration carried out by a basidiospore germ tube (GT) without appressorium formation. SbC, subsidiary cell; IS, infection structure; GC, guard cell. ×920. (Longo et al., 2000.) Fig. 14.6. Transmission electron micrograph of Cronartium flaccidum on a cotyledon of Pinus pinea: the same penetration as Fig. 14.5. Below the stromatal aperture, in the substomatal chamber, the hypha from the germ tube (GT) becomes the ‘substomatal infection structure’ (IS), which appears not to be diametrically sectioned. GC, Guard cell; L, lip. ×6800. (Longo et al., 2000.) 166 A. Ragazzi et al.

epidermal cell wall by a narrow neck and, at tube and the appressorium adhere to the distal end, by a hypha-like structure the epidermal cell wall by means of (infection hypha) consisting of two parts, extracellular material. one wide, and one narrow and long. These 2. Penetration is direct: a thin penetration parts are divided by septa. The infection peg comes out of the appressorium and structure enlarges within the penetrated epi- grows through the epidermal cell wall, pro- dermal cell. In many cases it runs along the ducing an intracellular infection structure cell wall and then turns back to its penetra- consisting of a vesicle and a septate hypha. tion point (Figs 14.7 and 14.8). The fully The branches of this septate hypha grow out developed infection structure generally has of the epidermal cells and produce both some branches that develop in succession intercellular hyphae in the mesophyll and, from the distal end of the vesicle, near the directly, hypha-like haustoria in the contig- septum, then from its wider part, and finally uous epidermal cells and the underlying from its terminal, narrower end. These mesophyll. branches extend towards the anticlinal wall 3. A matrix can be found between the of the cell, growing into contiguous epider- plasma membrane of the host cell ensheath- mal cells, or towards the lower periclinal ing the invading fungus and the fungal wall. wall of the epidermal cell that was invaded, The plasma membrane encloses all the intra- from which they grow out. The intracellular cellular fungal structures (infection hypha, branches that form terminal structures branches of the infection hypha, haustoria) (haustoria) in contiguous epidermal cells are except the intra-epidermal vesicle. more numerous in infections on the lower- leaf epidermis. These intracellular branches Collars with a host cell wall-like struc- become infection hyphae that grow out ture encircle the neck of the intra-epidermal through the lower periclinal walls of the vesicle in the penetration pore, and the epidermal cells, and form intercellular intracellularly growing rust structures hyphae in the spongy parenchyma. (Longo et al., 1994). These hyphae produce haustoria in the The formation of appressoria and the spongy parenchyma cells. The intracellular direct germ tube penetration at any point branches in the contiguous epidermal cells in the epidermal cell wall, including the are less numerous in infections on the stomatal subsidiary and guard cells, do upper-leaf epidermis, where numerous not occur frequently. Many researchers intracellular branches appear to grow out described that penetration through the epi- of the epidermal cells and form haustoria dermis is more likely to occur near the place in the palisade cells. The still young where the cell walls come together, towards haustoria produced in the epidermal cells which the germ tube directs itself in growth or in the cells of the spongy and palisade (Gold and Mendgen, 1984; Hopkin et al., parenchyma, although partially developed 1988; Morin et al., 1992). into the host cells, are similar in shape Differences in the behaviour of germ to infection hyphae, with a larger proximal tubes between different species can be part, and a distinct and thinner distal part. ascribed to characteristics of the epidermal These intracellular structures have not been tissue of the host. Even in direct penetration seen to grow out of the host cells. from basidiospores (monokaryotic), there Some of the most important features are some plant–fungus interactions based relating to the basidiospore germ tube on contact stimuli and chemical signals penetration and the infection structures of (Desprez-Loustau and Le Menn, 1989; Gold M. pulcherrima on leaves of M. annua are as and Mendgen, 1991), which are generally follows: typical of indirect penetration by dikaryotic urediniospores into the stomata (Hoch and 1. Germ tubes are short and random- Staples, 1991). growing, with a swollen apex that is an The basidiospore appressorium which undifferentiated appressorium. The germ is not separated from the germ tube by septa, Basidiospore-derived Penetration by Cronartium and Melampsora 167

appearing as a slightly enlarged apex of the hypothesized that speciation is taking place germ tube, as in M. pulcherrima, is com- in the M. populnea complex, and that monly described for basidiospore germlings the above-mentioned entities are evolving (Gold and Mendgen, 1984; Mims and towards two new species, reproductively Richardson, 1989; Morin et al., 1992). This isolated from their sympatric progenitor type of structure is very different from (Longo et al., 1997, 2002). the differentiated appressorium produced The basidiospore germ tubes of these during urediniospore germination which two rusts on their respective aecial hosts gives rise to indirect penetration (Staples (M. pinitorqua on Pinus spp. and M. larici- and Macho, 1984). tremulae on Larix decidua) generally In M. pulcherrima, the extracellular measure 5–10 mm, rarely more. Germ tube material can be seen around the appres- growth on the host proceeds in a random sorium and around most of the basidiospore manner. The apex of the basidiospore germ germ tube (Fig. 14.8). This is thought to be an tube forms an appressorium that appears exudate that characteristically accompanies to be the undifferentiated, slightly enlarged basidiospore germination (Gold and apex of the germ tube itself (Fig. 14.9). When Mendgen, 1991) and is generally associated P. sylvestris is inoculated with M. larici- with the fungal structures involved in direct tremulae, in contrast, most basidiospore penetration (Nicholson and Epstein, 1991). germ tubes are longer (50 mm and more), It not only protects the appressorium twisted, with irregular swellings and but also serves as a reserve for enzymatic branchings, and without apparent appres- penetration (Gold and Mendgen, 1984). soria. In this particular host–parasite inter- Extracellular material probably plays the action, secondary basidiospores are also same enzymatic role in the case of M. frequently observed (Longo et al., 1997). pulcherrima, the penetration peg of which The morphology of the direct type of produces a pore with a sharply defined edge, penetration and of the intra-epidermal infec- which might be the result of enzyme activity tion structures of both rusts on their respec- (Longo et al., 1994). tive hosts and of M. larici-tremulae on P. In other rusts, the basidiospore pene- sylvestris is similar. In the host epidermis, tration peg is very thin between the epi- the penetration peg derived from the appres- cuticular wax and the cuticle, and generally sorium is narrow at the point where it pene- enlarges where it passes through the cell trates the cuticle, and more expanded in the wall (Metzler, 1982; Gray et al., 1983; Gold cell wall. The intracellular infection struc- and Mendgen, 1984; Longo et al., 1991), ture derived from the penetrating peg looks while in M. pulcherrima it is of relatively somewhat like an ovoid, vacuolated vesicle, uniform diameter (Fig. 14.8). linked to the epidermal cell wall by a narrow In conclusion, ultrastructural studies neck, while its distal part becomes elon- (Longo et al., 1994) of M. pulcherrima on the gated, forming the infection hypha (Figs leaves of M. annua confirmed the behaviour 14.10–14.12). In the epidermal cell this and type of direct penetration typical of infection hypha is generally straight. Its ram- basidiospore germ tubes. ifications, branching off in succession from Melampsora pinitorqua Rostr. is the the proximal part towards the distal part, causal agent of twist rust of pine shoots, and grow intercellularly into the first layers of M. larici-tremulae Kleb. is the agent of rust the underlying parenchyma, where they of larch needles. They are morphologically form both intercellular and intracellular very similar. They both have uredial and hyphae (Fig. 14.10). According to Longo telial stages on the same range of poplar et al. (1988, 1991) the intracellular hyphae species, P. tremula L., P. alba L. and and the haustoria are characteristic of P. canescens Sm. and are considered to the first and last stages of rust mycelium be formae speciales of a single species, development in the host cell, respectively. M. populnea (Pers.) Karst. (Boerema and The general morphology of the first Verhoeven, 1972). More recently, it has been infection stages of the two rust agents in 168 A. Ragazzi et al.

Figs 14.7–12. Basidiospore-derived Penetration by Cronartium and Melampsora 169

their respective hosts is typical of a rust The stages of the dikaryotic infection infection from basidiospores (Gold and process are mediated by a series of physical Mendgen, 1991), as has already been and chemical stimuli that are released by reported for M. pinitorqua by Longo et al. the host surfaces (Hoch and Staples, 1991; (1991) and, more recently, by Morin et al. Mendgen et al., 1996; Epstein and Nichol- (1992) and Longo et al. (1994). son, 1997), particularly by the stomata (Terhune et al., 1991). These stimuli, which condition the behaviour of the fungal struc- Concluding Remarks tures on the host surface, are more effective when the fungus is more strongly adhered to host surface. Adhesion is secured by muci- In this chapter, we have reviewed the laginous exudates produced by the fungus literature on direct and indirect penetration and containing numerous lytic enzymes, at the monokaryotic and the dikaryotic especially cutinases, important factors in stage of the Uredinales. It reveals that ensuring the adhesion of the rust structures the penetrating processes and the and causing them to differentiate after adhe- structural elements of these two types sion (Mendgen et al., 1996; Howard, 1997; are fundamentally different. Mendgen, 1997). More specifically, accord- ing to Mendgen et al. (1996) and Epstein and Nicholson (1997), each enzyme changes the Penetration at the dikaryotic stage host cuticle from hydrophobic to hydro- philic, like the fungal surface itself, and in Indirect penetration of the host by the this way promotes adhesion. The hydropho- dikaryotic stage of the fungus has the bic nature of the host surface therefore seems following characteristics: the germ tubes to condition the entire pre-penetration pro- direct themselves purposely towards the cess by the fungus. In indirect penetration, stomata; the appressorium is separated the adhesion of the appressorium is very from the germ tube by a septum; and the important since it must remain correctly stomatal opening is entered by a penetra- placed on the guard cells of the stoma tion peg (Longo et al., 2000) (Fig. 14.1B). (Epstein and Nicholson, 1997).

Fig. 14.7. Melampsora pulcherrima on a leaf of Mercurialis annua. Intracellular infection structures from direct penetrations of basidiospore (B) germlings into cells of lower epidermis. The roundish naked vesicles (V) and developing infection hyphae (IH) are visible. ×310. (Longo et al., 1994.) Fig. 14.8. Transmission electron micrograph of Melampsora pulcherrima on a leaf Mercurialis annua. Penetration peg (P) through the epidermal cell wall and the naked vesicle (V) of an intracellular infection structure. The appressorium (A) is visible on the epidermal cell wall (ECW). E, Exudate; N, vesicle neck. ×8900. (Longo et al., 1994.) Fig. 14.9. Scanning electron micrograph of Melampsora pinitorqua on a primary needle of Pinus pinea. Germinated basidiospores (B) with short germ tubes (GT): a basidiospore germ tube shows the appressorium (A) at the penetration point on the epidermal cell wall (ECW). ×1000. (Longo et al., 1991.) Fig. 14.10. Transmission electron micrograph of Melampsora pinitorqua on a shoot of Pinus sylvestris. Two infection structures in two epidermal cells (EC). Penetration point (PP) through the epidermal cell wall and naked vesicle (V) (left structure); a vacuolated vesicle (V) and the transcellularly growing infection hypha (IH) from epidermal into underlying parenchyma cell (PC) (right structure). N, vesicle neck. ×2800. (Longo et al., 1997.) Fig. 14.11. Transmission electron micrograph of Melampsora larici-tremulae on a needle of Larix decidua. Penetration point (PP) through the epidermal cell wall and the naked vesicle (V) of an infection structure. EC, Epidermal cell; N, vesicle neck. ×4000. (Longo et al., 1997.) Fig. 14.12. Transmission electron micrograph of Melampsora larici-tremulae on a shoot of Pinus sylvestris. Penetration point (PP) through the epidermal cell wall and the naked vesicle (V) of an infection structure. The ‘cell wall-like apposition’ (WA) in the epidermal cell (EC) completely encases the infection structure. ×3350. (Longo et al., 1997.) 170 A. Ragazzi et al.

The characteristics of the dikaryotic mind that in direct penetration the turgor stage after penetration into the stoma are: pressure inside the appressorium plays a the substomatal vesicle is separated from the crucial role, and mechanical action com- penetration peg by a septum; and the infec- bined with enzymatic action then renders tion hypha has a distal septum that separates the cell wall penetrable (Mendgen and it from the haustorial mother cell, which Deising, 1993; Howard, 1997). is terminal and gives rise to the first The characteristics of the monokaryotic haustorium (Longo et al., 2000) (Fig. 14.1B). stage after direct penetration are: (i) an In the substomatal chamber all the sig- intra-epidermal vesicle that is not separated nals that regulate the interaction between from the penetration peg by a septum, but is fungus and host are present along the entire separated by a septum from the infection structure of the rust fungus, from the vesicle hypha; and (ii) an infection hypha which to the first haustorium which, in the dikary- forms intracellular hyphae and haustoria, otic stage, is the first intracellular structure and also intercellular hyphae that then give (Mendgen et al., 1988; Heath, 1989, 1995). rise to haustoria in the parenchyma cells (Longo et al., 2000) (Fig. 14.1A). Signals that regulate the host–parasite interaction after penetration can be found at Penetration at the monokaryotic stage the monokaryotic stage too, but unlike the dikaryotic stage, all such signals occur at Direct penetration of the host surface by the epidermal cells, where penetration takes the monokaryotic stage of the fungus place (Heath, 1989, 1995). has the following characteristics: the germ The infection process at the dikaryotic tubes grow indiscriminately, moving in stage of the Uredinales produces a series of an undirected manner over the leaves; the distinct and well-differentiated structures appressorium is poorly differentiated; the from both a morphological and functional penetration peg penetrates the epidermal point of view, and each of these structures cell wall (Longo et al., 2000) (Fig. 14.1A). reacts in a specific way with its host. This The signals between the host surface indicates that infection at the dikaryotic and the fungus during pre-penetration at the stage of the fungus is evolutionarily a monokaryotic stage are not as well known more complex process than infection as at the dikaryotic stage (Mendgen, 1997). at the monokaryotic stage (Gold and Some authors have postulated the occur- Mendgen, 1991; Mendgen and Deising, rence at the monokaryotic stage of some 1993; Heath, 1995; Mendgen et al., 1996; signals that condition the structures formed Mendgen, 1997). by the rust (Desprez-Lousteau and Le Menn, On the whole, the first stages of 1989; Gold and Mendgen, 1991; Longo et al., the monokaryotic infection process of C. 1994). It has been observed that the germ flaccidum investigated by Longo et al. (2000) tubes of some rusts show preferential growth (Fig. 14.13) bear a resemblance to the lines along the anticlinal walls of the epider- infection process of rusts at the dikaryotic mal cells (Gold and Mendgen, 1984; Hopkin stage. C. flaccidum also penetrates its host et al., 1988; Morin et al., 1992). On the other indirectly and forms a substomatal infection hand, a close adhesion of the fungus to the structure and a first haustorium in a paren- host surface is thought to be important, espe- chyma cell. However, this resemblance is a cially between appressorium and the host matter of behaviour only, since the morpho- epidermis (Epstein and Nicholson, 1997). logical and functional characteristics that According to Gold and Mendgen (1991), emerge with this type of infection process the mucilaginous matrix commonly present are essentially those of the monokaryotic around the external structures of the rust is stage. And this is particularly true as the not only useful to favour rust adhesion, but C. flaccidum germ tubes on the host surface above all serves as an enzyme reservoir do not grow in a specific direction; it is only to assist penetration. It should be borne in by chance that they grow into a stomatal Basidiospore-derived Penetration by Cronartium and Melampsora 171

Fig. 14.13. Diagrammatic representation of the monokaryotic infection process of Cronartium flaccidum. Indirect penetration (Longo et al., 2000). opening. When they do so, they do not dif- indicating its haustorial function (Longo ferentiate an appressorium or a penetration et al., 2000). peg; the infection structures in the sub- The penetration process of C. flaccidum stomatal chamber have only one septum at the monokaryotic stage differs from both between the vesicle and the infection hypha. the monokaryotic penetration of most other All haustoria, including those in the subsid- rust fungi and from the penetration of iary cells of the stoma, are typical mono- a dikaryotic stage. It is initiated by the karyotic structures, especially the first, germ tube without the formation of an which, on account of its particular shape, appressorium, and hence without a true acts even more like a hypha. Moreover, the penetration peg. Since the purpose of distinctive features of the first haustorium the appressorium is to cause the fungus to are also seen in the monokaryotic intra- adhere to the host, the lack of this special- cellular structures of M. pinitorqua (Longo ized structure has a biological significance et al., 1988, 1991) which, deriving directly that becomes clear considering that C. flacci- from the infection hypha in the epidermal dum does not penetrate the intact epidermis cell, colonize both the epidermal cells and of the host directly (as is characteristic of the contiguous parenchyma cells. The first monokaryotic penetration), nor indirectly haustoria of C. flaccidum can therefore be through a stoma (typical dikaryotic penetra- considered an extension of the substomatal tion) with a germ tube that grows purposely infection hypha, the behaviour of which towards a stoma (Longo et al., 2000). they maintain until they become covered It is interesting to consider the similar with a haustorial membrane and a matrix behaviour of C. flaccidum and rusts at the and assume the new function of exchanging dikaryotic stage in regard to the growth substances between the host and the fungus. towards a cutinized surface. Indeed, for the The same thing happens with the intra- dikaryotic penetration process, the hydro- cellular infection hypha, which in the case phobic nature of the cutinized host-cell of direct, monokaryotic penetration derives walls, which extend from the outer surface from an intra-epidermal vesicle: this hypha, of the epidermis to the substomatal chamber unlike the vesicle, is covered with a matrix, due to the continuity of the cuticle, seems to 172 A. Ragazzi et al.

stimulate the penetration process (Mendgen infection process; this holds true even et al., 1996; Epstein and Nicholson, 1997) though the cytological and histological and the formation of intercellular infection characteristics of the host organ, which this structures in the substomatal chamber rust fungus is adapted to infect in nature, (Terhune et al., 1991). As for the intra- condition its mode of penetration. cellular colonization, the hydrophobic nature of the cutinized inner tangential wall of the epidermal cells seems to prevent the Acknowledgements differentiation of the haustorial mother cells and hence the formation of haustoria, as We are grateful to Gabriele Tani, Department demonstrated in Uromyces appendiculatus of Plant Biology, University of Florence, for on Phaseolus vulgaris by Terhune et al. skilful assistance with the preparation of (1991). photographic plates. Appreciation is also In regard to C. flaccidum at the mono- expressed to Mrs E. Bruno, Department karyotic stage, it is not known what signals of Agricultural Biotechnology, University of are exchanged between the host cell walls Florence, for technical assistance with the and the fungal pre-penetration and infection scanning electron microscopy. structures, nor what is the role of the enzymes in the exchange of these signals. It has, however, been noted that C. flaccidum at this stage, like the rusts at the dikaryotic References stage, does not enter through the epidermal cell walls, but produces its first intracellular Bauer, R. (1983) Experimentell-ontogenetische und structures in the parenchyma (Longo et al., karyologische Untersuchungen an Uredinales. 2000). It does not produce any such struc- Doctoral dissertation, Universität Tübingen, tures in the epidermal cells. Cutin is scarce Tübingen, Germany. Bergdahl, D.R. and French, D.W. (1985) Penetration or absent in parenchyma cell walls and the of the primary tissues of Pinus banksiana by epidermal cells are impregnated with cutin. Cronartium comandrae. In: Barrows-Broaddus, As a final comment, it can be said that J. and Powers, H.R. Jr (eds) Proceedings of the basidiospore germ tubes of C. flaccidum the IUFRO ‘Rusts of hard Pines’ Conference, in nature infect organs such as the secondary October 1984, University of Georgia, Athens, needles of Pinus spp., that are strongly pp. 179–192. cutinized but also densely covered with Boerema, G.H. and Verhoeven, A.A. (1972) Check- stomata. Since these germ tubes are unable list for scientific names of common parasitic to exert a mechanical action (which, in fungi. Series la: fungi on trees and shrubs. monokaryotic penetration, is always com- Netherland Journal of Plant Pathology 78 (suppl. 1). bined with enzymatic activity), in order to Bushnell, W.R. and Roelfs, A.P. (eds) (1984) succeed in colonizing the secondary needles The Cereal Rusts, Vol. I. Origins, Specificity, they changed their mode of penetration from Structure and Physiology. Academic Press, direct, characteristic of the Uredinales at Orlando, Florida. their monokaryotic stage, to indirect. The Desprez-Loustau, M.L. and Le Menn, R. (1989) penetration structures of this rust at Epicuticular waxes and Melampsora pinitorqua the monokaryotic stage nevertheless still Rostr. pre-infection behaviour on maritime retain the morphological and functional pine shoots. A scanning electron microscope characteristics of the typical monokaryotic study. European Journal of Forest Pathology 19, structures, even if their behaviour is remi- 178–188. Epstein, L. and Nicholson, R. (1997) Adhesion of niscent of dikaryotic structures (Longo et al., spores and hyphae to plant surfaces. In: Carroll, 2000). G.C. and Tudzynski, P. (eds) The Mycota, Vol. V It is concluded that with C. flaccidum (part A). Springer, Berlin, pp. 11–25. at the monokaryotic stage it is the nuclear Gold, R.E. and Mendgen, K. (1984) Cytology of set that determines the morphology and basidiospore germination, penetration and early functions of the structures involved in the colonization of Phaseolus vulgaris by Uromyces Basidiospore-derived Penetration by Cronartium and Melampsora 173

appendiculatus var. appendiculatus. Canadian Forestry Centre, Forestry Canada, Edmonton, Journal of Botany 62, 1989–2002. Canada, pp. 120–127. Gold, R.E. and Mendgen, K. (1991) Rust basidiospore Longo, N., Naldini, B., Drovandi, F., Gonnelli, T. germlings and disease initiation. In: Cole, G.T. and Tani, G. (1994) Penetration and early and Hoch, H.C. (eds) The Fungal Spore and colonization in basidiospore-derived infection Disease Initiation in Plants and Animals. Plenum of Melampsora pulcherrima (Bub.) Maire on Press, New York, pp. 67–99. Mercurialis annua L. Caryologia 47, 207–222. Gray, D.J., Amerson, H.V. and Van Dyke, C.G. (1983) Longo, N., Naldini, B., Paolillo, A., Drovandi, F., Tani, Ultrastructure of the infection and early coloni- G. and Gonnelli, T. (1997) Morphological zation of Pinus taeda by Cronartium quercuum f. aspects of early host–parasite interactions in sp. fusiforme. 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Chris C. Mundt Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, OR 97331-2902, USA

Introduction on the potential to use mixtures for control of Melampsora rust of willow. The simultaneous culture of plant geno- types differing in their reactions to pathogen genotypes is one of several (Mundt, 1994b) strategies for disease Specialized Foliar Pathogens of control and management of host plant Small Grains resistance. Multiline cultivars (mixtures of lines bred for phenotypic uniformity The majority of studies regarding disease of agronomic traits) and cultivar mixtures control through use of host mixtures have (mixtures of agronomically compatible, been with rusts (Puccinia spp.) and pow- cultivated varieties, with no additional dery mildews (Blumeria graminis f. spp.) breeding for phenotypic uniformity) are of small grains, or with rice (Oryza sativa) being used increasingly in commercial blast, caused by Magnaporthe grisea. Sev- production, and have been the subject of eral mechanisms have been postulated to recent reviews (Garrett and Mundt, 1999; explain the reduction in severity of such Finckh et al., 2000; Mundt, 2002). It has diseases caused by mixtures (Browning and been emphasized that randomly chosen Frey, 1969; Chin and Wolfe, 1984b; Mundt mixture components will not necessarily and Browning, 1985b; Garrett and Mundt, provide adequate disease control. Instead, 1999; Koizumi, 2001). Inoculum dilution mixtures must be relevant, or functional, caused by the increased distance between against the specific pathogen population plants of the same genotype is often the in question (Schmidt, 1978; Mundt and most important mechanism (Burdon and Browning, 1985b; Wolfe, 1985). Effects of Chilvers, 1977; Chin and Wolfe, 1984b; mixtures on disease levels as compared to Wolfe, 1985). However, other mechanisms their component pure stands can range can be important as well. For example, from a disease increase to nearly complete induced resistance is predicted to be impor- disease control (Smithson and Lenne, tant by simulation models (Lannou et al., 1996). This review will discuss factors 1995) and accounted for about 30% of the impacting the level of disease control in total reduction of wheat (Triticum aesivum) mixtures, the effects of host diversity on yellow rust (caused by Puccinia striiformis) pathogen evolution, and comment briefly (Calonnec et al., 1996). In another wheat

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 175 176 C.C. Mundt

yellow rust study, cultivars resistant to all 1979; Elliot et al., 1980; Koizumi and Kato, inoculated races compensated for suscepti- 1987). Similarly, field evidence suggests ble cultivars through increased tillering, that disease levels may decline when an which sometimes substantially increased increasing number of host genotypes is the proportion of healthy host tissue in added to equiproportional mixtures mixtures (Finckh and Mundt, 1992a,b). (Mundt, 1994a; Newton et al., 1997). Approximately 25% of the total powdery From a disease control standpoint, mildew reduction observed in a barley one would expect diminishing returns for (Hordeum vulgare) cultivar mixture was decreasing the frequency of a host genotype attributed to disruptive selection caused in a mixture or to increasing the number of by quantitative adaptation of the pathogen host genotypes in a mixture (Leonard, 1969; to the genetic background of the different Mundt, 1990). On the other hand, incorpo- cultivars in the mixture (Wolfe et al., 1981). rating a larger number of genotypes in a A highly successful mixture approach for mixture could be useful in countering patho- control of rice blast involves row mixtures gen evolution towards complex virulence of a tall, susceptible cultivar with a shorter, (Marshall, 1989). In practical applications of more resistant one (Zhu et al., 2000). Height mixtures, considerations such as agronomic differences between cultivars resulted in performance of potential mixture compo- reduced leaf wetness, which may have nents, agronomic compatibility among mix- reduced pathogen infection (Y. Zhu, ture components, and characteristics of the Yunnan Agricultural University, personal seed-handling infrastructure may dominate communication). over other considerations. Multiline culti- vars have included as many as 8–12 lines (Browning and Frey, 1981; Allan et al., 1983). In contrast, cultivar mixtures often Mixture Composition include only the most agronomically competitive genotypes and, thus, usually There must usually be a correspondence contain only 2–5 components. between the resistance genes incorporated in the mixture and the avirulence genes present in the target pathogen population if diversity is to be functional. This can be Epidemic Intensity accomplished in either formal or informal ways (Mundt, 2002), but often involves The effect of epidemic intensity on mixture attempts to minimize the percentage of the efficacy can vary. Theoretically, one would host population that a given race will be expect the difference in disease levels virulent against. If there is functional host/ between pure stands and mixtures to pathogen diversity present, disease severity increase with increasing number of patho- will decrease with decreasing frequency of gen generations (Leonard, 1969). The level a host genotype in mixture. This has been of initial infection, the generation time demonstrated numerous times with rusts of the pathogen, and the rate of epidemic and powdery mildews of small grains development will, in turn, determine the (Browning and Frey, 1969; Mundt and number of pathogen generations that occur Browning, 1985b; Wolfe, 1985), and with in a given epidemic. In the field, disease rice blast (Mundt, 1994b; Koizumi, 2001). levels initially diverge over time, and then Leonard (1969) predicted that the infection converge as the host’s carrying capacity for rate of the pathogen will decline logarith- disease is approached (e.g. see Wolfe and mically with the proportion of a genotype Barrett, 1980). The rate of approach to in the mixture, and this relationship has carrying capacity will greatly impact the been demonstrated in the field for several effectiveness of mixtures for disease control small grain diseases (Leonard, 1969; (Mundt, 1990), and a fast approach can Burdon and Chilvers, 1977; Luthra and Rao, substantially reduce mixture effectiveness. Host Diversity, Epidemic Progression and Pathogen Evolution 177

Large amounts of external inoculum can expanded rapidly to eventually incorporate greatly reduce the ability of mixtures to con- 92% (360,000 ha) of the barley area. This trol disease. For example, potato mixtures large-scale mixture adoption was associated provided better late blight control in areas with a sharp decline in both mildew levels with little or no outside inoculum, than in and fungicide use (Wolfe, 1992; Wolfe et al., an area where external spore showers are 1992). In China, mixtures of sticky rice culti- heavy and frequent (Garrett and Mundt, vars highly susceptible to blast were mixed 2000; Garrett et al., 2001). Thus, high with non-glutinous cultivars that were more disease pressure caused by fast epidemics resistant. A total of 812 and 3342 ha of con- and large amounts of outside inoculum tiguous rice fields were grown to mixtures may reduce mixture efficacy, while a large in 1998 and 1999, respectively. Observation number of pathogen generations may both plots of non-diversified rice indicated that increase disease pressure and increase the mixtures reduced panicle blast severity mixture effectiveness. by an average of 94% on the susceptible sticky rice cultivars (Zhu et al., 2000). This practice expanded to 550,000 ha in 2003 (Y. Zhu, Yunnan Agricultural University, Spatial Scale personal communication).

Mixtures may provide greater disease control in production-scale situations than is observed in small-scale experimental Autoinfection and Host Geometry plots (Wolfe, 1985; Mundt, 1994b, 2001), for several reasons. Unnaturally high inocu- The impact of mixtures on disease levels is lation rates and the effects of interplot expected to decline with increasing pro- interference can reduce observed levels portions of autoinfection (Barrett, 1980; of disease control in experiments, and Mundt et al., 1986; Goleniewski and mixtures are especially sensitive to such Newton, 1994), i.e. infections ‘in which the effects (Wolfe, 1985; Mundt, 1994b, 2002). donor (infector) host individual is the same Mixtures reduce the velocity of spread as the recipient (infected) host individual’ of disease from an initial focus (Van den (Robinson, 1976). Although the degree of Bosch et al., 1990), and the nature of this autoinfection can be influenced by patho- expansion is such that the velocities of pure gen characteristics such as dispersal and stands versus mixtures may diverge as lesion expansion (Garrett and Mundt, 1999; epidemics expand in time and space Mundt, 2002), this section will focus on (Mundt, 2001, 2002). For spatially uniform the effects of host geometry. Everything else epidemics, mixtures may reduce inoculum being constant, autoinfection would be levels regionally, resulting in increased expected to increase with increasing geno- levels of disease control at larger spatial type unit area (GUA), i.e. the ground area scales (Mundt et al., 2002). occupied by an independent, genetically Some evidence exists to support the homogeneous unit of host tissue (Mundt view that mixtures perform better at and Browning, 1985a). The GUA can be larger spatial scale. Field observations, approximated as the ground area occupied though lacking replication or controls, have by an individual plant in a random mixture sometimes suggested that mixtures provide of plants, and the area occupied by a row greater levels of disease control in commer- and field for row mixtures and interfield cial production than in small experimental diversification, respectively. field plots (Wolfe and Barrett, 1980; Brown- Computer simulations and field studies ing and Frey, 1981; Wolfe, 1985; Mundt, have shown that mixture efficacy will 1994b). In the early 1980s, the use of barley decrease when the degree of aggregation of cultivar mixtures to control powdery mil- plant genotypes is changed to alter GUA dew in the German Democratic Republic (summarized in Mundt, 2002). However, 178 C.C. Mundt

GUA interacts with other spatial variables to will the extensive use of mixtures select determine disease levels in mixtures. Com- over time for complex pathogen genotypes puter simulations (Mundt et al., 1986) and able to attack multiple host genotypes in a field studies with rust pathogens (Mundt mixture? In small-scale experimental plots, and Browning, 1985b; Mundt and Leonard, the relative frequency of complex powdery 1985, 1986) showed that GUA had little mildew genotypes has been found to be influence on mixture efficacy if initial greater in barley cultivar mixtures than in inoculum was distributed in a single focus, pure-stand controls (Munk, 1983; Chin and rather than uniformly over plots. Later Wolfe, 1984a; Huang et al., 1994). However, theoretical work suggested that the number a different view may emerge when one con- of host units may be more important than siders both relative and absolute genotype their size, and that mixtures of large geno- frequencies. For example, Chin and Wolfe type units can provide substantial disease (1984a) found that the increased relative control, even when inoculum is distributed frequency of complex powdery mildew uniformly, provided that the total number genotypes in barley cultivar mixtures was of host units is sufficiently large (Mundt due to their effectiveness in controlling and Brophy, 1988). However, a field test of simple races, and that the absolute fre- this concept provided inconclusive results quency of the most complex genotype was (Mundt et al., 1996). less in a three-way barley cultivar mixture Spatial manipulations may demon- than in the component pure stands. A very strate effects of GUA when compared under similar result was found when examining constant conditions, or in simulation mod- race frequency data after several years of els. However, such constancy rarely occurs extensive commercial use of barley cultivar in the real world, and there is large variation mixtures in the German Democratic Repub- in reported levels of disease control for lic (Wolfe et al., 1992). Thus increased mixtures of crops with large plants (Mundt, relative frequency of complex races in a 2002). Nevertheless, it appears possible to mixture does not necessarily imply loss of have substantial disease reductions in mix- disease control. It may be more difficult to tures even for tree crops, despite the earlier determine the selective influence of mix- cautions to the contrary by van der Plank tures for clonal pathogens, since virulence (1968). Evidence for disease reductions of genes will not be randomly associated with greater than 50% in mixtures has been other genes that influence pathogen fitness reported for apple (Malus × domestica) scab (Mundt, 1994a, 2002). (caused by Venturia inequalis) (Blaise and Pathogen complexity in mixtures has Gessler, 1994; MacHardy et al., 2001) and been the subject of many modelling efforts Melampsora rust (caused by Melampsora (Mundt, 2002). Most of these models assume spp.) of willow (Salix spp.) (McCracken and that a fitness cost associated with virulence Dawson, 1998; Hunter et al., 2002). More is the only mechanism to counter selection detailed descriptions of the use of mixtures for increased virulence complexity in the for control of Melampsora rust are presented pathogen. In general, the models suggest that elsewhere in this volume. it will be difficult to prevent complex races from eventually dominating the pathogen population, although the process may be sufficiently slow such that the pathogen Pathogen Evolution population can be managed. Although models of pathogen evolution Pathogen populations tend to be more usually require a cost of virulence for main- diverse in mixtures than in pure stands tenance of pathogen diversity in mixtures, (Browning and Frey, 1981; DiLeone evidence supporting such costs is very and Mundt, 1994; Muller et al., 1996; weak (Parlevliet, 1981). Parlevliet (1981) McCracken et al., 2000; Koizumi, 2001), but explained the apparent lack of association Host Diversity, Epidemic Progression and Pathogen Evolution 179

between virulence and fitness with a Conclusions conceptual model consisting of mutation to virulence, which is often associated with Large plant size, and its effect on auto- a fitness reduction initially, followed by infection, is the factor most often cited as a selection for fitness modifiers that eventu- deterrent to the use of mixtures to control ally ameliorate the initial fitness cost. Such tree diseases effectively (e.g. van der Plank, fitness modifiers have been shown to reduce 1968; Miot et al., 1999). As noted above, or eliminate fitness reductions initially there are reports of substantial control of associated with antibiotic resistance in tree diseases with mixtures, including bacteria (Cohan et al., 1994; Morrell, Melampsora rust of willow (McCracken and 1997; Levin et al., 2000), but have been Dawson, 1998; Hunter et al., 2002). On the largely ignored in plant pathology. other hand, much smaller disease reduc- Selection for fitness modifiers is likely tions were noted for Melampsora rust in to be slower than selection for virulence poplar mixtures (Miot et al., 1999). There genes themselves (Wolfe and Barrett, 1980), are several differences in biology, environ- thus slowing pathogen evolution towards ment or experimental design that could complexity. explain the discrepant results between In addition, there are mechanisms other these similar pathosystems. Regardless, the than a cost of virulence that may counter contrasting results emphasize the need for selection for pathogen complexity in mix- designing mixtures to attain functional tures (Leonard and Czochor, 1980; Mundt diversity, and for field experiments that and Browning, 1985b; Mundt, 1994a, 2002; are designed appropriately to provide an Lannou and Mundt, 1997). For example, accurate evaluation of mixture efficacy for quantitative adaptation to host genetic back- the epidemiological conditions in question. ground can induce disruptive selection on Lack of agronomic and end-product complex races able to attack multiple host uniformity have often been raised as limita- components, thus reducing their fitness rel- tions to the use of field crop mixtures, ative to simple pathotypes (Chin and Wolfe, though these issues have caused little or 1984a; Lannou and Mundt, 1996; Villareal no difficulty in many cases (Wolfe, 1985; and Lannou, 2000; Lannou, 2001). Other Mundt, 1994b; Finckh et al., 2000). Unifor- mechanisms include density-dependent mity is likely to be even less crucial for selection, frequency-dependent selection, crops produced primarily for biomass. In a selection in heterogeneous environments, hybrid poplar mixture, competition reduced founder effects and migration (Mundt, the diameter of some clones. However, other 2002). clones compensated with increased growth, Although more data are needed, there and the mean diameter and mean biomass is no evidence to date for strong selection was no different in the mixture as compared of complex and highly aggressive pathogen with its component monocultures (Berthe- genotypes in host mixtures. Similarly, in lot, 2001). Under disease conditions, there is natural ecosystems, complex pathogen potential for more resistant willow clones to races can be the most frequent, but do not compensate for yield loss of diseased ones, dominate the pathogen population (Segal as was found for a soybean (Glycine max) et al., 1982; Dinoor and Eshed, 1984) nor mixture in the presence of Phytophthora cause substantial damage to the host (Segal sojae (Wilcox and St Martin, 1998). et al., 1980, 1982). None the less, pathogen With field crops, it is generally assumed evolution should be managed by ensuring that a successful mixture will need to be that no single mixture is grown exclusively composed of genotypes that yield well in in either time or space (Wolfe and Barrett, monoculture (Pfahler and Linskens, 1979; 1980), which can be a natural result of Wolfe and Barrett, 1980; Mundt, 1994b), and normal crop management in many cases the same is likely to be true for willow (Mundt, 2002). mixtures. Though small yield increases 180 C.C. Mundt

sometimes occur in the absence of disease Browning, J.A. and Frey, K.J. (1969) Multiline cultivars (Wolfe and Barrett, 1980; Finckh and as a means of disease control. Annuual Review. Mundt, 1992a; Smithson and Lenne, 1996), Phytopathology 14, 355–382. increased yield stability is more consistently Browning, J.A. and Frey, K.J. (1981) The multiline concept in theory and practice. In: Jenkyn, J.F. associated with mixtures (Smithson and and Plumb, R.T. (eds) Strategies for the Control of Lenne, 1996). Stability of production may be Cereal Disease. Blackwell, Oxford, pp. 37–46. even more important for a perennial crop, Burdon, J.J. and Chilvers, G.A. (1977) Controlled for which the catastrophic loss of a single environment experiments on epidemic rates of genotype to disease or other stress could barley mildew in different mixtures of barley and occur after several years of economic wheat. Oecologia 28, 141–146. investment in the plantation. Calonnec, A., Goyeau, H. and de Vallavieille-Pope, As with field crops, Melampsora popu- C. (1996) Effects of induced resistance on lations were more diverse in mixtures than infection efficiency and sporulation of Puccinia in monocultures, and there was no evidence striiformis on seedlings in varietal mixtures and on field epidemics in pure stands. European for rapid selection of super-races. However, Journal Plant Pathology 5, 733–741. the life expectancy of a willow plantation is Chin, K.M. and Wolfe, M.S. (1984a) Selection on expected to be in the order of 25 years, Erysiphe graminis in pure and mixed stands of which makes the potential for selection of a barley. Plant Pathology 33, 535–546. super-race more critical, and calls for incor- Chin, K.M. and Wolfe, M.S. (1984b) The spread of porating a wide array of resistance within Erysiphe graminis f. sp. hordei in mixtures of mixtures (McCracken et al., 2000). Mixtures barley varieties. Plant Pathology 33, 89–100. of differing composition could be deployed Cohan, F.M., King, E.C. and Zawadzki, P. (1994) spatially, perhaps even within the same Amelioration of the deleterious pleiotropic plantation. Further, as plantations will be effects of an adaptive mutation in Bacillus subtilis. Evolution 48, 81–95. established over a period of years within a DiLeone, J.A. and Mundt, C.C. (1994) Effect of region, regional diversity can be attained by wheat cultivar mixtures on populations of altering mixture composition over time. Puccinia striiformis races. Plant Pathology 43, As with field crops (Mundt, 2002) this 917–930. will likely occur naturally as growers incor- Dinoor, A. and Eshed, N. (1984) The role and porate new, higher performing clones into importance of pathogens in natural plant mixtures. communities. Annual Review Phytopathology 22, 443–466. Elliot, V.J., MacKenzie, D.R. and Nelson, R.R. (1980) Effect of number of component lines on powdery References mildew epidemics in a wheat multiline. Phytopathology 70, 461–462 [Abstract]. Allan, R.E., Line, R.F., Peterson, C.J. Jr, Rubenthaler, Finckh, M.R. and Mundt, C.C. (1992a) Plant competi- G.L., Morrison, K.J. and Rohde, C.R. (1983) tion and disease in genetically diverse wheat Crew, a multiline wheat cultivar. Crop Science populations. Oecologia 91, 81–92. 23, 1015–1016. Finckh, M.R. and Mundt, C.C. (1992b) Stripe rust, Barrett, J.A. (1980) Pathogen evolution in multi- yield, and plant competition in wheat cultivar lines and variety mixtures. Zeitschrift für mixtures. Phytopathology 82, 905–913. Pflanzenkrankheiten und Pflanzenschutz 87, Finckh, M.R., Gacek, E.S., Goyeau, H., Lannou, C., 383–396. Merz, U., Mundt, C.C., Munk, L., Nadziak, J., Berthelot, A. (2001) Mélange de clones entaillis à Newton, A.C., de Vallavieille-Pope, C. and courtes rotations de peuplier: influence sur Wolfe, M.S. (2000) Cereal variety and species la productivité et l’homogénéité des produits mixtures in practice. Agronomie 20, 813–837. recoltes. Canadian Journal of Forest Research Garrett, K.A. and Mundt, C.C. (1999) Epidemiology 31, 1116–1126. in mixed host populations. Phytopathology 89, Blaise, P. and Gessler, C. (1994) Cultivar mixtures in 984–990. apple orchards as a means to control apple scab? Garrett, K.A. and Mundt, C.C. (2000) Host diversity In: Butt, D.J. (ed.) Integrated control of pome can reduce potato late blight severity for focal fruit diseases. Norwegian Journal Agricricultural and general patterns of primary inoculum. Science Supplement 17, 105–112. Phytopathology 90, 1307–1312. Host Diversity, Epidemic Progression and Pathogen Evolution 181

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for the Control of Cereal Disease. Blackwell, Zhu, Y., Chen, H., Fan, J., Wang, Y., Li, Y., Oxford, pp. 73–80. Chen, J., Fan J.-X., Yang, S., Hu, L., Wolfe, M.S., Brandle, U., Koller, B., Limpert, E,, Leung, H., Mew, T.W., Teng, P.S., Wang, Z. McDermott, J.M., Muller, K. and Schaffner, D. and Mundt, C.C. (2000) Genetic diversity (1992) Barley mildew in Europe: population bio- and disease control in rice. Nature 406, logy and host resistance. Euphytica 6, 125–139. 718–722. This page intentionally left blank 16 Short-rotation Coppice Willow Mixtures and Rust Disease Development

Alistair R. McCracken1, W. Malcolm Dawson2 and Diane Carlisle3 1Applied Plant Science Division, Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, UK; 2Applied Plant Science Division, Northern Ireland Horticultural and Plant Breeding Station, Department of Agriculture and Rural Development, Loughgall, Co. Armagh BT61 8JB, UK; 3Department of Applied Plant Science, Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK

Introduction This often occurred after 8–10 years’ growth in mono-plots. For example, in N. Ireland Rust, caused by Melampsora epitea, was S. burjatica ‘Korso’ was rust free for 10 years first reported on short-rotation coppice but rapidly succumbed to the disease during (SRC) willow growing in N. Ireland, UK 1986. Similarly 38% of Salix viminalis during the summer of 1985 (McCracken and ‘Bowles Hybrid’ stands examined in the UK Dawson, 1998). Salix burjatica ‘Korso’ had in 1987 were free of rust and the remaining been grown in large mono-plots in the 62% showed only low to moderate levels. province for at least 10 years with no evi- However, when the same stands were exam- dence of disease. In that same year rust was ined in 1988 only 22% had no symptoms, also observed in SRC willow plantations in 67% had low to moderate levels and other parts of the UK and the Republic of 13% were severely infected (Dawson and Ireland (McCracken and Dawson, 1998). In McCracken, 1994). Currently S. viminalis subsequent seasons rust infections occurred ‘Bowles Hybrid’ becomes heavily infected as early as mid-June, especially on regrowth with rust wherever it is grown. from freshly coppiced stools. These early Early work (Dawson and McCracken, infections resulted in premature leaf fall 1994) demonstrated that rust could be con- which, in turn, was followed by invasion of trolled with the intensive use of fungicides. the stems by secondary dieback organisms, In trials carried out at the Northern Ireland including Fusarium sambucinum Fuchel Horticulture and Plant Breeding Station, and Glomerella miyabeana (Fukushi) von Loughgall (54°24′N, 7°35′W) benodanil, Arx., which caused high levels of stem myclobutanil and captan/penconazol were dieback and ultimately stool death applied to first year regrowth of S. burjatica (McCracken, 1989). ‘Korso’. When benodanil was applied at There was evidence to suggest changing 14-day intervals starting in mid-May, before susceptibility of certain Salix spp. varieties the first appearance of rust pustules, there to rust over a number of years. Hence was a significant reduction in rust at all varieties that were ostensibly free of rust for disease assessment dates. This was accom- several years, became increasingly infected. panied by increased dry matter yield at the

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 185 186 A.R. McCracken et al.

end of the year and only 12% shoot death at In the early 1980s White (1982) reported the time of flushing the following spring, that when barley was grown in cultivar mix- compared to over 60% in unsprayed plots tures there was a decrease in the incidence (Dawson and McCracken, 1994). Similar but of powdery mildew caused by Erysiphe less pronounced trends were observed when graminis, with an associated increase in the other fungicides were used. yield. Wolfe (1985) reviewed the benefits of Hence the use of large amounts of fungi- using multi-line cultivars and variety mix- cide could result in good disease control tures of cereals as a disease-control strategy, with associated increase in dry matter yield particularly for rusts and mildews. The use and delayed leaf fall which, in turn, resulted of varietal mixtures of cereals with as few as in less shoot and stool death at flushing the three components resulted in significantly following spring. However, it was clearly reduced levels of diseases, which was in recognized that such use of fungicides was turn associated with increased yields. Cer- not a sustainable option, for a number of tain barley mixtures gave increased yield in reasons: the presence of scald and net blotch (Mundt et al., 1994), and where the mixture con- • Economic: SRC willow is a low-value tained a susceptible cultivar along with rela- crop on which there are small financial tively resistant cultivars the disease severity margins. The cost of regular repeated was reduced by up to 20–32%. However, fungicide applications throughout the in this trial some of the barley mixtures growing season could not be sustained. resulted in slight yield reduction, indicating • Environmental: often willow is grown the importance of careful evaluation of the close to water courses or in areas where mixture components, in order to choose the intensive use of agrochemicals those with a positive effect on disease would be unacceptable. control and yield (Mundt et al., 1994). • Energy: one of the end uses for SRC Similar results were obtained using wheat willow is as a source of renewable cultivar mixtures, with reduced leaf rust energy. It is therefore unacceptable to severity and increased yield (Mahmood be applying agrochemicals which have et al., 1991). high energy production demands. It was therefore proposed to test the con- • Practical: while it is relatively easy cept of clonal/varietal/genotype mixtures of to apply fungicide to an SRC willow Salix spp. as a disease-control strategy for crop in its first few weeks regrowth, foliar rust caused by M. epitea. It should after coppicing it becomes impractical be noted that in early publications (e.g. to get effective chemical cover of plants McCracken and Dawson, 1994) the terms which are up to 3 m high (McCracken ‘clonal’ or ‘mixed-clonal’ were used. Subse- and Dawson, 1994). quently the terms ‘varieties’ or ‘species vari- It was therefore essential that a practi- eties’ have been used (e.g. McCracken et al. cal, cost-effective disease-control strategy be 2001), and most recently individual mixture developed. In the early 1980s high yielding, components have been called ‘genotypes’ rust-resistant Salix varieties were becoming (McCracken and Dawson, 2003). The term commercially available from a breeding pro- ‘genotype’ will therefore be used throughout gramme in Sweden, and laterally from a UK this, and the following, chapter. breeding programme. However, the experi- ence with S. burjatica ‘Korso’ (Dawson and McCracken, 1994) and other Salix varieties suggested that, when grown in monoculture Mixtures with Few Components of in conditions of high disease pressure, Older Genotypes previously rust-resistant varieties could become increasingly susceptible. The In 1987 five Salix spp. genotypes, S. vimi- projected life of a willow plantation is nalis ‘Bowles Hybrid’, S. viminalis ‘683’, approximately 30 years. S × dasyclados, S. burjatica ‘Germany’ and Coppice Willow Mixtures and Rust Disease Development 187

S. burjatica ‘Korso’, were planted in a single 2–4 weeks after they were prevalent in replicate block, with a minimum plot size mono-plots. of 0.5 ha, as both mono-genotype and 2. The build-up of disease was slower: intimate mixtures (Site 1). In 1988 a 3.3 ha even when genotypes growing in mixtures site was planted using S. viminalis ‘Bowles developed rust, the speed of progress of the Hybrid’, S. viminalis ‘683’, S. × dasyclados, disease was less. S. burjatica ‘Germany’ and S. mollissima- 3. Lower total rust scores at the end of the undulata ‘SQ83’, in two replicate blocks growing season: the outcome of the delay both in mixture and mono-plots (Site 2). and slower build-up was that the level of In 1989, a third area was planted using S. disease at the end of the season was low viminalis ‘Bowles Hybrid’, S. viminalis enough not to induce premature leaf fall. ‘683’, S. × dasyclados, S. burjatica ‘Ger- This, in turn, meant that there was little or many’, S. mollissima-undulata ‘SQ83’ with no infection with secondary pathogens that the addition of S. × calodendron in two could potentially kill the stools (McCracken, replicate blocks, both in mixture and 1989). mono-plots (Site 3). In all cases the planting density was 20,000 cuttings/ha and stools However, when Salix × calodendron, were cut back at the end of their first year’s which only developed low levels of disease growth and then allowed to grow in a 3-year late in the growing season, was incorporated harvest cycle (McCracken and Dawson, into a mixture, no benefits, in terms of dis- 1998). Random leaf samples were collected ease reduction, were observed. In these trials at fortnightly intervals from each Salix S. viminalis ‘683’ performed atypically. genotype growing in mono-plots or as In most seasons the levels of rust on S. part of the intimate mixtures in each site, viminalis ‘683’ were very similar irrespec- starting in mid-May and continuing until tive of whether it was growing in mono-plots mid-September. Rust levels were measured or incorporated within a mixture. It has been using a rust assessment key (McCracken suggested (McCracken and Dawson, 1997) and Dawson, 1992) and cumulative rust that one reason for this could be that S. scores (Dawson and McCracken, 1994) viminalis ‘683’ is made up of both male calculated. This enabled disease progress and female components and hence could curves to be drawn, in order to determine achieve some of the benefits of a mixture. the effect of inclusion of the Salix In 1993 at least 11 pathotypes of M. genotypes in a mixture. epitea had been identified (Pei et al., 1993). When relatively rust-susceptible Salix At this time it was thought probable that a genotypes, e.g. Salix × dasyclados, S. bur- genotype mixture would only be effective in jatica ‘Germany’ and S. viminalis ‘Bowles reducing the impact of disease if the individ- Hybrid’, were grown as part of an intimate ual constituents of the mixture were not all mixture, the level of disease was signifi- susceptible to the same pathotype (Anon., cantly reduced as the season progressed. 1994). Hence, genotypes, which were avail- Furthermore, the impact of the disease on able for commercial SRC planting in the the growth of stools was also greatly early 1990s, were attributed an ‘incompati- reduced. These disease reductions and bility code’ (Anon., 1994). These codes had increased growth in mixtures were similar been worked out using a differential set on to that achieved with the intensive use of willow genotypes. It was recommended that fungicides (McCracken & Dawson, 1997). genotypes with the same incompatibility Three aspects of disease reduction were code should not be planted together in small identified when susceptible genotypes were (2–3 component) mixtures, whereas in larger incorporated into mixtures: mixtures only half of the genotypes included should have the same incompatibility 1. Delay in the onset of disease: often the code (McCracken and Dawson, 1997). As first significant numbers of rust pustules the European Salix breeding programmes were observed on genotypes in mixtures released new planting material during the 188 A.R. McCracken et al.

1990s and into 2000, their susceptibility detected. On S. burjatica ‘Germany’ only to similar pathotypes became an important pathotypes LR1 and LR2 were detected issue. During this period almost all of (McCracken et al., 2000). There was no the new genotypes were S. viminalis or significant difference between the mean S. viminalis hybrids, which were all (Shannon–Weaver) diversity indices for all theoretically susceptible to the same range of the genotypes in each of the two years, of pathotypes. 1994 (0.435) and 1995 (0.493). However, A further concern relating to the use there was a significant difference between of genotype mixtures as a disease-control means in the mono-plots (0.372) and mix- strategy was the potential for the selection tures (0.556), indicating that there was a of ‘super-races’ or ‘super-pathotypes’. The greater diversity of pathotypes on genotypes term refers to the pathotypes which are capa- growing in mixtures (McCracken et al., ble of infecting all of the components of the 2000). One pathotype clearly dominated on mixture, thus making disease control even each of the Salix genotypes. Nevertheless, more difficult. Using mathematical models, throughout the growing season, in each year, Groth (1976) indicated that, in mixtures, there was progression of pathotypes, with complex races could develop, and that for multiple pathotypes present on the same polycylic pathogens such as rust, this could genotype or even the same leaf at the same occur quickly. When random mixtures of time. spring barley were grown, complex patho- It is recognized that there is a real possi- types (‘super-pathotypes’) of the mildew bility that pathogens may be able to adapt to pathogen, Erysiphe graminis f. sp. hordei mixtures (Groth, 1976; Finckh and Mundt, were preferentially selected (Huang et al., 1992), resulting in populations dominated 1994). If similar cultivar mixtures were by ‘super-races’ or ‘super-pathotypes’. This employed over long periods and large areas, phenomenon has been reported with bio- then such super-races would eventually trophs such as rust and powdery mildews on dominate the mildew population (Huang cereal crop mixtures (Finckh and Mundt, et al., 1994). The pathogen adaptation 1992). The results from the willow mixture in mixtures may be slow (Wolfe, 1985), trial would seem to indicate that the although if the same mixture is grown over evolution of such ‘super-races’ or ‘super- several seasons there will be selection for the pathotypes’ is unlikely. Within Salix geno- modifier genes (Wolfe, 1985). This, in turn, type mixtures there is a greater diversity of would improve the performance of a patho- pathotypes, with the associated increase in gen genotype with complex virulence on competition between them, which in turn that particular range of hosts. This would decreases the evolutionary pressure on each suggest that diversification of mixture pathotype (McCracken et al., 2000). Within components both in time and space will any Melampsora rust population there is a be a significant feature in the long-term wide diversity of individuals of carrying vir- sustainability of a willow plantation. ulence genes that are capable of overcoming The pathotype composition of the their corresponding host resistance genes. It M. epitea populations associated with the was therefore recognized that if genotype mixed and monoculture plots in this willow mixtures were to continue as a commercially trial was analysed over 2 years, 1994 and applicable disease-control strategy, then it 1995 (McCracken et al., 2000). Six LET was essential to have a range of components (larici-epitea typica) pathotypes and two which contained as wide an array of LR (larici-retusae) pathotypes (Pei et al., multiple resistance genes as possible. 1999) of M. epitea and M. caprearum were There were a number of key issues detected during the course of the study. In relating to the construction of Salix geno- 1994, LET4, LET5 and LET6 were recorded type mixtures that contributed to the in N. Ireland willow plantations for the first management of rust disease, which gave time. Furthermore, three new pathotypes, increased yields and could be predicted to designated NI5, LET7 and LET8, were be sustainable over the average life of 7–10 Coppice Willow Mixtures and Rust Disease Development 189

harvest cycles of an SRC willow plantation. Two criteria were used to select geno- These were: types from clonal selection trials conducted in N. Ireland – high yield and reduced rust • optimum number of Salix genotypes susceptibility. Three genotypes, S. burjatica included in a mixture ‘Germany’, S. dasyclados × aquatica ‘V7511’ • how improved Salix genotypes, specifi- and S. dasyclados × caprea ‘V794’ were cally bred for SRC willow production, known to be rust susceptible, but were would perform within mixtures included in the trial as a means of testing • planting configuration mixtures as a disease-control strategy. All • planting density varieties were planted in large mono- • effect of harvesting on disease develop- genotype plots, which were large enough to ment and yield. ensure that they functioned as mono-plots. Each plot was a split plot with cuttings being planted at three densities (10,000; 15,000 Mixtures With Larger Numbers of and 20,000 cuttings/ha) (McCracken et al., Components, Including Improved 2001). Four mixture combinations (5-, 10-, Genotypes 15- and 20-way) were employed, each of which was planted as a totally random A large two-block plantation was estab- mixture at the same three densities and lished at Castlearchdale, Co. Fermanagh, which were also in proportionally larger Northern Ireland (54°28′N, 7°43′W). The plots. site was 60–70 m above sea level and com- Leaf samples were collected, at prised heavily gleyed soils with impeded 2-weekly intervals, starting in mid-May drainage. Block 1 was planted in spring through to mid-September in each of the 1994 using standard planting protocols 6 years, from each of the Salix genotypes (Dawson and McCracken, 1995). It was cut growing in mono-plots and wherever they back at the end of its establishment year appeared within a mixture. Samples were in winter 1994/95. Block 2 was planted in assessed for rust (McCracken and Dawson, spring 1995 and both blocks cut back 1992) and disease progress curves were con- in winter 1995/96. Hence in the 1996 grow- structed for each genotype in each year at ing season both blocks were at the same each density when growing in a mono-plot stage of growth – first year regrowth from or when incorporated into a mixture. freshly coppice stools. Twenty Salix geno- There were considerable differences in types (Table 16.1) were selected for inclu- the patterns of rust development on geno- sion within the trial. types growing in mono-plots, in the 6 years

Table 16.1. Twenty Salix genotypes included in 5-, 10-, 15- and 20-way mixtures.

Included in 5; 10; 15; 20-way mixtures Included in 15; 20-way mixtures

S. burjatica ‘Germany’ S. viminalis × caprea ‘V789’ S. mollissima-undulata ‘SQ83’ S. viminalis ‘77683’ S. dasyclados × aquatica ‘V7511’ S. viminalis ‘78101’ S. viminalis ‘77082’ S. viminalis ‘78195’ S. dasyclados × caprea ‘V794’ S. schwerinii × aquatica ‘V7534’

Included in 10; 15; 20-way mixtures Included in 20-way mixture

S. viminalis × aquatica ‘V7503’ S. viminalis ‘77699’ S. viminalis ‘78118’ S. viminalis ‘Gigantea’ S. viminalis ‘78183 S. viminalis ‘Gustav’ S. schwerinii × viminalis × dasyclados ‘V7531’ S. schwerinii × aquatica ‘V7533’ S. viminalis ‘870146 ULV’ S. viminalis ‘870082 ORM’ 190 A.R. McCracken et al.

Table 16.2. Cumulative rust scores on 14 September on each of 20 Salix genotypes growing in mono-plots, 1996–2001. Least significant differences (LSDs: P = 0.05) between means within years are given (McCracken and Dawson, 2003. Reproduced with permission from the Annals of Applied Biology).

1996 1997 1998 1999 2000 2001

Germany 8767 1733 3016 11319 676 2321 SQ83 5110 5607 5102 340 310 907 V7511 9110 1839 5287 11842 672 1835 77082 616 622 868 1993 800 358 V794 11458 1754 6565 14301 1306 2178 V7503 1868 283 59 2633 400 106 78118 2308 1082 762 740 693 317 78183 297 1035 1820 1219 1652 898 V7531 49 0 0 10 0 2 ULV 331 743 1360 266 1012 402 V789 926 2898 1451 2038 2300 105 77683 156 358 306 132 208 21 78101 1384 309 1243 305 1127 878 78195 8 375 370 82 239 12 V7534 365 0 5 493 1 47 77699 557 246 1516 1027 475 330 Gigantea 43 4 23 17 63 13 Gustav 5453 1442 2881 3447 3184 1117 V7533 80 0 0 240 2 23 ORM 741 184 172 856 85 209 LSD (P = 0.05) 5905 1299 2590 3347 569 1344

(Table 16.2). Salix schwerinii × viminalis pathotype becoming less prevalent. Rust × dasyclados ‘V7531’, S. schwerinii × levels on the remaining 12 genotypes grow- aquatica ‘V7533’ and S. viminalis ‘Gigantea’ ing in mono-plots were relatively low in all remained virtually rust free throughout the years and had little or no impact on growth 6 years of the trial. However, there are still or survival (Chapter 17). On the basis of benefits in the inclusion of such genotypes rust development on genotypes growing in within mixtures, not only to increase mix- mono-plots, the 20 genotypes were ranked ture diversity, but also their inclusion in in terms of their relative susceptibility mixtures should ensure that their rust (Table 16.3). resistance does not break down during the Inclusion of rust-susceptible Salix 30-year life of the willow plantation. genotypes in mixtures generally resulted in High levels of rust were recorded on S. reduced rust, compared to the mono-plots. burjatica ‘Germany’, S. dasyclados × caprea At the August assessment dates in 1996 and ‘V794’, S. dasyclados × aquatica ‘V7511’ 1999, the rust scores on S. burjatica ‘Ger- and S. viminalis ‘Gustav’. In general, the many’ were significantly higher on plants final level of rust was much higher in the growing on mono-plots than on plants in year immediately following coppicing (1996 any of the mixtures. In September 1997 and and 1999) than in subsequent years. In con- 1998, when there were high levels of rust on trast, rust levels on S. mollissima-undulata S. mollissima-undulata ‘SQ83’ significantly ‘SQ83’ were highest during the first harvest lower rust scores were recorded on plants cycle (1996–98) and low during the second growing in the 5-, 10-, 15- and 20-way mix- cycle (1999–2001) to a point where the dis- tures. On S. dasyclados × aquatica ‘V7511’ ease was having no impact on growth. This in 1997, 1998, 1999 and 2001 there was indicated that there had been a change in significantly more rust on plants in mono- the balance of pathotype composition, with plots compared to those in mixtures (Fig. the previously dominant highly damaging 16.1). In 1996 the difference was only Coppice Willow Mixtures and Rust Disease Development 191

significant for the 20-way mixture. Rust Mixtures with Limited Diversity levels on S. dasyclados × caprea ‘V794’ were generally highest in years immediately Concern had been expressed that almost following coppicing, i.e. 1996 and 1999. all of the improved genotypes available for commercial planting were S. viminalis or S. Table 16.3. Relative susceptibility to rust of the viminalis hybrids (McCracken et al., 2000; 20 Salix genotypes growing in mono-plots. McCracken and Dawson, 2001). In 2000 a trial was planted at the Northern Ireland Rank Genotype Horticulture and Plant Breeding Station, 1 V7531 Most resistant Loughgall, Co. Armagh. Seven S. viminalis 2 Gigantea genotypes were planted: S. viminalis 3 V7533 ‘77082’, S. viminalis ‘Ulv’, S. viminalis 4 V7534 ‘Orm’, S. viminalis ‘Jorr’, S. viminalis 5 78195 ‘Jorunn’, S. viminalis ‘Gigantea’ and S. 6 77683 viminalis ‘78195’ in large mono-genotype 7 ORM plots. S. viminalis ‘77082’ and S. viminalis 8 V7503 ‘Ulv’ were known to have a limited suscep- 9 77699 10 ULV tibility to M. epitea (McCracken and 11 78101 Dawson, 2003) and so were both incorpo- 12 77082 rated into a 2-way mixture, five 3-way mix- 13 78118 tures with each of the other genotypes and a 14 78183 7-way mixture including all of the geno- 15 V789 types. As in previous trials, leaf samples 16 SQ83 were collected at 2-week intervals, from in 17 Germany mid-May to mid-September, during each of 18 Gustav the growing seasons 2000–2003. 19 V7511 In all 3 years, the highest levels of rust 20 V794 Most susceptible were recorded on S. viminalis ‘77082’. Rust

Fig. 16.1. Rust disease (June–September 2001) development on Salix dasyclados × aquatica ‘V7511’ grown in mono-plots and as part of 5-, 10-, 15- and 20-way mixtures. 192 A.R. McCracken et al.

was normally observed in mid-July and mechanism functioning in this trial, other- levels increased steadily during subsequent wise the greatest reduction should have weeks. Highest levels of rust were observed been observed in the 7-way mixture and in mono-plots in 2002 on S. viminalis there should have been little difference ‘77082’ and S. viminalis ‘Orm’. In 2002 and between each of the 3-way mixtures. It 2003 there was a significant reduction in seems possible that another mechanism may rust on S. viminalis ‘77082’ in the 7-way be responsible. It is clear from the yield data mixture compared to levels in the mono- (Chapter 17) that individual components plot. A similar improvement was observed can have a significant impact on the in 2002 on S. viminalis ‘Orm’. growth of neighbouring components, either S. viminalis ‘77082’ had been included beneficially or detrimentally. It is therefore in seven different combinations: in mono- postulated that the presence of a particular plots, a 2-way mixture with S. viminalis component, e.g. S. viminalis ‘Gigantea’ or S. ‘Ulv’, 3-way mixtures with S. viminalis ‘Ulv’ viminalis ‘Jorunn’, induces a response in and one of S. viminalis ‘Jorr’, S. viminalis S. viminalis ‘78195’. This may be some form ‘Jorunn’, S. viminalis ‘Gigantea’ or S. of induced systemic resistance to rust, but viminalis ‘78195’, or in a 7-way mixture. this hypothesis needs further investigation. This meant that it was possible to identify whether the presence of certain willow genotypes influenced the degree of suscepti- bility of the rust susceptible genotype S. Conclusions viminalis ‘77082’. When growing with S. viminalis Growing SRC willow genotypes in mono- ‘Gigantea’ there was significantly less rust culture in temperate wet climates carries a on S. viminalis ‘77082’ compared to rust high risk, which these authors regard as levels in the mono-plots. This occurred unacceptable. While there are a number consistently in all 3 years, with differences of high-yielding, rust-resistant Salix geno- being evident in late August and remaining types which may perform well over many throughout the rest of the growing season. harvest cycles, there does seem to be a gen- Similar reductions were observed with uine risk of previously resistant genotypes S. viminalis ‘78195’ and to a lesser extent developing susceptibility over a relatively S. viminalis ‘Orm’. Salix viminalis ‘Jorunn’ short period of time. Inclusion of such was inconsistent with rust levels on S. genotypes in a diverse mixture should viminalis ‘77082’ being reduced in 2001 guarantee their survival throughout the and 2003 but not in 2002. Inclusion with life of the plantation. In addition there are S. viminalis ‘Jorr’ resulted in no changes well-documented yield benefits. in amount of rust disease developing. The The greater the diversity of the mixture, 2-way mixture with S. viminalis ‘Ulv’ had no incorporating inter- and intra-species geno- effect on rust and the 7-way mixture contain- types, it would seem the greater the benefits ing all of the genotypes had a limited effect and long-term sustainability of the planta- in reducing rust on S. viminalis ‘78195’. tion. However, straight S. viminalis mix- It has been speculated (Dawson and tures with a range of susceptibilities have McCracken, 1995; Mundt et al., 2002; Chap- reduced rust impact over the first 3-year har- ter 15, this volume) that one of the primary vest cycle. This trial needs to be run for at mechanisms to explain the effectiveness of least two more harvest cycles to determine mixtures in reducing disease impact is a the effectiveness in the long-term. dilution of susceptible genotypes with less The mechanism of the effectiveness of susceptible individuals, i.e. since the mix- mixtures in reducing rust also merits further ture contains a number of resistant compo- research. While the dilution effect is an nents, the spread of the pathogen is slowed obvious factor, it does seem that other, more down. This does not seem to be the only complex mechanisms are operating. Coppice Willow Mixtures and Rust Disease Development 193

Acknowledgements rust disease. Norwegian Journal of Agricultural Sciences Suppl. 18, 101–109. The authors gratefully acknowledge McCracken, A.R. and Dawson, W.M. (1997) Using mixtures of willow clones as a means of control- funding for part of this work from the ling rust disease. In: Bullard, M.J., Ellis, R.G., Framework5 R & D Programme of the Health, M.C., Knight, J.D., Lainsbury, M.A. and European Union (QLRT-1999-01585). They Parker, S.R. (eds) Aspects of Biology 49; Biomass are also grateful to the Department of Agri- and Energy Crops. Association of Applied culture and Rural Development (Northern Biologists, Cirencester, UK, pp. 97–103. Ireland) for their support to the R & D McCracken, A.R. and Dawson, W.M. (1998) Short programme over a number of years. rotation coppice willow in Northern Ireland since 1973: development of the use of mixtures in the control of foliar rust (Melampsora spp.). European Journal of Forest Pathology 28, References 241–250. McCracken, A.R. and Dawson, W.M. (2001) Disease Anon (1994) Plantation Design: Combating Disease effects in mixed varietal plantations of willow. and Pests. Agriculture and Forestry Fact Sheet In: Bullard, M.J., Christian, D.G., Knight, J.D., Short Rotation Coppice no. 14. Department of Lainsbury, M.A. and Parker, S.R. (eds) Aspects Trade and industry, Energy Technology Support of Biology 65 – Biomass and Energy Crops II. Unit, London. Association of Applied Biologists, Warwick, UK, Dawson, W.M. and McCracken, A.R. (1994) Effect pp. 255–262. of Melampsora rust on the growth and develop- McCracken, A.R. and Dawson, W.M. (2003) Rust ment of Salix burjatica ‘Korso’ in Northern disease (Melampsora epitea) of willow (Salix Ireland. European Journal of Plant Pathology spp.) grown as short rotation coppice (SRC) 24, 32–39. in inter- and intra-species mixtures. Annals of Dawson, W.M. and McCracken, A.R. (1995) The per- Applied Biology 143, 381–393. formance of polyclonal stands in short rotation McCracken, A.R., Dawson, W.M., Watson, S. and coppice willow for energy production. Biomass Allen, C.Y. (2000) Pathotype composition and Bioenergy 8, 1–5. in Melampsora epitea populations occurring Finckh, M.R. and Mundt, C.C. (1992) Stripe rust, on willow (Salix) grown in mixed and mono- yield and plant competition in wheat cultivar culture plantations. European Journal of Plant mixtures. Phytopathology 82, 905–913. Pathology106, 879–886. Groth, J.V. (1976) Multi-lines and super- McCracken, A.R., Dawson, W.M. and Bowden, G. races: a simple model. Phytopathology 66, (2001) Yield responses of willow (Salix) grown 937–939. in mixtures in short rotation coppice (SRC). Huang, R., Kranz, J. and Welz, W.G. (1994) Selection Biomass and Bioenergy 21, 311–319. of pathotypes of Erysiphe graminis f. sp. hordei in Mundt, C.C., Hayes, P.M. and Schon, C.C. (1994) pure and mixed stands of spring barley. Plant Influence of barley mixtures on severity of scald Pathology 43, 458–470. and net blotch and on yield. Plant Pathology 43, Mahmood, T., Marshall, D. and McDaniel, M.E. 356–361. (1991) Effect of winter wheat cultivar mixtures Mundt, C.C., Cowger, C. and Garrett, K.A. (2002) on leaf rust severity and grain yield. Phyto- Relevance of integrated management to pathology 81, 470–474. resistance durability. Euphytica 124, 245–252. McCracken, A.R. (1989) SEM study of fungi associated Pei, M.H., Royle, D.J. and Hunter, T. (1993) Identity with cankers on willow. In: Rushton, B.S. and and host alternation of some willow rusts Flynn, A.M. (eds) Proceedings of Irish Botanists (Melampsora spp.) in England. Mycological Meeting 1988. University of Ulster at Coleraine, Research 97, 845–851. p. 42. Pei, M.H., Hunter, T and Royle, D.J. (1999) Occur- McCracken, A.R. and Dawson, W.M. (1992) Clonal rence of Melampsora rusts in biomass willow response in Salix to Melampsora rust in short plantaions for renewable energy in the United rotation coppice plantations. European Journal Kingdom. Biomass and Bioenergy 17, 153–163. of Forest Pathology 22, 19–28. White, E.M. (1982) The effect of mixing barley culti- McCracken, A.R. and Dawson, W.M. (1994) vars on incidence of powdery mildew (Erysiphe Experiences in the use of mixed-clonal stands graminis) and on yield in Northern Ireland. of Salix as a method of reducing the impact of Annals of Applied Biology 101, 539–549. 194 A.R. McCracken et al.

Wolfe, M.S. (1985) The current status of multiline resistance. Annual Review of Phytopathology cultivars and variety mixtures for disease 23, 251–273. 17 Short-rotation Coppice Willow Mixtures and Yield

W. Malcolm Dawson1, Alistair R. McCracken2 and Diane Carlisle3 1Applied Plant Science Division, Northern Ireland Horticultural and Plant Breeding Station, Department of Agriculture and Rural Development, Loughgall, Co. Armagh BT61 8JB, UK; 2Applied Plant Science Division, Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, UK; 3Department of Applied Plant Science, Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, UK

Introduction just to improve productivity levels, but also to improve pest and disease resistance and The principle on which crop yield is based plant form, is a major input into the area of is the conversion of light energy into the yield from short-rotation coppice systems. chemically bound energy contained in the Genotype evaluation has been an ongo- economically important part of the crop, ing commitment from the beginning of the whether that is in root tubers, cereal grains programme on biomass. Initially this was or, in the case of short-rotation coppice with relatively unimproved genotypes, willows, the above-ground woody stems. which had often been selected for purposes Not all of the total organic matter produced other than biomass production for energy. In by the crop as a result of its photosynthetic 1989, a more formal link was forged with the activity is harvested. In short-rotation breeding programme in Sweden and a trial coppice, the stool, root system and leaves protocol was agreed with the International remain unharvested and, together with the Energy Agency. In 1996 a second dedicated woody stems (economic yield), make up the breeding programme was initiated in the UK total biological yield. In willow coppice it and potential operational genotypes were has been estimated that the harvested stems included in a revised testing protocol, represent 60% of the total biological yield, drawn up with the Forestry Commission, to with the leaves (10%) and stool/root system facilitate their inclusion in recommended (30%) making up the remainder. For listings for planting. short-rotation coppice the economic yield is normally quoted as an annual figure of tonnes of dry matter per hectare. To maxi- Yield Expectations mize the economic yield the crop canopy (leaf area index) must be maximized as When the work on short rotation coppice as early as possible and maintained for as long a renewable energy crop began, in the mid- as possible through the control of negative 1970s, yields of 12–15 tonnes DM/ha/year biological influences, particularly pests and were recorded from experimental plots diseases. As a consequence, breeding, not using the relatively non-specific and ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 195 196 W.M. Dawson et al.

unimproved willow species and varieties genotypes from these breeding programmes available. Of the candidates assessed in were tested at four sites, Yorkshire, N. these trials Salix burjatica ‘Korso’ emerged Ireland, Devon and Avon, located across the as the most productive over a range of sites, UK (Lindegaard et al., 2001), initially using a producing yields up to 17 tonnes DM/ha/ protocol designed by the International year at high density (40,000/ha) planting. Energy Agency and later, in 1996, convert- However, total dry matter (DM) yields from ing to a trial protocol agreed with Forestry small experimental plots have to be treated Commission. The current protocol for carry- with caution and early work indicated ing out these comparative trials involves that these yields could be overestimated by establishing the cuttings and cutting them as much as 30% (Stott et al., 1987). This back at the end of the first growing season. would objectively reduce these early yield Subsequently a 2-year yield and a 3-year estimates from 12–15 tonnes DM/ha/year to yield are taken before consideration for 8.4–10.5 tonnes DM/ha/year. inclusion in the recommended listing In the earliest collaborative trials, (Tabbush and Parfitt, 1996). These trials, carried out at Long Ashton Research Station, using agreed standard varieties, gave a much Somerset, and in Northern Ireland (Anon., more accurate picture of potential yield than 1980), yield estimates for a range of Salix the earlier trials. Initially the standard was S. spp. genotypes indicated that S. burjatica viminalis ‘78183’, the most widely planted ‘Korso’ and S. × dasyclados produced the species in Sweden, and later S. viminalis × highest yields across all sites. Fresh weight S. schwerinii ‘Tora’ as representative of the yields from S. burjatica ‘Korso’ ranged from newer, high-yielding hybrids. 19.0 to 20.5 tonnes/ha/year equivalent to When the mean yield of all varieties 8.7–9.4 tonnes DM/ha/year. Similarly S. × was compared, with the Avon site at 100%, dasyclados gave 23–29 tonnes fresh Devon produced 79%, N. Ireland 75% and weight = 10.6–13.3 tonnes DM/ha/year. Yorkshire 68%. These differences were However, both of these have shown an likely to be due to local topographical, increasing susceptibility to Melampsora soil and climatic factors. The yields from rust, to the extent that they can not now be these four sites are collated and used by recommended for planting. the Forestry Commission to produce a In an effort, in particular, to improve recommended listing for planting in the dry matter yield and disease susceptibility, UK (Tabbush and Parfitt, 1996). and more generally plant form, to facilitate Most of the new operational varieties mechanical harvesting, commercial willow produced significantly higher yields than breeding programmes were initiated in the S. viminalis ‘78183’ standard. The earli- Sweden in 1987 and in the UK in 1996. est commercially available releases from The Swedish programme has focused on S. the Swedish programme, S. viminalis ‘Orm’, viminalis, S. schwerinii and S. dasyclados of ‘Rapp’ and ‘Ulv’, produced dry matter yields European and central Russian and Siberian of 177%, 141% and 154%, respectively, origin (Larsson, 1997, 2001). The Russian of the standard. Later introduction of material has provided a good source of S. viminalis ‘Jorr’ and ‘Joruun’ and S. increased productivity and resistance to viminalis × S. schwerini ‘Tora’ and ‘Sven’ Melampsora rust. The UK programme has also gave improved yields, representing involved a much wider species range 156%, 142% and 202% and 167%, respec- including S. rehderiana, S. udensis, S. dis- tively. The most productive introductions color and S. aegyptica as well as S. viminalis from the UK programme, S. × stottii ‘Ashton and S. schwerinii. The first output from this Stott’ and ‘Ashton Parfitt’, have similarly breeding programme entered the final stages given 243% and 220% of the standard. of comparative yield trials in 2002, with the Even when compared with the new, higher- earlier S. viminalis × S. burjatica crosses yielding standard, ‘Tora’, they show a S. × stottii ‘Ashton Stott’ and ‘Ashton Parfitt’ significant increase in dry matter yield of coming into trials in 1992. Operational 120% and 109%, respectively (Lindegaard Short-rotation Coppice Willow Mixtures and Yield 197

et al., 2001). Figure 17.1 shows the dry species have been supplied with optimal matter yield from two of these trials, nutrients and water, yields of over 30 tonnes established in 1996 and 1997, with 3-year DM/ha/year have been achieved (Stott, yields in 2001 and 2002, respectively. 1984; Christersson, 1987) from experimental These yields show the highly signifi- plots, indicating that further improvements cant improvements in dry matter yield not can be made before the biological potential just on the early standard variety, S. of the species is approached. viminalis ‘78183’, but also on the new It is generally accepted that, under the standard variety S. viminalis × S. schwerinii current commercial and energy supply ‘Tora’. For example, in 2002 S. viminalis × conditions, that energy from short-rotation S. schwerinii × S. viminalis ‘Sven’ (930824) willow coppice is only marginally economic recorded 110% of the annual yield of S. at best. However, a range of economic mod- viminalis × S. schwerinii ‘Tora’. Addition- els haa shown (McBurney and Brigg, 1996; ally, these data also illustrate that annual dry Rosenquist and Dawson, 2004) that yield matter yields from a first coppicing cycle are has an important part to play in improving generally in the region of 20% lower than this situation and ensuring the economic those from the second cycle. In this particular sustainability of the enterprise. Rosenquist case, the improved yield in the second cycle and Dawson (2004) demonstrated that, in is in cumulative response to site capture and Northern Ireland, assuming a £40 per tonne to the fact that increasing harvesting interval dry matter return, a yield increase of 20% to gives increased annual dry matter increment 14 tonnes DM/ha/year would produce a (McElroy and Dawson, 1986). 40% increase in gross margin. This sustained improvement in dry matter (DM) and the use of a wide range of Salix species in the breeding programmes has given a situation currently where com- Yield Losses due to Rust mercially sustainable yields of 10–12 tonnes DM/ha/year can be achieved, with the short- To assess the effect of Melampsora rust on term objective of raising that to 14–15 tonnes the productivity of short-rotation coppice, DM/ha/year. In conditions where Salix willow fungicides have been used on the

18 2 year 3 year

16

14

12

10

8

6 Yield (tonnes DM/ha/year) Yield 4

2

0 930722 930725 930769 930824 78183 Jorr Tora Mean Genotype Fig. 17.1. Dry matter yields (tonnes DM/ha/yr) from two comparative trials, showing annual yields from a first, 2-year, cycle and a second, 3-year, cycle. 198 W.M. Dawson et al.

rust-sensitive variety Salix burjatica Yield losses in rust-affected varieties ‘Korso’. Unlike other major agricultural result from premature leaf fall and a reduc- commodities, the use of fungicides for the tion in photosynthetic area, but also from control of foliar rust in short-rotation shoot death in the years following infection. coppice willow for energy is not an option Leaf fall can reach significant levels early in principally on a cost basis. The economic the growing season, with the rust-sensitive burden of regular fungicide application variety S. burjatica ‘Korso’ showing in could not be justified on such a high- excess of 50% defoliation by mid-July volume, low-value crop. Additionally, the (McCracken and Dawson, 1994). Where density of the growing crop, particularly in serious rust infection occurs in the year the second and third years of the rotation, following coppicing, shoot death has been produced a major practical problem in recorded at levels approaching 60% (Fig. obtaining effective fungicide coverage. 17.3). This was reduced to 5% in the Also, the use of significant volumes of fungicide-treated plots in this single-variety pesticide on an environmentally sensitive planting. However, where fungicides were crop, and often in or near environmentally not applied, the resulting weakening of the sensitive areas, would not be tolerated. stool, and subsequent increased competi- In these fungicide trials, benodanil tion from weed growth, resulted in complete (Calirus™) and myclobutanil (Systhane™) crop failure. provided effective control of the rust, but only when applied in a protectant programme at 14-day intervals, beginning in mid-May before the rust had an Mixtures: Early Trials opportunity to establish itself (McCracken and Dawson, 1998). In these circumstances, These serious reductions in yield and DM yield from the sprayed plots was subsequent crop failures were not totally significantly greater than from the unexpected, given the dynamic nature of unsprayed plots. In the Systhane™-treated the pathogen and the clonally propagated plots, total DM yield was 78% higher than nature of the crop. However, if the concept the unsprayed control, indicating the of producing a carbon dioxide neutral devastating effect on productivity of source of renewable energy from short- Melampsora rust on susceptible Salix rotation coppice willow was to progress, varieties (Fig. 17.2). an alternative disease-control strategy was

250

200

150

Yield (kg) Yield 100

50

0 Calirus Systhane Topas Unsprayed Treatment Fig. 17.2. Yield (kg DM) of Salix burjatica ‘Korso’ treated with a range of rust fungicides. Short-rotation Coppice Willow Mixtures and Yield 199

70

60

50

40

30 % Shoot death 20

10

0 Calirus Systhane Topas Unsprayed Treatment Fig. 17.3. Percentage shoot death in the year following coppicing in Salix burjatica ‘Korso’ treated with a range of rust fungicides. required, and the potential of introducing 2001). There is strong evidence to support diversity, and hence stability, into the sys- the proposition that clonal yield response tem was investigated, and the use of mix- is highly site-specific; however, evidence tures began. Initially, these were limited from a range of sites indicates that the mixtures made up of 4–6 components, and yield response of mixtures is maintained they were planted as structured intimate irrespective of site (Armstrong, 2000). mixtures, i.e. the components were always Having demonstrated, over a number of planted in the same order rather than totally harvesting cycles, the yield advantages of randomly (Anon., 1998). The first trials mixtures containing relatively small num- were established in 1987. Yield estimates bers of components, work initiated in 1996 taken in the dormant season throughout the began to evaluate the effects of mixtures life of the coppice system showed that final with up to 20 components, and planted dry weight yields from the mixtures were at three densities: 20,000, 15,000 and always significantly higher (up to 40%) 10,000/ha. A 5-way mixture was established than the mean of its component varieties with three components known to be highly grown in monoculture. These differences rust susceptible. These were combined with were not apparent in the first year after a further five genotypes with reduced sus- coppicing, but became significant in year 2, ceptibility, in a 10-way mixture. These ten maintaining and enhancing this difference were included with five more genotypes in a until harvest in year 3 (Fig. 17.4) 15-way mixture, and a further five to form a (McCracken and Dawson, 2001). 20-way mixture (McCracken and Dawson, When the contribution of the individual 2001). Hence, the susceptible varieties component species genotypes was analysed, formed 60% of the 5-way and 30%, 20% and it was apparent that whereas the mixture, in 25% of the 10-, 15- and 20-way mixtures, total, outyielded its component parts, they respectively. The varieties included in the individually varied in their response to investigation were representative of breed- inclusion in mixtures. In the early trials, ing programmes in Finland and Sweden, S. viminalis ‘Bowles Hybrid’, S. burjatica and older varieties from various sources, ‘Germany’ and S. viminalis ‘683’ all showed which had been screened in selection trials improved yields in mixtures, whereas at the Northern Ireland Horticulture and S. × dasyclados did not show the same Plant Breeding Station. The 20 varieties positive response (McCracken and Dawson, comprised 11 straight S. viminalis varieties, 200 W.M. Dawson et al.

including S. viminalis ‘78183’ as the stan- aquatica ‘V7534’, S. schwerinii × aquatica dard. The varieties and their yields are listed ‘V7533’ and S. schwerinii × viminalis × in Table 17.1. dasyclados ‘V7531’. These yields were Grown in monoculture, the highest equivalent to 29.9, 25.9 and 24.9 tonnes yields were obtained from S. schwerinii × DM/ha. At the other end of the scale,

2.5 year 1 year 2 year 3

2.0

1.5

Yield (kg) Yield 1

0.5

0 S. × dasyclados S. burj. Germany S. vim. 683 Mixture Genotype Fig. 17.4. Mean dry matter yields (kg per stool) from four Salix varieties grown in monoculture and as a 4-way mixture.

Table 17.1. Dry matter yield (first rotation) from 20 Salix species/varieties grown at three planting densities (kg 100 m−2).

Genotype 10,000/ha 15,000/ha 20,000/ha Mean % of 78183

S. schwerinii × aquatica ‘V7534’ 306.8 81.2 308.6 298.9 173 S. schwerinii × aquatica ‘V7533’ 262.1 239.6 274.8 258.9 150 S. schwerinii × viminalis × dasyclados‚ ‘V7531’ 280.1 254.0 215.6 249.9 145 S. viminalis ‘78195’ 249.0 227.0 236.6 237.5 137 S. viminalis ‘77683’ 227.9 207.1 242.4 225.8 131 S. viminalis × aquatica ‘V7503’ 211.9 177.6 228.1 209.2 121 S. viminalis ‘Orm’ 241.6 191.0 188.8 207.1 120 S. viminalis ‘Gigantea’ 212.2 173.6 208.4 98.0 115 S. viminalis ‘77699’ 97.5 169.8 192.8 186.7 108 S. viminalis ‘Ulv’ 180.3 165.7 196.8 180.9 105 S. viminalis ‘78101’ 167.0 159.5 196.5 174.3 101 S. viminalis ‘78183’ 174.0 151.7 192.3 172.7 100 S. viminalis ‘781181’ 151.4 157.4 162.4 157.1 91 S. molissima undulata ‘SQ83’ 193.3 139.2 133.4 155.3 90 S. viminalis ‘77082’ 160.2 125.4 166.9 150.8 87 S. viminalis × caprea ‘V189’ 105.4 136.8 139.4 124.9 72 S. viminalis ‘Gustav’ 105.1 96.1 123.5 108.8 62 S. dasyclados × aquatica ‘V7511’ 81.3 66.0 83.1 76.8 44 S. burjatica ‘Germany’ 71.1 64.9 78.4 71.5 41 S. dasyclados × caprea ‘V794’ 53.3 54.8 75.4 61.2 35 Short-rotation Coppice Willow Mixtures and Yield 201

S. dasyclados × aquatica ‘V7511’, S. fact, due to competitive effects of the burjatica ‘Germany’ and S. dasyclados × less-susceptible components and their caprea ‘V794’ produced yields equivalent to proximity in randomly planted mixtures, 7.7, 7.1 and 6.1 tonnes dry matter. These their survival and productivity were often varieties were also the three rust-susceptible poorer in mixtures than in monoculture. genotypes included in the mixtures. When the contribution of the various The yields of these 20-component geno- components to the mixture was analysed, it types grown in monoculture were compared was clear that not all contributed positively with equivalent areas grown in various to the increased yields obtained from mixture configurations. The yield from all of mixtures. In all cases, the rust-susceptible the mixtures was greater than the mean yield genotypes produced significantly less in the from an equivalent area of the constituent mixture than their predicted value, calcu- varieties grown in monoculture. Thus the lated from the percentage of the mixture the results from these mixtures, containing up to component occupied. For example, it was 20 constituents, mirror the results from the postulated that an individual genotype earlier mixture trials. The increases in yield within a 20-way mixture would contribute from the mixtures represented 163% in the 5% of the total yield of that mixture. Where case of the 5-way mixture, 111% in the the productivity of a mixture was calculated 10-way mixture, 126% in the 15-way as the yield from an equivalent area of mixture and 119% in the 20-way mixture the component genotypes grown in mono- (Fig. 17.5). The yield increases recorded in culture, these rust-susceptible genotypes the mixture plots were independent of performed marginally better. This was planting density. attributable to the competitive effects of A number of other important issues also the remaining, less-susceptible genotypes became apparent from this investigation. within the mixture (McCracken and Where component genotypes were rust sus- Dawson, 2003). ceptible prior to their inclusion, in mixtures Other varieties gave significantly higher they did not improve their performance. In yields than their predicted contribution, e.g.

350 Mixture Mono

300

250

200

Yield (kg) Yield 150

100

50

0 ×5 ×10 ×15 ×20 Mixture Fig. 17.5. Dry matter yield from 100 m2 of 5-, 10-, 15- and 20-way mixtures, compared to an equivalent area of the components grown in monoculture. 202 W.M. Dawson et al.

S. viminalis ‘Orm’ contributed 11.5% of the were unable to compensate fully for the yield in the 20-way mixture, compared with losses of the three rust-susceptible compo- its expected 5%. Similarly S. schwerinii × nents. Although they grew more vigorously, aquatica ‘V7533’, S. schwerinii × aquatica they were unable to make up the shortfall ‘V7534’, S. viminalis ‘78195’, S. viminalis (Fig. 17.5). However in the 10-way mixture, ‘78683’ all contributed a significantly greater where the rust-susceptible varieties consti- proportion than expected of the yield in the tuted 30% of the components, the remain- 20-way mixture. These varieties are well ing varieties not only compensated for suited to exploiting the diversity of growth their loss but also produced a significantly habit within mixtures. They also have a higher dry matter yield, than the 5-way greater vigour, being the highest yielding mixture. The same was true for the 15- and varieties in monoculture. The remainder of 20-way mixtures, where susceptible geno- the components contributed at, or about, types occupied progressively less of the their expected levels, and although they did space. The inclusion of more than 10 not add to the productivity of the mixture in components in the mixture did not have terms of DM yield, they contributed to its any significant effect on final productivity. sustainability by increasing the diversity However, there is a strong argument that of the mixture and thereby reducing the the increased diversity in the 15- and 20- selection pressure in the disease way mixture would increase sustainability (McCracken and Dawson, 2001). Although more detailed work requires to be done, it is clear that where losses of components in a Yield Compensation mixture, for whatever reason, remain below 30%, the remaining components can fully The dry matter yield figures for 5-, 10-, 15- compensate in terms of dry matter yield. and 20-way mixtures, compared with yield This is confirmed by earlier studies in from an equivalent area of their compo- planting density, where high planting den- nents grown in monoculture, illustrates a sities of 20,000/ha tended, over a period further advantage of diverse mixtures. The of two or three rotations, to self-thin 5-way mixture contained three genotypes, to 13,000–15,000/ha without materially which were known to have significant sus- affecting their productivity. ceptibility to rust. Subsequently, their sus- It is important to maximize this ability ceptibility increased until the impact of the of mixtures to compensate for the loss of disease was sufficient to reduce their con- individual components through disease or tribution to final dry matter figures to unac- other factors. In this respect, the planting ceptable levels. Although their inclusion in pattern of the mixture has an important part mixtures with more components reduced to play. Ideally, the mixture should be disease levels, DM yields still remained planted totally randomly, with each compo- below acceptable levels and these varieties nent as widely separated from itself as the effectively made little or no contribution to number of genotypes in the mixture allows. overall mixture yields. In the case of the However, in commercial practice a compro- 5-way mixture, these susceptible varieties mise is necessary to deal with planting made up 60% of the components planted mechanization. Here, where full planting and this level was successively ‘diluted’ to rods are used in automatic planters, ran- 30%, 20% and 15%, with the addition of 5, domly planted short runs (8–10 cuttings) of 10 and 15 less-susceptible genotypes to the the same genotype offer an acceptable alter- mixtures. The 5-way mixture was signifi- native. Although line mixtures or mosaics cantly less productive than the 10-, 15- or may be equally productive, they do not offer 20-way mixture, but the 10-, 15- and 20-way the same opportunities for yield compensa- mixture yields did not differ significantly tion following the loss of component(s). from each other. Thus, in the 5-way mixture Hence such mixtures will be less sustainable the remaining two productive genotypes in the long term (Pei et al., 1996). Short-rotation Coppice Willow Mixtures and Yield 203

Stool Survival indicated a genotype which was not well adapted to mixtures (Anon., 1998). This was The ability of a genotype to survive within a due to its reduced competitive ability as mixture is obviously an important aspect of a consequence of its later leafing up and its contribution to overall yield. In early shorter growth habit. trials it became obvious that survival in Similar survival data were recorded on monocultures was significantly higher later mixture plantations and, in these cases, than in mixtures in all of the genotypes the response varied with the genotypes’ investigated. Figure 17.6 shows the compar- inherent susceptibility to rust. For resistant ative survival rates for six component genotypes, or where rust has only been genotypes used in the early mixture recorded late in the season and at very investigations. low levels, e.g. S. schwerinii × viminalis × In monoculture, where there are dasyclados ‘V7534’, survival remained uniform competitive effects from adjacent at over 80%, with no difference between stools of the same genotype, survival is good the first and second harvests in 1999 – 90% for all varieties after the first harvest- and 2002. Where the genotype had a low ing cycle. However, unless the genotype is to moderate susceptibility to rust, e.g. well adapted to exploiting the diversity of S. viminalis 77118, and its inclusion in growth habit and conditions within a mix- mixtures had the effect of reducing rust to ture, survival will be affected. All of these acceptable levels, survival was over 90%. varieties showed a significant reduction in Although reduced at the second harvest, it survival in mixtures. However, this reduc- still maintained levels above 80% in mix- tion was at or below the critical 30% for tures. However, survival of highly suscepti- yield compensation. These trials were ble varieties, e.g. S. dasyclados × caprea planted at an initial density of 20,000/ha and ‘V794’ was reduced after the first harvesting survival of over 70% in all genotypes except cycle, in some instances below the critical S. × calodendron resulted in a reduced 70% where yield compensation can not density of 14,000/ha. In the case of S. × make up the loss. This was further reduced calodendron the mixture survival of 40% after the second harvest (Fig. 17.7), and in all

100 mono mix

75

50 Survival

25

0 S. vim BH S. × dasy- S × calod- S. burj S. vim. 683 S. mol. 83 clados endron GermanyBH Genotype Fig. 17.6. Percentage survival after the first 3-year harvest of willow genotypes grown in monoculture and mixtures. 204 W.M. Dawson et al.

100 1999 2002

75

50 % Survival

25

0 ×5 ×10 ×15 ×20 Mono Mixture Fig. 17.7. Percentage survival of Salix dasyclados × caprea ‘V794’ in 1999 and 2002 after one and two harvesting cycles grown as a monoculture and in mixtures. cases the survival rate was below 70% it was not at all clear whether mixtures (McCracken et al., 2001). composed of the commercially available, The competitive ability of highly rust- high-yielding, improved varieties, which susceptible genotypes within mixtures is were almost exclusively S. viminalis or S. so impaired that survival is reduced below viminalis hybrids, would function in the the level at which the remaining mixture same way as more diverse mixtures. There components can compensate for the yield are indications that, because of the wide loss. Not only is survival adversely affected, range of susceptibility within these new but the impact of the disease reduces the varieties and the improved resistance capacity of those surviving to contribute introduced via S. schwerinii, these largely effectively to overall yield. S. viminalis mixtures may function in the same way as more diverse mixtures. This is particularly so where the disease popula- tion is dominated by asexual reproduction Mixture Diversity and where, as a consequence, there is less opportunity for diversity through recombi- It has already been observed that diversity nation as in sexually derived populations within mixtures is important in terms of the (Samils et al., 2002). To evaluate mixtures sustainability of the cropping system, par- containing genetically less diverse constitu- ticularly in respect of reducing the selection ents, the focus of the work has turned to pressure on the rust disease organism. How- S. viminalis-only mixtures. These investiga- ever, the Swedish Breeding programme, tions included seven S. viminalis varieties in particular, has tended to focus on S. from varying sources, including four of the viminalis and S. viminalis hybrids in commercially available improved varieties producing new operational varieties for bio- from the Swedish Breeding Programme (S. mass production. In the early work carried viminalis ‘Orm’, ‘Ulv’, ‘Jorr’ and ‘Jorunn’). out on genotype susceptibility to M. epitea S. viminalis ‘Ulv’ was known to be rust pathotypes, all varieties, which had S. susceptible, as was S. viminalis ‘77082’. viminalis in their parentage, were suscepti- The remaining two varieties S. viminalis ble to the same range of pathotypes (Pei ‘Gustav’ and S. viminalis ‘78195’ had low et al., 1996). It was apparent that the patho- levels of susceptibility. The trial was estab- type composition of the pathogen popula- lished in 2000 and in addition to the seven tion varied from region to region. However, varieties grown as monocultures, they were Short-rotation Coppice Willow Mixtures and Yield 205

also grown as a 7-way mixture. The rust- significant yield advantage has not been susceptible genotypes were combined in a recorded. range of 3-way mixtures with each of the It was noted in these early mixture trials remaining five genotypes (McCracken et al., (Anon., 1998) that, where varieties were 2004). Total dry matter yields after the first grown in monoculture, stool size and vigour harvesting cycle in 2003 are shown in were uniform but the same stools in mixed Fig. 17.8. plantings were much more variable and Although the 7-way mixture was more were influenced by the size and vigour of productive than an equivalent area of adjacent stools. Additionally, the mixture the constituent varieties grown as mono- provided a much more diverse canopy in cultures, this difference was not statistically terms of leaf characteristics and length and significant. This is in contrast to previous number of rods. This allowed the mixture to results where, without exception, mixtures exploit the growing space to the full. How- were more productive statistically than ever in the S.viminalis ‘mixtures’ the situa- the mean of their components grown in tion was more similar to the monoculture, monoculture. with uniform stools and even canopy. In the earliest mixture trials, established In another respect the S viminalis ‘mix- in Northern Ireland in 1987, the level of tures’ behaved in a similar way to the more species diversity was high, with only two of diverse mixtures: when the contribution of the component varieties being S. viminalis. the various components to the mixture was These trials indicated a highly significant analysed, it was clear that not all contributed increase in dry matter yield in mixtures, positively to the yield (Fig. 17.9). even when disease levels were generally low It was assumed as before that an indi- (Anon., 1998). This improvement in yield vidual genotype within a 3-way mixture was also recorded in later trials, established could be expected to contribute 33.3% of in 1996, where the number of mixture com- the total yield of that mixture, because it ponents was higher, but the overall level occupied one third of the space. Similarly of species diversity lower (McCracken and in a 7-way mixture individual components Dawson, 2003). However, in these current could be expected to contribute 14.3% of the trials, where there is no species diversity, a DM yield. However, this was not the case

350 Monoculture Mixture

300

250

l 200 rviva u S 150 %

100

50

0 2-way 3-way(1) 3-way(2) 3-way(3) 3-way(4) 3-way(5) 7-way Mixture Fig. 17.8. Comparative dry matter yields (kg) from 100 m2 of a range of Salix viminalis mixtures, compared with equivalent areas of the components grown in monoculture. 206 W.M. Dawson et al.

60 Gen 47 Gen 48

40

20

0 % Difference −20

−40

2-way 3-way(1) 3-way(2) 3-way(3) 3-way(4) 3-way(5) 7-way −60 Mixture Fig. 17.9. Differences between expected and actual contribution to mixture yields from two Salix viminalis varieties included in 2-way (T8), 3-way (T9, 10, 11, 12 and 13) and 7-way (T14) mixtures. with some varieties, e.g. S. viminalis oceanic conditions in western areas of the ‘77082’, which consistently contributed UK, the use of mixtures has been shown more to the mixture than expected in a range to offer a sustainable approach to disease of mixtures, and S. viminalis ‘Ulv’, contri- control. With this in mind, the main buting less than expected. Generally only conclusions are: S. viminalis ‘Orm’ and S. viminalis ‘Jorr’ • Evidence is clear that where disease made positive contributions to the mixture pressure is high, the planting of large above their expected value, with the others areas of a single genotype, even where either contributing at or below their that genotype is less susceptible or expected values. These types of genotype resistant to rust, is not to be still make a positive contribution in terms of recommended. the diversity and their influence in reducing • The yield of the newer genotypes from the impact of disease. the dedicated breeding programmes Further work will be necessary to in Sweden and the UK show highly sig- unravel the interaction between mixture nificant yield increases over selected components and its influence on yield, dis- standards. Consequently, only these ease impact and sustainability. It should be improved genotypes should be used in noted that non-uniform mixtures containing commercial developments. a number of heavy stools randomly within • Generally, yield from diverse mixtures the plantation may be a practical difficulty is greater than the equivalent yield of for mechanical harvesting. their component genotypes grown in monoculture. • However, with less diverse mixtures Conclusions these yield increases have not been recorded, but the disease-suppression Where climatic conditions put additional aspect of the use of mixtures remains. disease pressure on short-rotation coppice This latter is important given that willow plantations, such as the cool, wet most of the improved genotypes Short-rotation Coppice Willow Mixtures and Yield 207

commercially available are S. viminalis Christersson, L. (1987) Biomass production by or S. viminalis hybrids. irrigated and fertilised Salix clones. Biomass • Increased diversity in mixtures 12, 83–95. resulted in yield compensation when Dawson, M. and McCracken, A. (1998) Clonal selection in willow (Salix) grown as short rotation components of the mixture were lost coppice for energy production. Tests of Agro- due to disease or other factors. It chemicals and Cultivars 19. Annals of Applied has been shown that where a mixture Biology 132 (suppl.), 56–57. loses up to 30% of its component geno- Larsson, S. (1997) Commercial breeding of types it can fully compensate for the willow for short rotation coppice. Aspects of potential yield loss. Consequently, the Applied Biology – Biomass and Energy Crops inclusion of, at least, six genotypes in a 49, 215–218. mixture would appear to be prudent. Larsson, S. (2001) Commercial varieties from • Where genotypes have been shown to the Swedish breeding programme. Aspects of be significantly rust susceptible their Applied Biology – Biomass and Energy Crops II. 65, 173–182. inclusion in mixtures does not improve Lindegaard, K., Parfitt, R., Donaldson, G., Hunter, T., their performance or increase their Dawson, M., Forbes, G., Carter, M., Whinney, sustainability. C., Whinney, J. and Larsson, S. (2001) Compara- • Completely random planting configu- tive trials of elite Swedish and UK biomass rations in mixtures would seem to willow varieties. Aspects of Applied Biology – provide the greatest opportunity for Biomass and Energy Crops II 65, 183–198. yield compensation, rather than line McBurney, S. and Brigg, J. (1996) Financial analysis mixtures or small block mosaic mix- of willow coppicing. In: Proceedings of the Con- tures. In practical commercial terms, ference ‘Willow a Commercial Opportunity’, the short line random mixtures October, Enniskillen, Northern Ireland. McCracken, A.R. and Dawson, W.M. (1994) Effects of achievable with available commercial Melampsora rust on the growth and develop- planters would seem to be the best ment of S. burjatica ‘Korso’ in Northern Ireland. practical compromise. European Journal of Forest Pathology 24, 32–39. • There are differences in how individ- McCracken, A.R. and Dawson, W.M. (1998) Short ual component genotypes contribute to rotation coppice willow in Northern Ireland the overall yield of mixtures. However, since 1973: development of the use of mixtures provided this is not significantly in the control of foliar rust (Melampsora negative, their inclusion is justified spp.) European Journal of Forest Pathology by the diversity they contribute to the 28, 307–311. plantation and the positive effect they McCracken, A.R and Dawson, W.M. (2001) Disease effects in mixed varietal plantations of willow. therefore have on sustainability. Aspects of Applied Biology – Biomass and Energy Crops II 65, 255–262. McCracken, A.R. and Dawson, W.M. (2003) Rust dis- ease (Melampsora epitea) of willow (Salix spp.) References grown as short rotation coppice (SRC) in inter- and intra-species mixtures. Annals of Applied Anon. (1980) The willow as a genetic resource. Biology 143, 381–393. Annual Report of Long Ashton Research Station, McCracken, A.R., Dawson, W.M. and Bowden, G. Bristol, pp. 45–49. (2001) Yield responses of willow (Salix) grown Anon. (1998) Establishment and monitoring of in mixtures in short rotation coppice (SRC). large-scale trials of short rotation coppice for Biomass and Bioenergy 21, 311–319. energy. Contractor report for Energy Technology McCracken, A.R., Dawson, W.M. and Carlisle, D. Support Unit on behalf of the Department of (2004) Benefits of growing short rotation Trade and Industry ETSU B/W2/00541/REP. coppice (SRC) willow in genotype mixtures. Armstrong, A. (2000) National trials network – Proceeding of 2nd World Conference on preliminary results and update. Proceedings Biomass for Industry and Energy, Rome, May. of the Short Rotation Coppice and Wood Fuel McElroy, G. and Dawson, M. (1986) Biomass from Symposium – From Research to Renewable short rotation coppice willow on marginal land. Energy. Alice Holt, Surrey, UK, pp. 12–18. Biomass 10, 225–240. 208 W.M. Dawson et al.

Pei, M.H., Royle, D.J. and Hunter, T. (1996) Stott, K. (1984) Improving the biomass potential of Pathogenic specialisation in Melampsora willow by selection and breeding. In Ecology epitea var. epitea on Salix. Plant Pathology 45, and Management of Forest Biomass Systems. 679–690. Report 15. Swedish University of Agricultural Rosenquist, H. and Dawson, M. (2004) Economics of Sciences, Uppsala, Sweden, pp. 233–260. willow growing in Northern Ireland. Biomass Stott, K., Parfit, R., McElroy, G. and Abernethy, W. and Bioenergy (in press). (1987) Productivity of Coppice Willow in Bio- Samils, B., McCracken, A., Dawson, M. and Gullberg, mass trials in UK. Occasional publication of Long U. (2003) Host specific genetic composition Ashton Research Station, Bristol. of Melampsora larici-epitea populations on Tabbush, P. and Parfitt, R. (1996) Poplar and Willow two Salix viminalis genotypes in a mixture Clones for Short Rotation Coppice. Research trial. European Journal of Plant Pathology 109, Information Note 278. Forestry Commission 183–190. Research Division, Surrey, UK. 18 Effect of Preventative Fungicide Sprays on Melampsora Rust of Poplar in the Nursery

R.C. Sharma, S. Sharma and A.K. Gupta Department of Mycology and Plant Pathology, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan-173 230 Himachal Pradesh, India

Introduction planted in nursery beds at 33°N77°E and 1500 m above sea level, in the first week of Poplars are prone to many insect pests and February 1999 and 2000. Twenty cuttings diseases which may account for losses of up were planted at 45 × 45 cm spacing in each to one-third total yield. Melampsora ciliata plot, replicated three times. Fungicides, Barclay, a microcyclic autoecious rust carbendazim (0.05%), benomyl (0.05%), confined to the Indian and Nepal Himala- difenoconazole (0.02%), penconazole yas, is serious not only on Populus ciliata, (0.06%), hexaconazole (0.1%), captan an indigenous species, but also on exotic (0.2%), copper oxychloride (0.3%), foltaf hybrids and species of Populus (Singh (0.25%), mancozeb (0.25%), dinocap et al., 1983; Vannini et al., 1995; Sharma (0.1%), Nimbecidine (0.2%), Neem Jeevan and Sharma, 2000, 2001). The pathogen is Spray (0.3%) and Kranti spray oil (0.4%) distributed between 700 and 3130 m above were applied as sprays at fortnightly inter- sea level and can cause moderate to severe vals. In all, six sprays were applied during losses in nurseries and plantations (Sharma the period 24 June to 2 September. and Sharma, 2002). The disease causes premature defoliation c. 2–3 months prior to normal leaf fall, thereby affecting plant Disease incidence and severity growth (Sharma et al., 2001). In light of the seriousness of the disease, the efficacy The percentage disease incidence was of five systemic and eight non-systemic calculated by counting the infected and fungicides, including three neem-oil total number of leaves on each seedling. formulations, was tested against leaf rust Disease severity was rated on a 0–7 scale of P. ciliata. on the basis of leaf area covered by rust pustules, as 0, 0.1–1.0, 1.1–5.0, 5.1–10.0, 10.1–25.0, 25.1–40.0, 40.1–65.0 Methodology and > 65%. Uredinial pustules per leaf were calculated by counting the uredinial Plant material and fungicides pustules of 20 tagged leaves selected from top, middle and bottom positions Cuttings (18–23 cm) of the highly rust- of plants in each replication, and means susceptible clone P. ciliata ‘Theog’ were calculated. ©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 209 210 R.C. Sharma et al.

Apparent infection rate Results and Discussion The apparent infection rate (r) for different treatments was calculated according to the Minimum disease incidence was recorded method of Van der Plank (1963), using the following treatment with hexaconazole logistic equation: (30.39%) and was not significantly different (P = 0.05) on penconazole-treated plants − − r = [2.3/(t2 t1)] log10[x2/(1 x2)] (Table 18.1). The three ergosterol- − log10[x1/(1 x1)] biosynthesis inhibiting (EBI) fungicides, where r denotes the apparent infection rate hexaconazole, penconazole and difeno- conazole were superior to all other treat- per unit per day; t2 − t1 is the time interval ments. Ruaro and May (1996) reported the between first and last observations; x1 efficacy of difenoconazole, captan and and x2 are the proportions of diseased plant mancozeb against urediniospore germina- parts; and 1 − x1 and 1 − x2, the proportion tion of Melampsora medusae under in vitro of healthy plant parts at t1 and t2, respectively. conditions. Among the three neem-oil for- mulations, Nimbecidine was most effective, Area under the disease progress curve with 50.64% disease incidence compared to 76.50% for the control. From the severity data, the area under Significant reductions in uredinial pus- the disease progress curve (AUDPC) was tules per leaf were recorded when plants calculated in accordance with Shaner and were sprayed with any of the fungicides Finney (1977), as follows: (Table 18.2). The least number of uredinial pustules per leaf (3.8) was recorded on n AUDPC = ∑ ()()YYXxiii++12+−/ 12 plants treated with hexaconazole, which i + 1 was not significantly different from where Yi denotes disease severity (%) at the numbers on plants sprayed with the ith observation, Xi denotes time (days) difenoconazole, penconazole, dinocap, at the ith observation, and n denotes total Nimbecidine or Kranti spray oil. Foltaf was number of observations. least effective, with 22.1 uredinial pustules

Table 18.1. Effect of preventive sprays of fungicides on leaf rust of nursery-grown poplar in nursery.

Disease incidence (%)

Fungicide 1999 2000 Mean

Carbendazim 27.62 (31.69) 69.20 (56.35) 48.41 (44.02) Benomyl 22.10 (32.29) 75.92 (60.69) 55.40 (48.49) Difenoconazole 29.49 (32.88) 51.75 (46.01) 40.42 (39.31) Penconazole 29.49 (32.88) 41.47 (40.04) 35.48 (36.46) Hexaconazole 28.90 (32.51) 31.87 (34.36) 30.39 (33.44) Captan 56.60 (48.83) 70.61 (57.18) 63.61 (53.01) Copper oxychloride 44.00 (41.55) 51.78 (46.02) 47.89 (43.79) Foltaf 49.62 (44.79) 69.05 (56.24) 59.34 (50.51) Mancozeb 38.75 (38.50) 54.55 (47.62) 46.65 (43.06) Dinocap 47.00 (43.28) 37.90 (37.98) 42.45 (40.63) Nimbecidine 40.27 (39.37) 61.01 (51.43) 50.64 (45.40) Neem Jeevan spray 70.67 (58.13) 79.28 (63.34) 74.98 (60.74) Kranti spray oil 58.00 (49.61) 76.94 (61.31) 67.47 (55.46) Control 67.50 (55.26) 85.50 (67.77) 76.50 (61.51)

CD0.05 3.60

Figures in parentheses are arcsine transformed values. CD0.05 = critical difference at 5% level of significance. Preventative Fungicide Sprays on Melampsora of Poplar 211

per leaf compared to 35.1 on the control. The effect of fungicide sprays on the Earlier efficacy of cyproconazole against poplar leaf rust apparent infection rate and M. larici-populina and carbendazim and area under the disease progress curve is pre- cyproconazole against M. ciliata has been sented in Table 18.3. The least apparent demonstrated (Khan et al., 1988; Boudier, infection rate (0.0044/unit/day) was recor- 1992; Khan, 1994). ded on plants treated with hexaconazole; this was not significantly different (P = 0.05) from that on plants treated with pencona- Table 18.2. Effect of preventive sprays of zole or difenoconazole. Maximum apparent fungicides on number of urediniopustules per infection rate was on foltaf-treated plants leaf in nursery-grown poplar. (0.0138/unit/day) compared to 0.0171/unit/ day on control plants. The losses from dis- Urediniopustules eased areas were also checked, by reducing per leaf the disease at the beginning of season or Fungicide 1999 2000 Mean through decreasing the apparent infection rate during the growing period (Van der Carbendazim 5.9 12.5 9.2 Plank, 1963). Measurement of the apparent Benomyl 15.6 13.2 14.4 infection rate was useful for rapid compari- Difenoconazole 4.2 4.8 4.5 son of the effectiveness of various treatments Penconazole 6.5 5.3 5.9 Hexaconazole 5.3 2.2 3.8 (Sharma and Sharma, 1999). Captan 16.8 16.5 16.7 The area under the disease progress Copper oxychloride 14.0 6.0 10.0 curve (AUDPC) was least on hexaconazole- Foltaf 13.4 30.8 22.1 treated plants (4.01), and was not signifi- Mancozeb 7.5 16.5 12.0 cantly different from penconazole- (4.67) Dinocap 15.1 2.5 8.8 or difenoconazole-treated plants. Of the Nimbecidine 10.6 7.2 8.9 neem-oil formulations, AUDPC was least on Neem Jeevan spray 17.8 3.0 10.4 plants sprayed with Nimbecidine (6.87) Kranti spray oil 12.5 5.5 9.0 as compared to 11.33 in unsprayed Control 26.2 44.0 35.1 plants. AUDPC also provides a quantitative CD 5.3 0.05 measure of disease development and its

Table 18.3. Effect of preventive sprays of fungicides on poplar leaf rust apparent infection rate (r) and area under the disease progress curve (AUDPC).

Apparent infection rate (r) AUDPC

Fungicide 1999 2000 Mean 1999 2000 Mean

Carbendazim 0.0127 0.0077 0.0102 3.71 13.30 8.51 Benomyl 0.0054 0.0130 0.0092 4.84 10.22 7.53 Difenoconazole 0.0042 0.0063 0.0052 3.87 7.14 5.51 Penconazole 0.0049 0.0056 0.0052 4.16 5.19 4.67 Hexaconazole 0.0045 0.0043 0.0044 3.89 4.14 4.01 Captan 0.0091 0.0137 0.0134 7.43 10.25 8.84 Copper oxychloride 0.0034 0.0164 0.0099 5.97 7.09 6.53 Foltaf 0.0191 0.0086 0.0138 6.87 10.08 8.45 Mancozeb 0.0105 0.0046 0.0075 5.45 8.42 6.93 Dinocap 0.0116 0.0079 0.0098 6.68 4.65 5.67 Nimbecidine 0.0124 0.0025 0.0074 5.68 8.07 6.87 Neem Jeevan spray 0.0089 0.0042 0.0066 8.83 10.69 9.76 Kranti spray oil 0.0082 0.0056 0.0052 8.16 10.35 9.23 Control 0.0136 0.0207 0.0171 9.27 13.40 11.33

CD0.05 0.0081 0.87 212 R.C. Sharma et al.

intensity (Lee Compbell, 1998; Sharma Lee Compbell, C. (1998) Disease progress in time: and Sharma, 1999). Lower AUDPC values modelling and data analysis. In: Jones, D.G. (ed.) in hexaconazole and penconazole are due The Epidemiology of Plant Diseases. Kluwer to slow disease progress and poor terminal Publishers, Dordrecht, pp. 181–206. Ruaro, L. and May, L.L. (1996) Characterization of rust disease severity. in poplar (Populus species) and the efficacy of fungicides in vitro over the germination of urediniospores. Revista do Sector de Ciencias Conclusions Agrarias 15, 77–82. Shaner, G. and Finney, R.E. (1977) Effect of nitrogen fertilization on the expression of slow mildewing 1. Hexaconazole was the most effective resistance in Knox wheat. Phytopathology 67, treatment in reducing leaf rust of Populus 1051–1056. ciliata, followed by penconazole and Sharma, R.C. and Sharma, S. (1999) Effect of difenoconazole. fungicides on the development of Cladosporium 2. Among neem-oil formulations tested, leaf spot of poplar in western Himalaya. Acta Nimbecidine was better in checking the Phytopathologica et Entomologica Hungarica disease. 34, 57–62. 3. The least number of uredinial pustules Sharma, R.C. and Sharma, S. (2000) Status and distri- bution of foliar diseases of poplar in Himachal per leaf were recorded in hexaconazole- Pradesh. Indian Phytopathology 53, 57–60. treated plants. Sharma, R.C., Khan, Y., Sharma, S. and Malhotra, R. 4. Apparent infection rates and area (2001) Development of Melampsora ciliata rust under the disease progress curve were on nursery grown poplars in north western least in hexaconazole-, penconazole- and Himalayas. Forest Pathology 31, 313–319. difenoconazole-treated plants. Sharma, S. and Sharma, R.C. (2001) Epidemiology of Melampsora ciliata leaf rust of poplars in India. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 108, 337–344. References Sharma, S. and Sharma, R.C. (2002) Prevalence of poplar leaf rust in Himachal Pradesh. Indian Boudier, B. (1992) Rusts of Salicaceae: remarkable Phytopathology 55, 81–83. efficacy of tebuconazole and cyproconazole. Singh, S., Khan, S.N. and Misra, B.M. (1983) Status of Revue Horticulture 323, 29–30. Melampsora rusts of poplars in India. Indian Khan, Y. (1994) Studies on Melampsora leaf rust of Forester 109, 743–747. Populus species. MSc thesis, Dr Y.S. Parmar Uni- Van der Plank, J.E. (1963) Plant Diseases: Epidemics versity of Horticulture and Forestry, Solan, India. and Control. Academic Press, New York. Khan, S.N., Rehill, P.S., Tiwari, R.K., Rawat, D.S. and Vannini, A., Cecco, D., Monaci, L. and Anselmi, N. Misra, B.M. (1988) Control of poplar rust, (1995) Main tree pathogens of western Himala- Melampsora ciliata in nurseries. Indian Journal of yan forests in Nepal: description and risk of Forestry 11, 253–255. introduction. Bulletin OEPP 25, 455–461. 19 Biocontrol of Rust Fungi by Cladosporium tenuissimum

Salvatore Moricca1, Alessandro Ragazzi2 and Gemma Assante3 1Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira 4, 50121 Firenze, Italy; 2Dipartimento di Biotecnologie Agrarie, Sezione di Patologia Vegetale, Università di Firenze, Piazzale delle Cascine 28, 50144 Firenze, Italy; 3Istituto di Patologia Vegetale, Università di Milano, Via Celoria 2, 20133 Milano, Italy

Introduction the microbial community on the plant rhizosphere and phyllosphere, and play a The use of chemical pesticides in agri- pivotal role in regulating many interactions culture is under increasing public scrutiny, between plants and parasitic microorgan- with mounting concern over possible isms (Jeffries, 1997). Since they are an inte- harmful effects on the environment and gral part of the ecosystem, no alien microbe on human health. Agrochemicals pollute species, toxic substances or chemicals are groundwater, enter the food chain, have introduced into the environment by their a deleterious impact on many living use, and hence they appear to be more organisms and may bring about pesticide environmentally sustainable than other, resistance in plant parasites. Increasing more intrusive, control methods. As a awareness of such possible hazards is result, fungal biocontrol agents (BCAs) prompting a heightened interest in alter- are becoming a promising means to control native strategies (Boland and Kuykendall, fungal diseases and to reduce dependence 1998). on chemical pesticides (Butt et al., 2001). One of the most promising approaches However, developing a reliable and to control economically important crop effective method for the biocontrol of a pests and diseases is by exploiting naturally fungal pathogen is not a straightforward pro- occurring antagonists. Over the past few cess. Several biological control experiments years unprecedented advances have been that were successful in vitro produced made in the biological control of many inconsistent results in the field. Moving damaging insect pests and phytopathogenic from the laboratory or the greenhouse to microorganisms (Butt and Copping, 2000). large-scale field-testing is difficult because, Many fungal pathogens throughout the in the field, the antagonist is subject to envi- world have natural enemies that limit the ronmental influences. Plant disease is the harm they cause. Some of these competitors result of a dynamic interaction over time are non-fungal hyperparasites such as between a pathogen, a plant and the environ- bacteria (Yuen et al., 2001) or mycoviruses ment. The environmental component of the (Brasier, 1990), but most are other fungi. disease triad is crucial for the success of a Antagonistic fungi are a major component of BCA, the limited ecological amplitude of

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 213 214 S. Moricca et al.

which might represent a major constraint. Rust epidemics became particularly While an antagonist has to actively control a severe in the 20th century, after extensive pathogen, over a period that may take from a monocultures had been aggressively estab- week to several months, it must be able to lished in agriculture and forests as a result withstand fluctuations in the physical envi- of advances in plant genetics and the ronment and resist the competing action modernization of agriculture. But such of other, established microorganisms. The developments ignored a basic principle insufficient ecological fitness of the antago- in epidemiology, which is that both the nist inoculum may lower its effectiveness as number of diseases and disease incidence a control agent, while a poor shelf-life may increase in proportion to host abundance. cause lack of persistence, which means that The advantage of identifying, planting, har- the control achieved does not last over a suf- vesting and marketing particular genetically ficiently long term (Whipps and Lumsden, homogeneous crops was therefore, in many 2001). cases, nullified by sudden disease out- This chapter examines whether Clado- breaks, caused directly by low crop diver- sporium tenuissimum Cooke, a destructive sity. The limitless potential for pathogen hyperparasite of rust spores, can be spread in monocultures led to the rapid exploited as a BCA of rust fungi. It reports on selection of rust pathotypes able to over- tests carried out to evaluate C. tenuissimum come host defences. As a consequence, effectiveness against rusts in the genera destructive fungi such as the coffee leaf Melampsora, Cronartium, Peridermium, rust Hemileia vastatrix, the wheat stem Uromyces and Puccinia, focusing on its rust Puccinia graminis, the Melampsora leaf modes of action, the fine-level analysis of rusts of Salicaceae (Populus and Salix), the the fungal host–hyperparasite interface, the Cronartium stem rusts of hard pines, to antifungal compounds it produces, and its name just a few, spread epidemically over ability to reduce disease, both in vitro and in vast areas, causing enormous losses and planta. Since effective biological control is often making it necessary to replace impossible without due consideration of the susceptible crops entirely with non-host ecology of the BCA, as well as of the other species (Littlefield, 1981). partners involved, and an examination of At present, the main strategies to limit their spatial relationships and interaction populations of rust pathogens and maintain with the environment, attention is also paid yield stability are the continual breeding for to the biological nature of the fungus, new disease resistances and the search for in particular to those characteristics that new fungicides (to deal with the selection of enable it to survive in natural habitats and new pathogen genotypes that attack resis- retain activity under varying environmental tant crops and that are resistant to existing conditions. fungicides), as well as crop rotation and the adoption of spatial and/or temporal crop diversity (Wolfe, 1985; Zhu et al., 2000). However, the effectiveness of such The Rust Disease Problem measures is subject to a number of limit- ing factors. Constant improvements in crop The rust fungi (Uredinales) are one of the resistance and the never-ending need for largest groups in the Basidiomycota, with new fungicides are costly to the farmer, the about 5000–6000 species described on a consumer and to society at large. Economic, wide range of hosts, including ferns, gym- environmental and technical reasons often nosperms, and mono- and dicotyledonous make it impractical to use fungicides to angiosperms (Alexopoulos et al., 1996). combat rusts, especially rusts of forest trees. Rust fungi occur worldwide, under varied Other measures, such as the eradication climates, and include destructive species of alternate hosts, scheduled harvesting that are responsible for costly diseases in (particularly of hazard-rated crops and agriculture and forestry. stands), pop-up spacing, crop rotation with Biocontrol of Rust Fungi by Cladosporium tenuissimum 215

non-hosts and standard sanitation proce- These additional criteria are used to sup- dures (removal of infected branches, burn- port the traditional classification because ing of inoculum-bearing debris, etc.) are the inadequacy or lack of description for deficient because of the large geographic several members, and the phenotypic plas- distribution of the pathogens, their wide ticity within individual taxonomic entities, host ranges, their high spore dispersal make typification of species difficult or capabilities, and the autoecism of some impossible. Substrate differences, climate rusts. Planting mixed genotypes with differ- and geographic variations also influence ent disease-resistance profiles may be costly morphological expression. As a conse- and may require particular expertise in quence, morphological features such as cultivation practices, not to mention the fact conidium size, shape, septation, pigmenta- that resistant plant varieties and fungicides tion, surface characteristics (smooth or are the major selective agents of pathogen with ornamentation), conidiophore size virulence. Any single method of control and morphology are often variable and thus has its limitations and it seems that a inconsistent (Morgan-Jones and McKemy, combination of measures in an integrated 1990; Ho et al., 1999). disease-management strategy is needed to Members of this genus display a variety counteract rust fungi. of lifestyles: some commonly occur on their Biological control is a non-chemical hosts as epiphytes, endophytes, or as patho- measure, exploiting microbiological agents gens; others thrive as saprophytes, or even as to provide an additional method to combat hyperparasites (Ellis, 1976; Petrini, 1991; rust disease. Among the common phyllo- Moricca et al., 1999; Dugan and Lupien, plane mycobiota many fungi have been 2002; Larran et al., 2002; Abdel-Baky and reported since the first half of the 20th Abdel-Salam, 2003). century to be associated with rust fructifi- Several taxa in this large genus are prev- cations (Arthur, 1929). Some of these fungi, alently associated with rust sori, and some eg. Sphaerellopsis filum (Biv.-Bern.F) are assumed to be invariably hyperparasites Sutton (teleomorph: Eudarluca caricis (Fr.) of Uredinales (Table 19.1). Cladosporium O. Erikss.), Scytalidium uredinicola uredinicola Speg. is a common necrotrophic Kuhlman et al., Aphanocladium album hyperparasite that destroys rust hyphae and (Preuss) W. Gams, and several species causes coagulation and disintegration of the of Cladosporium, Tuberculina and Verti- cell cytoplasm of a number of hosts, such cillium, have already demonstrated a pro- as Puccinia cestri Dietel and P. Henn. nounced hyperparasitism against different (Spegazzini, 1912), Puccinia recondita spore states of a number of rust fungi Roberge ex Desm. (Ellis, 1976), Cronartium (Kuhlman et al., 1978; Tsuneda et al., 1980; quercuum (Berk.) Miyabe ex Shirai f. sp. Sharma and Heather, 1981a; Wicker, 1981; fusiforme (Morgan-Jones and McKemy, Allen, 1982). 1990), Puccinia violae (Schum.) DC (Traquair et al., 1984) and Puccinia horiana (Srivastava et al., 1985). Spegazzini (1922) reported that Cladosporium uredinophilum Cladosporium Species Hyperparasitic colonized and destroyed Uredo cyclotrauma on Rusts Speg. propagules in Paraguay. Steyaert (1930) described Cladosporium hemileiae The genus Cladosporium is one of the most Steyaert as an effective hyperparasite of widespread and prevalent genera of fungi, the coffee rust fungus Hemileia vastatrix containing over 500 species Some of its spe- in Zaire (Democratic Republic of the Congo). cies have teleomorphs in the ascomycete Powell (1971) encountered Cladosporium Mycosphaerella (Dothideales), but a vast gallicola in galls of Cronartium comandrae majority of taxa are known by their Pk. on Pinus contorta var. latifolia and con- anamorphic state or by criteria such as host sidered the fungus parasitic on aeciospores association or presumed host specificity. and responsible for lowering aeciospore 216 S. Moricca et al.

production. Sutton (1973) reported a and Heather (1981a,b) reported that C. close association between C. gallicola tenuissimum was a common phylloplane and Endocronartium harknessii (J.P. Moore) fungus that actively colonized uredinio- Hiratsuka on Pinus banksiana, and observed spores of the rust Melampsora larici- hyphae of the hyperparasite penetrating populina Kleb. on Populus × euro- the rust aeciospores. Tsuneda and Hiratsuka americana. More recently C. tenuissimum (1979) found that C. gallicola parasitized was also detected in collections of aecio- E. harknessii both by simple contact, disin- spores of the two-needle pine stem rust tegrating the cell walls of the spores, and by Cronartium flaccidum and its non-host actual penetration of the spores walls, with alternating form Peridermium pini (Moricca or without the formation of appressoria, and and Ragazzi, 1998; Moricca et al., 1999). causing the coagulation and disappearance This list of Cladosporium species of the host cytoplasm. These authors also parasitic on rusts is not exhaustive and is found evidence that the rust spores, acting likely to increase in the near future. The lack by contact, secreted enzymes in order to dis- of genuine morphological structures and the integrate the cell walls. A similar behaviour great instability of such characters not only was noted for Cladosporium aecidiicola, hamper the placement of Cladosporium spe- a fairly common hyperparasite of rusts cies in a congruent evolutionary context, but in Europe and in the Mediterranean area also make it impossible to identify consis- (Hulea, 1939; Rayss, 1943), parasitizing tently many individual taxa and distinguish E. harknessii on Pinus contorta, Pinus them from related ones. As new molecular muricata and Pinus radiata in California techniques become increasingly employed (Byler et al., 1972). This hyperparasite to confirm and refine known taxonomic rela- also heavily parasitized aecia of Puccinia tionships, it is predictable that new taxa now conspicua (Arth.) Mains. in Arizona undistinguishable or considered synonyms, (Keener, 1954) and urediniospores of will be added to the list. The number of Melampsora medusae Thüm under storage hyperparasites is also expected to grow once conditions (Sharma and Heather, 1980). it is shown that some Cladosporium species, Srivastava et al. (1985) found that Puccinia now thought to be common saprophytes horiana was regularly parasitized by because they are found on aged host Cladosporium sphaerospermum. Sharma structures, are in fact true hyperparasites.

Table 19.1. Cladosporium species hyperparasitic on rust fungi.

Parasite Host

C. aecidiicola Thüm Endocronartium harknessii (J.P. Moore), (Byler et al., 1972); Melampsora medusae Thüm (Sharma and Heather, 1980); Puccinia conspicua (Arth.) Mains., (Keener, 1954) C. gallicola Sutton Endocronartium harknessii (J.P. Moore) (Sutton, 1973; Tsuneda and Hiratsuka, 1979); Cronartium comandrae Pk. (Powell, 1971) C. hemileiae Steyaert Hemileia vastatrix Berk. et Br. (Steyaert, 1930) C. sphaerospermum Penzig Puccinia horiana Henn. (Srivastava et al., 1985) C. tenuissimum Cooke Melampsora larici-populina (Sharma and Heather, 1978); Cronartium flaccidum (Alb. et Schwein.) G. Winter; Peridermium pini (Pers.) Lév. (Moricca et al., 1999) C. uredinicola Speg. Puccinia cestri Dietel and P. Henn. (Spegazzini, 1912); Puccinia recondita Roberge ex Desm (Ellis, 1976); Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme (Morgan-Jones and McKemy, 1990); Puccinia violae (Schum.) DC (Traquair et al., 1984); Puccinia horiana (Srivastava et al., 1985) C. uredinophilum Speg. Uredo cyclotrauma Speg. (Spegazzini, 1922) Biocontrol of Rust Fungi by Cladosporium tenuissimum 217

Identification and Distribution of Since the abundance of heterogeneous Cladosporium tenuissimum taxonomic elements makes traditional clas- sification at species level difficult, represen- The dematiaceous hyphomycete Clado- tative European isolates of C. tenuissimum sporium tenuissimum has long been known were identified by matching mycological as a polyphagous saprophyte occurring in characteristics with nucleotide sequences the air, on the soil and on plant surfaces from coding and non-coding regions of the (Ellis, 1976). It is a frequent colonizer of ribosomal RNA operon (Moricca et al., 1999) senescent or dead plant material and is and by chemotaxonomic profiling (Moricca common on the phyllosphere and rhizo- et al., 2001). Molecular differences in sphere of plant species, but it is also nucleic acid sequences were instrumental reported as an endophyte (Fisher and particularly in vis-à-vis species recognition, Petrini, 1992) and a facultative plant patho- since congeneric, morphologically similar gen, causing blights, leaf spots, and seed, taxa (C. herbarum, C. cladosporioides and fruit and blossom-end rots (Pandey and other unidentified Cladosporia) were Gupta, 1983; Xiang et al., 1989; Dohroo and syntopic to C. tenuissimum, often sharing Sharma, 1992; Sharma and Majumdar, the same ecological niche (rust sori). 1993; Fujii et al., 1995; Dhal et al., 1997). Several Cladosporium members have Together with other Moniliales, it is the not yet been comprehensively described, aetiological agent of human mycoses many of its taxa are still ill-defined and known as chromoblastomycosis, phaeo- the appropriateness of their separate recog- hyphomycosis and eumycotic mycetoma nition remains a vexed question (Morgan- (Gugnani and Okeke, 1989). The hyper- Jones and McKemy, 1990). Many species are parasitic nature of C. tenuissimum was first not considered good species because they reported, as already mentioned, by Sharma have not been identified with sufficient con- and Heather (1981a,b) on the poplar rust fidence. As a result, a number of ecological, M. larici-populina in Australia. It has phytopathological and biomedical studies recently attracted the attention of research- identify Cladosporia only at the generic ers because it suppresses rust under level (Bolland, 1973; McKenzie and laboratory conditions and in glasshouses, Hudson, 1976; Heather and Sharma, 1977; and because it secretes compounds that Sharma and Heather, 1978; Hamada and are biologically active against various Fujita, 2002; Chew et al., 2003). This means phytopathogens (Sharma and Heather, that information on the geographic distri- 1981a; Moricca et al., 2001; Assante et al., bution of C. tenuissimum in particular is 2004). scanty. Direct and indirect elements suggest, The culture characteristics of this mito- however, that it is an ubiquitous fungus with sporic fungus (colony appearance, texture a worldwide distribution. It is known to and morphology, growth/temperature rela- be cosmopolitan and has been recovered tionships, etc.), its micro-morphology (type from various matrices. In Europe it has of conidia and conidiogenesis) and other been found on rust aeciospore samples of distinguishing features, are summarized in two-needled pines from various countries Moricca et al. (1999). The effect of nutrient (Moricca et al., 2001). On the other hand, a composition on the production of secondary high incidence of airborne Cladosporium metabolites has also been investigated in inoculum was reported in the London region vitro on different agar media and liquid by Ainsworth (1952). More importantly, cultures (Moricca et al., 2001). Irrespective that author also found that Cladosporium of the culture medium, the fungus produces inoculum reached a peak in summer, coin- typical, geniculate and sympodially elon- ciding with maturation of most rust spore gated conidiophores, by virtue of which it is stages. The possibility for the hyper- unambiguously ascribed to the anamorph parasite to colonize spores and control genus Cladosporium Link (Domsch and their numbers at developmentally just the Gams, 1980). right time is particularly attractive in the 218 S. Moricca et al.

light of its potential exploitation for rust inoculated with a mixture of conidia from biocontrol. different antagonist isolates displayed, after 24 h, reductions in germination of 19% and 21%, respectively, compared with the controls (Torraca, Italy, personal The Hyperparasitic Relationship communication).

In vitro antagonism Timing of infection

Interaction on glass slides The reduction in spore germination might also depend on the time of initial infection. The great potential of C. tenuissimum as Experiments on C. flaccidum and P. pini a BCA has been evident since the initial indicated that the order in which the rust experiments of Heather and Sharma (1977) and hyperparasite were deposited was the and Sharma and Heather (1978, 1981a,b, main factor causing variability in aecio- 1983, 1988). These authors observed both spore germination. In these experiments direct parasitism of urediniospores of M. three deposition sequences were tested: larici-populina by this hyperparasite, and (i) aeciospores deposited 1 h prior to inhibition of rust spores without any the hyperparasite conidia; (ii) conidia physical contact, suggesting that antibiosis deposited 1 h prior to the aeciospores; and might also occur. This evidence induced (iii) aeciospores and conidia deposited sim- these researchers to postulate that C. tenuis- ultaneously. Maximum inhibition of aecio- simum had significantly reduced the spore germination was achieved when incidence and severity of rust disease in conidia were inoculated 1 h before the rust poplar plantations in the Canberra district aeciospores (Moricca et al., 2001). This out- for several years (Sharma and Heather, come suggests that, in nature, control is 1981a). most effective if the antagonist establishes The antagonistic capability of C. tenuis- itself early on the host surfaces, before the simum was further confirmed when it was rust, in order to build up a mass of inocu- found to inhibit, in vitro, the germination lum sufficient to parasitize rust propagules of propagules of other rust fungi. Selected as they burst from the plant epidermis isolates of C. tenuissimum significantly somewhat later (Kranz, 1969a; Moricca reduced average percentage germination of et al., 2001). However, the research data do aeciospores of C. flaccidum and P. pini at 12, not present a coherent picture. Other stud- 18 and 24 h from inoculation with conidial ies on Puccinia recondita, Cronartium fusi- suspensions of the hyperparasite (33, 39 and forme and Cronartium strobelinum found 46% versus controls, respectively) (Moricca that with these rusts, infections became most et al., 2001). The germination of uredinio- severe when the antagonist Darluca filum spores of the bean rust fungus Uromyces was inoculated before them (Swendsrud appendiculatus treated simultaneously and Calpouzos, 1972; Kuhlman et al., 1978). with a conidial suspension of the antagonist isolate ‘Itt21’ was reduced significantly from Interactions in storage 56.4% to 36.9% after just 3 h of contact, a reduction of 35%. After 6 h, urediniospore The recovery of C. tenuissimum at various germination was still lower than in the latitudes, from separated geographic areas control (69.5 versus 83.3%), and at the with varying climates, suggests that the end of the observations the difference was fungus can survive and remain active 17% (Assante et al., 2004). Beyond this time, under disparate environmental conditions. U. appendiculatus urediniospores no longer This adaptability is also shown by its ability germinated. Freshly collected aeciospores of to survive and parasitize aeciospores of C. the pine twist rust Melampsora pinitorqua flaccidum and P. pini in storage in a range and of the common rust Puccinia sorghi of temperatures. In tests it was effective at Biocontrol of Rust Fungi by Cladosporium tenuissimum 219

−20, 4 and 20°C. Control was greatest at dusted with aeciospores of two-needled 20°C but spore viability was decreased at all pine rust fungi and incubated in the dark at test temperatures, including −20°C. Viabil- 22°C in moist Petri dishes, strongly reduced ity also gradually decreased with storage spore germination after 12, 18 and 24 h, as time (Moricca et al., 2001). Antagonist- compared with the controls. The rust prove- treated spore lots were visibly discoloured. nances, the antagonist isolates and the Stereoscope observations revealed deterio- interaction term (rust provenance × antago- ration of the rust propagules, which were nist isolate) were not significant variables, densely intertwined with hyphae of the indicating an absence of physiological spe- antagonist. The hyperparasite had prolifer- cialization in the hyperparasite (Moricca ated extensively, giving rise to an apprecia- et al., 2001). Several of the spores examined ble mycelial biomass. Spore deterioration individually under the microscope for via- was the first indication that an exogenous bility were barely recognizable, with their enzymatic effect was probably involved in spinules displaced and scattered all over the disintegration process. The long shelf- the mounting medium, indicating an action life of C. tenuissimum inoculum at low tem- of proteolytic enzyme(s) secreted into the peratures provides evidence of ecological medium. tolerance, a characteristic that is common to The pronounced sterility of rust spores other hyperparasites (Kranz, 1969b). As in treated with C. tenuissimum culture filtrates other fungal host–hyperparasite interac- suggested that the hyperparasite was a tions, the antagonist thrives on the host source of extracellular antifungal antibiot- spores, which represent an ideal pabulum ics, as it is in other hyperparasite–parasite (Swendsrud and Calpouzos, 1970). How- interactions (Jackson et al., 1997; Rodriguez ever, the host propagules are not vital for and Pfender, 1997; Trejo-Estrada et al., the antagonist which, being also a faculta- 1998). The toxicity of the culture filtrates tive saprophyte, a plant and animal parasite was immediate, from the first inspection and an unspecialized hyperparasite, can (after 12 h), and remained fairly constant for survive on various materials such as plant the duration of the experiment, with a slight debris, foliar exudates, small insects and increase in germination after 18 and 24 h. other organic substances occurring in the environment. Hyperparasitic secondary metabolites: Rust spores collected during adverse a band of killers weather (high humidity, rainfall) and not properly dehydrated before storage, soon C. tenuissimum actively produces meta- clumped together, and were impaired and bolites with antifungal properties. These discoloured. Many of these clumped spores include a major pure common metabolite were soon heavily overgrown with hyper- with the molecular formula C20H16O6, and parasite mycelium, which proliferated and corresponding to Mr 352, and a series of sporulated profusely on them, markedly related compounds, all of which were iso- decreasing spore viability (Moricca et al., lated from the ethylacetate crude extracts 2001). High humidity is therefore favourable (EtOAc CEs) of several antagonist isolates to infection, pathogenesis and sporulation of (Moricca et al., 2001). This metabolite was C. tenuissimum. already known as cladosporol, a dimeric decaketide which had been isolated from Effect of C. tenuissimum culture filtrates C. cladosporioides (Fukushima et al., 1993; Sakagami et al., 1995). Cladosporol Treatment of all rust spores with culture induces hyphal malformations in Phyto- filtrates showed that enzyme(s) and/or phthora capsici when tested at 10 mg/disc toxic agent(s) had a role in C. tenuissimum (Fukushima et al., 1993). It is also an inhibi- parasitism. Aliquots (25 ml) of culture fil- tor of b-1,3-glucan synthetase, the enzyme trates from four antagonist isolates spread that synthesizes the fungal cell wall compo- separately on sterilized water-agar slides, nent b-1,3-glucan. In an in vitro assay with 220 S. Moricca et al.

labelled UDP-glucose and b-1,3-glucan cladosporol C, reaching an inhibition synthetase prepared from Saccharomyces value higher than 80% at the highest cerevisiae, cladosporol showed an IC50 concentration. activity on the enzyme at 50 mg/ml The cladosporols reduced radial growth (Sakagami et al., 1995). of colonies of the phytopathogenic fungi The series of related compounds Alternaria alternata, Botrytis cinerea, Cerco- purified from EtOAc CEs of C. tenuissimum spora bieticola, Cercosporella herpotrich- cultures, consists of cladosporols B, C, D oides, Colletotrichum lindemuthianum, and E (Assante et al., 2002; Nasini et al., Fusarium roseum, Helminthosporium 2004). The major metabolite, now named oryzae, Mucor sp., Rhizoctonia solani cladosporol A, was isolated as a white and Septoria tritici; of the Oomycota powder and represented more than 30% of Phytophthora capsici, P. cinnamomi, P. the crude extract. A second metabolite had erytroseptica, P. nicotianae and Pythium the same 1H and 13C nuclear magnetic ultimum; and of human-pathogenic strains resonance (NMR) spectra as cladosporol A, of Candida sp. (Moricca et al., 2001; Assante except that it had a 4-oxo function instead of et al., 2002; Nasini et al., 2004; T. Kasuga, the C(4)HOH grouping. This metabolite was California, 2001, personal communication; named cladosporol B. A third compound Aloi and Fossati, Italy, 2002, personal com- had the same basic skeleton as cladosporol munication). The strongest antagonistic A, but with a C(2)H2-C(3)H2 unit instead of effect was against the Oomycota. the 2,3-oxirane ring. This compound was Sensitivity of tested fungi varied in compatible with the molecular formula relation to concentration and differences in C20H18O5 and was named cladosporol C. the functional groups bound in this family of A fourth metabolite, cladosporol D, was metabolites to the tetralone skeleton. The a cream-coloured solid with the formula antifungal activity of described cladosporols 1 13 C20H18O6. Its H and C NMR data, when is likely to reside, as reported for other compared with those of cladosporol C, indi- similar compounds (Arnone et al., 1986; cated that it contained a C(3)HOH fragment Fukushima et al., 1993), in the intrinsic tox- instead of CH2, the remaining signals being icity of the 2-tetralone chromophore and the quite similar. The last metabolite, clado- occurrence of highly reactive substituents, sporol E, was isolated as a brown solid like the epoxy group b-1,3-glucan, as a con- 1 13 with the formula C20H18O7. Its H and C stituent of the fungal cell wall skeleton. By NMR spectra were very similar to those of inhibiting the enzyme that synthesizes this cladosporol D, the only important difference constituent, the cladosporols directly affect being that this cladosporol had an additional the biochemistry and structural organiza- hydroxy group at C-2 (Nasini et al., 2004). tion of the fungal cell. They thus strongly Cladosporols A–C, produced in an condition the pathogenicity of C. tenuis- amount sufficient to enable some assays on simum and play a major role in its their biological activity, inhibited, in vitro, hyperparasitism. a number of rust fungi, non-rust fungi, Oomycota and yeasts. They suppressed germination of urediniospores of U. append- In vivo antagonism iculatus and of aeciospores of M. pinitorqua, C. flaccidum, P. recondita and P. sorghi,ina In planta assays range between 75 and 100%, when tested at 100 mg/ml. Cladosporol B was the most Inoculation tests on whole rust-infected active of the group, completely suppress- plants in a controlled environment (green- ing germination of U. appendiculatus at house or laboratory) give a first indication 50 ppm, reducing it by more than 90% at of how the rust may be controlled in nature. 25 ppm, and lowering it even at 12.5 ppm. They therefore represent an important step Cladosporol A, through less inhibitory in studying the mode of action of the hyper- than cladosporol B, was more active than parasite. If a natural inoculation procedure Biocontrol of Rust Fungi by Cladosporium tenuissimum 221

and an objective disease evaluation proto- and simultaneously treated with a suspen- col are followed, in planta assays can, in sion of C. tenuissimum conidia, or with a just a few weeks, provide valuable data on MPGG (malt extract, peptone, glucose, glyc- the biocontrol effectiveness of a tested erol) culture filtrate of a 2-week-old liquid microorganism. Furthermore, in such an stationary culture of the hyperparasite. Con- artificial system, the tri-trophic interaction trols were healthy bean plants inoculated between host plant, rust parasite and with: (i) a water suspension of C. tenuis- hyperparasite can be more accurately simum conidia; (ii) rust urediniospores, investigated, since the effect of the environ- then treated with sterile water/Tween 20; or ment, which in the field can positively or (iii) only a sterile uninoculated MPGG broth. negatively affect each interacting partner, is Disease severity was scored by the number of eliminated. pustules per square centimetre in a total of In two consecutive growing seasons, eight 1-cm2 leaf areas on digitalized primary 1999 and 2000, spermogonia of C. flaccidum leaves, after 13 d from inoculation. After that had developed on 2-year-old, rust- 1 month, plants inoculated simultaneously infected pine seedlings, were inoculated with U. appendiculatus and the conidial with mixed conidial suspensions from suspension developed rust in the normal different C. tenuissimum isolates. Rust- way. By contrast, treatment with the antago- infected control seedlings were sprayed nist culture filtrate provided total protec- only with sterile water/Tween 20. Disease tion: the urediniospores did not germinate evaluation, based on a standardized proce- and the bean plants did not develop any dure, was completed after 5 months, and the infection (Assante et al., 2004). incidence and severity of the rust infection A possible explanation of these findings were defined. The percentage of infected on bean rust is that in the simultaneous seedlings and the number of infections per infections the time available for inter-fungus seedling stem were significantly lower in interaction was too short to enable the the antagonist-treated seedlings than in the antagonist to establish a parasitic relation- untreated controls. Percentage mortality ship with the pathogen. The specialized, was significantly lower in seedlings with biotrophic agent found refuge by rapidly antagonist inoculation than in those with- penetrating into the living host tissues, out. A mycelial biomass attributable to the occupying the ecological niche it has hyperparasite and detectable as a felty, dark evolved since primordial times to colonize greenish-brown mycelium was observed on in order to gain access to nutrients and pro- spermatial and aecial fructifications on the tection from natural enemies. The antifungal bark of some treated seedlings. The fungus compounds and enzymes in the culture fil- was positively identified by microscope trate, on the other hand, acted immediately examination of the sporulating structures and prevented propagule germination and (erect, straight, regularly septate conidio- rust development. phores; holoblastic, conidiogenous cells; cylindrical to clavate ramo-conidia with Detached leaves 2–3 flattened, thickened scars; intercalary and terminal conidia of various shapes and A simple test in a strictly controlled envi- sizes) (Moricca et al., 2001). ronment, using Petri dishes and a water- In an experiment exploring the saturated atmosphere, can give an insight hyperparasitism of C. tenuissimum on U. into the type of hyperparasitic interaction. appendiculatus, a classical disease escape Primary leaves of bean rust-infected plants, mechanism may have prevented the anta- inoculated as above with a conidial suspen- gonist from parasitizing the rust. Primary sion or a culture filtrate of C. tenuissimum, leaves of bean plants of P. vulgaris L. cv. were immediately detached and incubated ‘Borlotto nano Lingua di fuoco’ were in 15-cm diameter Petri dishes. The leaf inoculated on their lower surface with a sus- stems were dipped in a medium containing pension of U. appendiculatus urediniospore 1% water-agar (WA) supplemented with 222 S. Moricca et al.

5 ppm gibberellic acid (GA). Hyperparasite together with the germ tubes and hyphae colonization of rust pustules and disease growing from them, were attracted to the severity were monitored daily under a rust propagules, to which they became stereoscope, starting 1 week after hyper- firmly attached. parasite inoculation and continuing until The contact stimulus between the the end of the experiment. Rust regularly host and the hyperparasite – a reflection of formed appressoria at the precise location recognition events among them – mediated of the stomata (indirect-type, dikaryotic the secretion of different substances at the penetration) but these appressoria began to host–parasite interface. These substances collapse a few hours after formation. In are believed to play a fundamental role in spite of the positive tropism of C. tenuis- pathogenesis, either in interactions between simum conidia towards the rust propagules, plants and parasitic fungi (Chaubal et al., as indicated by the many conidia closely 1991; Braun and Howard, 1994; Jones, 1994; attached to rust spores and appressoria, Carver et al., 1995; Nicholson, 1996) or penetration of the bean leaves by the rust between those fungi and their hyper- could not be prevented completely, and this parasites (Carling et al., 1976; Tsuneda and can explain why, compared with the con- Skoropad, 1977; Moricca et al., 2001). Some trols, disease severity was curtailed by 13% of these substances, visible as a dense net- only. As in the experiment with the whole work of amorphous, fibrous material, were plants, the culture filtrate had a toxic effect the direct product of hyperparasite meta- on the detached leaves that prevented any bolism and served to ensure close adhesion rust spores from germinating (Assante et al., of the hyperparasite to the host cell wall 2004). (Moricca et al., 2001). Amorphous material from a different source was observed adjacent to shrunken and eroded parts of the spore wall. This second type of LM, SEM and TEM Examination of the extracellular, amorphous material charac- Host/Parasite Interface teristically accompanied spore penetration and was associated with the hyperparasite Examination of the interface between C. structures involved in the process, i.e. the tenuissimum and the rust agents C. variously shaped appressoria formed on flaccidum, P. pini and U. appendiculatus the host surface and the infection hyphae. with light (LM), scanning (SEM) and Ultrastructural examination at contact transmission electron microscopy (TEM) points provided evidence that lytic enzymes showed the strong antagonistic action of the caused degradation of the host cell walls hyperparasite, and the multiple strategies and released the amorphous material at the it employed to parasitize the rust fungi. The rust–hyperparasite interface. A decrease in reproductive capacity of C. tenuissimum electron density from the outer to the inner appeared greatly enhanced by the proxim- layers of the spore wall sometimes made the ity of rust spores, most of which were inac- fibrillar chitin structure of the wall visible tivated and overgrown by the antagonist (Assante et al., 2004). mycelium. The hyperparasite sporulated An adhesive matrix pad intimately profusely on the spores, producing on their connected the host and the hyperparasite to surface tufts of fructifications bearing num- each other. This pad serves a double func- bers of conidiophores. These conidiophores tion, to attach and support the appressorium generated a multitude of asexual propa- and the penetrating hypha, and to be a gation units, represented by ellipsoidal reservoir for enzymatic penetration. Such to limoniform ramo-conidia, oblong or functions have already been reported both in fusiform intercalary conidia, and mostly inter-fungus parasitism and in host plant– globose or subglobose terminal conidia. parasite interactions (Gold and Mendgen, Prolific antagonist sporulation gave rise 1984, 1991; Benhamou and Chet, 1996; to an enormous mass of conidia which, Askary et al., 1997; Moricca et al., 2001). The Biocontrol of Rust Fungi by Cladosporium tenuissimum 223

growth of the antagonist on a synthetic While the inner wall layer of the rust spores medium containing the polymer laminarin therefore remains almost intact, the cell con- as the sole carbon source, on the other hand, tent is probably completely digested by the shows that it produces extracellular b-1,3- combined action of the b-1,3-glucanases and glucanases. There is extensive enzymatic other lytic enzymes. It is supposed these degradation of the matrix wall poly- enzymes work in cooperation with b-1,3- saccharides in which the polymeric chitin glucanases, there being no chitinase microfibrils in rust fungi are embedded production by the antagonist when grown (Locci et al., 1971; Trocha and Daly, 1974; on medium with chitin as the sole carbon Trocha et al., 1974; Humme et al., 1981; source. Moreover, the great number of unger- Maxemiuc-Naccache and Dietrich, 1981; minated spores suggests that, especially Freytag and Mendgen, 1991), especially in in the early stages of penetration, toxic the early stages of rust–hyperparasite inter- metabolites are secreted whose role is action, when nutrients are vital for the antag- important since they kill the host cells onist. The involvement of b-1,3-glucanases and thus facilitate the colonization process in another hyperparasite interaction, that (Moricca et al., 2001; Assante et al., 2004). between Fusarium solani and Puccinia In brief, the parasitization of rust spores arachidis, has recently also been reported by the antagonist, as shown under the and discussed (Mathivanan, 2000). Other microscope, is divided into the following elements suggesting enzymatic activity sequential events: besides the dissolution of the host cell wall, • pre-penetration (signal interplay are the lack of indentation of the host wall with recognition, contact, adhesion, at the contact site, and the minimal swelling antibiosis, formation of infection of the infecting hyphal tip (Moricca et al., structures); 2001; Assante et al., 2004). • penetration (production of degrading Like congeneric hyperparasites enzymes, spore entry by mechanical (Tsuneda and Hiratsuka, 1979; Traquair pressure); et al., 1984; Srivastava et al., 1985), C. • post-penetration (evasion from the host tenuissimum is endowed with alternative cell, sporulation). modes of penetration, since it can also invade propagules by physical destruction of the spore wall. This type of direct penetra- tion uses a simple mechanical process of Ecological Fitness of C. tenuissimum physical pressure against the host cell wall. Hyphae often coil around the rust spores, A precondition for the biological control of displacing the spinules as they advance, in plant parasites is a full understanding of some cases producing a swollen structure, how the control agent operates. C. tenuis- and gaining access to the cell by breaching simum showed itself to be a destructive, the spore wall, with or without the produc- unspecialized hyperparasite of rust fungi. tion of appressoria (Moricca et al., 2001). A Research has elucidated some of the basic histological examination of the infection principles underlying inter-fungus para- process reveals that when appressoria are sitism, clarified the fine structure of the produced, they generate penetration pegs rust–hyperparasite interface, shown that that pierce the spore wall. Penetration pegs the hyperparasite inhibits rust propagules are narrower at the point where they pass in vitro and rust diseases in planta under through the spore wall, but once they have glasshouse conditions, and explored how entered the cell lumen they swell out again. the hyperparasite affects the target host. The hyperparasite destroys the protoplast of This last part of the research effort has led the host cells it invades and its mycelium to the discovery of some lytic enzymes and proliferates inside the cells. Degradation of toxic metabolites that are important patho- the spore contents is also evident from the genicity determinants. Among the antago- many shrunk, collapsed and empty spores. nist’s ‘weapons’, these toxic metabolites are 224 S. Moricca et al.

probably of prime importance. They are may represent a crucial bottleneck for the the cladosporols, a family of related hyperparasite. compounds with strong antifungal activity, The occurrence of C. tenuissimum at of which cladosporols B, C, D and E are various latitudes and altitudes indicates, how- described for the first time and reported as ever, that the physical environment does not being produced by C. tenuissimum (Nasini particularly affect its survival. Similarly, et al., 2004). The basic role of these nutrient availability is not a problem for this substances in nature is to preserve the microorganism, since it has a sufficiently ecological niche of the hyperparasite: broad range to exploit alternate hosts. It they protect the fungus against competing thrives on several rust species, on plant or microorganisms; they prevent the growth animal hosts, it survives saprophytically on of saprophytic microbes; and they displace moribund or dead plant material, and it can plant pathogens from the plant surface overwinter on dead leaves, fallen flowers or (Vey et al., 2001). The enormous potential fruits, or in necrotic spots. Such versatility of such molecules to control fungal para- indicates that its life in natural habitats sites is underlined by recent findings in is quite stable, as all these substrates are pharmacotherapy, where two compounds permanent or semi-permanent trophic reser- (caspofungin and micafungin), having the voirs from which the microbe can disperse same biological activity as the cladosporols into the target host at its first appearance. (inhibition of the synthesis of b-1,3-glucan), Among the attributes a good hyperpara- were reported as a new generation of anti- site must have to be a successful BCA are, fungal drugs (Letscher-Bru and Herbrecht, according to Wicker and Shaw (1968): a 2003; Pawlitz et al., 2003). These anti- wide range (overlapping with that of its fungal compounds have been patented and hosts); ecological amplitude (ensuring per- launched on the market by pharmaceutical sistence within the host range); the produc- companies Merck and Fujizawa as the tion of abundant inoculum (necessary for first commercial inhibitors of b-1,3-glucan epiphytotics to break out); an effective mode synthesis, and are claimed to be effective of action (to restrict the target disease); high against several fungal infections in humans. infectivity; and virulence. An important However, the fact that C. tenuissimum is attribute that should be added for the partic- a destructive hyperparasite that attacks and ular control of rust fungi is that the period of disintegrates rust spores, remains viable maximum sporulation of the hyperparasite over a wide range of temperatures, possesses should coincide with the maturation time of several aggression mechanisms, produces the rust spores. The aerobiological study of fungicidal metabolites and strongly sup- Ainsworth (1952) on the amount of Clado- presses rust development in planta does not sporium inoculum in the air at different guarantee it will be effective under natural times of year, showed that it reached a peak conditions. A thorough understanding of in the spring and summer. These findings hyperparasite biology, ecology and fitness is were confirmed by Cammack (1955), who needed to obtain effective disease control reported that the release of Cladosporium and avoid inconsistency in efficacy. The air-spores from senescent or dead leaves antagonist has first to survive application, was favoured by alternating wet and dry then to establish itself in the environment weather, a condition that frequently occurs (forest or agroecosystem), and finally it must in temperate regions of the world as a result remain active until it is required for control. of the diurnal variation in late spring and This means that it has to spend a significant early summer. The peak of C. tenuissimum period of time in a permanent habitat where sporulation therefore coincides with multi- it has to cope with environmental con- plication of rust spores (aecidio-, uredinio-, straints and live side by side with the teleuto- and basidiospores) and this is indigenous, competing microbial commu- further evidence that rust biocontrol with nity. Inability to overcome such limitations C. tenuissimum is feasible. Biocontrol of Rust Fungi by Cladosporium tenuissimum 225

Authors sceptical about biocontrol environment. Such selection pressure plays usually assert that antagonists already occur a prominent role in shaping pathogen in natural habitats, and yet epidemics still population structure, the strong alteration continue to break out (Kranz, 1981). If we of which may have dramatic effects on the accept this argument, it would mean that the epidemiology of the disease. Reduction of whole concept of disease control was sporulation, infection period and dissemi- vitiated, not only that of plants but that of nation causes, in fact, a restriction of the all living organisms. Fortunately, however, pathogen (Moricca et al., 2001). If it is the real successes achieved in controlling also considered that hyperparasites have a number of important diseases shows a beneficial effect on the fitness of the that those suspicions are unfounded. The host plants, which regain vigour when freed upsurge of a disease over time and space from fungal diseases (Kiss, 2001), it is clear depends on a repeated cycle of infection, that C. tenuissimum has an important role production of inoculum, and dispersal of in the evolution of both the tree host and inoculum to new sites. Pathogen inoculum the rust pathogen. spread is central to the development of Detailed examination of inter-fungus any disease epidemic, and in the same way parasitism indicated that C. tenuissimum antagonist inoculum spread is fundamental has potential to significantly curtail rust in achieving control. Fluctuating climatic diseases and, as a consequence, the use factors (temperature, relative humidity, of chemicals. This prospect is held out by precipitation, solar radiation, wind) and knowledge of the biology and behaviour of soil characteristics (texture, organic matter C. tenuissimum in perennial habitats and content, cation exchange capacity, moisture, agricultural settings. Nevertheless, recent pH) strongly affect propagule persistence research demonstrates that BCAs are not a in the epigeal and hypogeal milieu, as well cure-all to control all phytosanitary disor- as the dynamics of fungal populations. It is ders. A single control measure can rarely because of climatic and edaphic variations provide effective and economically feasible that the distribution of diseases in stands levels of disease control. C. tenuissimum is a is patchy and the occurrence of hyperpara- valuable resource to be used in an integrated sites erratic. For this reason the hyper- pest management (IPM) framework where, parasite may need a long time to build up together with other practices and control and maintain its biomass at levels that will strategies, it can become a long-term, stable control a target pathogen. If conditions con- control measure. ducive to high levels of infection are not forthcoming, the hyperparasite will be slow-acting and the pathogen will have time Acknowledgements to cause disease symptoms and to reproduce on the crop. Purely epidemiological factors, The authors acknowledge the valuable therefore, often explain BCA ineffective- information provided by Gianluca Nasini, ness. A correct assessment of these, as well CNR-ICRM, Milan, Italy, on the character- as of biological, ecological and technical ization of secondary metabolites; Dario (application methods) factors, are essential Maffi and Marco Saracchi, University for the successful exploitation of BCAs in of Milan, and E. Bruno, University of plant diseases. Florence, for providing technical assistance with SEM and TEM observations; Novella Fossati and Claudio Aloj, Isagro, Italy, Concluding Remarks for data on the inhibition of Puccinia recondita and Septoria tritici; and G. C. tenuissimum strongly reduces both the Torraca, CNR-IPP, Florence, Italy, for data number and the longevity of rust spores, on the inhibition of Melampsora pinitorqua and also the amount of spores in the and Puccinia sorghi. 226 S. Moricca et al.

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Mirko Liesebach1 and Irmtraut Zaspel2 1Federal Office and Research Centre for Forests, Department of Forest Genetics, Hauptstrasse 7, A-1140 Vienna, Austria; 2Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Eberswalder Chaussee 3A, D-15377 Waldsieversdorf, Germany

Introduction Melampsora spp. rusts of Salicaceae, whereas no reports exist on the appearance Sphaerellopsis filum (Biv.-Bern. ex Fr.) of the teleomorph, E. caricis, on this plant Sutton is a widely distributed hyperparasite family. of rust fungi, occurring naturally on about The efficacy of S. filum is based on its 30 genera. It is specific to rust fungi ability to degrade uredial sori, which stops (Uredinales), but has an extremely broad the micro-cycle of propagation of uredo- host range within this diverse group of spores and so prevents new rust infections. important plant pathogens. A host list of The potential of the fungus as a method of rusts, compiled from literature and herbar- controlling rust diseases, which cause great ium material, comprises 369 species (Kranz economic damage, was soon recognized. and Brandenburger, 1981). Most records, by The first experiments using artificial far, were made on species of the genus inoculations of the hyperparasite to con- Puccinia, but it was also common on trol Puccinia triticina were carried out by Uromyces spp., Melampsora spp. and Schroeder and Hassebrauk (1957). Phragmidium spp. More recently, the use of S. filum as a Most commonly, the hyperparasite has biological protectant to control rust diseases been recorded in its imperfect stage on has again raised interest. Its use may uredial sori and seldom in its perfect stage, be of particular value in the cultivation of Eudarluca caricis (FR.) Eriksson (Ascomy- fast-growing tree species, e.g. willows and cotina, Dothideomycetidae, Pleosporales, poplars used for biomass production in Venturiaceae). Sphaerellopsis filum has short-rotation coppice plantations, where been recorded on several or single species of chemical control methods are not applicable the genera Puccinia, Uromyces, Schroeter- for technical and environmental reasons, iaster, Sphaerophragmium, Phragmidium and hence there is a need for alternative and the form-genus Uredo (Kranz, 1973; rust disease control strategies (Whelan et al., Yuan et al., 1998). The anamorphic stage of 1997). Quantitative investigations on the the hyperparasite has been observed only on suppressive effect of S. filum on willow rust,

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 231 232 M. Liesebach and I. Zaspel

carried out by Yuan et al. (1999) and Pei et al. hybrids (478 samples) infected with Melam- (2003) demonstrated its potential for disease psora spp. rusts was assessed for control. These studies on the efficacy of hyperparasitism by S. filum. The leaf sam- S. filum against willow rust showed that rust ples were collected from willows growing spore production decreased as the S. filum in clone collections, short-rotation coppice conidia inoculum increased. Under experi- (SRC) plantations and several natural sites mental conditions certain strains of the in Germany and related regions (Table 20.1). hyperparasite were able to reduce rust spore They were viewed under a light microscope production by up to 98%. (Olympus SZH10 Research Stereo) at 25- to Despite the evidence for the widespread 30-fold magnification and the conidiomata distribution of S. filum, there were only of S. filum were detected as black spots limited records for its occurrence in central inside the yellow uredia (Fig. 20.1). Europe on willow rusts. Furthermore there were no references found describing its complete biological cycle, causes of patho- genicity and efficiency of rust control under natural conditions. Therefore a screening of rust samples was started to determine the frequency of the mycoparasite on rusts of the genus Salix. The mode of action of S. filum on willow rusts and the genetic diversity of the hyperparasite were also investigated.

Occurrence and Frequency of S. filum on Willow Rusts in Central Europe Fig. 20.1. Pycnidia of Sphaerellopsis filum formed In summer and autumn 2000–2002 leaf on willow rust uredias on Salix daphnoides material from 36 willow species and (bar = 10 mm).

Table 20.1. Sites where leaf samples were collected from willow plants and the number of Sphaerellopsis filum isolates obtained.

Site Latitude Longitude Elevation (m asl) Isolates (number)

I. Clone collections: Waldsieversdorf 52°32′N 14°05′E 46 20 Großhansdorf 53°39′N 10°15′E 50 8 Eberswalde 52°49′N 13°47′E 35 26 Hann. Münden 51°28′N 09°38′E 140 3 Arnsberg 51°24′N 08°03′E 210 7 Long Ashton (UK) 51°25′N 02°35′W 40 1 II. SRC plantations: Berlin-Buch 52°39′N 13°28′E 50 2 Berchem (Belgium) 51°10′N 04°50′E 5 2 III. Natural sites: district MOL 52°38′N 14°07′E 50 1 river site Elbe 53°04′N 11°21′E 20 2 Wächtersbach 50°14′N 09°15′E 170 1 Germering 48°08′N 11°20′E 540 2 Merbachfeld 51°29′N 09°37′E 120 1 Arnsberg 51°24′N 08°03′E 210 2 asl, above sea level; SRC, short-rotation coppice. Biology and Genetic Diversity of Sphaerellopsis filum 233

The levels of infection for each of the 3 Extract Agar, Potato Dextrose Agar), using years, 2000–2002, were 15%, 25% and 26%, standard microbiological practices. Often respectively. There was no correlation the isolation of S. filum was complicated between the level of infection in any given by the occurrence of secondary saprophytic year and the number of Sphaerellopsis bacteria. To reduce the growth of such bac- pycnidia detected on uredia, which differed teria the agar was supplemented with the significantly from sample to sample. While antibiotics chloramphenicol (10 mg/ml) and the overall infection levels were low in the oxytetracyclin (15 mg/ml), which did not population assessed, 75% of willow species inhibit the germination of S. filum conidia and hybrids investigated were infected with and enabled the establishment of single- S. filum. On rust on only nine species/ spore isolates of the fungus. hybrids was there no infection with the A culture collection of S. filum com- hyperparasite visible (Table 20.2). Addition- prised 77 isolates from Melampsora spp. ally, S. filum was observed in uredia of causing rust on willow, supplemented with the stem-infecting form of rust on hybrids, four isolates from Melampsora spp. causing S. caprea × viminalis and S. × sericans. poplar rust and six isolates from Puccinia spp. (Liesebach and Zaspel, 2004). The hyperparasite was cultured successfully on Malt Extract Agar Complete Medium Cultural and Microscopic Studies (SERVA) containing 3% malt extract and 0.3% peptone from soy, with the pH Some authors have classified S. filum as an adjusted to 7.0. Cultures were maintained at obligate, biotrophic hyperparasite, depend- 16°C under continuous ultraviolet (UV) light ent on its host for its development and at 370 nm (Blacklight Blue Philips TLD unable to be be cultured on artificial media 36W/08). The growth of S. filum isolates in (Leinhos and Buchenauer, 1992). However, vitro was slow, with a mean radial growth older studies (Keener, 1933; Schroeder and of 40–50 mm in 25 days. After 5–7 months Hassebrauk, 1957), as well as more recent in culture many isolates had restricted ones (Pei et al., 2003) and those of our own growth with the fungus building up a black (Zaspel and Liesebach, 2004), have demon- plectenchyma, without any production of strated that the fungus can be cultivated on conidia. At this stage mycelial production of the most common mycological media (Malt a range of isolates could be stimulated by

Table 20.2. Frequency of occurrence of S. filum on rusts on willow species and hybrids.

Frequently (50% or more of Rarely (< 50% of assessed assessed samples) samples) No findings

S. purpurea S. alba + hybrids S. adenophylla S. daphnoides S. aurita S. aurita hybrids S. × aquatica S. caprea + hybrids S. cinerea + hybrids S. acutifolia S. cordata S. kazbekensis S. megeriana S. fragilis + hybrids S. purpurea hybrids S. magnifica S. nigra S. schwerinii S. nigricans hybrids S. triandra hybrids S. pentandra S. × smithiana S. repens + hybrids S. triandra S. viminalis + hybrids S. × dasyclados S. × hirtei S. × sericans S. × stipularis 234 M. Liesebach and I. Zaspel

cultivation in liquid medium, Malt Extract Broth Complete Medium (SERVA) on a shaker with slow rotation (80 rpm) under similar conditions. The colour and growth behaviour in vitro were extremely variable between isolates. Schroeder and Hassebrauk (1957) reported that the appearance of S. filum isolates obtained from Puccinia spp. rusts of cereals and isolates from Melampsora rusts could be categorized into three groups. Group I had white aerial mycelium and pale pink- or brown-coloured submerged myce- lium, with sparse sporulation. Group II had whitish, pale pink, or pale beige superficial Fig. 20.2. Conidia formed by Sphaerellopsis filum m mycelium without or with restricted sub- (bar = 20 m). merged mycelium, and formation of conidia was sparse. Group III showed grey or black aerial mycelium and a black submerged disorganization of cytoplasma in penetrated mycelium. This group showed regular rust spores suggested rapid diffusion of toxic conidia formation in whitish drops on the substances, as well as degrading enzymes, surface. The appearance of some isolates and confirmed the observations on the changed following longer cultivation peri- Puccinia graminis/S. filum host–parasite ods and therefore an assignment of isolates relationship (Carling et al., 1976). to a determined growth type was not possible. In vivo the S. filum isolates developed Mode of Action of Parasitism of black pycnidia emerging from well- Sphaerellopsis filum developed rust uredia. Under conditions of high relative humidity, whitish cirrhi The mode of action of the parasitization and drops of conidia appeared from the by S. filum is unclear for most parts. pycnidial conidiomata. Conidia were Older studies described appressoria-like ellipsoid, 1-septate, rarely 2-septate, structures of the fungus penetrating uredo- hyaline and later pale brown with a size spores of Puccinia sp. rust (Schroeder and of 16–18 m × 5–6 mm (Fig. 20.2). These data Hassebrauk, 1957). Later works indicated confirm the descriptions given by Schroeder biochemical interactions of the rust– and Hassebrauk (1957). They differ margin- mycoparasite combinations and suggested ally from the description given by Sutton lytic exoenzymes and toxic metabolites (1980) in that the conidia were marked by (Koc and Defago, 1983; Leinhos and an apical, fan-shaped, gelatinous appendage Buchenauer, 1992). Two antibiotics of that disappeared with proliferation, making the xanthocillin type, Darlucin A and B, this trait unsuitable as a specific character. which exhibited antibacterial, antifungal Scanning electron micrographs (SEMs) and weak cytotoxic activity, have been illustrated the infection process of rust isolated (Zapf et al., 1995). Furthermore, uredia by S. filum. A fine net of non- it can presumed that activity by degrading specialized hyphae passed through the exoenzymes, such as chitinase, cellulase, rust uredium, and conidia became directly xylanase, and/or 1,3-b-glucanase com- fastened on rust spores. In an advanced stage plexes, can contribute to the efficacy of the of parasitism, rust spore walls and spore mycoparasitic fungus. In order to determine content showed enzymatic degradation the relative importance of enzymes, Wirth and the invaded uredia subsequently and Wolf (1992) used a method based on collapsed (Fig. 20.3A–D). This extreme the colorimetric assay for the determination Biology and Genetic Diversity of Sphaerellopsis filum 235

Fig. 20.3. Scanning electron micrographs of the infection process of a willow rust uredinium by Sphaerellopsis filum. (A) Section of a uredial sorus of Melampsora spp. willow rust bearing a S. filum pycnidium. (B) Long cirrhi of conidia are developing from a pycnidium. (C) Conidia of S. filum, with their appendages fixed on uredospores. (D) Late stage of parasitism, a uredium with collapsed rust spores is embedded in mucous substances. of the polysaccharide endo-hydrolases, cel- at 600 nm for substrates labelled with RBB, lulase (E.C.3.2.1.4), xylanase (E.C.3.2.1.8), and at 550 nm with RBV. The fresh weight 1,3-b-glucanase (E.C.3.2.1.39) and chitinase of S. filum isolates was determined in order (E.C.3.2.1.14). The substrates used in these to calculate enzyme production for each 1 g assays were carboxymethyl-substituted, fungal fresh weight. and water-soluble polysaccharide deriva- Enzyme activity was measured for 17 tives labelled with Remazol Brilliant Blue S. filum isolates obtained from Melampsora R (CM-Cellulose-RBB, CM-Curdlan-RBB, spp. willow rust and one from Melampsora CM-Xylan-RBB) or Remazol Brilliant Violet sp. poplar rust. Chitinase showed the high- 5R (CM-Chitin-RBV) (Loewe Biochemical, est level of activity per gram mycelia fresh Sauerlach, Germany). The determination of weight. Furthermore, differences in activity the enzyme activity was carried out on cul- of enzyme complexes, xylanase, cellulase ture filtrates from isolates grown in liquid and 1,3-b-glucanase could be demonstrated culture at 21°C for 20 days. Assays were (Table 20.3). performed by adding an aqueous solution There was a high level of variation of dye-labelled substrate to culture filtrates between the enzyme activities of individual and buffer, followed by incubation at 35°C. isolates. These large differences could have The reaction was terminated with HCl been the consequence of the artificial and cooling, and the supernatants con- cultivation techniques that resulted in the taining soluble dye-labelled degradation development of mutants lacking the ability products measured spectrophotometrically to produce exoenzyme. On the other hand, 236 M. Liesebach and I. Zaspel

Table 20.3. Sample mean of enzyme activity g−1 fungal fresh weight of the S. filum collection isolated from Melampsora willow and poplar rusts and comparisons between enzyme complexes (U test).

Wilcoxon rank sum testa

Substrate Mean (SD) Cellulase 1,3-b-Glucanase Xylanase

Cellulase 1.45 (2.30) – 1,3-b-Glucanase 1.36 (2.56) < 0.201 – Xylanase 1.45 (1.84) < 0.045 < 0.151 – Chitinase 2.55 (1.56) < 0.0001 < 0.0001 < 0.0001 aData analysis was carried out by SAS program (SAS Inc, Cary, NY). Calculated data of enzyme activities of several enzyme complexes were computed for comparison with the Wilcoxon rank sum test

(U test) and small P values for |Z| to support the alternative hypothesis of H0 were described. differences have been demonstrated to be Protocols for PCR amplification of ITS due to S. filum populations being composed regions and RFLP analysis are described by of pathogenically specialized isolates differ- Liesebach and Zaspel (2004). The primer ing widely in their virulence (Yuan et al., pair ITS1 (5′ TCC GTA GGT GAA CCT GCG 1999). The correlation between mycelial G3′) and ITS4 (5′ TCC TCC GCT TAT TGA fresh weight in liquid culture and TAT GC 3′) universal primers (White et al., absorbance for chitinase (r = 0.429***, 1990) were used for the amplification of the P < 0.0001) and for xylanase (r = 0.532***, ITS regions based on the repetitive units of P < 0.0001) showed only a weak relation cal- the nuclear rDNA consisting of conserved culated for all isolates tested. A significant coding and variable non-coding sequences. correlation was demonstrated between fresh This method permitted the amplification of weight and activity of enzyme complexes a single DNA fragment containing ITS1, of cellulase and xylanase (r = 0.885***, ITS2, 5.8S and partial sequences of 18S and P < 0.0001). 25S rDNA genes, of a total size between 494 and 537 bp. The RFLP analyses were carried out by restriction digestion of the ITS region with the enzymes TaqI, EcoRI, HhaI, RsaIor Genetics MboI. Using the clear patterns of the five restriction enzymes in combination, in total The use of polymerase chain reaction five RFLP profiles could be distinguished (PCR)-based genetic techniques, such as clearly. Table 20.4 shows the frequency of internal transcribed spacer-restriction frag- the multilocus patterns. The length of the ment length polymorphism (ITS-RFLP) and uncut amplicon using the primer pair ITS1 sequencing of determined DNA fragments, and ITS4 and the number of the fragments offered the possibility of studying the generated by restriction digestion with TaqI, genetic diversity at the molecular level EcoRI, MboI, RsaI and HhaI are listed in and the phylogenetic relationships of Table 20.5. isolates of the mycoparasite S. filum. For A set of 19 DNA samples, representing this investigation, DNA was extracted from the patterns detected by RFLP and the the collection of 87 isolates (Liesebach and resulting subgroups, were sequenced. The Zaspel, 2004), which included 77 isolates identified ITS1, ITS2 and 5.8S regions of from willow rust. For comparative studies Sphaerellopsis filum isolates were used to four isolates from Melampsora spp. poplar infer phylogenetic relationships among rust and six from Puccinia spp. rusts were these isolates. The sequence data were included. Of the latter, one was obtained aligned using the ‘CLUSTALX’ program from P. obscura on Bellis perennis and (Thompson et al., 1997). For calculating five from P. abrupta on Parthenium a tree the neighbour-joining (N-J) method hysterophorus from Ethiopia. was used (Saitou and Nei, 1987). Bootstrap Biology and Genetic Diversity of Sphaerellopsis filum 237

Table 20.4. RFLP profiles of ITS region, frequency, host plant species of isolates, number of sub-groups and sampling sites.

RFLP S. filum from rusts from species/hybrids Isolated from collections/ Number of profile Frequency (number of isolates) sites sub-groups

I 27 S. alba (1); S. caprea hybrid (1); S. Waldsieversdorf; Eberswalde; 3 (A, B,C) cordata (1); S. daphnoides + hybrid Hann. Münden; Long (9); S. fragilis (1); S. purpurea (2); Ashton/UK; district MOL; S. triandra (1); S. viminalis + hybrid riversite Oder, riversite (8); Populus sp. (3) Elbe; Germering II 6 Bellis perennis (1); Parthenium District MOL; Ethiopia 1 (L) hysterophorus (5) III 44 S. alba + hybrid (2); S. aurita (2); S. Waldsieversdorf; 2 (D, E) acutifolia (1); S. caprea + hybrids (6); Großhansdorf; Eberswalde; S. daphnoides (5); S. fragilis + hybrid Hann. Münden; Arnsberg; (3); S. megeriana (1); S. nigra (1); district MOL; S. nigricans hybrid (1); S. purpurea Wächtersbach; Berlin-Buch; (1); S. viminalis (8); S. × aquatica (4); Berchem/B S. × dasyclados (4); S. × sericans (2); S. × stipularis (2); Populus sp. (1) IV 9 S. caprea (1); S. daphnoides (2); S. Eberswalde; Arnsberg; 5 (G, H, I, pentandra (1); S. viminalis + hybrid (5) Germering; Berchem/B J, K) V 1 S. repens hybrid (1) Eberswalde 1 (F)

RFLP, restriction fragment length polymorphism; ITS, internal transcribed spacer.

Table 20.5. Lengths of the uncut amplicons and number of the fragments generated by restriction digestion with TaqI, EcoRI, MboI, RsaI and HhaI.

Number of fragments Number of Uncut Profile Sub-group isolates amplicon TaqI EcoRI MboI RsaI HhaI

I A 2 520 4 2 4 2 6 I B 2 519 4 2 3 2 6 I C 1 520 3 2 3 2 6 III D 3 536 4 2 4 2 6 III E 1 537 4 2 3 3 6 V F 1 497 3 3 3 1 2 IV G 2 495 6 2 4 1 3 IV H 1 495 5 2 4 1 4 IV I 1 499 5 2 4 1 3 IV J 1 498 5 2 4 1 3 IV K 1 494 5 2 3 1 3 II L 3 512 4 2 4 2 5

sampling was carried out on 1000 replicate Based on the sequence analyses, two data sets to assess the confidence limits main lineages were distinguished. The pro- of branch pattern (Felsenstein, 1985). The files between these main groups differed at program TREEVIEW (Page, 1996) was used 32% or more of their nucleotide positions to construct phylogenetic trees, which were (Table 20.6). Within the first main group rooted using the sequences of ITS1, ITS2 and there were three profiles (I, II, III). The differ- 5.8S of Colletotrichum acutatum (GenBank ences between those profiles were about accession EM_FUN:CAC536212) as the 12%. The variation within each profile was outgroup. very low (less than 2%). The second main 238 M. Liesebach and I. Zaspel

Table 20.6. Percentage divergence between two sequences of 19 Sphaerellopsis isolates (I–V: RFLP profiles).

s27 s42 s23 s45 s15 s13 s68 s101 s18 s40 s76 s21 s9 s71 s99 s16 s14 s17 s8 s27 I s42 > 00 s23 > 00 > 00 s45 > 02 > 02 01 s15 > 01 > 01 01 02 s13 > 11 > 11 12 13 11 II s68 > 11 > 11 12 13 11 00 s101 > 11 > 11 12 13 11 00 00 s18 > 11 > 11 11 12 11 11 11 11 III s40 > 11 > 11 11 12 11 11 11 11 > 00 s76 > 12 > 12 12 13 12 11 11 11 > 00 > 00 s21 > 12 > 12 12 13 12 11 11 11 > 00 > 00 01 s9 > 33 > 34 34 35 33 34 34 34 > 34 > 34 34 34 IV s71 > 34 > 34 34 36 34 34 34 34 > 34 > 34 34 34 01 s99 > 32 > 32 32 33 32 33 33 33 > 33 > 33 32 33 05 05 s16 > 32 > 32 33 34 32 33 33 33 > 33 > 33 32 33 05 05 03 s14 > 32 > 32 33 34 32 33 33 33 > 33 > 33 33 33 05 04 02 01 s17 > 33 > 33 33 34 33 34 34 34 > 33 > 33 33 33 05 04 03 01 > 00 s8 > 39 > 39 40 40 39 39 39 39 > 40 > 40 40 40 16 16 14 13 > 12 12 V

group comprised two profiles (IV, V), which into both main types. Isolates from the same differed in between 12 and 16% of their willow clones at the same site in different nucleotide positions. The larger of the two years belonged partly to the same profile and profiles (IV) had a higher variation (up to partly to another profile compared to the 5%) than those of the first main group. The previous year. Isolates from leaves and from other profile (V) was only represented by a the stem infection form of the same willow single isolate. clone all belonged to profile III. Only profile A phylogenetic tree considered these II was the exception because this group results (Fig. 20.4). The analysis showed, on contained unique strains from Puccinia the one hand, two clear lineages and, on the rusts originating from the African continent other hand, a separation of the five profiles and from Germany. detected by RFLP. There was also a close A high genetic diversity could be phylogenetic relationship of S. filum iso- detected within a restricted population of lated from rust on Salix spp., Populus spp., S. filum isolates. This indicated specific Bellis perennis and Parthenium hystero- rust–hyperparasite relationships and could phorus. The division into the two main be associated with the high number of groups, with the large distance, suggested Melampsora rust genotypes on willow the existence of two separate genetic units, (Chapter 6). Until now, we have not been indicating different species or sub-species able to detect any relations between them, (Liesebach and Zaspel, 2004). Furthermore, and currently it has been impossible to the results demonstrate, that there is no geo- establish any specific relationship between graphical concentration in a special profile incidence of specific willow rust and S. of the mycoparasite, or any relation to the filum genotypes. The sequence analyses of rust provenence (Liesebach and Zaspel, selected strains showed that the S. filum 2004). S. filum isolates from Melampsora isolates belonged to two main types, which spp. rusts on Salix and Populus are grouped differed in more than a third of their Biology and Genetic Diversity of Sphaerellopsis filum 239

Fig. 20.4. Phylogenetic tree of 19 isolates of Sphaerellopsis. The tree was constructed by the neighbour- joining method on the basis of the ITS1, ITS2 and 5.8S region sequences. The sequence of Colletotrichum acutatum was used for rooting the tree. Bootstrap values for 1000 re-samplings are indicated on each node. nucleotide positions. This finding indicated conspicuous difference was the profile II, the existence of two separate taxonomic which contained all strains obtained from forms of the hyperparasite, which existed Puccinia rusts. side by side. It is possible that these are Since hyperparasitic mechanisms are two separate species or sub-species. There diverse and complex, the development was no evidence indicating either a host of the mycoparasite is closely dependent preference of these types or a different on the development of rust fungi. It geographic distribution connected with was evident that strains of S. filum were different ecological demands. The most able to produce polysaccharide-degrading 240 M. Liesebach and I. Zaspel

exoenzymes, which could destroy the Station). Dr F. Ehrig (BAZ Aschersleben) is important polymers of cellulose and chitin thanked for SEM photographs. of the cell walls of rust spores. SEM micrographs gave insight into the host– mycoparasite interface and showed clearly References the degradation of rust uredia by the destruc- tive hyperparasitic fungus. In contrast Carling, D.E., Brown M.F. and Millikan D.F. cellulase-degrading enzymes did not appear (1976) Ultrastructural examination of the to have a role in the parasitizing process on Puccinia graminis–Darluca filum host–parasite rust uredia. Furthermore, the enzyme com- relationship. Phytopathology 66, 419–422. plex played a certain role for a possible Felsenstein, J. (1985) Confidence limits on phylo- saprophytic phase on plant surfaces during genies: an approach using the bootstrap. overwintering and would be needed for the Evolution 39, 783–791. phyllosphere competence of S. filum in the Keener, P.D. (1933) Some characteristics of Darluca absence of a rust host. in culture. Proceedings Academy of Natural In conclusion, the occurrence of S. Sciences (Philadelphia, Pennsylvania) 7, 1–8. Koc, N.K. and Defago, G. (1983) Studies on the host filum on rusts of Salicaceae in Germany and range of the hyperparasite Aphanocladium neighbouring regions was evident, as well as album. Phytopathologische Zeitschrift 107, the appearance of a range of genotypes of the 214–218. hyperparasite. The existence of two differ- Kranz, G. (1973) A host list of the rust parasite ent taxonomic, species or sub-species, units Eudarluca caricis (Fr.) O. Eriks. Nova Hedwigia can be assumed, but to date it has been 24, 169–180. impossible to assign these to host plant spe- Kranz, G. and Brandenburger, W. (1981) An amended cies and/or rust genotypes. This is an area host list of the rust parasite Eudarluca caricis. needing further investigation. To exploit Journal of Plant Disease Protection 88, this information as a means of of biocontrol, 682–702. Leinhos, G.M.E. and Buchenauer, H. (1992) Hyper- it is important to establish multiclonal parasitism of selected fungi on rust fungi of short-rotation coppice (SRC) plantations as a cereal. Zeitschrift für Pflanzenkrankheiten und way of increasing the biodiversity of fungal Pflanzenschutz 99, 482–498. communities of the hyperparasite complex. Liesebach, M. and Zaspel, I. (2004) Genetic diversity This would seem to be more effective than of the mycoparasite Sphaerellopsis filum acting those biomass plantations with a limited on Melampsora willow rusts. Forest Pathology number of willow genotypes. 34, 292–305. Page, R.D.M. (1996) TREEVIEW: an application to display phylogenetic trees on personal comput- ers. Computer Applications in the Biosciences Acknowledgements 12, 357–358. Pei, M.H., Hunter, T., Ruiz, C., Bayon, C. and Harris, J. (2003) Quantitative inoculation of willow rust The research was funded by the EC and Melampsora larici-epitea with the mycoparasite is part of the joint project ‘Integrated, non- Sphaerellopsis filum (teleomorph Eudarluca fungicidal control of Melampsora rusts caricis). Mycological Research 107, 57–63. in renewable energy willow plantations’ Saitou, N. and Nei, M. (1987) The neighbour-joining (QLK5–1999–01585). Ms H. Mattauch is method: a new method for reconstructing phylo- gratefully acknowledged for technical assis- genetic trees. Molecular Biology and Evoution 4, tance with this work. The fungal material 406–425. from Parthenium hysterophorus was kindly Schroeder, H.V. and Hassebrauk, K. (1957) Beiträge zur Biologie von Darluca filum (Biv.) Cast. und supplied by Dr T. Tessema (Humboldt Uni- einigen anderen auf Uredineen beobachteten versity Berlin), the samples from Belgium Pilzen. Zentralblatt für Bakteriologie und were supplied by Drs R. Ceulemans and Parasitenkunde II, 110, 676–696. I. Laureysens (University of Antwerpen), Sutton, B.C. (1980) The Coelomycetes. Common- those from Long Ashton by Dr M.H. Pei and wealth Mycological Institute, Kew, UK, T. Hunter (IACR Long Ashton Research pp. 470–471. Biology and Genetic Diversity of Sphaerellopsis filum 241

Thompson, J.D., Gibson, T.J., Plewniak, F., amylase extracted from forest soil horizons. Soil Jeanmougin, F. and Higgins, D.G. (1997) The Biology and Biochemistry 24, 511–519. ClustalX windows interface: flexible strategies Yuan, Z.W., Pei, M.H., Hunter, T. and Royle, D.J. for multiple sequence alignment aided by (1998) Eudarluca caricis, the teleomorph of the quality analysis tools. Nucleic Acids Research mycoparasite Sphaerellopsis filum on blackberry 24, 4876–4882. rust Phragmidium violaceum. Mycological Whelan, M.J., Hunter, T., Parker, S.R. and Royle, D.J. Research 102, 866–868. (1997) How effective is Sphaerellopsis filum Yuan, Z.W., Pei, M.H., Hunter, T., Ruiz, C. and Royle, as a biocontrol agent of Melampsora willow D.J. (1999) Pathogenicity to willow rust, rust? Aspects of Applied Biology 49, Melampsora epitea, of the mycoparasite 143–148. Sphaerellopsis filum from different sources. White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Mycological Research 103, 509–512. Amplification and direct sequencing of fungal Zapf, S., Hossfeld, M., Anke, H., Velten, R. and ribosomal RNA genes for phylogenetics. In: Steglich, W. (1995) Darlucin-A and Darlucin-B, Innis, M.A., Gelfand, D.H., Sninsky, J.J. and new isocyanide antibiotics from Sphaerellopsis White, T.J. (eds) PCR Products: a Guide to filum (Darluca filum). Journal of Antibiotics 48, Methods and Applications. Academic Press, 36–41. San Diego, California, pp. 315–322. Zaspel, I. and Liesebach, M. (2004) Interaction of Wirth, S.J. and Wolf, G.A. (1992) Micro-plate rust fungi and rust hyperparasite Sphaerellopsis colourimetric assay for endo-acting cellu- filum. Acta Physiologia Plantarum 26, Suppl. 3, lase, xylanase, Chitinase. 1,3-b-glucanase and 129–130. This page intentionally left blank 21 Mycoparasite Sphaerellopsis filum and its Potential for Biological Control of Willow Rust

Ming Hao Pei1 and Zhiwen W. Yuan2 1Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK; 2Institute of Applied Ecology, Academia Sinica, PO Box 417, Shenyang, China

Background species of Melampsora on willows (Kranz and Brandenburger, 1981). Our notice was Sphaerellopsis filum (Biv.-Bern. ex Fr.) first drawn to the mycoparasite in 1990, Sutton (Darluca filum (Biv.-Bern. ex Fr.) when a rust epidemic in an established Cast.) is a mycoparasite of rust fungi plantation of S. viminalis ‘Bowles Hybrid’ at (Uredinales). The fungus can be seen, often Long Ashton became suppressed without in large numbers, on rust pustules (sori). external intervention. Subsequent studies Since some of the most important plant showed that S. filum can reduce the spore diseases are caused by rust fungi, the production in willow Melampsora by more possibility of using the mycoparasite for than 50% (Morris et al., 1995; Whelan et al., biological control has long been of interest. 1997). We are interested in S. filum as a possible In this chapter, we give a brief review of means of biological control of Melampsora the existing information on S. filum and dis- rusts of willows (Salix spp.). When grown cuss the potential of using the mycoparasite in short-rotation coppice (SRC) plantations, for biological control of willow rusts in willow is a potential renewable source of short-rotation coppice plantations. energy and fibre. In coppice plantations, Melampsora spp. cause the most wide- spread and damaging disease of willows, Nomenclature and Taxonomy reducing biomass yields by up to 40% in susceptible clones (Parker et al., 1993). The fungus was first described by Because of economic, technical and envi- Bivona-Bernadi in 1813 as Sphaeria filum ronmental considerations, control of rust (Bivona-Bernadi, 1813). Two of the hosts using fungicides is not favoured and alter- described were rusts. Later, the fungus was native approaches, including biological transferred to the genus Phoma by Fries control, are needed. (1823) and given the name Phoma filum. S. filum was first reported on Melamp- The genus Darluca was established by sora spp. on Salix in 1873 in Germany by Castagne in 1851 (Castagne, 1851) using the Fuckel (cited by Morelet and Pinon, 1973). previously named Sphaeria filum as type Since then, it has been reported on 28 species and accordingly, the fungus was

©CAB International 2005. Rust Diseases of Willow and Poplar (eds M.H. Pei and A.R. McCracken) 243 244 Ming Hao Pei and Z.W. Yuan

named Darluca filum (Biv-Bern. ex Fr.) Morphology Cast. Before 1851, the same fungus had been sent out from the Castagne herbarium Anamorph to several European botanists as Darluca vagans. Since the name Darluca vagans as The fungus forms pycnidia on rust sori. used by Castagne prior to 1851 was not Occurrence of S. filum is most common on published, it cannot be recognized as uredinia but it can frequently be found on valid. Darluca filum was the most widely telia and aecia. The pycnidia of S. filum used name for the fungus until Sutton are black when mature, sub-globose, (1977) proposed adopting the genus 90–200 mm in diameter, within or upon the name Sphaerellopsis Cooke (Cooke, 1883, rust sori, and often had distinct ostioles Grevillea 12:23) for the fungus. through which the spores are exuded in The genus Eudarluca was established thin, white mucilaginous cirrhi. Conidia by Spegazzini (1908) for a pyrenomycete are hyaline, 1-septate, fusiform, 13–18 × occurring on uredinia of a rust on Canna 3–5 mm and often with a small gelatinous sp. in Brazil, citing Eudarluca australis as cap at one or both ends. Small (2–3 × 1 mm) type species. It was assumed that E. australis microconidia can sometimes be found was the perfect state of Darluca filum. mixed with conidia in herbarium collec- The connection between imperfect and tions of the fungus (Eriksson, 1966). Yuan perfect states of the fungus had not et al. (1998) described microconidia in been proven until Keener (1951) produced cultures derived from ascospores of pycnidia and conidia of S. filum from E caricis from Phragmidium violaceum cultures derived from ascospores of Eudar- on blackberry Rubus fruiticosus. The luca from Puccinia extensicola oenotherae fresh microconidia were cylindrical to (Mont.) Arth. on Carex sp. in Pennsylvania, oblong–ellipsoid, hyaline, non-septate and USA. measured 4.0–5.5 × 1.5–2.0 mm. Eriksson (1966) examined a collection described by Fries (1823) as Sphaeria caricis and found asci and ascospores which were identical to the type collection of Teleomorph E. australis (Eriksson, 1966). The same collection contained typical pycnidia of Blackish-brown stromata of E. caricis can S. filum on the leaves of Carex sp. be found on rust sori which often contain heavily infected by Puccinia caricina pycnidia of S. filum. Stromata are promi- DC. After studies of some 300 herbarium nent in var. indica but poorly developed in collections and extensive literature search, var. caricis. Stromata are comprised of com- Eriksson (1966) combined several synonyms pact cells, brown to dark-coloured outside of the perfect state of Darluca into Eudarluca and hyaline inside. Pseudothecia occur in a caricis (Fr.) O. Erik. He listed the following few to numerous locules and are variable in as synonyms of E. caricis: Dothidea size. Pseudothecia are completely or partly genistalis Pers. ex. Fr., Dothidella appen- immersed, sub-globose or ampulliform, diculata deLacr. ex. Br. & Har., Eudarluca often with protruding, conical necks, and australis Speg., Eudarluca indica Ramakr., measure 100–260 mm across. Asci have Leptosphaeria nigrificans Bub. & Wróbl., two layers of wall, cylindric-clavate, Myrmecium cannae Dearn. & Barth., and short-stalked, 55–90 × 7–11 mm, 8-spored. Uleodothis pasipali Stev. Ascospores are irregularly biseriate, Two varieties of E. caricis were spindle-shaped, slightly inequilateral, 1- recognized by Eriksson (1967): var. caricis septae in var. indica and 2–3 septate in with poorly developed stromata and 2–3 var. caricis, hyaline in var. indica and pale septate ascospores, and var. indica with honey-yellow in var. caricis, and measure well-developed stromata and 1-septate 15–24 × 3.5–5 mm. Pseudoparaphyses are ascospores. branched and 2–3 mm thick. Sphaerellopsis filum and Biological Control of Willow Rust 245

Natural Occurrence of S. filum in the same planting in 1992 showed 60–80% of the uredinia on stems were The fungus is distributed globally, occur- colonized by S. filum by the end of July ring in all parts of the world except (Morris et al., 1995). Antarctica. It appears that the teleomorph of the fungus is more common in tropical regions (Eriksson, 1966). Life Cycle and Overwintering Much information on the behaviour of natural populations of S. filum came from Occurrence of the sexual stage field surveys or observations. Early studies on Peridermium peckii on hemlock (Tsuga canadensis) indicated that natural infection From available information, teleomorph of of aecia by S. filum prevented their maturing the fungus appears to be rare in temperate and suppressed spore release (Adams, regions. No perfect state of S. filum was 1920). In a survey of ten maize fields in a known in Europe until 1962 when Eriksson location in the highlands of Kenya, S. filum found E. caricis at two locations in Sweden occurred widely on uredinia of Puccinia (Eriksson, 1966). In Finland, only four sorghi, colonizing up to 40% of rust uredinia samples of E. caricis, three mature and (Kranz, 1969a). In a pure forest stand in one immature, were found (Eriksson, 1967). Florida, USA, Kuhlman et al. (1978) found Relatively recently, E. caricis was found that S. filum colonized 93% of uredinia of on Phragmidium violaceum on blackberry Cronartium strobilinum on Quercus minima Rubus fruticosus in south-west England and less than 1% of the uredinia contained (Yuan et al., 1998). The morphology of telia. In contrast, in an adjacent mixed stand, the E. caricis on Ph. violaceum conformed more sparsely populated with the same oak, to var. caricis offered by Eriksson (1966). only 32% of rust uredinia were infected and Cultures derived from ascospores produced 26% of them had telia. pycnidia and conidia typical of S. filum, In a larch forest stand in north-eastern confirming the connection between the China, field assessments showed that the sexual and asexual stages of the fungus. frequency of rust sori colonized by S. filum Microconidia were also found together increased over the 3 consecutive years of with conidia in the cultures derived from study, 38% for 1986, 57% for 1987 and 62% ascospores of E. caricis from Ph. violaceum for 1988 (Yuan et al., 1988). At the same (Yuan et al., 1998). Actual functions of the time, there was a decrease in the frequency microconidia of S. filum are not certain but of needles bearing rust sori, 68% for 1986, they appear to be equivalent to spermatia, 58% for 1987 and 37% for 1988. Yuan and which play a significant role in sexual Han (2000) reported that, over a period of 3 recombination in fungi. years, the frequency of plants infected by Uromyces hedysari-mongolici in a stand of Hedysarum mongolicum decreased from Life cycle study in willow plantations 65% to 27% while rust pustules colonized by S. filum increased from 36% to 65%. In willow plantations, asexually cycling S. In England, the stem-infecting form filum conidia are responsible for epidemics (SIF) of Melampsora rust (Pei et al., 1995) of S. filum on rusts in the growing season. caused severe infections in an established Morris et al. (1995) also showed that S. coppice planting of Salix viminalis ‘Bowles filum is capable of overwintering as conidia Hybrid’ in May–July 1990. By mid-August, on infected rust pustules of the stem- however, the great majority of rust pustules infecting form (SIF) of Melampsora on Salix became discoloured and inactive. Close viminalis. It is not yet clear whether conidia inspections revealed that over 90% of the are the main form of spores that cause pri- rust uredinia were colonized by S. filum mary infections early in the season. A low (M.H. Pei, unpublished results). Assessment frequency of suspected E. caricis stromata 246 Ming Hao Pei and Z.W. Yuan

was observed in rust pustules on midribs extensive host range raises the question and stems of a biomass willow, Salix whether S. filum is composed of patho- viminalis L. ‘Bowles Hybrid’ (Morris et al., genically specialized groups that are 1995). To determine whether the sexual equivalent to the formae speciales in stage occurs during its life cycle, monthly biotrophic fungi such as rusts and powdery samples were taken between August 1994 mildews. The issue of host specificity bears and March 1995 and examined after great significance in biological control as sectioning. The result was inconclusive more virulent strains of S. filum would be because all the stromata examined were more effective in suppressing rust disease immature and no recognizable asci or development. ascospores were found. Keener (1934) carried out experiments In a separate study, abundant S. filum in a greenhouse, in which 19 species of rust pycnidia containing microspores were belonging to five genera were inoculated found on the leaves of S. burjatica ‘Germany’ with 11 S. filum isolates obtained from heavily infected by M. larici-epitea in late Puccinia spp. and Uromyces spp. Of a total autumn in 1999 and in 2000 (M.H. Pei and T. of 175 inoculations, 101 (58%) resulted in Hunter, unpublished results). These leaves formation of S. filum pycnidia on the rusts. were collected and placed outside in muslin Although there were indications that S. bags in late autumn. At the same time, filum from different sources may differ in suspected stromata on the remains of rust their pathogenicity to different rusts, firm pustules on ‘Bowles Hybrid’ stems were also evidence of pathogenic specialization was collected from the field and placed outside lacking because of ill-defined experimental in 20-ml Sterilin tubes (bottom ends were conditions. As acknowledged by Keener, cut off and both ends were wrapped with many factors, such as the age and type of rust muslin). The fruiting bodies on the leaves sori, inoculation methods, greenhouse tem- and the stromata on ‘Bowles Hybrid’ stems perature and the condition of plant hosts were examined by squashing the fruiting may have contributed to the failed infections bodies on slides or making hand sections at by S. filum. In a review on the hyper- 1–2-monthly intervals until July. Each time, parasitism of biotrophic fungi, Kranz (1981) at least ten fruiting bodies from the leaves or summarized that ‘all attempts have failed to the stems were examined. Again, no recog- reveal such a specificity’. nizable asci or ascospores were found over Conclusive evidence on the host speci- the 2-year period of study. ficity in S. filum came to light more recently. Amplification fragment length poly- Yuan et al. (1999) examined pathogenicity morphism (AFLP) analyses of the S. filum of nine isolates of the mycoparasite to isolates collected from willow and poplar M. larici-epitea under strictly controlled rusts in the UK showed that there is consid- experimental conditions. Five of the tested erable variation (Nei and Li’s similarity coef- isolates were derived from Melampsora spp. ficients as low as 0.52) and that the S. filum on willows and poplar, one from Puccinia on Salicacea does not have a clonal lineage coronata on couch grass (Agropyron (M.H. Pei and C. Ruiz, unpublished results). repens), two from Phragmidium viola- To date, it is not certain whether the field cearum on blackberry (Rubus fruticosus) populations of S. filum occurring on willow and one from Triphragmiopsis laricinum Melampsora undergo a sexual life cycle. on larch (Larix kaempferi). Two inoculation experiments were carried out. In the first, S. filum and rust were applied simultaneously on to leaf discs of Salix burjatica ‘Korso’. In Pathogenic Variation the second, leaf discs were inoculated with the rust initially, and then resulting rust The fungus is found associated with at least uredinial pustules were inoculated with 369 species of 30 genera of rusts worldwide S. filum. In both experiments, all S. filum (Kranz and Brandenburger, 1981). Such an isolates from Melampsora spp. and that from Sphaerellopsis filum and Biological Control of Willow Rust 247

P. coronata, developed pycnidia on the envelop them, no penetration of germ tubes willow rust. No pycnidia were produced was found (von Schroeder and Hassebrauk, from the other three isolates. Figure 21.1 1957). Using scanning and transmission shows the results from the first experiment, electron microscopy, Carling et al. (1976) in which Isolate E from M. larici-epitea on S. observed that, after inoculation with S. viminalis reduced rust spore production by filum, the majority of mycelial development 98% compared with the control (without occurred in the sori below the exposed application of S. filum), while Isolate H from layers of urediniospores. As mycelia of Puccinia coronata on couch grass reduced S. filum and rust develop inside plant rust spore production by 64%. These experi- tissue (see Kranz, 1981), it is certain that ments clearly demonstrated that S. filum the mycoparasite absorbs nutrients from is composed of pathogenically specialized rust sori. However, the actual metabolism populations differing widely in their viru- involved in the colonization by S. filum is lence. When a further inoculations were not clear. carried out with 13 S. filum isolates derived from Melampsora on willow and poplars, all the isolates proved to be effective against Conditions for Colonization of Rust M. larici-epitea, reducing rust sporulation by 88–99% 13 days after simultaneous High humidity appears to be essential inoculation with the rust and S. filum for infection of rusts by S. filum. Optimum (M.H. Pei and C. Ruiz, unpublished results). incubation time under moist conditions for S. filum infection was 16–24 h for Cronartium flaccidum on oak leaves and Mode of Action 48 h for Puccinia recondita on wheat seedlings (see Kranz, 1972). The mode of infection of the mycoparasite S. filum can establish infections at is not yet well understood. Hulea (1939) a range of temperatures, 8°C as minimum, suggested that the fungus colonizes rust sori 22–24°C optimum and 30°C maximum simply through the opening of host epider- (Kranz, 1981). Inoculation experiments of mis resulting from the development of the M. larici-epitea with S. filum showed that sori. Although S. filum hyphae can attach the fungus can readily establish infection of themselves to rust germ tubes or even rust pustules at 10–26°C (Morris et al., 1995).

100 re 90 po

s 80 t s 70 u n r o

i 60 of n

uct 50 o d

o 40 r p

ressi 30 20 upp s

10

% 0 EBDCA HF G I S. filum isolate

E - from Salix viminalis ‘Bowles Hybrid’ (willow) B - from S. triandra ‘Black Maul’ (willow) D - from Populus x euramericana ‘Dorskamp’ (poplar) C - from S. x hirtei ‘Reifenweide’ (willow) A - from S. triandra ‘Black Maul’ (willow) H - from Agropyron repens (grass) F - from Rubus fruticosus (blackberry) G - from Rubus fruticosus (blackberry) I - from Larix kaepferi (larch) Fig. 21.1. Suppression of rust spore production of Melampsora larici-epitea in simultaneous inoculation with the rust and nine S. filum isolates (from Pei et al., 1999). 248 Ming Hao Pei and Z.W. Yuan

Survival on Plant Surface and Timing poplar leaves carrying S. filum were dried of Infection at room temperature for 2–3 days and stored at −15°C. To recover the S. filum, dry cirrhi It was reported that with or without the were suspended in water and spread over presence of rust, S. filum can infect wheat agar or inoculated on to rust sori. Some − ° leaves outside uredinia (von Schroeder of the S. filum samples stored at 15 C for and Hassebrauk, 1957; Kranz, 1972). When several years proved to be viable. applied on to leaf discs of a willow (S. burjatica), S. filum remained viable on the willow leaves in the absence of rust for up Growth and Sporulation on Media to 14 days (Whelan et al., 1997). Examina- tion using scanning electron microscopy S. filum can be grown readily on agar media revealed that S. filum spores germinated such as Potato Dextrose Agar (PDA). It also and produced a hyphal network, which grows well on chemically defined media spread across the leaf surface. and utilizes a wide range of carbon and Swendsrud and Calpuzos (1972) nitrogen compounds, including inorganic reported that application of S. filum to wheat nitrogen sources (Calpouzos et al., 1957; seedlings 3 days prior to inoculation with Bean, 1968). Mycelial cultures of the P. recondita resulted in less infection by fungus can also be grown in liquid media S. filum compared with simultaneous (T. Hunter, unpublished results). inoculation with both S. filum and rust Near-ultraviolet (UV) light was consid- spores. With M. larici-epitea, more rust ered to be helpful for sporulation of S. filum pustules were colonized by S. filum and (Kranz, 1969a). However, we often experi- fewer rust spores were produced when enced difficulties in inducing sporulation S. filum and rust were inoculated simulta- on agar media with relatively old cultures of neously, compared to the inoculations of S. filum. In such cases, mycelial cultures of rust pustules with S. filum 3–4 days after S. filum were homogenized and applied, rust pustules had appeared (Yuan et al., together with rust urediniospores, on to 1999). It was reported that S. filum failed to detached leaves of hosts (see Pei et al., 2003). infect P. recondita in inoculation experi- After 2 weeks of incubation in a growth ments with S. filum later than 4 days cabinet, the leaves were re-inoculated by after rust inoculation (von Schroeder and brushing the newly produced conidial cirrhi Hassebrauk, 1957). Keener (1934), Kranz over the same leaves. After a further 2 weeks, (1972) and Kuhlman et al. (1978) found that fresh cirrhi were removed using fine sterile infections by the mycoparasite increased forceps under a stereomicroscope and used with age of rust sori. for inoculation.

Storage of the Fungus Incubation Period

On dried rusted wheat leaves, S. filum Incubation periods (time lapse between remained viable for 36 weeks in a refrigera- inoculation and appearance of pycnidia) for tor, 10 weeks in the laboratory and from S. filum have been reported in a range of 6 to 12 weeks on water in the laboratory 4–9 days, mostly 5–7 days at summer tem- (Kranz, 1969b). With S. filum on willow peratures (see Kranz, 1981). When S. filum and poplar rusts, we found that keeping isolates obtained from Melampsora spp. on dried leaf samples carrying both S. filum willow and poplars were inoculated simul- and rust sori in a freezer is an effective way taneously with rust, pycnidia developed of long-term storage of the fungus (M.H. Pei, 6–7 days after inoculation (Yuan et al., unpublished results). In our lab, willow or 1999). Sphaerellopsis filum and Biological Control of Willow Rust 249

‘Infection Efficiency’ in S. filum

It appears that relatively small numbers of S. filum are required to establish infection of rust pustules. Kranz (1972) inoculated P. recondita on wheat seedlings with S. filum using seven spore concentrations between 30 × 103 and 3000 × 103 spores/ml and pro- duced infections on 17–63% rust pustules. Kuhlman et al. (1978) inoculated uredinia and telia of Cronartium fusiforme on water oak (Quercus nigra) with six concentrations of S. filum, ranging from 31 × 103 to 1000 × 103 spores/ml, and obtained 6–73% infection of uredinia. In a recent study, S. filum was applied using different spore concentrations on to willow leaf discs inoculated with M. larici- epitea (Pei et al., 2003). In this experiment, willow leaf discs were inoculated using a standard inoculum of rust (inoculum Fig. 21.2. Effects of inoculum densities (the 2 density = 262 viable spores per 0.95 cm number of conidia applied per leaf disc) of leaf disc) and, as a result, an average of 46 Sphaerellopsis filum on (a) the frequency of rust uredinia formed on each leaf disc. Results uredinia infected and (b) the number of S. filum from this study showed that the ratios of pycnidia produced, per leaf disc 13 days after infected rust pustules/S. filum conidia inoculation. Sqrt, square root. (From Pei et al., applied were in a range of 0.25–0.31 when 2003.) less than 20 S. filum spores were inoculated on to a leaf disc. The ratios were 0.8–0.12 when 60–200 S. filum spores ratio of the number of pycnidia/S. filum were inoculated on to a leaf disc. There conidia applied is not entirely synonymous was a close correlation between S. filum with the infection efficiency in plant–rust inoculum and percentage infection of rust systems. uredinia/the number of pycnidia (Fig. 21.2). Suppressive effects of S. filum on rust spore production were more obvious in the second assessment, carried out 23 days Effects of S. filum on Rust Spore after inoculation. Production and Disease Severity In plant–rust pathogen systems, the proportion of inoculum that produces Both rust fungi and S. filum produce large pustules/lesions is referred to as infection numbers of spores during the season. Quan- efficiency (see Zadoks and Schein, 1979). titative inoculations with M. larici-epitea With rusts, it has been proven that one rust suggested that each uredinium can produce spore produces only one pustule on the up to 13 × 103 urediniospores per day (Pei hosts (Zadoks and Schein, 1979; Pei et al., et al., 2002). When assessed 13 days after 2002) and, therefore, the ratio between the inoculation of M. larici-epitea with S. filum, inoculum density and the number of rust the average number of S. filum conidia pustules produced is equivalent to the infec- produced per pycnidium was 8920 ± 880 tion efficiency. With S. filum, however, it is (P = 0.05) (Pei et al., 2003). Given the not certain whether a conidium can produce assumption that the average incubation more than one pycnidium. Therefore, the period of S. filum had been 8 days, more 250 Ming Hao Pei and Z.W. Yuan

than 1000 conidia would have been colonized and rust spore production was produced per pycnidium per day. suppressed by 64–98%. In the second, the Kranz (1981) observed that, compared same figures were 33–97% and 53–73%, with the uredinia free of S. filum, one respectively. S. filum pycnidium per uredinium of P. In quantitative inoculations of M. recondita could reduce urediniospore pro- larici-epitea with S. filum (Pei et al., duction by 10–70%, and nearly complete 2003), there was a reasonable linear corre- coverage of rust uredinia by pycnidia lation (y = −2.9353x + 423.75, % variance reduced urediniospores by 50–95%. accounted for = 51.1) between the square Kuhlman et al. (1978) reported 25–50% root number of rust spores produced (y) and reduction in basidiospore production in the frequency of rust uredinia infected by Cronartium flaccidum compared to the telial S. filum (x). According to the relationship, leaves free of S. filum. With larch rust the % reduction in spore production (com- Triphragmiopsis laricinum, Yuan (1991) pared with the control) can be calculated as found that germination of teliospores from in Table 21.1. the telia colonized by S. filum was reduced by some 50% compared with those from the telia free of S. filum pycnidia. Spread in Plantations Hau and Kranz (1978) described a com- puter model using P. recondita f. sp. tritici How S. filum spreads in a plantation is to assess the effectiveness of S. filum in an important question to be answered in suppressing rust epidemics. The model deployment of the mycoparasite for bio- comprised functions for green leaf biomass control. The fungus colonizes rust pustules of wheat corrected for phase-dependent and produces masses of conidia (cirrhi), susceptibility, interactions between host which are easily suspended in water. The growth and disease development, etc. Using gelatinous caps of conidia may indicate a the model, various assumptions were tested, splash-borne mechanism of spore dispersal. i.e. the extent of colonization of rust sori Such a structure may also help conidia to by S. filum, time to reach certain coverage be attached to rust spores or to insects to (e.g. 40% of pustule area), the time between facilitate airborne dispersal. In nature, dry the first appearance of rust and the first weather conditions are certainly detrimen- appearance of S. filum, and the effect of tal to the spread of S. filum. In southern rust density. The model estimated that rust India, S. filum is absent during the dry severity would be reduced by 87% when season but may occur after heavy showers 40% of pustule area is covered by S. filum (Ramakrishnan and Narasimhalu, 1941; 2 days after rust pustules appeared. If 40% of Sundaram, 1962). pustule area was covered by S. filum 4, 6, 8 or 10 days after rust pustules appeared, rust severity would be reduced by 78%, 64%, 42% or 24%, respectively. Hence the model Table 21.1. Estimated percentage reduction showed that early establishment of infection in rust spore production at different levels of by S. filum may be more effective in infection by Sphaerellopsis filum 13 days after suppressing disease levels. simultaneous inoculations with rust and S. filum. Yuan et al. (1998) conducted two inocu- Frequency of uredinia % reduction in rust lation experiments with M. larici-epitea and colonized by S. filum spore production S. filum. In the first experiment, S. filum and rust were applied simultaneously on to leaf 0 0 discs of Salix burjatica ‘Korso’. In the second 20 26 experiment, leaf discs were inoculated 40 48 with rust initially and then resulting rust 60 66 uredinia were inoculated with S. filum.In 80 80 100 91 the first experiment, 55–99% pustules were Sphaerellopsis filum and Biological Control of Willow Rust 251

To investigate the spread of S. filum in Potential for Biological Control of Rust plantations, Morris et al. (1995) conducted in Coppice Plantations a field experiment using three sub-plots in a S. viminalis ‘Bowles Hybrid’ plantation at Laboratory inoculation experiments have Long Ashton, south-west England. Early in consistently proved that, given optimum the season (May), S. filum was introduced conditions, S. filum can be remarkably to two of the sub-plots by placing willow effective in suppressing rust disease on plants previously inoculated with both M. willow. It was also revealed that some larici-epitea and S. filum. A lattice of 12 × 12 genotypes of S. filum were more virulent stools from each subplot was examined for than others. As knowledge on the patho- the presence of S. filum at 6- to 10-day genic variation in S. filum became available intervals until late July, and the data were only very recently, it has not been possible analysed using two-dimensional distance to verify whether the numerous reports that class analysis (2D analysis) (Grey et al., rust disease was suppressed markedly by S. 1986). It was shown that S. filum disperses filum were associated with the occurrence in a non-random, concentric pattern from of highly virulent populations of S. filum in the central inoculum source (introduced) as the field. opposed to the random pattern found in the In a low-input crop system such as control sub-plot where S. filum occurred SRC willow, sustaining the mycoparasite naturally without introduction. The average populations in the field is important in the rate of S. filum dispersal from the inoculum deployment of S. filum for biocontrol. Previ- source was slow initially (in the first 4 weeks ous work by Morris et al. (1995) and our of observation) and became more rapid unpublished results showed that S. filum thereafter. is capable of overwintering as conidia on The dispersal of S. filum in willow infected rust pustules of the stem-infecting plantations was further examined using form of Melampsora (SIF) on S. viminalis. molecular markers (M.H. Pei and C. Ruiz, Melampsora rusts on SRC willows are unpublished results). As S. filum repro- highly specialized in their pathogenicity duces asexually in the growing season, DNA (Pei et al., 1996). For example, the pathoge- fingerprinting provides an effective way of nicity of SIF is confined to certain clones examining the genotype distribution in within S. viminalis. By carefully designing plantations. Sampling was done from S. host genotype mixtures, it is possible to min- burjatica ‘Germany’ in a willow planting imize the impact of certain forms of rust on that was surrounded by cereal crops. Natural which S. filum can overwinter. Rapid spread infections were detected in the early stages of the mycoparasite to keep pace with rust of rust epidemics (July 1998). S. filum sam- development can only be achieved in mixed ples were made consecutively from 46 stools clonal plantings, in which the clones carry- (0.5 m apart) of ‘Germany’ in the autumn ing S. filum are planted in intimate mixtures (September). A single-spore isolate was with others. The ‘carrier’ of S. filum may not made from each sample and fingerprinted be confined to the clones that are susceptible using AFLP. A preliminary analysis showed to SIF, since some other Melampsora spe- that two genotypes were most frequent, one cies, such as M. capraearum, can cause stem of which was found on 43% of the stools. infections and may harbour overwintering The same genotypes often occurred in S. filum. adjacent stools, indicating that rain splash Morris et al. (1994, 1995) successfully or leaf contact may be an important means introduced S. filum into willow planting of spread in S. filum. However, somewhat using potted willow plants. These studies scattered distribution of the same genotypes showed that effective biocontrol can be also suggested that other mechanisms, achieved only if the mycoparasite is dispersal by insects or rust spores, for introduced or sustained at many sources example, might play a significant role in throughout the plantation. Practical ways of the spread of S. filum. achieving this require further investigation. 252 Ming Hao Pei and Z.W. Yuan

Concluding Remarks Calpouzos, L., Theis, T. and Buller, C.M.H. (1957) Culture of the rust parasite, Darluca filum. In this chapter, we have given a brief review Phytopathology 47, 108–109. Carling, D.E., Brown, M.F. and Millikan, D.F. of some of the existing information on (1976) Ultrastructual examination of the taxonomy, life-cycle, pathogenicity, and Puccinia graminis–Darluca filum host parasite suppressive effects of S. filum on rusts. We relationship. Phytopathology 66, 419– 422. have also discussed the potential of using Castagne, L. (1851) Catalogue des Plantes qui the mycoparasite for biological control of Croissent Naturellement aux Environs de willow rusts in short-rotation coppice plan- Marseille. Supplément. Aix, pp. 52–56. tations. The development of a practicable Eriksson, O. (1966) On Eudarluca caricis (Fr.) O. rust control strategy without relying on Eriks., Comb. nov., a cosmopolitan uredini- fungicides requires integrated approaches colous pyrenomycete. Botaniska Notiser 119, involving selection and breeding for 33–69. Eriksson, O. (1967) On graminicolous pyrenomycetes resistance, and planting clonal mixtures. from Fennoscandia, 2. Phragmosporous and By designing mixtures to encourage the scolecosporous species. Arkiv For Botanik 6, spread of the mycoparasite, the suppressive 390–392. effects of mixtures on rust disease can be Fries, F. (1823) Systema Mycologicum 2, 547. enhanced. Grey, S.M., Moyer, J.W. and Bloomfield, P. (1986) In all, there is a strong potential for a Two-dimensional distance class model for significant degree of biological control of quantitative description of virus-infected rust by deploying the mycoparasite S. filum plants distribution lattices. Phytopathology 76, in SRC willow plantations. Compared with 243–248. annual crops, perennial crops, such as SRC Hau, B. and Kranz, J. (1978) Modellrechnungen zur Wirkung des Hyperparasiten Eudarluca caricis willows, which are harvested at 2–5-year of Rostepidemien. Zeitschrift für Pflanzenkrank- intervals, are better suited for deploying heiten und Pflanzenschutz 85, 131–141. mycoparasites for biological control, Hulea, E.E. (1939) Contributions à la connaissance because they allow inoculum to be carried des champignons commensaux des uredinea. over from 1 year to the next. Bulletin de la Section Scientifique de l’Academie Roumaine 22, 1–19. Keener, P.D. (1934) Biological specialization in Acknowledgements Darluca. Bulletin of Torrey Botanical Club 61, 475–490. Keener, P.D. (1951) An ascigerous stage of Darluca This study was funded by the Department filum (Biv.) Castagne. Plant Disease Reporter 35, for Environment, Food and Rural Affairs 86–87. (DEFRA), UK, and the European Union. Kranz, J. (1969a) Zur natülichen Verbreitung des Rothamsted Research receives grant-aided Rostparasiten Eudarluca caricis (Fr.) O. Erikss. support from the Biotechnology and Phytopathologische Zeitschrift 65, 43–53. Biological Sciences Research Council of Kranz, J. (1969b) Das Verhalten von Darluca filum the UK. (Biv.-Bern.) Cast. in vitro unter verschiedenen Versuchsbedingungen. Phytopathologische Zeitschrift 65, 325–331. Kranz, J. (1972) Zur Infection mith Eudarluca caricis References (Fr.) O. Erikss. Phytopathologische Zeitschrift 74, 13–20. Adams, J.F. (1920) Darluca on Peridermium peckii. Kranz, J. (1981) Hyperparasitism of biotrophic Mycologiga 12, 309–315. fungi. In: Blakeman, J.P. (ed.) Microbial Ecology Bean, G.A. (1968) Growth of the hyperparasite of the Phylloplane. Academic Press, London, Darluca filum on a chemically defined medium. pp. 327–352. Phytopathology 58, 252–253. Kranz, J. and Brandenburger, W. (1981) An amended Bivona-Bernardii, A. (1813) Stirpium rariorum in host list of the rust parasite Eudarluca caricis. Sicilia provenienthium descriptions. Manipulus Journal of Plant Diseases and Protection 88, 3, 12. 682–702. Sphaerellopsis filum and Biological Control of Willow Rust 253

Kuhlman, E.G., Mathews, F.R. and Tillerson, H.P. Spegazzini, C. (1908) Fungi Aliguot Paulistani. (1978) Efficacy of Darluca for biological control Revista del Museo de La Plata 15, 22. of Cronartium fusiforme and C. strobelinum. Sundaram, N.V. (1962) Studies on the parasites of the Phytopathology 68, 507–511. rusts. Indian Journal of Agricultural Sciences 32, Morelet, M. and Pinon, J. (1973) Darluca filum hyper- 266–276. parasite du genre Melampsora sur peuplier et Sutton, B.C. (1977) Coelomycetes. VI. Nomenclature saule. Revue Forestière Française 25, 378–379. of generic names proposed for Coelomycetes. Morris, R.A.C., Royle, D.J., Whelan, M.J. and Arnold, Mycological Papers 141, p. 196. G.M. (1994) Biocontrol of willow rusts with the Swendsrud, D.P. and Calpouzos, L. (1972) Effect of hyperparasite Sphaerellopsis filum. Proceedings inoculation sequence and humidity on infection of the Brighton Crop Protection Conference of P. recondita by the mycoparasite Darluca – Pests and Diseases, vol. 3. Brighton, UK, filum. Phytopathology 62, 931–932. pp. 1121–1126. von Shroeder, H. and Hassebrauk, K. (1957) Beiträge Morris, R.A.C, Royle, D.J. and Whelan, M.J. (1995) zur Biologie von Darluca filum (Biv.) Cast. und Potential for biological control of willow rust einigen anderen an Uredi beobachteten Pilzen. with the hyperparasite Sphaerellopsis filum. Zentralblatt für Bakteriologie Parasitenkunde Final Report of Project ETSU B/W6/00230/REP. Infektionskrankheiten und Hygiene II Energy Technology Support Group, Department Abteilung-Naturwissenschaftliche-Mikrobiologi of Trade and Industry, UK. e Der Landwirtschaft Der Technologie 110, Parker, S.R., Royle, D.J. and Hunter, T. (1993) Impact 676–696. of Melampsora rust on yield of biomass willows. Whelan, M.J., Hunter, T., Parker, S.R. and Royle, D.J. In: Abstracts of the 6th International Congress of (1997) How effective is Sphaerellopsis filum Plant Pathology, Montreal, Canada, 28 July– as a biocontrol agent of Melampsora 6 August 1993. National Research Council willow rust? Aspects of Applied Biology 49, Canada, Ottawa, p. 117. 143–148. Pei, M.H., Royle, D.J, and Hunter, T. (1995) A Yuan, J.Y., Yuan, Z.W. and Li, L.Z. (1988) Studies on comparative study of stem-infecting and leaf- the biological control of larch brown rust with a infecting forms of Melampsora rust on Salix rust parasite – 1. Morphological and cultural viminalis in the UK. Mycological Research 99, characteristics of the rust parasite [in Chinese]. 357–363. Journal of Shenyang Agricultural University 19, Pei, M.H., Royle, D.J. and Hunter, T. (1996) Patho- 17–22. genic specialisation in Melampsora epitea var. Yuan, X.Y. and Han, Y.J. (2000) Hyperparastism and epitea on Salix. Plant Pathology 45, 679–690. biocontrol of Hedysarum mongolicum rust Pei, M.H., Ruiz, C., Hunter, T., Arnold, G.M. and by Sphaerellopsis filum [in Chinese]. Forest Bayon, C. (2002) Quantitative relationships Research 13, 103–106. between inoculum of Melampsora larici-epitea Yuan, Z.W. (1991) Observations on a pathogen and corresponding disease on Salix. Plant parasitizing larch brown rust, Triphragmiopsis Pathology 51, 443–453. laricinum [in Chinese]. Chinese Journal of Pei, M.H., Hunter, T., Ruiz, C., Bayon, C. and Harris, J. Biological Control 7, 61–63. (2003a) Quantitative inoculation of willow rust Yuan, Z.W., Pei, M.H., Hunter, T. and Royle, D. J. Melampsora larici-epitea with the mycoparasite (1998) Darluca caricis, the teleomorph of the Sphaerellopsis filum. Mycological Research 107, mycoparasite Sphaerellopsis filum, on black- 57–63. berry rust Phragmidium violaceum. Mycological Pei, M.H., Ruiz, C., Hunter, T. and Bayon, C. (2003b) Research 102, 866–868. Rust resistance in Salix induced by inoculations Yuan, Z.W., Pei, M.H., Hunter, T., Ruiz, C. and Royle, with avirulent and virulent isolates of D.J. (1999) Pathogenicity to willow Melampsora Melampsora larici-epitea. Forest Pathology 33, epitea, of the mycoparasite Sphaerellopsis filum 383–394. from different sources. Mycological Research Ramakrishinan, T.S. and Narasimhalu, I.L. (1941) 103, 509–512. The occurrence of Darluca filum (Bev.) Cast. on Zadoks, J.C. and Schein, R.D. (1979) Epidemiology cereal rusts in South India. Current Science 17, and Plant Disease Management. Oxford Univer- 215–216. sity Press, Oxford, UK. This page intentionally left blank Index

Abies Biological control 213–229, 243–252 Melampsora – alternate hosts 3, 19, bacteria 213 37, 53, 54, 58, 100 difficulties in transfer to field 213–214 Aecia and aeciospores fungi 213, 243 Melampsora on Salicaceae 3, 13–16, Melampsora ciliata 115 18, 32, 55–58 mycoviruses 213 AFLP Sphaerellopsis filum 243–252 Melampsora 93–96, 127 Sphaerellopsis filum 251 Agrochemicals 213 Caeoma salicis-miyabeana 16, 24 Aigeiros – black poplars 51–54, 101 Castlearchdale 94, 95, 189 Allium Chamaetia 11 Melampsora – alternate hosts 19, 21, Chelidonium 24, 32, 46, 53, 144 Melampsora – alternate hosts 34, 35, Alternate hosts 108 influence on genetic structure 95 Chemical control Melampsora on Populus 53–55, 58 Melampsora ciliata 116, 209–212 Melampsora on Salix 19, 21–24, 75 Melampsora epitea 185, 186, 198 AMOVA 94, 96 Chrysomyxa abietis – Picea abies 161 Appressorium 163, 165–167, 169–171 Cladosporium aecidiicola 216 Arum Cladosporium gallicola 215 Melampsora – alternate hosts 59, 144 Cladosporium hemileiae 215 Aspen, transgenic hybrid 156 Cladosporium species – hyperparasitic on Auricularia 4 rusts 215 Autoecious 2, 3, 19, 33 Cladosporium sphaerospermum 216 Autoinfection, mixtures 177 Cladosporium tenuissimum distribution 217 ecological fitness 223 b-1,3-glucanases 223 effect of culture filtrates 219 Basidiospore identification 217 germ tubes 165, 166, 167 in vitro antagonism 218 penetration 161–174 in vivo antagonism 220 Basidiospores and spermagonia LM, SEM, TEM 222 Melampsora on Populus 55 secondary metabolites 219 Melampsora on Salix 18 timing of infection 218

255 256 Index

Cladosporium uredinicola 215 of rust genera 2 Cladosporium uredinophilum 215 Sphaerellopsis filum 246 Cladosporols 220, 224 Human transportation 128 Coleosporium spp. on Pinus spp. 161 Hyperparasite 224 Corydalis, Melampsora – alternate hosts 19, 21, 24, 34, 35, 48, 53, 58, 106, 108 Incompatibility codes 187 Cronartium flaccidium Infection efficiency haustoria 171 Melampsora larici-populina 145 penetration 163 Spaerellopsis filum 249 Pinus nigra subsp. laricio 163 Infection process Pinus pinaster 163 dikaryotic 162, 169 Pinus pinaster – micrographs 164 monokaryotic 162, 170 Cronartium ribicola – Pinus strobus 161 Infection rate 210 Infection, relative 143 Inoculum density Darluca filum = Eudarluca caricis 244 disease scoring 131–137 Differential set, Populus 68 Melampsora larici-epitea 134 Dikaryotic stage – penetration 169 Melampsora larici-populina 133 Disease scoring 131–137, 187, 209, 210 uredinial numbers 132, 133 inoculum density 134, 135 uredinial pustule area 133–136 uredinial pustule area 133–136 Inoculum exchange Divergence, ITS of Melampsora 84 intercontinental 128, 129 Diversity, host composition in long-distance 128, 129 mixtures 175–183 Inoculum pressure 132

Erysiphe graminis 186, 188 Larch Eudarluca caricis 231, 244 alternate host of Melampsora 18, 19, Evolution, rust fungi 2–7 21–23, 53, 54, 58, 68, 69, 124, 150 host of Triphragmiopsis laricinum 246 influence on genetic structure of Flavonoids 155 Melampsora 95 Fumaria – Melampsora – alternate Larix see Larch hosts 99 Latency, relative 143 Fungicides 185, 186, 198, 209–212 Leuce – aspen and true white poplars 52 hexaconazole 210, 211 Life cycle rust control 209 evolution 5, 6 Fusarium sambucinum 185 Melampsora on Populus 58 Melampsora on Salix 18, 19 Long distance migration, rust Gene flow – rust populations 125, 128, 129 populations 125, 128, 129 Genotype evaluation 195 Genotype Unit Area (GUA) 177, 178 Glomerella miyabeana 185 Marssonina brunnea 139 Melampsora abieti-caprearum 18, 20, 21, 25, 81 Haustoria 166, 171–173 abietis-canadensis 54, 100–103 Heteroecious 1–3 abietis-populi 53, 55, 100–102, 109 Host specificity allii-fragilis 13, 17, 19, 20, 21, 32, 33, Melampsora 2, 20, 92 37, 47, 79, 80, 85 Index 257

allii-fragilis f.sp. galanthi-fragilis 32, kamikotica 15, 17, 22, 25, 43 33, 37 kiusiana 15, 17, 22, 36 allii-populina 53, 55, 58, 63–66, 70, kuperviczii 15, 43 100–103, 144–147, 150 lapponum 15 interaction with poplar larici-caprearum 45 clones 144 see also M. caprearum pathogenicity 144 larici-epitea 15, 17–19, 22, 25, 26, 45, poplar susceptibility 150 94, 96 virulences 145 biology 91 amygdalinae 13, 17, 19–21, 33, 37, 85 Castlearchdale, NI 94, 95 artica 13, 17, 20, 21, 25, 38, 39, 75 dispersal 92 capraearum see M. caprearum diversity 92, 93 caprearum 13, 19–21, 25, 26, 33, 34, gene flow 96, 97 36, 188, 251 genetic variation 96 caprearum, Chinese form 21 host-specificity 92 caprearum, Japanese form 21 larch 95 chelidonii-pierotii 13, 17, 21, 34, 35, life cycle 18, 91–93 48 populations 91–98 choseniae 14, 17, 21, 25, 35, 43 Sweden 94, 95 ciliata 52, 53, 55, 56, 113–116 larici-pentandrae 15, 17, 20, 22, 37, biological control 115 43–45, 75 chemical control 116 larici-populina 53, 56, 58, 59, 63–70, epidemiology 114 101, 104–107, 114, 132–135, host genotype 115 139–151 variability 115 aggressiveness 142 coleosporioides 14, 17, 20, 36, 128 disease scoring 132–139 ¥ columbiana 53, 56, 59, 66, 67, 131 interaction with poplar dimorphospora 14, 17, 21, 35 clones 142 epiphylla 14, 17, 22, 26, 35, 36 pathotype groups 141 epitea 36, 73, 131 pathotypes 140 alternate host – Chile 124 poplar susceptibility 150 life cycle 74, 75 variability 140 pathogenic variation 131 virulences 139, 140 pathogenicity 120 larici-tremulae = M. laricis 52, 53, 55, pathotypes 73, 120, 121 56, 58, 63, 101, 103, 104 epitea – complex 14, 17 larici-urbaniana 15, 17, 23, 44, 45 epitea f.sp. abieti-capraearum 36, 37 magnusiana 52, 53, 56, 58, 63, 100, epitea f.sp. artica 38 101, 104, 106–110 epitea f.sp. euonymi 39 medusae 54–60, 63–70, 145, 146 epitea f.sp. lapponum 39 distribution 54, 58, 59, 145 epitea f.sp. larici-epitea 40 interaction with poplar epitea f.sp. rebesii-purpurae 41, 42 clones 146 epitea f.sp. repentis 40 pathogenicity 146 epitea f.sp. reticulatae 41 medusae-populina 54, 55, 59, 67 epitea f.sp. tsugae 14, 42 microsora 15, 17, 22, 36 euonymi-caprearum 14 microspora 52, 54, 55, 57 euphorbiae 2, 4, 99 multa 54, 55, 57–59, 101, 105, 108 galanthi-fragilis 13, 17, 19, 20, 22, 33, occidentalis 54, 55, 57–60, 64–67, 75 101, 109 helioscopiae 4 paradoxa 15, 20, 23, 25, 45 humilis 14, 21, 36 pinitorqua 52, 54, 57, 58, 63, 167 hypericorum 4 haustoria 171 258 Index

Melampsora continued uredinia and urediniospores 13–17, Larix decidua 162 75–79 populnea 109, 114 Melampsora, overwintering 20, 58 pruinosae 52, 54, 55, 57, 100, 101,109, Melampsora, phylogenetic position 4, 5 110 Melampsora, relationships to other rusts 6, pulcherrima 54, 55, 58, 106, 165, 167, 7 169 Melampsora spp. penetration 165, 167 isolate divergence 84–86 penetration, micrographs 169 ITS of rDNA 64–66, 81–85 repentis 15, 23, 41, 75 multi-virulence 122 reticulatae 16, 17, 23, 25, 41 on Populus ribesii-epitea 16, 24, 42, 75, 81 diversity 64, 65 ribesii-purpureae 16, 17, 19, 24, 26, interspecific hybrids 66, 67 41, 42, 75 on Salix 11–28, 73–89 ribesii-viminalis 17, 24, 44, 46, 75 Chile 119–130 rostrupii 52, 54, 55, 57, 58, 63, 100, Northern Europe 119–131 101, 106, 108, 110 pathotypes 120 salicis-albae 16, 20, 24, 46, 47, 75 penetration 165 salicis-cavaleriei 16, 17, 24, 47 Melampsora, timing of divergence 7 salicis-cupularis 16, 17, 24, 47 Mercurialis, Melampsora – alternate salicis-warburgii 16, 17, 47 hosts 99 stem infecting form (SIF) 18–20, 26, Mixtures 185–194 245 autoinfection 177 yezoensis 16, 17, 24, 44, 48 Blumeria graminis 175 Melampsora, aecial hosts of poplar composition 176 rusts 53, 54, 58 contribution of components to Melampsora, aecial hosts of willow yield 201 rusts 19, 21–24 cultivar 175 Melampsora, host alternation 19, 58 diversity 204 Melampsora, host specificity 20, 25 effect on disease 175, 187 Melampsora, interspecific epidemic intensity 176 hybridization 59 limited diversity 191, 192 Melampsora, introduction 1, 2 Magnaporthe grisea 175 Melampsora, life cycle 18, 58 mechanisms 192 Melampsora, on Populus 51–62, 52–54, Melampsora rusts 179, 180 63–72, 99–112 multi-virulent pathotypes 122 aecia and aeciospores 55 numbers of components 199 basidiospores and spermagonia 55 Oryza sativa 175 distribution 58 pathogen complexity 178 host alternation 58 pathogen evolution 178 morphology 52, 56, 57 poplar clones 151 telia and teliospores 55 potato blight 177 uredinia and urediniospores 55 Puccinia spp. 175 Melampsora, on Salix 21–24 rows 176 aecia and aeciospores 13–16, 17 Salix viminalis 191, 192, 204–206 basidiospores and spermagonia 18 small grains 175 distribution 21–24 spatial scale 177 host alternation 19 stool survival 203, 204 morphology 13–18, 29–48 Venturia inaequalis 178 shrub and dwarf willow 25 Molecular clock 7 telia and teliospores 13–17 Molecular variance – AMOVA 94, 96 tree willow 20 Monoculture 186 Index 259

Monokaryotic, penetration 170, 171 India 113–117 Multiline, cultivars 175 Population biology Multilines 186 Melampsora larici-populina 68–70 MYB genes Population structure phenylpropanoid metabolism 155 Melampsora epitea 123, 124 PttMYB46 156, 157, 158 Melamspora larici-epitea 91–97 PttMYB76 156, 158 Populus antisense constructs 156 adenopoda 104, 109 alba 52–54, 58, 60, 63, 103, 106, 110, 113–115, 165, 167 Overwintering, Melampsora 20, 58 alba ¥ davidiana 103 alba var. pyramidalis 106, 107 alba var. vitellina 26, 45, 47 Papaver, Melampsora – alternate hosts 99 angustifolia 52, 54 Pathogen evolution, mixtures 178 balsamifera 53, 54 Pathogenicity tests 80 ¥ berolinensis 104 Pathotypes ¥ canescens 52–54, 167 aggressiveness 120 capsica 114 Chile 123, 124, 126 casale 114 coding 122 cathayana 100, 102–104, 109 diversity 188 ciliata 53, 113–116, 209 evolution of population 148, 149 ciliata ¥ maximowiczii 114, 115 Melampsora 73, 87, 201 ciliata ‘Theog’ 114, 219 population 188 davidiana = tremula var. super race 188 davidiana 52, 102–106, 109 Sweden 121, 126 deltoides 51–54, 63, 64, 67, 104, virulences 139 113–115, 141, 142, 146, 152 Penetration deltoides ‘19’ 115 direct 161 deltoides ‘Lux’ 114, 141, 146 indirect 161 deltoides ¥ trichocarpa 53 signalling 170 deltoides ¥ yunnanensis 54 Phenolic compounds euphratica 52, 104, 106, 109, 110 defence chemicals 157 ¥ euramericana 52–54, 59, 63, 64, 68, plant response 158 70, 100, 104–106, 108, 113, Salicaceae 155 114, 141, 144, 149, 150 Phenylpropanoid pathway 158 ¥ euramericana ‘Gaver’ 64, 141, 146 Phylogeny of rusts 1–7 ¥ euramericana ‘Ghoy’ 64, 140, 141, Picea, Melampsora – alternate hosts 54, 144, 146 58, 99 ¥ euramericana ‘I 45-51’ 64, 146 Pinaceae, aecial hosts 3 ¥ euramericana ‘I 488’ 64, 146 Pinus ¥ euramericana ‘I-214’ 54, 108, 141, Melampsora – alternate hosts 54, 58, 144–146, 149 99 ¥ euramericana ‘Marilandica’ 109 nigra subsp. laricio 163 ¥ euramericana ‘Robusta’ 54, 106, pinaster 163 108, 114, 141–144, 147–152 Planting configuration ¥ euramericana ‘Rubra Poiret’ 114 line mixtures 176, 202 ¥ euramericana ‘Serotina’ 54, 108 random mixtures 202 ¥ euramericana var. regenerata 104 short run mixtures 202 fremontii 53, 54 Pollacia radiosa 157 ¥ gansuensis 104, 106 Poplar rust grandidentata 54 China 99–111 hopeiensis 106 260 Index

Populus continued usbekistanica 54, 59 ¥ interamericana = trichocarpa ¥ ussuriensis 53, 106 deltoides 63 wilsonii 53, 100, 102, 103 ¥ interamericana ‘Baupré’ 64, ¥ xiaozuanica 106, 109 139–147, 149, 151, 152 Populus, Aigeiros – black poplars 51, 52 ¥ interamericana ‘Boelare’ 64, 140, Populus, Leuce – aspen and true white 141, 144, 148, 149, 151 poplars 52 koreana 52, 53, 104, 109 Populus, Tacamahaca – balsam poplars 52 lasiocarpa 53, 105 Populus, Turanga and Leucoides 52 laurifolia 52, 53, 64, 102–104, 107 manshurica 105 maximowiczii 52–54, 113, 152 Races, physiological 139 maximowiczii ¥ balsamifera 64 RAPD markers 91 maximowiczii ¥ berolinensis 113 Resistance nigra 51–54, 63, 68–70, 105, 113–115, breakdown 146, 147, 152 141, 142, 144, 145 chemical basis 157 nigra ‘Italica’ 54, 100, 105, 107, 109, complete 141 110, 141, 144, 146 durable 139–154 nigra ‘Vereecken’ 132, 135, 146 pathotype-specific 141 nigra ¥ deltoides ‘Spijk’ 132, 135, 140, virulences 147 141, 146 RFLP analysis 81–85 nigra ¥ hopeiensis 105 Ribosomal DNA, Sphaerellopsis nigra ¥ trichocarpa 114, 115 filum 236–239 nigra var. italica 54, 100, 105, 107, Ribosomal DNA, Uredinales 109, 110, 141, 144, 146 internal transcribed spacer (ITS) nigra var. thevestina 105 region 64–66, 81–85 opera 103, 105 large subunit (LSU) 3–7, 84–86 oxfordii 114 small subunit (SSU) 3–7 pioneer 106 Row mixtures 176 pruinosa 54 Rust pseudo-simonii 106 disease control strategies 214 pseudo-simonii ¥ deltoides 106 infection development 79 purdomii 106 susceptibility 191 robusta 114 shanxiensis 106 sieboldii 53, 54, 108 simonii 52, 53, 106 Salicaceae 11 simonii ¥ davidiana 106 Salicin 156 suaveolens 53 Salicylates 155, 158 talassica 102, 103 Salix tremula 52–54, 63, 103, 110, 113, 114, acutifolia 23, 76, 77, 80, 82, 233, 237 167 alba 24, 26, 36, 45, 47, 75, 77, 79, 80, tremula var. davidiana see P. 82, 124, 125, 233, 237 davidiana alpicola 23 tremula ¥ tremuloides 156 amygdaloides 45 tremuloides 52, 54 anglorum 38 trichocarpa 52–54, 63, 64, 67, 106, appendiculata 21, 24, 37 114, 115, 141, 142, 144, 149, ¥ aquatica 77, 78, 80–82, 84, 87, 237 151, 152 aquilonia 38 trichocarpa ¥ deltoides ¥ deltoides arbuscula 24, 42 ‘75028’ 132 arctica ¥ cuneata 44 trichocarpa ‘Trichobel’ 132, 141 argyrocarpa 38 Index 261

aurita 21–25, 34, 38–42, 75, 77, 78, delnortensis 45 80, 82, 84, 233, 237 discolour 26, 45, 196 aurita ¥ viminalis ¥ caprea disperma 23, 122 ‘Valdivia’ 122 disperma ‘Himalayas’ 122 babylonica 21, 45, 47, 124, 125 disperma ‘Pte. Chanco’ 122 bakko 21, 25 elegans 24 balsamifera 38 exigua 20, 21, 28, 45 barclayi 45 fendleriana 45, 47 barrattiana 23 flavescens 45 bebbiana 38, 41 fluviatilis 45 bicolour 42 foetida 23 bigelowii 45 fragilis 19–22, 26, 32, 33, 37, 43, 75, bonplondiana 45 77, 79–82, 84, 85, 124, 125, brachycarpa 41, 45 233, 237 brachypoda 22, 43 fuscescens 38 breweri 41, 48 futura 22 burjatica ‘Cascadas’ 122 geyriana 45, 47 burjatica ‘Germany’ 186, 187, 189, 190 gilgiana 22 burjatica ‘Korso’ 97, 122, 125, 132, glabra 23 185–187, 196, 198, 199, 246, glandulosa 24, 47 250 glauca 38, 42 candida 45 glaucops 38, 45 caprea 21, 22, 25, 34, 37–42, 45, 75, gracilistyla 45 77, 78, 80–82, 122, 124, 125, grandifolia 34, 42 233, 237 groenlandica 21, 38 caprea ¥ cinerea ¥ viminalis ‘L. de hastata 23 Aculeo’ 122 hegetschweileri 23 caprea var. sinica 25 helvetica 23 cavaleriei 24, 47 herbacea 21, 23, 38, 45 chamissoni 38 hibernica 23 chanomeloides 21 ¥ hirtei 77, 80, 82, 233, 247 chilensis 21 humboldtiana 12, 36, 38, 46, 124, 125 Chosenia arbutifolia = Salix humilis 38, 45 arbutifolia 11, 21, 22, 25, 35, imprimis 34 43 incana 21, 38, 39 cinerea 21, 22, 25, 38–40, 45, 124, integra 22 125, 233 interior 38 commutate 21, 38 irrorata 44 cordata 38, 45, 77, 80, 233, 237 kazbekensis 77, 233 coulteri 41 kinuyanagi 22 cupularis 16, 17, 24, 47 koriyanagi 22, 35 daphnoides 17, 23, 26, 34, 42, 75–82, lanata 38 84, 86, 122, 125, 232, 233, 237 lapponum 22, 39 daphnoides ‘78139’ 122 lasiandra 20, 23, 47, 48 daphnoides ‘San Javier’ 122 leucopithecia 21, 25 dasyclados 23, 34, 77, 78, 80, 82, 84, longifolia 38, 47 87, 92, 122, 186, 187, 199, 200, lucida 33 203, 233, 237 mackenzieana 45 dasyclados ¥ aquatica magnifica 77, 82, 233 ‘V7511’ 189–191, 200, 201 megeriana 77, 233, 237 dasyclados ¥ caprea ‘V794’ 189–191, missourensis 45 200, 201, 203, 204 miyabeana 19, 23, 40 262 Index

Salix continued schwerinii ¥ aquatica ¥ mollissima ‘V7534’ 189–191, 200, 202, = ¥ mollissima-undulata 97, 132, 203 187, 189, 190 schwerinii ¥ viminalis ¥ dasyclados ¥ mollissima ‘Chuyaca’ 122 ‘V7531’ 189–191, 200 ¥ mollissima ‘Q83’ scouleriana 21, 22, 38, 42, 45, 46, 48 = ¥ mollissima-undulata ¥ sericans 77, 78, 80, 82, 84, 233, 237 ‘SQ83’ 97, 121, 132, 187, sericea 38 189, 190, 191 serphyllifolia 23 monticola 45 silesiaca 45 myrsinites 38 sitchensis 22, 23, 38, 45, 47 myrtillifolia 38 smithiana 34, 78, 233 nigra 37, 38, 45, 77, 78–80, 82, 84, spathulifolia 21, 25 233, 237 ¥ stipularis 80, 82, 84, 122, 132, 233, nigricans 21, 23, 45, 76, 77 237 nigricantes 21, 23 stolonifera 38 ovalifolia 38 subcaerulea 45 oxycarpa 40 subfragilis 23, 24, 47 parksiana 45 subopposita 22 pedicellaris 38 subreniformis 21, 38 pentamira 33 tetrasperma 34, 47 pentandra 19–22, 26, 32, 33, 43, 47, tortuosa 124, 125 75, 77, 80, 82, 233, 237 triandra 19, 21, 33, 37, 46, 75, 77, 78, petiolaris 38 80, 82, 84, 85, 233, 237, 247 petrophila 23, 38 ¥ undulata 76, 77 pet-susu 22 urbaniana = Toisusu urbaniana 23, 44 phlomoides 34 uva-ursi 41 phylicifolia 34, 45 vestita 23 pierotii 21, 24, 34, 35, 47 viminalis 18, 20, 22, 24, 25, 39, 40, 46, piperi 41 74, 75, 77, 78–82, 84, 87, planifolia 45 92–97, 122, 124, 125, 128, 188, pseudocordata 45 191, 192, 196, 199, 204–207, purpurea 24, 38, 41, 42, 75–78, 80, 82, 233, 237, 251 84, 85, 124, 125, 233, 237 viminalis ‘77082’ 189–192, 200, 204, pyrifolia 28, 47 206 reinii 21 viminalis ‘77683’ 189–191, 200 repens 21–23, 38, 40, 41, 45, 75, 77, viminalis ‘77699’ 189–191, 200 80, 233 viminalis ‘78101’ 189–191, 200 repens var. argentea 22, 40 viminalis ‘78112’ 93, 94 reptans 38 viminalis ‘78118’ 94, 95, 189–191, 200 reticulata 23, 41, 45 viminalis ‘78183’ 94, 125, 132, retusa 23, 40, 46, 81 189–191, 196, 197, 200 rosmarinifolia 22, 34 viminalis ‘78195’ 189–192, 200, 202, rosmarinifolia var. brachypoda 22 204 rostrata 45 viminalis ‘870082 ORM’ 189–192, 200 rotundifolia 38 viminalis ‘870146 ULV’ 189–192, 200 rubra 42 viminalis ‘Chimbarongo’ 122 sachalinensis 22, 36, 40 viminalis ‘Gigantea’ 189–192, 200 schwerinii 23, 76, 77, 79, 196, 204, viminalis ‘Gustav’ 189–191, 200, 204 233 viminalis ‘Mullatin’ 122, 132 schwerinii ¥ aquatica viminalis ‘Rapp’ 93, 94, 196 ‘V7533’ 189–191, 200, 202 viminalis ‘Santiago’ 122 Index 263

viminalis ¥ aquatica ‘V7503’ 189–191, Tacamahaca – balsam poplars 52 200 rusts 53, 53, 63 viminalis ¥ caprea ‘78149’ 122 Taphrina 2, 4 viminalis ¥ caprea ‘Llanquihue’ 122 Telia and teliospores viminalis ¥ caprea ‘V789’ 189–191 Melampsora on Populus 55 vitellina see alba var. vitellina Melampsora on Salix 13–17 waldsteiniana 23 sub-cuticular 30 warburgii 24 sub-epidermal 30 wardi 45 Teliospores yessoensis 24 micrographs 102, 105, 107, 110 yestita 45 pedicelled 4–6 yezoalpina 21, 38 sessile 4, 5, 7 Salix, genus 11, 12 Tsuga, Melampsora – alternate hosts 99 Salix, geographical distribution 12 Turanga and Leucoides 52 Sphaerellopsis filum 231–241, 243–253 anamorph morphology 244 conidia 234 Uredinales 1 cultural characteristics 233 co-evolution with rusts 2 disease severity 249, 250 evolution 1, 2, 5, 6 disease suppression model 250 Uredinia, Melampsora 75 effect on rust spore production 249, Uredinia and urediniospores 250 Melampsora on Populus 55 enzyme activity 234–236 Melampsora on Salix 17 frequency 232 micrographs 102, 105, 107, 110 genetic diversity 238, 239 host specificity 238, 239, 246, 247 hyperparasitism 231 Venturia inaequalis 178 incubation period 248 Venturia tremulae 157 infection efficiency 249 Vetrix 11 infection process 234 Virulence in vitro growth 234, 248 components 124 in vivo growth 234 cost 142 life cycle 245, 246 fitness 179 lineages 237–239 frequency in M. allii-populina 147, Melampsora on willow 243 148, 149 mode of action 247 patterns 80, 81 mycoparasitism 243 occurrence 232, 233, 245 parasitism – mode of action 234 Willow rusts pathogenic variation 246 morphology 12, 13–16 PCR analysis 236, 237 taxonomy 12 rust colonization 247 Wind transportation 128 sexual stage 245, 246 spread in willow plantation 250, 251 Xanthomonas populina 139 SRC willow plantation 245, 246 sub-species 238–240 survival 248 Yield 195–208 teleomorph morphology 244 comparisons 196 variation in ITS region of compensation 202 rDNA 236–239 components 195 Superpathogen 67 components of mixtures 199 264 Index

Yield continued expectations 195, 196 dry matter 197 increase in mixtures 200 effect of rust 197, 198 Salix viminalis 204, 205