Download Fungal viruses, hypovirulence, and biological control of Sclerotinia

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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
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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.
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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
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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
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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
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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.
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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. Evidence to date suggests that an innundative,
population approach to target plant pathogens with reduced
asexual and sexual reproduction, and a reduced diversity of
VCGs in agricultural populations may be most effective for
biocontrol success.
16
Acknowledgements
I would like to acknowledge the insightful discussions
and suggestions provided by M.S. Melzer and F. Deng during the preparation of this manuscript, and Ginette Fortier
for her contributions as technical editor. Funding to support
this research and publication was provided by the Natural
Sciences and Engineering Research Council of Canada, the
Ontario Ministry of Agriculture, and The Canadian
Phytopathological Society.
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