Chestnut Blight Fungus Research Articles

Antifungal properties of chitinases from Castanea sativa against hypovirulent and virulent strains of the chestnut blight fungus Cryphonectria parasitica

Four proteins (C1, C2, C3 and C4) with a chitin binding domain have been purified from European chestnut (Castanea sativa Mill.) plantlets grown in vitro . Three of the proteins C2, C3 and C4 have chitinase activity. C3 from its N-terminal sequence is a Class I basic chitinase with high sequence homology with other Class I chitinases from chestnut cotyledons and other plants. Antifungal tests showed that C1, C3 and C4 inhibit hyphal growth of the chestnut blight fungus Cryphonectria parasitica in culture. The proteins were much more effective in inhibiting the hypovirulent form of the fungus than the virulent form, suggesting that the Hypovirus induced effect on virulence may be due to changes in the susceptibility of C. parasitica to host defense responses.

Infectious cDNA clone of hypovirus CHV1-Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica.

We report the construction of a full-length infectious cDNA clone for hypovirus CHV1-Euro7, which is associated with reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica. Field strains infected with CHV1-Euro7 are more virulent and exhibit less severe phenotypic changes (hypovirulence-associated traits) than strains infected with the prototypic hypovirus CHV1-EP713, for which the first infectious cDNA clone was developed. These differences exist even though the two hypoviruses show extensive sequence identities: 87 to 93% and 90 to 98% at the nucleotide and amino acid levels, respectively. The relative contributions of viral and host genomes to phenotypic traits associated with hypovirus infection were examined by transfecting synthetic transcripts of the two hypovirus cDNAs independently into two different virus-free C. parasitica strains, EP155 and Euro7(-v). Although the contribution of the viral genome was clearly predominant, the final magnitude and constellation of phenotypic changes were a function of contributions by both genomes. The high level of sequence identity between the two hypoviruses also allowed construction of viable chimeras and mapping of the difference in symptom expression observed for the two viruses to the open reading frame B coding domain. Implications of these results for engineering enhanced biological control and elucidating the basis for hypovirus-mediated attenuation of fungal virulence are discussed.

Mutagenesis of putative acylation sites alters function, localization, and accumulation of a Gi alpha subunit of the chestnut blight fungus Cryphonectria parasitica.

Targeted disruption of cpg-1, a gene encoding the G protein Gi alpha subunit, CPG-1, in the chestnut blight fungus, Cryphonectria parasitica, results in reduced mycelial growth, reduced orange pigmentation, loss of virulence, loss of asexual sporulation, and female infertility. We report the development of a complementation system for cpg-1 null mutants and its use to evaluate the in vivo consequences of mutating conserved putative CPG-1 myristoylation (G2) and palmitoylation (C3) sites. Independent mutations of the two putative acylation sites differentially altered complex fungal biological processes, including virulence, and modified CPG-1 membrane association. Results of combined Northern (RNA) and Western (immunoblot) analysis also indicated a role for lipid modification in post-transcriptional regulation of CPG-1 accumulation.

Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the '447' cytoplasmic male sterility trait in Vicia faba.

The entire nucleotide sequence of the double-stranded (ds) RNA associated with the unconventional '447' cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17,635 nt long, while the positive-strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14,900 nt, with the smaller RNA being located on the 5' side. The entire positive-strand could encode a single in-frame ORF starting at the first AUG at position 42-44 and ending with a TGA at 17,517-17,519. This long potential polypeptide with a predicted molecular mass of 654,109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by self-cleavage being the determinant of the CMS trait.

Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan

ABSTRACT Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.

Genetics of vegetative incompatibility in cryphonectria parasitica

Vegetative incompatibility in the chestnut blight fungus, Cryphonectria parasitica, in Europe is controlled by six unlinked vic loci, each with two alleles. Four previously identified vic loci (vic1, vic2, vic3, and vic4) were polymorphic in European vegetative compatibility (vc) types. Two new loci, vic6 and vic7, also were identified among European vc types. In one cross, vic genes segregated independently at five loci, and 194 progeny were assigned to 32 vc types; none of these loci were linked. A total of 64 vc types were identified from all crosses. All 64 genotypes possible from six vic loci, each with two alleles (2(6) = 64), were identified and assigned to vc types. Based on our model, vc types v-c 5 and v-c 10, which had been used in previous genetic studies, differ by only five vic genes. Future studies of vc types in C. parasitica can use knowledge of vic genotypes for analysis of population genetic structure based on vic allele frequencies and to determine the effect of each vic gene on virus transmission between vc types.

Hypovirus Transmission to Ascospore Progeny by Field-Released Transgenic Hypovirulent Strains of Cryphonectria parasitica

ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed.

Seasonal variation of peroxidase activity in chestnut trees

Peroxidase (E.C.1.11.1.7) activity in twigs of American ( Castanea dentata), Chinese ( C. mollissima), and Japanese ( C. crenata) chestnut was determined monthly over an eight month period. The total amount of peroxidase activity was highest in December and April for most trees. An inhibitor of peroxidase activity was present in the extracts which could be removed by gel filtration. Extracts from healthy twigs had consistent major bands on IEF gels. However, a new band was detected in bark of American chestnut after wounding or infection with chestnut blight fungus ( Cryphonectria parasitica).

The Acquisition of Hypovirulence in Host‐Pathogen Systems with Three Trophic Levels

A major focus of research on the dynamics of host-pathogen interactions has been the evolution of pathogen virulence, which is defined as the loss in host fitness due to infection. It is usually assumed that changes in pathogen virulence are the result of selection to increase pathogen fitness. However, in some cases, pathogens have acquired hypovirulence by themselves becoming infected with hyperparasites. For example, the chestnut blight fungus Cryphonectria parasitica has become hypovirulent in some areas by acquiring a double-stranded RNA hyperparasite that debilitates the pathogen, thereby reducing its virulence to the host. In this article, we develop and analyze a mathematical model of the dynamics of host-pathogen interactions with three trophic levels. The system may be dominated by either uninfected (virulent) or hyperparasitized (hypovirulent) pathogens, or by a mixture of the two. Hypovirulence may allow some recovery of the host population, but it can also harm the host population if the hyperparasite moves the transmission rate of the pathogen closer to its evolutionarily stable strategy. In the latter case, the hyperparasite is effectively a mutualist of the pathogen. Selection among hyperparasites will often minimize the deleterious effects, or maximize the beneficial effects, of the hyperparasite on the pathogen. Increasing the frequency of multiple infections of the same host individual promotes the acquisition of hypovirulence by increasing the opportunity for horizontal transmission of the hyperparasite. This effect opposes the usual theoretical expectation that multiple infections promote the evolution of more virulent pathogens via selection for rapid growth within hosts.

Identification of a Cryphonectria parasitica laccase gene promoter element involved in cycloheximide-inducible, hypovirus-repressible transcriptional activation

The gene lac-1, encoding the enzyme laccase, is the best characterized of a number of genes in the chestnut blight fungus, Cryphonectria parasitica, that are repressed by hypoviruses, a group of virulence-attenuating mycoviruses. lac-1 has also been shown to be transcriptionally activated by low concentrations of the translational inhibitor cycloheximide (CHX) and by the immunosuppressant cyclosporin A. We now report the identification of a CHX responsive element within the lac-1 promoter region. Gel-mobility shift analysis revealed a 111-bp fragment located 1.8 kb upstream of the lac-1 transcriptional start point that exhibited protein binding activity. Insertion of this element within a basal lac-1 promoter sequence conferred CHX responsive transcriptional activation. Moreover, this activation was prevented by hypovirus infection. A 22-bp sequence with an imperfect dyad symmetry located within the 111-bp element was found to be essential for sequence-specific protein binding and, thus, represents a putative target for interactions between the lac-1 promoter and proteins that are involved in mediating CHX inducible activation of lac-1 transcription.

The Acquisition of Hypovirulence in Host-Pathogen Systems with Three Trophic Levels

A major focus of research on the dynamics of host‐pathogen interactions has been the evolution of pathogen virulence, which is defined as the loss in host fitness due to infection. It is usually assumed that changes in pathogen virulence are the result of selection to increase pathogen fitness. However, in some cases, pathogens have acquired hypovirulence by themselves becoming infected with hyperparasites. For example, the chestnut blight fungus Cryphonectria parasitica has become hypovirulent in some areas by acquiring a double‐stranded RNA hyperparasite that debilitates the pathogen, thereby reducing its virulence to the host. In this article, we develop and analyze a mathematical model of the dynamics of host‐pathogen interactions with three trophic levels. The system may be dominated by either uninfected (virulent) or hyperparasitized (hypovirulent) pathogens, or by a mixture of the two. Hypovirulence may allow some recovery of the host population, but it can also harm the host population if the hyperparasite moves the transmission rate of the pathogen closer to its evolutionarily stable strategy. In the latter case, the hyperparasite is effectively a mutualist of the pathogen. Selection among hyperparasites will often minimize the deleterious effects, or maximize the beneficial effects, of the hyperparasite on the pathogen. Increasing the frequency of multiple infections of the same host individual promotes the acquisition of hypovirulence by increasing the opportunity for horizontal transmission of the hyperparasite. This effect opposes the usual theoretical expectation that multiple infections promote the evolution of more virulent pathogens via selection for rapid growth within hosts.

Molecular Mapping of Resistance to Blight in an Interspecific Cross in the Genus Castanea

ABSTRACT A three-generation American chestnut x Chinese chestnut pedigree was used to construct a genetic linkage map for chestnut and to investigate the control of resistance to Endothia parasitica (chestnut blight fungus). DNA genotypes for 241 polymorphic markers (eight isozymes, 17 restriction fragment length polymorphisms [RFLPs], and 216 random amplified polymorphic DNAs [RAPDs]) were assayed on an F(2) family consisting of 102 individuals. Of these markers, 196 were segregating as expected and, subsequently, used for primary linkage mapping. Two isozymes, 12 RFLPs, and 170 RAPDs were mapped to 12 linkage groups spanning a total genetic distance of 530.1 Kosambi centimorgans. F(2) plants were evaluated for a response to E. parasitica infection by directly inoculating them with two unique fungal isolates and measuring canker expansion over a period of 3.5 months. Results were compared with the marker genotype data, thereby identifying genomic regions significantly associated with a resistance response. Single-marker or nonsimultaneous analyses of variance identified seven genomic regions that appear to have an effect on host response. Multiple-marker or simultaneous models suggest that three of these regions have a significant effect on host response, together explaining as much as 42.2% of the total variation for canker size. At each of the three putative resistance loci, alleles derived from the Chinese chestnut grandparent were associated with smaller canker size, or higher levels of resistance.

Distinct roles for two G protein alpha subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption.

Reduced accumulation of the GTP-binding protein G(i)alpha subunit CPG-1, due either to hypovirus infection or transgenic cosuppression, correlates with virulence attenuation of the chestnut blight fungus, Cryphonectria parasitica. The role of G protein-mediated signal transduction in fungal virulence was further examined by targeted disruption of the gene cpg-1, encoding CPG-1, and a second Galpha gene, cpg-2, encoding the subunit CPG-2. Disruption of cpg-1 resulted in a set of phenotypic changes similar to, but more severe than, those associated with hypovirus infection. Changes included a marked reduction in fungal growth rate and loss of virulence, asexual sporulation, female fertility, and transcriptional induction of the gene lac-1, encoding the enzyme laccase. In contrast, cpg-2 disruption resulted in only slight reductions in growth rate and asexual sporulation and no significant reduction in virulence, female fertility, or lac-1 mRNA inducibility. These results provide definitive confirmation of previous correlative evidence that suggested a requirement of CPG-1-linked signaling for a number of fungal processes, including virulence and reproduction, while demonstrating that a second Galpha, CPG-2, is dispensable for these processes. They also significantly strengthen support for the apparent linkage between hypovirus-mediated disruption of G protein signal transduction and attenuation of fungal virulence.

Using Hypoviruses to Probe and Perturb Signal Transduction Processes Underlying Fungal Pathogenesis.

Imagine the task of designing a reagent to explore regulatory mechanisms that govern fungal pathogenesis. The assignment triggers recollection of a group of cytoplasmically transmissible RNA mycoviruses that have the property of attenuating fungal virulence. Utility of these viruses is significantly enhanced by the construction of an infectious cloned cDNA copy of the viral RNA genome. Subsequent studies reveal that virus infection attenuates fungal virulence by altering cellular signal transduction processes, thus exposing a role for G protein-linked, cAMP-mediated signaling in fungal pathogenesis. Synthetic transcripts derived from the infectious viral cDNA are used to establish virus infection in pathogenic fungi other than the natural host, thereby expanding opportunities for probing and manipulating a broader range of plant-pathogen interactions. As a bonus, the infectious viral cDNA is found to provide enhanced potential for biological control of fungal disease. Elements of the account presented above represent actual developments derived from recent molecular analyses of mycoviruses, taxonomically grouped within the genus Hypovirus of the family Hypoviridae, that attenuate virulence of the chestnut blight fungus Cryphonecrria parasitica. Details of the intriguing biology and potential applications offered by this system have been the subject of several recent reviews (MacDonald and Fulbright, 1991; Nuss, 1992; Heiniger and Rigling, 1994). This article discusses recent progress on two major fronts. The first involves advances in the development of hypovirus molecular biology and the extended experimental and practical utility provided by these advances. The second focuses on progress in elucidating mechanisms by which hypoviruses alter fungal gene expressionNirulence and the insights that these surprising results present for further understanding of signal transduction processes that underlie fungus-plant pathogenic interactions.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155.

In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155.

Cloning and targeted disruption of enpg-1, encoding the major in vitro extracellular endopolygalacturonase of the chestnut blight fungus, Cryphonectria parasitica.

The gene enpg-1, encoding the major extracellular endopolygalacturonase (endoPG) purified from culture filtrates of the chestnut blight fungus, Cryphonectria parasitica, was cloned and characterized. The deduced mature enpg-1 protein product, ENPG-1, had a calculated molecular mass of 34.5 kDa and a pI of 7.2, consistent with empirically derived values for the purified enzyme, and had 66% identity with an endoPG from the maize pathogen Cochliobolus carbonum. Targeted disruption of enpg-1 was accomplished by homologous recombination with a cloned copy of the gene that contained the Escherichia coli hygromycin B phosphotransferase gene (hph) inserted into exon 1. enpg-1 disruption resulted in no reduction in canker formation on dormant American chestnut stems. Unexpectedly, the level of polygalacturonase (PG) activity measured in cankered bark tissue infected with enpg-1 disruptants was indistinguishable from that found in canker tissue infected with virulent strain EP155. Isoelectric focusing and activity gel analysis of PG activity extracted from canker bark tissue revealed ENPG-1 to be a minor (less than 5%) activity component in tissue infected with the virulent strain and to be absent in tissue infected with the disruption mutants. The predominant activity in both canker samples consisted of two previously undetected acidic PG forms that appear absent in C. parasitica culture filtrates. We conclude from these results that the major C. parasitica extracellular endoPG produced in culture, ENPG-1, does not play a significant role in fungal virulence. However, the identification of two acidic PG activities expressed predominantly, if not exclusively, in planta provides new opportunities for examining the importance of PGs in C. parasitica pathogenesis.

Intercontinental population structure of the chestnut blight fungus,Cryphonectria parasitica

The population structure of the chestnut blight fungus, Cryphonectria parasitica was analyzed using restriction fragment length polymorphisms (RFLPs). A total of 791 isolates were sampled from four regions, China, Japan, North America and Europe, and assayed for alleles at eight RFLP loci. Alleles at all eight loci segregated in simple Mendelian ratios, and most loci were unlinked. Gene diversity was decomposed hierarchically: 56% of gene diversity was attributable to diversity within subpopulations, compared to 7% among subpopulations within regions, and 37% among regions. Subpopulations of C. parasitica in China have distinctly different allele frequencies from subpopulations in other regions, including Japan. DNA fingerprint genotypes in China were also significandy different from those in the other regions. There was an average of 3.2 restriction fragments hybridizing to a DNA fingerprinting probe in Chinese isolates from 11 subpopulations compared to 8.6 in isolates from Japan; the exception was in one subpopulation in northeastern China where there was an average of 11.1 fragments per isolate. North American and European subpopulations were similar to each other, and more similar to Japan than to China, for both RFLP allele frequencies and DNA fingerprints. The results suggest that C. parasitica was introduced into North America from Japan, not China. The origin of C. parasitica for European subpopulations cannot be determined from these results, but eastern China was not a likely source. Analysis of population structure within China showed moderate differentiation, with 11% of gene diversity attributable to differences among subpopulations (ĜST = 0.11). Pairwise estimates of gene flow between sub-populations were negatively correlated to geographic distances between subpopulations in China. This result suggests that Chinese populations are in equilibrium, and that restricted gene flow and genetic drift shape these populations.

Intercontinental Population Structure of the Chestnut Blight Fungus, Cryphonectria parasitica

The population structure of the chestnut blight fungus, Cryphonectria parasitica was analyzed using restriction fragment length polymorphisms (RFLPs). A total of 791 isolates were sampled from four regions, China, Japan, North America and Europe, and assayed for alleles at eight RFLP loci. Alleles at all eight loci segregated in simple Mendelian ratios, and most loci were unlinked. Gene diversity was decomposed hierarchically: 56% of gene diversity was attributable to diversity within subpopulations, compared to 7% among subpopulations within regions, and 37% among regions. Subpopulations of C. parasitica in China have distinctly different allele frequencies from subpopulations in other regions, including Japan. DNA fingerprint genotypes in China were also significandy different from those in the other regions. There was an average of 3.2 restriction fragments hybridizing to a DNA fingerprinting probe in Chinese isolates from 11 subpopulations compared to 8.6 in isolates from Japan; the exception was in one subpopulation in northeastern China where there was an average of 11.1 fragments per isolate. North American and European subpopulations were similar to each other, and more similar to Japan than to China, for both RFLP allele frequencies and DNA fingerprints. The results suggest that C. parasitica was introduced into North America from Japan, not China. The origin of C. parasitica for European subpopulations cannot be determined from these results, but eastern China was not a likely source. Analysis of population structure within China showed moderate differentiation, with 11% of gene diversity attributable to differences among subpopulations (ĜST = 0.11). Pairwise estimates of gene flow between sub-populations were negatively correlated to geographic distances between subpopulations in China. This result suggests that Chinese populations are in equilibrium, and that restricted gene flow and genetic drift shape these populations.

Phenotypic Changes Associated with Wild-Type and Mutant Hypovirus RNA Transfection of Plant Pathogenic Fungi Phylogenetically Related toCryphonectria parasitica

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.