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6 Past-President’s contribution / Contribution du président sortant Fungal viruses, hypovirulence, and biological control of Sclerotinia species Greg J. Boland Abstract: Hypovirulence in fungal plant pathogens refers to the reduced ability of selected isolates within a population of a pathogen to infect, colonize, kill, and (or) reproduce on susceptible host tissues and is often associated with fungal viruses and associated double-stranded RNA elements. It has been reported to occur in numerous fungal plant pathogens, including Sclerotinia sclerotiorum, S. minor, and the disparate species S. homoeocarpa. In these fungi, hypovirulence has been associated with the presence of several fungal viruses, including one species of the genus Mitovirus, another species possibly belonging to the genus Hypovirus, and a satellite RNA. Sclerotinia spp. are primarily clonal in their life strategies, with varying degrees of diversity manifested as vegetative compatibility groups within naturally occurring populations. Vegetative compatibility groups can reduce the frequency of transmission of fungal viruses between isolates that are not compatible. Agricultural populations of S. sclerotiorum typically consist of numerous clones, although several clones often represent the majority of a population within individual fields. In contrast, populations of S. minor and S. homoeocarpa are characterized by relatively few clones and may represent more promising pathogens for hypovirulence as a biocontrol strategy. Biological control has been demonstrated through applications of hypovirulent isolates to diseased plant tissues in controlled and field environments. In S. minor, disease severity was suppressed by more than 50%, and the number of sclerotia produced on treated diseased tissues was reduced by up to 90%. These sclerotia were hypovirulent and contained double-stranded RNA characteristic of the hypovirulent isolate. In S. homoeocarpa, biocontrol efficacies of up to 90% and 80% have been achieved in controlled and field environments, respectively, and were comparable with treatment with a fungicide. Single applications of the hypovirulent isolate Sh12B, containing a strain of the species Ophiostoma mitovirus 3a (OMV3a) previously described from Ophiostoma novo-ulmi in Europe, were as effective as up to four applications of fungicide, and treatment efficacy persisted into the following year. Collectively, studies of fungal viruses and hypovirulence in Sclerotinia spp. can increase our understanding of molecular mechanisms influencing the expression of virulence in these plant pathogens and expand the potential of fungal viruses as a unique mechanism of action for biological control. Key words: double-stranded RNA, dsRNA, biological control, genus Hypovirus, genus Mitovirus, Sclerotinia sclerotiorum, Sclerotinia minor, Sclerotinia homoeocarpa. Résumé : Chez les agents phytopathogènes fongiques, le concept d’hypovirulence fait référence à la capacité réduite 18 de certains isolats d’une population d’un agent pathogène à infecter, coloniser, tuer ou se reproduire dans les tissus d’un hôte sensible, ces isolats contenant fréquemment des virus de champignons et des éléments associés d’ARN double brin. L’hypovirulence a été observée chez plusieurs champignons phytopathogènes tels que le Sclerotinia sclerotiorum, le S. minor et l’espèce disparate S. homoeocarpa. Chez ces champignons, l’hypovirulence a été associée à la présence de plusieurs virus de champignons, y compris une espèce du genre Mitovirus, une espèce appartenant probablement au genre Hypovirus et un ARN satellite. Les espèces de Sclerotinia ont des stratégies de vie principalement axées sur la reproduction clonale avec divers degrés de diversité qui se manifestent sous la forme de groupes de compatibilité végétative au sein des populations naturelles. Les groupes de compatibilité végétative peuvent réduire le taux de transmission des virus de champignons entre isolats incompatibles. Les populations agricoles de S. sclerotiorum comprennent habituellement de nombreux clones, quoique plusieurs clones forment habituellement la majorité d’une population dans les champs individuels. Au contraire, les populations de S. minor et de S. homoeocarpa sont caractérisées par un nombre relativement faible de clones et constituent des agents pathogènes plus prometteurs pour l’emploi de l’hypovirulence comme stratégie de lutte biologique. Ce type de lutte biologique a été démontré par l’ajout d’isolats hypovirulents à des tissus végétaux malades en environnement contrôlé ou au champ. Pour le S. minor, la gravité de la maladie a été réduite de plus de 50% et une diminution allant jusqu’à 90% du nombre de sclérotes produits sur le tissu malade traité a été observée. Les sclérotes produits sur le tissu malade traité étaient hypovirulents Accepted 15 December 2003. G.J. Boland. Department of Environmental Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. (e-mail: [email protected]). Can. J. Plant Pathol. 26: 6–18 (2004) Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. 7 et contenaient l’ARN double brin caractéristique de l’isolat hypovirulent. Pour le S. homoeocarpa, l’efficacité de la lutte biologique a atteint jusqu’à 90% et 80% respectivement en environnement contrôlé et au champ et a été comparable à celle de traitements avec un fongicide. Une seule application de l’isolat hypovirulent Sh12B, contenant une souche de l’espèce Ophiostoma mitovirus 3a (OMV3a) déjà décrite auparavant et provenant d’un Ophiostoma novo-ulmi européen, a été aussi efficace que jusqu’à quatre applications de fongicide, avec une efficacité qui a persisté jusque dans l’année subséquente. Ensemble, les études sur les virus de champignons et celles sur l’hypovirulence chez les espèces de Sclerotinia peuvent améliorer notre compréhension des mécanismes moléculaires qui agissent sur l’expression de la virulence chez ces agents phytopathogènes et accroître le potentiel représenté par les virus de champignons comme mode d’action unique en lutte biologique. Mots clés : ARN double brin, ARNdb, lutte biologique, genre Hypovirus, genre Mitovirus, Sclerotinia sclerotiorum, Sclerotinia minor, Sclerotinia homoeocarpa. Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. Introduction Sclerotinia spp., including S. sclerotiorum (Lib.) de Bary, S. minor Jagger, and the disparate species S. homoeocarpa F.T. Bennett, are an important group of fungal plant pathogens because of their widespread distribution in temperate and subtemperate regions of the world, and because they cause crop damage and economic losses in a wide variety of agricultural, horticultural, and ornamental crops (Purdy 1979; Walsh et al. 1999; Willetts and Wong 1980). Millions of dollars are lost each year because of crop damage and resulting reduced yield and quality in plants that are susceptible to these pathogens. Additional costs are associated with the management of these diseases, including applications of fungicides in many crops. Despite the apparent similarity in current nomenclature, these fungi include a diversity of lifehistory strategies (Andrews 1984), and knowledge of these strategies is key to improved management and biological control of these pathogens. One of the strategies of biological control being investigated for these diseases is the use of hypovirulence, including the unique mechanism of action of fungal viruses to reduce the virulence of individuals and populations of fungal plant pathogens. Sclerotinia spp. Following the monographic revision of this genus in 1979, three economically important species were retained: S. sclerotiorum, S. trifoliorum Eriks., and S. minor (Kohn 1979). In the same revision, S. homoeocarpa was excluded from this genus but, for reasons outlined below, is still referred to by this binomial. Sclerotinia sclerotiorum is worldwide in distribution, but occurs primarily in temperate and subtemperate regions, and is an important pathogen of numerous crops, including bean, canola or rapeseed, soybean, and lettuce. Crop losses vary considerably by crop, region, and year, but severe outbreaks can regularly reduce crop yields by up to 35% and also reduce the quality of harvested yield (Purdy 1979). Sclerotinia minor is also worldwide in distribution and causes severe outbreaks in all major lettuce-producing regions of Canada and the United States (Melzer and Boland 1994; Subbarao 1998). Sclerotinia homoeocarpa is an important plant pathogen that affects turfgrasses and causes the disease known as dollar spot. The pathogen is widely distributed through North America, Central America, Australia, New Zealand, Japan, the United Kingdom, and continental Europe (Walsh et al. 1999). The disease can cause considerable damage to highly maintained golf course putting greens, closely mown fairways, and bowling greens (Goodman and Burpee 1991) and on less intensively managed turfgrasses such as home lawns, recreational and athletic facilities, and educational or industrial properties. Dollar spot reduces the aesthetic and playing quality of infected turf, and the disease can also contribute to weed encroachment and plant death (Smith et al. 1989). With the exception of western Canada and the Pacific northwest region of the United States, dollar spot is the most common disease of turf in North America (Couch 1995). More money is spent to manage dollar spot than any other turfgrass disease on golf courses (Goodman and Burpee 1991). The taxonomic status of S. homoeocarpa remains controversial, and most authorities believe that this fungus will eventually be reclassified. However, difficulty in obtaining reproductive structures for study has prevented resolution of its taxonomic status. The history of systematics surrounding this fungus was reviewed by Walsh et al. (1999). Kohn (1979) concluded that S. homoeocarpa was not a true Sclerotinia sp. and, based on personal communication with Korf, proposed that it was more appropriately classified in Lanzia Sacc. or Moellerodiscus Henn. Vargas and Powell (1997) recently proposed that S. homoeocarpa was most closely related to Rutstroemia henningsiana (Ploettn.) Dennis and R. cuniculi (Boud.) Elliott, with up to 88% similarity based on nuclear ribosomal internal transcribed spacer (ITS) 1 sequence alignment. Continued assessment of the taxonomic classification of this pathogen has been prevented by the recalcitrant nature of isolates to produce teleomorph or anamorph reproductive structures. Fertile apothecia are rarely observed in nature, and cultures on artificial media often yield sterile apothecia (Baldwin and Newell 1992; Fenstermacher 1980; Jackson 1973). Recently, fertile apothecia were reported from stromata in field samples of Festuca sp. turf (Baldwin and Newell 1992), and this report may stimulate renewed interest in a taxonomic assessment of this pathogen. Proper taxonomic placement of the pathogen responsible for dollar spot remains unresolved (Rossman et al. 1987; Walsh et al. 1999), and because of its historical placement in the genus Sclerotinia, we refer to it as S. homoeocarpa in this article. Host ranges of Sclerotinia spp. The host ranges of S. sclerotiorum and S. minor were recently reviewed. The index for S. sclerotiorum includes 408 8 species, 278 genera, and 75 plant families (Boland and Hall 1994). With few exceptions, all reported hosts of this pathogen were classified within the subclass Dicotyledonae of the Angiospermae, but several hosts were classified within the Monocotyledonae. The index for S. minor was considerably smaller and contained 94 species, 66 genera, and 21 plant families (Melzer et al. 1997). Most hosts occurred within the subclass Dicotyledonae, but two hosts were classified within the Monocotyledonae. Sclerotinia homoeocarpa can cause diseases in at least 40 plant hosts (Couch 1995; Fenstermacher 1980; Vargas 1994; Walsh et al. 1999). Most hosts are classified within the grass family Poaceae, but additional hosts have been reported from Cyperaceae (sedge family), Caryophyllaceae (pink family), Convolvulaceae (morning-glory family), and Leguminosae (pea family) (Walsh et al. 1999). Can. J. Plant Pathol. Vol. 26, 2004 gen is primarily associated with asexual sclerotia, although limited plant-to-plant spread can also occur. Sclerotinia homoeocarpa is also a clonally reproducing fungus that rarely produces apothecia in nature, and disease is initiated by mycelium that arises from overwintering stroma of the pathogen (Britton 1969; Couch 1995; Smiley et al. 1992) and possibly also from systemic infections of perennial grass hosts (Fenstermacher 1980). Epidemics of disease are considered to be polycyclic, primarily through secondary spread of infected and infested grass clippings during mowing (Walsh 2000). Dissemination over longer distances is associated with the movement of infected and infested grass clippings on the bottom of shoes, golf equipment, and turf maintenance equipment (Walsh 2000; Walsh et al. 1999). Fertile apothecia have been reported from stromata in field samples of Festuca sp. turf in Scotland (Baldwin and Newell 1992). Life strategies of Sclerotinia spp. The life cycles of Sclerotinia spp. represent a range of sexual and asexual life strategies, but all three species produce apothecia and ascospores within the teleomorphic or sexual cycle, and sclerotia or stroma within their anamorphic or asexual cycle. Asexual spores, such as conidia, are not produced in any of these species, and the prevalence of sexual and asexual reproduction varies considerably within the disease cycles in field environments. Sclerotinia sclerotiorum can reproduce asexually by production of sclerotia or sexually through self-fertilization and production of apothecia and ascospores. This results in a largely clonal population structure with occasional genetic exchange and recombination (Kohli and Kohn 1998). Clonal diversity exists within individual fields where numerous clones can often be detected, and there is a similar profile of clone frequency distribution among fields (Cubeta et al. 1997; Kohn et al. 1991; Kohli et al. 1992, 1995), although this pattern of the clonal dynamic can vary between agricultural and wild populations (Kohn 1995). Ascospores released from apothecia are the primary inoculum for initiating epidemics of most diseases caused by this fungus and require senescing or dead foliar plant tissue to initiate saprophytic colonization, which is then followed by infection of healthy foliar plant tissues (Abawi and Grogan 1979; Boland and Hall 1987, 1988). There is limited asexual reproduction through the production of sclerotia that serve as survival and dissemination structures and by the secondary spread of the pathogen through mycelial growth from plant to plant. In some crops, such as sunflower and bean, direct infection of roots or leaves from germinating sclerotia can also occur (Abawi and Grogan 1979; Tu 1989). Disease epidemics caused by S. sclerotiorum are typically considered to be monocyclic, although in carrot, disease can be bicyclic with separate epidemics developing in the field and in storage (Kora et al. 2003). Sclerotinia minor has a disease cycle differing substantially from that of S. sclerotiorum although it is also considered primarily a clonally reproducing fungus with a monocyclic disease cycle. It rarely produces apothecia in nature, and epidemics are associated with sclerotia in and on soil as the primary inoculum for disease (Melzer and Boland 1994; Subbarao 1998). Dissemination of the patho- Hypovirulence of Sclerotinia spp. Hypovirulence refers to the reduced ability of selected isolates within a population of a fungal plant pathogen to infect, colonize, kill, and (or) reproduce on susceptible host tissues (Elliston 1982), but may also be associated with other phenotypic characters such as reduced growth rate or sporulation, altered colony morphology or color, etc. In many cases, hypovirulence has been associated with the presence of double-stranded ribonucleic acid (dsRNA) characteristic of fungal viruses, although other factors such as mitochondrial mutations, nuclear mutations, and plasmids have been, or may be, associated with hypovirulence (Brasier 1999; Mahanti et al. 1993; Nuss and Koltin 1990; Monteiro-Vitorello et al. 1995, 2000; Tavantzis 2002). Hypovirulence has been reported to occur in many plant pathogens, and several review articles and texts provide summaries of our current understanding of this phenomenon in various fungi (Buck 1986; Koltin and Leibowitz 1988; Lemke 1979; Nuss and Koltin 1990; Tavantzis 2002; Zhang et al. 1994). Hypovirulence has been reported to occur in S. sclerotiorum, S. minor, and S. homoeocarpa and, to varying degrees, has been also associated with the presence of dsRNAs. One isolate of S. sclerotiorum was reported as hypovirulent and contained varying numbers of dsRNA elements (Boland 1992). This isolate grew slowly in culture, developed an atypical colony morphology (Fig. 1A), produced significantly smaller lesions on celery than virulent isolates (Fig. 1B), and contained dsRNA (Fig. 2). Treatment with cyclohexamide and (or) heat, followed by hyphal tip subculturing, to recover an isolate that was free of dsRNA were not successful. The hypovirulent phenotype and dsRNA were transferred to vegetatively compatible recipient isolates through hyphal anastomosis, and recipient isolates developed the hypovirulent phenotype and contained dsRNA. Other isolates of the pathogen also contained dsRNA, but there was no correlation between the presence of these dsRNAs and reduced virulence (Boland 1992; Zhou and Boland 1998a). Therefore, associations between dsRNAs and hypovirulent phenotypes are specific to individual dsRNAs and preclude any general observations on Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. the presence or absence of dsRNA and their association with fungal phenotypes. Thirty isolates of S. minor were assessed for transmissible hypovirulence and the presence of dsRNA (Melzer and Boland 1996). Three to 5 isolates (10–17%) displayed a hypovirulent phenotype (Fig. 1) and 12 isolates, including both virulent and hypovirulent isolates, tested positive for dsRNA (Fig. 2). The detection of dsRNA in virulent and hypovirulent isolates was variable in both the concentration and presence of individual segments of dsRNA. Because of this variability in recovery and association of dsRNA with hypovirulent isolates, no conclusions were drawn as to the mechanism(s) of action responsible for hypovirulence in this pathogen. The hypovirulent phenotype was transmissible to compatible, virulent, recipient isolates of the pathogen in culture and on lettuce leaves. Successful transmission to recipient isolates (i.e., conversion) resulted in isolates that did not grow, or grew and displayed the hypovirulent phenotype. Transmission between incompatible isolates was also successful but at a lower percentage of attempted conversions. In some cases, the recipient isolate initially displayed the hypovirulent phenotype, but subcultures of these isolates displayed a typical wildtype phenotype, suggesting that transmission had been initially successful but was unstable during subsequent subculturing (Melzer and Boland 1996). One hundred and thirty-two isolates of S. homoeocarpa were evaluated for virulence on detached leaves and swards of creeping bent grass (Agrostis stolonifera L.) and for the presence of dsRNA. Thirteen of the 132 isolates (9.8%) did not initiate dollar-spot lesions 4 weeks postinoculation and were considered to be hypovirulent. Double-stranded ribonucleic acid was detected in 6 of these 13 (46.2%) isolates (Zhou and Boland 1997). Compared with typical wild-type isolates of S. homoeocarpa, these six isolates often grew slowly in culture, formed thin colonies with atypical colony margins, and failed to produce a typical black stroma. Hypovirulence and dsRNA were transmitted from one hypovirulent isolate, Sh12B (Figs. 1 and 2), to a virulent isolate, Ky-7, resistant to the demethylase-inhibitor class of fungicides, and the converted isolate was hypovirulent, contained dsRNA, and grew on fungicide-amended medium. Hypovirulence and dsRNA were also transferred to at least four other isolates of the pathogen. Other hypovirulent isolates were also detected in this study but were variable in expression of the phenotype or were not associated with detectable concentrations of dsRNA. Characterization of dsRNA elements in fungi Double-stranded RNA elements have been reported from many fungi (Buck 1986; Koltin and Leibowitz 1988; Lemke 1979; Nuss and Koltin 1990; Tavantzis 2002; Zhang et al. 1994), and the detection of dsRNAs in fungi is assumed to represent the genomes of fungal RNA viruses. Many of these dsRNAs do not appear to be associated with a known phenotype and, therefore, are thought to represent latent or benign infections by fungal viruses. This neutral influence is thought to be the result of coevolution between the viruses and their hosts, assumedly due to selection against 9 virulence in the viral parasite, or for tolerance or resistance in the fungal host (Rosewich and Kistler 2000; Milgroom 1999). Some dsRNAs have been associated with known phenotypes, such as reduced virulence, and the list of fungal plant pathogens with such associations continues to grow, with the best characterized research models being Cryphonectria parasitica (Murrill) Barr and Ophiostoma spp. (Brasier 1990, 2000; Buck and Brasier 2002; Nuss and Koltin 1990; Nuss et al. 2002). Patterns of association between selected phenotypes and specific dsRNAs are often confused by the presence of multiple segments of dsRNA in a single isolate, with some segments representing nonfunctional internal deletions of larger dsRNA segments, and others representing distinct genomes of different virus species in the same thallus (Cole et al. 1998; Paul and Fulbright 1988; Shapira et al. 1991a, 1991b; Sutherland and Brasier 1997). Variations in inheritance of dsRNA segments in conidia has allowed several researchers to recover isolates of a fungus with varying number and segments of dsRNA, and this method has been successful in discriminating the influence of distinct segments of dsRNA on fungal phenotypes (Cole et al. 1998; Sutherland and Brasier 1997). However, in fungi that do not sporulate, or only produce sexual spores such as ascospores, this method is less effective. The relationship between dsRNA and phenotypic variation in fungi, including reduced virulence, is primarily inferred from correlative evidence, supported in some cases by curing and transmission experiments. The causal relationship between dsRNA and hypovirulence was first demonstrated through the use of an infectious viral cDNA derived from a hypovirulent-associated dsRNA from a European hypovirulent isolate, Ep713, of C. parasitica (Choi and Nuss 1992). Protoplasts of a virulent isolate were transformed with a full-length cDNA copy of the large dsRNA from the European isolate EP713, and the resulting transformants were hypovirulent, exhibited the hypovirulent phenotype, and contained an integrated cDNA copy of the dsRNA in the chromosomal DNA. In addition, RNA was transcribed from the integrated copy of the cDNA, and a corresponding dsRNA was present in the cytoplasm of the transformed isolates. This dsRNA was transmissible to virulent isolates in the same manner as cytoplasmically infected hypovirulent isolates. Subsequent studies have confirmed this strategy as an effective approach to determining the relationship between dsRNA and hypovirulence, and the same infectious cDNA from EP713 has been employed to determine the host range of this infectious viral cDNA in related Cryphonectria spp. and other fungi (Chen et al. 1994, 1996; Nuss et al. 2002). Complete nucleotide sequences have been reported for dsRNA elements from several plant-pathogenic fungi, and these dsRNAs resemble viral RNA genomes in genetic organization and expression strategy. Based on their genome structure, presence or absence of an encoded coat protein, particle morphology, and cellular location, these viral RNAs have been classified into four virus families: Totiviridae (genus Totivirus), Partitiviridae (genus Chrysovirus), Hypoviridae (genus Hypovirus), and Narnaviridae (genus Mitovirus) (Ghabrial 1998; van Regenmortel et al. 2000). Additional hypovirulence-associated dsRNAs unrelated to 10 Can. J. Plant Pathol. Vol. 26, 2004 Fig. 1. (A) Virulent (top) and hypovirulent (bottom) cultures of Sclerotinia sclerotiorum (left), S. minor (centre), and S. homoeocarpa (right). All cultures were grown on potato dextrose agar medium for 7 days at 20–22 °C. Virulent isolate Ss357 (top left) and hypovirulent isolate Ss275 (bottom left) of S. sclerotiorum. Virulent isolate Sm28 (top centre) and hypovirulent isolate Sm23 (bottom centre) of S. minor. Virulent isolate Sh80 (top right) and hypovirulent isolate Sh70 (bottom right) of S. homoeocarpa. (B) Virulence assays of virulent and hypovirulent isolates of S. sclerotiorum (top panel), S. minor (middle panel), and S. homoeocarpa (bottom panel). Plant tissues were inoculated with colonized agar discs from the actively growing colony margins of individual pathogens, and photographs were taken 48 h postinoculation. Top panel: hypovirulent isolate Ss275 (left) and virulent isolate Ss357 (right) on celery. Middle panel: hypovirulent isolate Sm23 (left) and virulent isolate Sm38 (right) on romaine lettuce. Bottom panel: hypovirulent isolate Sh70 (top) and virulent isolate Sh80 (bottom) on bent grass. these four families also have been reported (Peever et al. 1997). The Helminthosporium victoriae 190S virus and the Helminthosporium victoriae 145S virus are associated with hypovirulence in H. victoriae F. Meehan & H.C. Murphy and have been classified in the genus Totivirus of the family Totiviridae and the genus Chrysovirus of the family Partitiviridae, respectively. These are typical viruses with a dsRNA genome encapsidated within a protein capsid (Ghabrial et al. 2002). The genus Hypovirus is a relatively new one and currently contains four species, referred to as Cryphonectria hypovirus 1 (CHV-1), Cryphonectria hypovirus 2 (CHV-2), Cryphonectria hypovirus 3 (CHV-3), and Cryphonectria hypovirus 4 (CHV-4) (International Committee on Taxonomy of Viruses 2002a); CHV-1, CHV-2, and CHV-3 are all associated with hypovirulence in C. parasitica, but CHV-4 is not associated with any discernable phenotype (Enebak et al. 1994). These viruses are not present as true virions but are located within pleomorphic vesicles containing dsRNAs of 9–13 kilo base pairs (kbp) in size and polymerase activity (van Regenmortel et al. 2000). Only one strand of the dsRNA genome is employed in transcription, and one or two polyproteins are encoded. The polyproteins are autocatalytically cleaved to produce functional protein products (Hillman et al. 1994; Shapira et al. 1991a; Smart et al. 2000). The genus Mitovirus is also relatively new and is characterized by a lack of intact virion particles. Double-stranded ribonucleic acid of 2–3 kbp in size are located within the mitochondria of infected isolates, and a single-stranded RNA element is present within infected tissues. Mitoviruses are predicted to be translatable only in mitochondria and only encode a RNA-dependent RNA polymerase (RdRp)like protein, which is required for replication of the RNA (van Regenmortel et al. 2000). These mitoviruses represent the simplest form of all known autonomously replicating viruses. Currently, there are five species in the genus Mitovirus: Cryphonectria mitovirus 1 (Polashock and Hillman 1994) from C. parasitica, and Ophiostoma mitovirus 3a, Ophiostoma mitovirus 4, Ophiostoma mitovirus 5, and Ophiostoma mitovirus 6 from Ophiostoma novo-ulmi (Brasier) (Hong et al. 1998, 1999; International Committee on Taxonomy of Viruses 2002b). The first report of dsRNAs associated with hypovirulence in Sclerotinia spp. was in S. sclerotiorum (Boland 1992). Although several dsRNAs were observed in isolates of this fungus, one hypovirulent, debilitated isolate contained a segment of dsRNA that was approximately 12 kbp in size. Other dsRNA segments were present in earlier subcultures and extractions made from this same isolate (Fig. 2) but, over time, a single dsRNA became the only detectable seg- ment (Zhou and Boland 1997). Differential digestion with DNase, and with RNase in 0.3 and 0.03 mol·L–1 NaCl, confirmed that this was RNA and a double-stranded segment. Ultrastructural examination of hyphal and sclerotial cells of one hypovirulent isolate of S. sclerotiorum indicated that the dsRNA was not associated with viral particles in this isolate but with vesicles bound by a double membrane (Boland et al. 1993). The double membrane surrounding these vesicles was similar in size and structure to the nuclear envelope, and the vesicles appeared to originate from the nuclear membrane. The results showed that the dsRNA in this isolate of S. sclerotiorum was not associated with a typical mycovirus but, based on several of its physical characteristics, may be an unencapsidated dsRNA typical of the genus Hypovirus (van Regenmortel et al. 2000). Further characterization of this dsRNA is required to confirm this classification. In S. minor, selected hypovirulent isolates contained variable numbers of dsRNA segments that ranged in size from 3 to 17 kbp (Fig. 2; Melzer 1993; Melzer and Boland 1996). The presence and number of segments of dsRNA was variable, with 0–8 dsRNA segments being detected in multiple extractions and purifications. Because of this variability in recovery and association of dsRNA with hypovirulent isolates, no conclusions were drawn as to the mechanism(s) of action responsible for hypovirulence in this pathogen. Treatment of infected isolates with heat and (or) cyclohexamide were not effective in curing isolates of this pathogen to verify the role of dsRNA in the phenotype. In S. homoeocarpa, one to three segments of dsRNA were detected in 15 of 132 isolates but were associated with hypovirulence in only 6 isolates (Zhou and Boland 1997). The size of dsRNA segments from different isolates varied, although there was one segment (2.6 kbp) in common to all hypovirulent isolates. Isolate Sh12B was selected for subsequent study, and this isolate contained two segments of dsRNA, the larger band of dsRNA of ca. 2.6 kbp (L-dsRNA), and a smaller band of ca. 0.7 kbp (S-dsRNA) (Fig. 2). Subsequent analyses of other hypovirulent isolates of S. homoeocarpa that contained similar segments of dsRNA established that the L-dsRNA was consistently present in all hypovirulent isolates while the S-dsRNA was only found in some hypovirulent isolates (Deng 2003). Virulence analysis established that there was no significant difference between isolates containing one dsRNA and two dsRNAs, indicating that only the L-dsRNA was associated with hypovirulence in S. homoeocarpa. Both the L- and S-dsRNAs in hypovirulent isolate Sh12B of S. homoeocarpa have been characterized at the molecular level. The L-dsRNA is 2632 bp long and, using mitochon- Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. 11 12 Fig. 2. Double-stranded RNA (dsRNA) elements from isolates of Sclerotinia sclerotiorum, S. minor, and S. homoeocarpa. Lane 1, molecular-mass marker; lane 2, dsRNA from isolate Ss275 of S. sclerotiorum; lane 3, dsRNA from isolate Sm23 of S. minor; lane 4, dsRNA from isolate Sh12B of S. homoeocarpa; lane 5, molecular-mass marker. Can. J. Plant Pathol. Vol. 26, 2004 Reports of the presence of dsRNA in fungi are primarily based on the detection of dsRNA with nucleic acid extraction, purification, and agarose-gel detection methods. Recent evidence suggests that dsRNA can be present in some isolates of fungi at concentrations below the level of detection of these methods. Lakshman and Tavantzis (1994) reported, in an isolate of Rhizoctonia solani Kühn, the spontaneous appearance of a distinct dsRNA element that was not detected in the parental isolate with gel electrophoresis or Northern blot hybridization, but was detected with polymerase chain reaction (PCR). Similarly, hypovirulenceassociated OMV3a in S. homoeocarpa was only detected in 4 of 116 isolates from eastern Canada with gel electrophoresis, and all of these dsRNA-positive isolates displayed the hypovirulent phenotype (Melzer et al. 2003). However, using reverse transcriptase (RT)-PCR, 57 of 116 isolates tested positive for the presence of OMV3a, but only 4 of these displayed the hypovirulent phenotype. Isolates that tested positive for OMV3a, and had typical colony growth and virulence, were considered to be latently infected. Mechanisms of action of dsRNA elements drial codon analysis, one strand of this RNA contained an open reading frame with the potential to encode a protein of 720 amino acids. The amino acid sequence contained conserved motifs typical of RdRps. Sequence analyses of the nucleotide and RdRp-like protein revealed that the LdsRNA is homologous with a previously characterized mitochondrial virus and, to a lesser extent, with dsRNAs from other phytopathogenic fungi. Moreover, this dsRNA shared 92.4% nucleotide and 95.1% amino acid sequence identities with the strain Ophiostoma mitovirus 3a-OnuLd (OMV3aOnuLd) from O. novo-ulmi, the causal agent of Dutch elm disease, indicating that these two dsRNAs are conspecific. This degree of homology means that these two viruses are the same species (van Regenmortel 2000). Therefore, this is the first report that a hypovirulence-associated virus occurs naturally in two taxonomically distinct fungi, and it indicates that horizontal transmission of this virus may have occurred between these fungi. These results are particularly surprising given that OMV3a-OnuLd was reported from the United Kingdom, and that L-dsRNA was reported from eastern Canada (Deng 2003; Deng et al. 2003). Because the L-dsRNA in isolate Sh12B of S. homoeocarpa is conspecific with a previously described virus, it is assigned the name Ophiostoma mitovirus 3a-Sh12B (OMV3a-Sh12B), based on the nomenclature rules of the International Committee on Taxonomy of Viruses (van Regenmortel 2000). The S-dsRNA in S. homoeocarpa is 732–738 bp long, and the nucleotide sequence implies that it is not directly derived from OMV3a-Sh12B and does not encode a RdRp (Deng 2003; Deng and Boland 2003). These observations are consistent with the biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently in any hypovirulent or virulent isolates. Therefore, the S-dsRNA can be considered a satellite RNA of the species Ophiostoma mitovirus 3a (OMV3a) in S. homoeocarpa. Little information is available on the mechanism(s) of action through which dsRNA elements contribute to hypovirulence in Sclerotinia spp. Several dsRNA-associated hypovirulent isolates of S. sclerotiorum were associated with reduced or delayed production of oxalic acid, or both, in comparison with virulent isolates, particularly during the first 3 to 7 days of growth (Zhou and Boland 1999). However, over longer periods of time, some hypovirulent isolates produced concentrations of oxalic acid that were comparable to those produced by virulent isolates. Ultrastructural examination of hypovirulent isolate Sh12B of S. homoeocarpa detected a range of morphology in mitochondria, ranging from typical mitochondria with well-defined outer membranes and inner cristae to swollen mitochondria with well-defined outer membranes but degraded or absent cristae within (Boland et al. 2000). Such swollen debilitated mitochondria were not observed in virulent, wildtype isolates. In addition, there was a fibrillar material within the swollen mitochondria of the hypovirulent isolate. Isolation of subcellular fractions through differential and sucrose-gradient centrifugation confirmed that the Land S-dsRNAs were localized in mitochondria of the fungal host isolate (Deng 2003; Deng et al. 2003). From these results, it appears that hypovirulence in this isolate of S. homoeocarpa was associated with the presence of a mitovirus that may interfere with the normal functioning of mitochondria. A yellow precipitate was associated with cultures of hypovirulent isolates of S. minor (Melzer 1993), and chemical characterization of this precipitate determined it to be a novel 1-hydroxy-2,6-pyrazinedione called sclerominol (Savard et al. 2003). One other 1-hydroxy-2,6-pyrazinedione, flutimide, has been reported and shown to have human pharmaceutical activity as an inhibitor of influenza virus endonuclease. Sclerominol was evaluated for related activity and displayed some activity against cancer cell lines but little activity against three influenza virus strains. The role Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. of sclerominol in the physiology of hypovirulent isolates of S. minor has not been determined, but the chemical has also been recovered from hypovirulent isolates of S. sclerotiorum. Transmission of hypovirulence and fungal viruses Intraspecific transmission The potential of using hypovirulent isolates of a fungal pathogen as a biocontrol strategy resides in the ability to transfer hypovirulence from individual hypovirulent isolates to virulent isolates within a population of the target pathogen and, thereby, reduce the mean disease severity of the population through reductions in mean virulence, growth, sporulation, and (or) survival. There are three restrictions to such transferral, including the absence of an externally infectious stage, the lack of transferral of dsRNA elements through ascospores (i.e., vertical transmission), and restricted transmission through vegetative compatibility groups (VCGs) (i.e., horizontal transmission). It is assumed that the latter one or two genetic restrictions may have evolved, at least in part, to restrict the spread of such agents through a fungal population (Caten 1972). Mycoviruses are not known to have an externally infectious stage and, therefore, cannot be inoculated in assays to determine infectivity (Nuss and Koltin 1990; Zhang et al. 1994). Infections are viewed as persistent, with the virus particles or dsRNA being located in the cytoplasm or mitochondria and transmitted during cell division, anastomosis, or induced forms of cytoplasmic mixing. Mycoviruses have no known vectors for transmission between isolates. In vertical transmission, dsRNAs in parental strains are not transmitted to progeny through ascospores (Anagnostakis 1984; Brasier 1986; Rogers et al. 1986a). The reasons for this lack of transmission are not readily apparent but mycovirus-infected isolates of C. parasitica tend to be female-sterile, while those of Ophiostoma ulmi (Buisman) Nannf. are less fecund (Anagnostakis 1984; Brasier 1986). Nontransmission of dsRNAs through ascospores appears to apply to both cytoplasmic (e.g., genus Hypovirus) and mitochondrial (e.g., genus Mitovirus) viruses, but does occur in the basidiospores of several basidiomycete fungi, including the rusts (Zhang et al. 1994). Horizontal transmission of dsRNAs primarily occurs through hyphal anastomosis, and the resulting cytoplasmic continuity allows such elements to be exchanged between or among isolates. This somatic compatibility is controlled through both allelic and nonallelic genetic systems. For example, in C. parasitica, vegetative incompatibility is controlled by 5–7 gene loci (i.e., vic loci), and incompatibility occurs if one or more alleles differ at the controlling loci (Anagnostakis and Day 1979). Interactions among these alleles can result in the characterization of VCGs that often serve as indirect markers of genetic diversity within individual fungal species (Leslie 1993). Although these markers have limitations for use in population biology, they are important characters in hypovirulence because they are a direct reflection of the potential for transmission of infectious agents within the population of the target pathogen. In many 13 studies, vegetative incompatibility is determined through mycelial interactions between isolates (i.e., mycelial compatibility groups) rather than through more detailed genetic studies. However, there is evidence that VCGs do not correspond directly to mycelial compatibility groups in some fungi (Ford et al. 1995). Vegetative compatibility groups are not an absolute barrier to the transmission of dsRNA among isolates. Liu and Milgroom (1996) demonstrated that a negative correlation exists between the number of vic genes among isolates of C. parasitica and the ability to transfer two hypoviruses, CHV1-EP43 and CHV2-NB58, between isolates. Hypovirus transmission occurred between all donor and recipient isolates that were vegetatively compatible. The frequency of transmission between donor and recipient isolates that differed by one vic gene was reduced to 0.48–0.50, depending on the hypovirus tested. Transmission frequencies decreased to 0.13–0.14 when donor and recipient isolates differed by two vic genes and to 0.03–0.04 when more than two vic genes were involved. Similar results have been reported from other fungi where transfer of cytoplasmic factors was more frequent between isolates that differed by fewer incompatibility genes (Brasier 1984; Caten 1972). Sclerotinia sclerotiorum is considered primarily a clonally reproducing fungus and is characterized by high genetic diversity, as reflected in VCGs, DNA fingerprints, and other genetic markers (Cubeta et al. 1997; Kohli and Kohn 1998; Kohli et al. 1992, 1995; Kohn et al. 1991; Kohn 1995). The presence and prevalence of VCGs within individual populations varies considerably among populations and can also vary between agricultural and wild populations (Kohn 1995; Kohn et al. 1991). Agricultural populations typically consist of many clones and have a similar profile of clone frequency distribution. Within individual fields, a few clones often represent the majority of the population, and a large number of additional genotypes are recovered once or twice (Anderson and Kohn 1995; Kohn 1995). In S. minor, the production of apothecia and ascospores is considered rare in the life cycle, and the epidemiology of disease in lettuce and other crops primarily involves the myceliogenic germination of sclerotia (Melzer and Boland 1994). Both of these factors favor the spread of hypovirulence-associated dsRNAs in populations of S. minor. In one study of a population of 30 isolates of S. minor from one field in Ontario, there were only three VCGs, suggesting a relatively low level of genetic variability in this population (Melzer and Boland 1996). Hypovirulence and dsRNA in selected isolates of S. minor were transmitted to virulent recipient isolates, and recipient isolates developed the hypovirulent phenotype and contained dsRNA. In many cases, recipient isolates did not grow, raising the possibility that these isolates died. In some cases, the recipient isolates contained more dsRNA segments than the donor isolates (Melzer 1993). Transmission also occurred across the three VCGs that were identified from Ontario. However, the percentage of successful transmissions was affected by the combination of donor and recipient isolates, and possibly also the dsRNA segment being transmitted (Melzer 1993). Results from these experiments confirmed that transmission of dsRNA and hypovirulence was successful across VCGs 14 but at a lower proportion of attempts than among isolates from the same compatibility group. Vegetative incompatibility has also been reported among isolates of S. homoeocarpa, and to date, up to eight VCGs have been identified in central and eastern North America (Deng and Boland 2002; Powell and Vargas 2001; Sonodoa 1989; Zhou and Boland 1995). In eastern Canada, four VCGs were detected from 10 locations, and the most commonly recovered VCG was present at 9 of the 10 locations and comprised 56% of the sampled isolates. The results were consistent with the hypothesis that there is limited diversity among VCGs in S. homoeocarpa. The presence of VCGs in S. homoeocarpa was assessed for the ability to restrict transmission of dsRNAs and found to range from fully to partially incompatible. Isolates that were fully incompatible strongly restricted transmission of hypovirulence-associated dsRNA whereas the partially incompatible reaction allowed limited spread between VCGs. Interspecific transmission Little information is available on the ability of dsRNAs to spread between species but interspecific transmission has been reported for a hypovirulence-associated dsRNA from S. sclerotiorum to S. minor (Melzer et al. 2002). Transmission of this dsRNA was associated with the development of hypovirulence and debilitated growth in the recipient species and established that S. minor is also a susceptible host to this dsRNA from S. sclerotiorum. In these laboratory experiments, transmission was associated with only one isolate of S. minor, which appeared to have more interspecific mycelial compatibility with the donor isolate of S. sclerotiorum than other isolates. In addition, the fungal mitovirus OMV3a-OnuSh12B, recently reported in S. homoeocarpa in eastern Canada, was the same species as previously reported from O. novo-ulmi from the United Kingdom, suggesting that horizontal transmission of this virus may have occurred between these fungi (Deng et al. 2003). Horizontal transmission of genetic elements, such as plasmids, introns, transposons, genes, gene clusters, and even whole chromosomes, is an increasingly recognized phenomenon (Rosewich and Kistler 2000), and transmission of dsRNA elements may also occur between fungal species. Interspecific transmission of dsRNA elements may play a role in the evolution of fungi affected by such transmission, but further information on the mechanisms of such interspecific transfer, and comparisons of a wider range of dsRNA sequences, such as RdRp, will be required to confirm this potential role (Rosewich and Kistler 2000). Biological control, using fungal viruses and dsRNA elements In contrast to the numerous molecular, physiological, and laboratory studies of hypovirulence and dsRNA in plantpathogenic fungi, there are relatively few investigations of hypovirulence as a biocontrol strategy. Most emphasis has been placed on C. parasitica and the biological control of chestnut blight in Europe. This story has become a classic example in plant pathology, and the primary example of biological control mediated through hypovirulence and dsRNA. Chestnut blight was first recorded in Genova, Italy, Can. J. Plant Pathol. Vol. 26, 2004 in 1938, and subsequently spread throughout Europe so that, by 1967, most chestnut-growing areas were infected with C. parasitica (Heiniger and Rigling 1994). Epidemics were consistently severe and resulted in high mortality of trees. Based on reports by Biragi (1953), Grente (1965) isolated white, hypovirulent strains of C. parasitica from trees in northern Italy that were diseased but had superficial, nonlethal cankers, often referred to as healing cankers. Grente (1965) demonstrated that hypovirulent isolates coinoculated with virulent isolates could reduce the severity of canker development on susceptible Castanea sativa Mill., and that hypovirulence could be transmitted to virulent strains of the pathogen. The recovery of chestnut trees in Europe was largely thought to be the result of natural spread of hypovirulent isolates and, as early as 1951, healing cankers were observed in many parts of Europe. No clear picture is available on the lag period between the spread of virulent and hypovirulent isolates in Europe but several authors have suggested that it may have been relatively short (Heiniger and Rigling 1994). Most authors agree that hypovirulent isolates spread rapidly through virulent populations of the pathogen. One of the important principles that has arisen from studies of hypovirulence in this pathogen is that virulent isolates of the pathogen do not disappear, but remain in the population and continue to cause lethal cankers on branches and stump sprouts. The incidence of diseased trees has remained high in many regions, but the severity of disease has been drastically reduced (Heiniger and Rigling 1994). Despite this reduction in disease severity, the frequency of active and healing cankers, and virulent and hypovirulent isolates, varies widely among regions. DsRNA viruses have also been reported to exert a role on the natural ecology of O. ulmi and O. novo-ulmi, and epidemics of Dutch elm disease. Hypovirulent isolates, referred to as diseased or d-infected isolates, have been reported to occur in both species and, at least in the aggressive subgroup of O. ulmi, have been shown to contain dsRNA-like segments, called d factors (Cole et al. 1998; Rogers et al. 1986a, 1986b). D-infected isolates tend to be slow growing, unstable, and have reduced spore viability (Brasier 1990), and d factors can prevent xylem infection by O. ulmi at the beetle-feeding wounds (Brasier 1990, 2000). In contrast to the epidemic that developed in North America, the first epidemic of Dutch elm disease in Europe declined around 1940. This decline may have been associated with the presence of d factors that spread rapidly through the largely clonal populations of the pathogen that were present at that time, and which were characterized by relatively few VCGs. Subsequently, the pathogen populations developed a more diverse VCG structure and became highly polymorphic for VCGs, then the percentage of isolates that contained d factors decreased. This pattern of population development suggests that d factors may play a role in exerting selection pressure on the rapid development of VCGs within a population (Brasier 1990, 2000). The second, current epidemics of disease do not appear to be affected as strongly as the first epidemics, and this may be due to a more rapid development of VCGs within the populations, which restricts the effect of d infection (Brasier 2000; Buck and Brasier 2002). Boland: hypovirulence and double-stranded RNA / Sclerotinia spp. Research on biological control using hypovirulence in Sclerotinia spp. has concentrated on S. minor and S. homoeocarpa. The prevalence of numerous VCGs in S. sclerotiorum suggests that hypovirulence and dsRNA would be restricted from spreading through populations of this pathogen although more detailed experimental analysis is required to confirm this prediction. In S. minor, mycelial suspensions of hypovirulent isolates were sprayed onto established lesions caused by virulent isolates with matching and nonmatching VCGs. When the lesions were caused by a compatible virulent isolate, the spray caused a reduction in lesion size and in the number of sclerotia in comparison with control treatments (Melzer and Boland 1996). Lesion sizes were up to 50% smaller, depending on the time elapsed after initial inoculation with the virulent isolate, and the number of sclerotia that developed on diseased lettuce leaves was reduced by more than 90%, depending on the time of initial inoculation. Subcultures of isolates were obtained from treated lettuce leaves and categorized as typical, atypical, without growth, or contaminated (i.e., microbial infestation). In samples treated with hypovirulent isolates, there was a decrease in the number of subultures that were considered typical and an increase in the number of isolates that were considered atypical, without growth, and contaminated. These results provide a unique example of the role of hypovirulence in the epidemiology of disease as hypovirulent isolates reduced both the amount of primary disease (e.g., lesion diameter) and the amount of secondary inoculum (e.g., sclerotia) that would contribute to subsequent epidemics of disease. An increase in the number of sclerotia contaminated by microorganisms also suggests a possible interaction between hypovirulence and sclerotial survival that may contribute to reduced survival. Parallel inoculations with hypovirulent and virulent isolates that were not compatible (i.e., from different VCGs) did not result in a difference in lesion diameter or number of sclerotia produced on lettuce leaves, or number of typical, atypical, without growth, or contaminated subcultures obtained from treated tissues (Melzer and Boland 1996). Similar experiments were repeated in field trials, and corresponding reductions in lesion diameters and numbers of sclerotia were observed (M.S. Melzer, unpublished data). Biological control, using hypovirulent isolates of S. homoeocarpa, has also been demonstrated in controlled environments and field conditions (Zhou and Boland 1998a, 1998b). Under growth room conditions, individual pots of creeping bent grass were treated with inoculum of virulent or hypovirulent isolates, or both. Hypovirulent isolates Sh12B, Sh09B, or Sh08D caused 3.4–30.4% diseased turf in comparison with virulent isolates, which caused 80.2– 90.2% disease. In treatments that received both virulent and hypovirulent isolates, only hypovirulent isolate Sh12B significantly reduced dollar spot by 51–90% compared with treatment with virulent isolates alone. These results indicate that hypovirulent isolates of this pathogen can cause a reduced level of disease when inoculated onto a susceptible host under favourable environmental conditions for disease, and that only some hypovirulent isolates were effective as biocontrol agents. In a field experiment conducted on swards of creeping bent grass, experimental plots were artificially inoculated 15 with a virulent isolate of S. homoeocarpa, then treated with the selected hypovirulent isolates in various formulations. At 10 days after treatment, the percent diseased turf for each formulation of a hypovirulent isolate Sh12B was 6.3%, 12.5%, and 20.8% for treatments applied as a mycelial suspension, granular mix, and alginate pellets, respectively, and was significantly lower than their respective formulation controls (31.2%, 23.8%, and 30.0%, respectively). Hypovirulent isolate Sh12B suppressed disease by up to 80%, and disease suppression was still evident 45 days after treatment. Residual disease suppression persisted until the next growing season (Zhou and Boland 1998a, 1998b). Parallel treatment with a second hypovirulent isolate, Sh09B, was not effective in suppressing disease. Comparable suppression of dollar spot by isolate Sh12B was observed in an experiment conducted in the following year. To determine the effects of a hypovirulent isolate on suppression of naturally occurring dollar spot, an experiment was established on a sward of creeping bent grass with a history of severe dollar spot. Hypovirulent isolate Sh12B was applied as a mycelial suspension or as alginate pellets and was applied once, twice, or four times from June to September. Treatments with a mycelial suspension and alginate pellets of hypovirulent isolate Sh12B significantly reduced dollar spot up to 58% compared with their respective formulation controls, and in most plots, disease suppression was equivalent to treatment with chlorothalonil (Zhou and Boland 1998b). Multiple applications of the hypovirulent isolate did not result in greater suppression of dollar spot as compared with a single application. Conclusions Research into hypovirulence and fungal viruses in fungal plant pathogens contributes to our understanding of physiological and molecular regulation of virulence in these pathogens and to our knowledge of the population genetics of plant pathogens and the role of these genetic elements in the ecology of fungi. In addition, the hypothesis that we can utilize these fungal viruses as biocontrol agents is appealing from both a scientific and disease-management perspective. During the past two decades, significant progress has been made in characterizing and understanding how hypovirulence systems operate in several fungal plant pathogens. Studies of hypovirulence and fungal viruses in Sclerotinia spp. have revealed and expanded on many of the same principles, including partial characterization of several fungal viruses and RNA elements, intra- and inter-specific transmission of hypovirulence and dsRNA, mechanisms of action, and biological control of disease. The results demonstrate that hypovirulence is a naturally occurring phenomenon in Sclerotinia spp., with potential as an effective management tool for the control of diseases caused by these pathogens. 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