Search

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Hypovirus-infected Cryphonectria parasitica strains were introduced in a large stand of American chestnut (>4,000 individuals) in western Wisconsin (USA) to evaluate whether hypoviruses can serve as biological control agents. They were deployed by treating cankers from 1992 to 1997 and again from 2004 to 2014. After 17 years of hypovirus introductions within an area of the stand with the longest history of disease, isolation of hypovirus-infected strains increased from 55% in 1994 to 86% in 2014 from cankers that were treated. During the same period, isolation from cankers that arose on trees with treated cankers increased from 29 to 72% and from 15 to 84% for cankers on nearby trees that received no treatment. Tree survivorship over the 23-year study period for trees with treated cankers was 51% compared with 31% for trees that were not treated. Introduction of hypovirus has resulted in the regrowth of the crowns of many large-diameter trees. Putative recovery of American chestnut in this stand provides evidence that prolonged hypovirus treatment can act as a biological control when limited numbers of vegetative compatibility types of C. parasitica exist.

Prospero, S., and Rigling, D. 2012. Invasion genetics of the chestnut blight fungus Cryphonectria parasitica in Switzerland. Phytopathology 102:73-82. Cryphonectria parasitica is the best-known example of an invasive forest pathogen in Europe. In southern Switzerland, chestnut blight was first reported in 1948 whereas, north of the Alps, it did not appear until the 1980s. Between 1995 and 2008, we sampled 640 C. parasitica isolates from nine populations south of the Alps and nine north of the Alps. Twelve historical isolates, collected between 1950 and 1972 in the south, were obtained from our collection. All 652 isolates were screened at 10 microsatellite loci to test for the existence of divergent genetic pools and to infer possible origins of haplotypes. In total, 52 haplotypes were identified. Structure software analysis indicated that 43 haplotypes (including all historical haplotypes) belonged to a main cluster, 6 haplotypes belonged to a different cluster, and 3 haplotypes had an intermediate allele pattern. All newly founded populations in northern Switzerland were initiated by one or just a few haplotypes from the main cluster, which probably came directly from the populations south of the Alps. Subsequently, genetic diversity increased through mutations, sexual reproduction, or new migrations. The highest increase in diversity was observed in populations where haplotypes from different genetic pools were encountered.

The role of Cpkk1, a mitogen-activated protein kinase from Cryphonectria parasitica, was investigated by generating a number of mutant strains that overexpress, under the control of the cryparin promoter, both the wild-type protein and its allele with an extensive deletion in the catalytic domain. Furthermore, a hairpin construct was built and expressed to cause specific silencing of Cpkk1 mRNA transcripts. Specific mRNA silencing or overexpression was confirmed on both Northern and Western blot analysis. Selected C. parasitica strains with Cpkk1 either silenced or overexpressed were evaluated for their biological characteristics, including virulence on European chestnut, growth on different substrates, conidial sporulation, and resistance to cell-wall-degrading enzymes. Silencing of Cpkk1 and the overexpression of a defective Cpkk1 correlated with a marked reduction in virulence on 3-year-old chestnut trees, with no statistically significant effect on fungal growth in the various conditions tested.

The potential host range of mycoviruses is poorly understood because of the lack of suitable inoculation methods. Recently, successful transfection has been reported for somatically incompatible fungal isolates with purified virus particles of two mycoviruses, the partitivirus RnPV1-W8 (RnPV1) and the mycoreovirus RnMyRV3/W370 (MyRV3), from the white root rot fungus Rosellinia necatrix (class Sordariomycetes, subclass Xylariomycetidae). These studies examined and revealed the effect of the mycoviruses on growth and pathogenicity of R. necatrix. Here, we extended the experimental host range of these two mycoviruses using a transfection approach. Protoplasts of other phytopathogenic Sordariomycetous fungi—Diaporthe sp., Cryphonectria parasitica, Valsa ceratosperma (Sordariomycetidae), and Glomerella cingulata (Hypocreomycetidae)—were inoculated with RnPV1 and MyRV3 viral particles. The presence of double-stranded RNA viral genomes in regenerated mycelia of Diaporthe sp., C. parasitica, and V. ceratosperma confirmed both types of viral infections in these three novel host species. An established RnPV1 infection was confirmed in G. cingulata but MyRV3 did not infect this host. Horizontal transmission of both viruses from newly infected strains to virus-free, wild-type strains through hyphal anastomosis was readily achieved by dual culture; however, vertical transmission through conidia was rarely observed. The virulence of Diaporthe sp., C. parasitica, and V. ceratosperma strains harboring MyRV3 was reduced compared with their virus-free counterpart. In summary, our protoplast inoculation method extended the experimental host range of RnPV1-W8 and MyRV3 within the class Sordariomycetes and revealed that MyRV3 confers hypovirulence to the new hosts, as it does to R. necatrix.

The Cryphonectria parasitica populations in two 6-year-old European chestnut (Castanea sativa) coppices were investigated in southern Switzerland over a period of 4 years. Occurrence of white isolates indicating an infection with Cryphonectria hypovirus, vegetative compatibility groups (VCGs), hypovirulence conversion capacity, and mating types were used to characterize the populations. Sampling of randomly chosen cankers in the first year yielded 59% white isolates in one and 40% in the other population. The distribution of the VCGs and mating types was similar among white and orange isolates, indicating a homogeneous infection of the two populations by the hypovirus. Fourteen VCGs were found in the first population, 16 VCGs in the second. Altogether, 21 VCGs were determined. The same three VCGs dominated in both populations, comprising more than 60% of all isolates. Several VCGs were represented only by white isolates. Five of the six most common VCGs were clustered in two hypovirulence conversion groups, with almost 100% hypovirus transmission within each cluster. Repeated sampling of the same cankers in 1990, 1992, and 1994 did not reveal an increase of white isolates. The portion of blighted stems rose from 37% to about 60% in both plots within 4 years. In this time, chestnut blight killed 15% and competition an additional 21% of the sprouts. Predominantly, sprouts with low diameters at breast height were killed. The growth rate of new cankers was high in their first year and decreased gradually in the following years. A role of hypovirulence in the decline of disease severity was evident since (i) cankers yielding white isolates grew slower and killed considerably fewer sprouts than cankers with orange isolates; and (ii) the majority of the cankers yielded white isolates at least once during the 4-year observation period.

Correlation between hypovirus transmission and the number of different vegetative incompatibility (vic) genes among isolates of Cryphonectria parasitica was estimated using isolates sampled from a natural population. We tested the hypothesis that transmission of hypoviruses among isolates is negatively correlated to the number of vic genes that are different between vegetative compatibility (vc) groups. In a sample of 58 isolates collected from a population in Finzel, MD, eight isolates, each in a different vc group, were randomly selected and infected with each of the Cryphonectria hypoviruses CHV1-EP43 and CHV2-NB58. The frequency and time required for virus transmission were estimated by pairing infected donor isolates with uninfected recipient isolates in vitro. The number of vic genes different between vc groups was estimated by crossing the donor and recipient isolates and determining the proportion of ascospore progeny that were vegetatively compatible with either parent. Hypovirus transmission occurred between all pairs of isolates that were vegetatively compatible. The frequencies of transmission between vc groups that differed by one vic gene were 0.50 and 0.48 when the donor isolates were infected with CHV1-EP43 or CHV2-NB58, respectively. Transmission frequencies decreased to 0.13 for CHV1-EP43 and 0.14 for CHV2-NB58 when vc groups differed by two vic genes. When vc groups were different by more than two vic genes, transmission of hypoviruses occurred in only one out of 37 pairs (3%) and one out of 25 pairs (4%) with CHV1-EP43 and CHV2-NB58 in the donor isolates, respectively. The transmission frequency was negatively correlated to the number of vic genes different between isolates (P < 0.01). In contrast, the time taken for transmission to occur was only weakly correlated (r = 0.40, P = 0.05) to the number of vic genes different between vc groups. Unidirectional transmission of hypoviruses was observed between six pairs of vc groups. This study provides evidence for a significant negative correlation between the frequency of hypovirus transmission and the number of vic genes different between isolates of C. parasitica from a natural population.

Double-stranded RNA viruses within the genus Hypovirus attenuate virulence of the chestnut blight fungus Cryphonectria parasitica. The recent development of an infectious synthetic hypovirus transcript has allowed the expansion of hypovirus infection and virus-mediated virulence attenuation to several fungal species not previously shown to harbor hypoviruses. This report compared the phenotypic changes resulting from transfection-mediated hypovirus infection of Cryphonectria parasitica, C. radicalis, C. havanensis, C. cubensis, and Endothia gyrosa. By comparing virus-mediated phenotypic changes in different fungal hosts transfected with wild-type and mutated viral transcripts, it was possible to examine the relative contribution of viral and host genetic backgrounds for some traits. For example, hypovirus infection reduced sporulation in all fungal hosts, while it suppressed pigmentation in some species and induced pigment production in others. The hypovirus-encoded protein p29 was found to have a significant impact on both sporulation and pigmentation in different fungal hosts. All fungal species tested were able to transmit virus by anastomosis within the same species. However, considerable differences were observed in the efficiency with which different fungal species transmitted hypoviruses through conidia. A molecular phylogenetic analysis based on nuclear ribosomal DNA nucleotide sequences revealed the five fungal species to be closely related. Possible relationships between taxonomic position and hypovirus host range were discussed. The ability to introduce hypoviruses into different fungal species holds promise for the expanded utility of virus-mediated hypovirulence for understanding and controlling fungal pathogenicity.

To examine the effect of double infection by hypovirulence-associated viruses on the phenotype of Cryphonectria parasitica, dsRNAs from two Michigan hypovirulent isolates were cytoplasmically transferred into a common nuclear background. The coinfected strain had a dsRNA banding pattern profile that represented the sum total of the dsRNA segments of the parental strains. The douby infected strain was less virulent than either of the singly infected strains. Asexual segregation of the dsRNA viruses was followed through single-conidial isolates. The resulting asexual progeny contained one or both parental dsRNAs without any detected exchange of dsRNA segments. These findings indicate that field isolates of C. parasitica containing more than one dsRNA segment may be coinfected and that asexual segregation patterns relating dsRNA segments to hypovirulence phenotypes should be pursued before assuming the observed hypovirulence phenotype is due to a single virus

Isolates of Cryphonectria parasitica were sampled from American chestnut trees in 13 locations in eastern North America to study genetic differentiation and gene flow among subpopulations of this pathogen. Using data from six unlinked restriction fragment length polymorphism loci, we found significant differences in allele frequencies at all loci among the 13 subpopulations. Thirty-one percent of the total gene diversity was attributed to differences among subpopulations (G ST = 0.31). Genetic differentiation was examined for two subsets of data: one from between two subpopulations in Michigan, which were outside the natural range of the host, and the other from among nine subpopulations from ecologically similar sites (disturbed sites resulting from clearcutting and burning were eliminated). There were significant differences in allele frequencies at two loci between the two Michigan subpopulations, even though these sites were only 16 km apart. Differentiation was high between these subpopulations because two loci were fixed or nearly fixed for different alleles (G ST = 0.81). There was also differentiation among the nine subpopulations from ecologically similar sites (G ST = 0.20); significant differences in allele frequencies were found at five of the six loci. We attribute this level of differentiation to restricted gene flow among subpopulations. We did not, however, estimate gene flow quantitatively from these data because populations did not appear to be at equilibrium. There was no correlation between the estimated average number of migrants per generation between each pair of nine subpopulations and geographic distance. This correlation would be expected to be negative if populations were at equilibrium and gene flow was restricted to short distances (isolation by distance). The lack of correlation was interpreted as non-equilibrium conditions; therefore, gene flow would be overestimated

The effects on colony morphology, sporulation, laccase production, and virulence of hypovirulence-associated double-stranded (ds)RNAs of the chestnut blight fungus, Cryphonectria parasitica, from a population of chestnut trees in eastern New Jersey were examined. Size and number of dsRNAs were determined by gel electrophoresis, and their genetic variability was determined by polymerase chain reaction (PCR) and nucleotide sequencing. The New Jersey hypovirulent isolates were similar in cultural phenotype and electrophoretic characteristics of their dsRNAs

A 12-kb segment of double-stranded (ds) RNA was associated with 25% of the isolates of Cryphonectria parasitica recovered from actively growing cankers in the central Appalachians. The relative virulence of these dsRNA-containing isolates ranged from a level comparable to that of the dsRNA-containing hypovirulent control isolate GH-2 to a level significantly greater than that of the dsRNA-free virulent control isolate Ep-155. When dsRNA-containing isolates and their dsRNA-free progeny were inoculated into Golden Delicious apples, excised dormant chestnut stems, or American chestnut sprouts, none of the lesions produced by the dsRNA-free progeny was significantly larger than those produced by the dsRNA-containing isolate from which they were derived [...]

The inheritance of mitochondrial DNA (mtDNA) in the chestnut blight fungus, Cryphonectria parasitica, was investigated with laboratory crosses and progeny from perithecia collected from a natural population. In a reciprocal cross, mtDNA haplotypes, determined from restriction fragment length polymorphisms (RFLPs), were inherited only from maternal parents. Another lab cross, in which the maternal strain was not known, showed uniparental inheritance for 43 ascospore progeny. All 87 progeny from eight perithecia collected in the field showed uniparental inheritance of mtDNA haplotypes identical to the maternal (canker) isolates. These data strongly support the hypothesis that mtDNA is maternally inherited in C. parasitica [...]

The main objective was to study the effect of ultraviolet (UV) light on vegetative compatibility in the fungus Cryphonectria parasitica. A procedure was developed to detect individual incompatible colonies from cultured lawns of compatible colonies. Incompatibility reactions were observed when protoplasts from two different vegetative compatibility (v-c) groups were mixed and plated on a suitable medium containing red food color (...)

Twenty-seven compounds were tested in vitro for their effect on mycelial of 20 g of Difco BiTek agar, 10 g of D-glucose, I g of L-asparagine, growth of Cryphonectria cubensis, the cause of canker of Eucalyptus 0.001 g a.i. of chlorothalonil, 0.0055 g a.i. of dicloran, 0.001 g a.i. of spp. Eighteen were subsequently tested for their ability to suppress mycelial vinclozolin, 2 g of gallic acid, 0.1665 g of streptomycin sulphate, and growth of 15 fungi commonly associated with C. cubensis in host tissue. I L of distilled water proved very effective for isolating C. cubensis from Gallic acid and dicloran, respectively, were the most effective inhibitors woody tissue and forest soil. of Trichoderma sp. and Mucor sp. A semiselective medium composed Cryphonectria cubensis (Bruner) C. S. Hodges causes a serious mycelial growth of 15 fungi associated with C. cubensis in host canker of Eucalyptus spp. in many tropical areas of the world tissue. Test fungi were Alternaria alternata (Fr.:Fr.) Keissl., (2,3,5-8,10,12,16). The recent discovery of C. cubensis in South Aspergillus niger Tiegh., Botryosphaeria sp., Botrytis cinerea Africa has prompted speculation about possible inoculum sources Pers.:Fr., Cryphonectria parasitica (Murrill) Barr, Cylindro(16). The fungus may have originated from native Myrtaceae (4), cladium scoparium Morg., Cytospora eucalypticola Van der and studies are necessary to determine whether these plants could Westhuizen, Endothia gyrosa (Schwein.:Fr.) Fr., Epicoccum be a source of inoculum. Similarly, the possible presence of C. purpurascens Ehrenb., Fusarium sp., Graphium sp., Mucor sp., cubensis inoculum in old tree stumps and in soils where Eucalyptus Pestalotia sp., Penicillium sp., and Trichoderma sp. Test fungi spp. are planted needs to be investigated, were grown on PDA for 4 days at 25 C. A 5-mm plug of agar The isolation of C. cubensis from soil and diseased wood is was transferred from the periphery of each fungal colony to each severely hampered by the presence of saprophytic fungi and bacteria on these substrates. To date, no selective medium for C. cubensis exists that may be used for such a study. This paper TABLE 1. Effect of different fungicides and chemicals on inhibition of describes a semiselective medium for the isolation of C. cubensis radial growth of Cryphonectria cubensisa from both soil and host tissue. Its efficacy is compared with that of a nonselective basal medium.

A rapid and reproducible method for testing the virulence of Cryptonectria parasitica using American chestnut (Castanea dentata) was developed. Bark- and wood-tissue samples excised from American chestnut tree stems were inoculated with virulent and hypovirulent C. parasitica strains. Samples were incubated at 25 C for 4 days, and virulence was assessed as the area of bark and wood tissue having brown, necrotic cells. Hypovirulent strains damaged an area 2.16 cm 2 or less of each tissue sample, while virulent strains consistently damaged a greater area (3.62-3.67 cm 2 ) []

Phenotypic expression by cultured isogenic virulent (EP155) and hypovirulent (EP713) strains of the chestnut blight fungus, Cryphonectria parasitica, was examined under different light regimens. Colony morphology and the levels of pigmentation, conidiation, oxalate accumulation, and phenol oxidase production were all influenced by light intensity, but to different degrees in the two strains. Significantly, exposure of strain EP713 to high light intensity partially relieved the hypovirulence-associated suppression of pigmentation, conidiation, and oxalate accumulation observed at low or intermediate light intensity (...)

Double-stranded RNA (dsRNA) was detected in hypovirulent but not in virulent strains of Cryphonectria (Endothia) parasitica isolated in Switzerland. To test for phenol oxidase activity we grew the strains on malt extract agar containing tannic acid (Bavendamm test). All virulent strains produced a strong color reaction, indicating phenol oxidase activity, whereas hypovirulent strains showed weak or no activity. (...)

Kuhlman, E. G. 1983. The effects of hypovirulence in Cryphonectria parasitica and of secondary blight infections on dieback of American chestnut trees. Phytopathology 73:1030-1034.

Kuhlman, E. G., Bhattacharyya, H., Nash, B. L., Double, M. L., and MacDonald, W. L. 1984. Identifying hypovirulent isolates of Cryphonectria parasitica with broad conversion capacity. Phytopathology 74:676-682. When 118 virulent (V) and 27 hypovirulent (H) isolates of Cryphonectria parasitica were paired in culture, 95% of the V isolates were converted to the hypovirulent condition by at least one of the H isolates. The 118V isolates in 54 vegetative compatibility (v-c) groups included representatives from West Virginia, North Carolina, Virginia, Tennessee, and Italy. The average conversion capacity of the 27 H isolates was 15% and ranged from 0 to 41 %. Eight cluster analyses based on different similarity functions were performed to group V isolates according to their susceptibility to conversion. The most useful cluster analysis was based on the square root of the number of sectors converted by individual H isolates and formed nine conversion groups and left only eight isolates ungrouped. Twenty-eight of the 54 v-c groups had more than one V isolate, and isolates in 15 of these v-c groups were also together in conversion groups. Clustering appears useful for determining relatedness among v-c groups. Conidial or mycelial slurries