Molecular genetic determinants of intraspecific polymorphism of the phytopathogenic fungus Cryphonectria parasitica

The review summarizes the current evidence on the phytopathogenic fungus Cryphonectria parasitica, which is a classic object for studying hypovirulence. Phenotypic manifestations of hypovirulence and themolecular mechanisms of action of the mycovirus Cryphonectria hypovirus (CHV) infecting the fungus are described in detail. Genetic determinants of vegetative incompatibility in C. parasitica (a phenomenon increasing polymorphism of the fungus and preventing CHV expansion) are considered. The data on C. parasitica polymorphism are correlated with the data on the distribution of different CHV species in the European, American, and Asian populations of the fungus.     

Invasion history and demographic pattern of Cryphonectria hypovirus 1 across European populations of the chestnut blight fungus

We reconstructed the invasion history of the fungal virus Cryphonectria hypovirus 1 (CHV‐1) in Europe, which infects the chestnut blight fungus Cryphonectria parasitica. The pattern of virus evolution was inferred based on nucleotide sequence variation from isolates sampled across a wide area in Europe at different points in time. Phylogeny and time estimates suggested that CHV‐1 was introduced together with its fungal host to Europe and that it rapidly colonized the central range along the south facing slopes of the Alps and the north‐east facing slopes of the Dinaric Alps. These central populations were the source for two waves of simultaneous invasions toward the southern Balkans and Turkey, as indicated by migration rates. Our results showed that the evolutionary scenarios for CHV‐1 and C. parasitica were spatially congruent. As infection with CHV‐1 reduces the pathogenicity of C. parasitica toward the chestnut tree, CHV‐1 invasions of the newly established C. parasitica populations probably prevented the development of devastating chestnut blight epidemics in Europe. We propose that in this, and supposedly in other pathosystems, geographic, vegetation‐related, demographic, economic, and political factors may help explain the correlated invasion pattern of a parasite and its host.     

Comparative acetylomic analysis reveals differentially acetylated proteins regulating fungal metabolism in hypovirus-infected chestnut blight fungus

Cryphonectria parasitica, the chestnut blight fungus, and hypoviruses are excellent models for examining fungal pathogenesis and virus–host interactions. Increasing evidence suggests that lysine acetylation plays a regulatory role in cell processes and signalling. To understand protein regulation in C. parasitica by hypoviruses at the level of posttranslational modification, a label-free comparative acetylome analysis was performed in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Using enrichment of acetyl-peptides with a specific anti-acetyl-lysine antibody, followed by high accuracy liquid chromatography–tandem mass spectrometry analysis, 638 lysine acetylation sites were identified on 616 peptides, corresponding to 325 unique proteins. Further analysis revealed that 80 of 325 proteins were differentially acetylated between C. parasitica strain EP155 and EP155/CHV1-EP713, with 43 and 37 characterized as up- and down-regulated, respectively. Moreover, 75 and 65 distinct acetylated proteins were found in EP155 and EP155/CHV1-EP713, respectively. Bioinformatics analysis revealed that the differentially acetylated proteins were involved in various biological processes and were particularly enriched in metabolic processes. Differences in acetylation in C. parasitica citrate synthase, a key enzyme in the tricarboxylic acid cycle, were further validated by immunoprecipitation and western blotting. Site-specific mutagenesis and biochemical studies demonstrated that the acetylation of lysine-55 plays a vital role in the regulation of the enzymatic activity of C. parasitica citrate synthase in vitro and in vivo. These findings provide a valuable resource for the functional analysis of lysine acetylation in C. parasitica, as well as improving our understanding of fungal protein regulation by hypoviruses from a protein acetylation perspective.     

A membrane technique for producing protoplasts ofCryphonectria parasitica

A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×10⁶ protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.     

Polymorphic sequence‐characterized codominant loci in the chestnut blight fungus,Cryphonectria parasitica

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we describe the development of 11 codominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were cloned and sequenced, and polymerase chain reaction (PCR) primers were designed flanking insertions/deletions. Primers labelled with fluorescent dyes were combined in multiplex reactions to assay five or six loci simultaneously in a capillary sequencing system. These codominant markers have the potential to complement RFLP methods for studying C. parasitica.     

Differential Transfer and Dissemination of Hypovirus and Nuclear and Mitochondrial Genomes of a Hypovirus-Infected Cryphonectria parasitica Strain after Introduction into a Natural Population

Biological control of plant diseases generally requires release of living organisms into the environment. Cryphonectria hypoviruses function as biological control agents for the chestnut blight fungus, Cryphonectria parasitica, and hypovirus-infected C. parasitica strains can be used to treat infected trees. We used naturally occurring molecular marker polymorphisms to examine the persistence and dissemination of the three genomes of a hypovirus-infected C. parasitica strain, namely, the double-stranded RNA genome of Cryphonectria hypovirus 1 (CHV1) and the nuclear and mitochondrial genomes of its fungal host. The hypovirus-infected strain was experimentally introduced into a blight-infested chestnut coppice forest by treating 73 of 246 chestnut blight cankers. Two years after introduction, the hypovirus had disseminated to 36% of the untreated cankers and to 35% of the newly established cankers. Spread of the hypovirus was more frequent within treated sprout clusters than between sprout clusters. Mitochondrial DNA of the introduced fungus also was transferred into the resident C. parasitica population. Concomitant transfer of both the introduced hypovirus and mitochondrial DNA was detected in almost one-half of the treated cankers analyzed. The introduced mitochondrial DNA haplotype also was found in three resident isolates from newly established cankers. The nuclear genome of the introduced strain persisted in the treated cankers but did not spread beyond them.     

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.     

Molecular genetic identification of the phytopathogenic fungus <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>

Mycological analysis of samples of sweet chestnut (Castanea sativa) collected in the northern Turkish zone of its habitat was carried out. A total of 300 strains of micromycetes belonging to 13 taxa were isolated in pure cultures. The phytopathogenic fungus Cryphonectria parasitica, the causal agent of chestnut blight, was identified among these strains. A technique for detection of C. parasitica in pure culture and host tissues using species-specific PCR markers was developed and tested. This technique can be used as an express method for detection of chestnut blight.     

Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway

The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (~35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.     

Using chimeric hypoviruses to fine-tune the interaction between a pathogenic fungus and its plant host

Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9, 904. By swapping domains within this region, it was possible to generate chimeric hypovirus-infected C. parasitica isolates that exhibited a spectrum of defined colony and canker morphologies. Several severe strain traits were observed to be dominant. It was also possible to uncouple the severe strain traits of small canker size and suppression of asexual sporulation. For example, fungal isolates infected with a chimera containing nucleotides 2363 through 5310 from CHV1-Euro7 in a CHV1-713 background formed small cankers that were similar in size to that caused by CHV1-EP713-infected isolates but with the capacity for producing asexual spores at levels approaching that observed for fungal isolates infected with the mild strain. These results demonstrate that hypoviruses can be engineered to fine-tune the interaction between a pathogenic fungus and its plant host. The identification of specific hypovirus domains that differentially contribute to canker morphology and sporulation levels also provides considerable utility for continuing efforts to enhance biological control potential by balancing hypovirulence and ecological fitness.     

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.     

Diversity of viruses in Cryphonectria parasitica and C. nitschkei in Japan and China, and partial characterization of a new chrysovirus species

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9 kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.     

The Cryphonectria parasitica mitochondrial rns gene: Plasmid-like elements, introns and homing endonucleases

The mt-rns gene of Cryphonectria parasitica is 9872 bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmid-like mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt-rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype.     

Control of chestnut blight by the use of hypovirulent strains of the fungus Cryphonectria parasitica in northwestern Spain

Chestnut blight is controlled in Europe by using Cryphonectria hypovirus CHV1, a non-encapsulated RNA virus. The chestnut blight fungus, Cryphonectria parasitica, is weakened by the virus, and healing tissue growth occurs in the host tree. Transmission of this cytoplasmic hypovirus is restricted by the incompatibility system of the fungus, so that the hypovirus can be transmitted only between isolates of the same or closely related vegetative compatibility (vc) types. Hypovirulent isolates of C. parasitica (all of the French subtype CHV1-F1) from Castilla y León (NW Spain) were compared with virulent isolates in both laboratory (cut stems) and field inoculations (in two orchards in the province of León and one orchard in the province of Zamora). The tests were performed with the most common vc types in the region, EU1 and EU11. The cut stem assay revealed that the hypovirulent isolates of vc type EU1 did not reduce the growth of virulent cankers. By contrast, four hypovirulent strains H1, H4, H5 and H6 (all vc type EU11) reduced the growth of virulent isolates in the cut stem assay. Field tests showed that hypovirulent isolates of EU1 and EU11 were effective in reducing canker in both orchards in León with all treatments tested; however, in Zamora, where only EU11 was tested, all the treatments failed except H1, which was able to reduce growth of the canker eighteen months after the inoculation. The development of hypovirulence suggests that hypovirus subtype F1 is well adapted in the province of León. Both naturally extended and inoculated hypoviruses appear to have reduced the incidence of the canker, thus improving chestnut stands. However, the inoculations were not as effective in the orchards in Zamora. This indicates that the disease could be controlled in Castilla y León by inoculation of trees with hypovirulent strains, but that more tests should be done in provinces where the hypovirus is still not present.     

Heterologous expression of a tannic acid-inducible laccase3 of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

A tannic acid-inducible and mycoviral-regulated laccase3 (lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.     

A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.