Bradley I. Hillman

A Reovirus of the Fungus Cryphonectria parasitica That Is Infectious as Particles and Related to the Coltivirus Genus of Animal Pathogens

ABSTRACT RNA viruses of filamentous fungi fall into two broad categories, those that contain double-stranded RNA (dsRNA) genomes in rigid particles and those that are more closely related to positive-sense, single-stranded RNA viruses with dsRNA replicative intermediates found within lipid vesicles. Effective infectivity systems have been described for the latter, using RNA transcripts, but not for the former. We report the characterization of a reovirus from Cryphonectria parasitica, the filamentous fungus that causes chestnut blight disease. The virus substantially reduces the virulence of the fungus and results in dramatically altered colony morphology, as well as changes in other associated fungal traits, relative to the virus-free isogenic strain. Virus particles from infected mycelium contained 11 segments of dsRNA and showed characteristics typical of the family Reoviridae. Sequences of the largest three segments revealed that the virus is closely related to the Coltivirus genus of animal pathogens, which includes the human pathogen Colorado tick fever virus. The introduction of purified virus particles into protoplasts from virus-free isolates of the fungus resulted in a newly infected mycelium with the same morphology and virus composition as the original virus-infected isolate. This represents the completion of Kochs postulates for a true dsRNA virus from a filamentous fungus and the description of a definitive fungal member of the family Reoviridae.

Unraveling evolutionary relationships among the divergent lineages of colletotrichum causing anthracnose disease in turfgrass and corn.

ABSTRACT Colletotrichum species cause anthracnose diseases on a number of grass hosts and are common inhabitants of many others. They are divided into four species: C. sublineolum is pathogenic to Sorghum spp.; C. caudatum is found on C4 grasses such as indiangrass and big bluestem; C. falcatum causes red rot of sugarcane; and C. graminicola sensu lato is a broadly defined species including isolates that attack maize, wheat, oats, and many forage, turf, and amenity grasses of the subfamily Pooideae. In this paper, a combination of hierarchal- and nonhierarchal-based analyses were employed to examine evolutionary relationships among the grass-infecting Colletotrichum species, with special emphasis on isolates from turf and other grasses in the subfamily Pooideae. Reconstructions performed with data sets from over 100 Colletotrichum isolates at three variable loci using phylogenetic and network-based methodologies unambiguously supported the taxonomic separation of maize-infecting isolates of C. graminicola from the pooid-infecting strains of Colletotrichum. To reflect the evolutionary relationships that exist between these distinct lineages, we propose the resurrection of the species name C. cereale to describe the pooid-infecting isolates. There was also support for further subdivision of C. cereale, but the current data are insufficient to confidently subdivide the species, as there was some evidence of recombination between lineages of this species.

What is the value of ITS sequence data in Colletotrichum systematics and species diagnosis? A case study using the falcate-spored graminicolous Colletotrichum group

Because the genus Colletotrichum is among the most important groups of plant pathogenic fungi worldwide, the ability to accurately diagnose species is vital for the implementation of effective disease control and quarantine measures. Although the long-standing, unresolved taxonomic issues in the genus have recently begun to be addressed through multi-locus phylogenetic research, the tools most commonly used for Colletotrichum species identification are either insufficiently variable (e.g. morphology), or homoplasic (e.g. morphology and host range criteria). In this study, using the systematically well-defined falcate-spored, grass-associated group (FG) of Colletotrichum as a model, we test the utility of ITS sequence data to diagnose species affiliations through similarity-based searches of the NCBI GenBank database or by means of gene trees constructed using phylogenetic methods. 43% of all Colletotrichum sequences accessioned by GenBank are from the ITS region, making it the single most common sequence curated by the community; however, 34% of the ITS accessions existed only as sequence data in the database, with no associatedPUBLICation. Using Colletotrichum ITS sequence data from 53 FG defined isolates and 16 falcate-spored, non-graminicolous isolates to perform GenBank BLASTN searches, we found that erroneous identifications occurred for 86% of the 14 species tested. In contrast, the phylogenetic tree generated by the ITS sequence data, although poorly supported by bootstrap values, correctly grouped most of the species, but 10% of the individual isolates were incorrectly placed. From this study, we conclude that the currently available infrastructure of Colletotrichum ITS sequence data may yield unreliable species diagnoses, particularly if sequence similarity alone is the only criterion applied.

Evidence for interspecies transmission of viruses in natural populations of filamentous fungi in the genus Cryphonectria

Interspecies transmission is a significant evolutionary event that has allowed a variety of pathogens to invade new host species. We investigated interspecies transmission of viruses between the chestnut blight fungus, Cryphonectria parasitica, and a sympatric unidentified Cryphonectria species in Japan. Two isolates of Cryphonectria sp. were found to contain Cryphonectria hypovirus 1 (CHV‐1), which has been typically found in C. parasitica. Three lines of evidence support the hypothesis of interspecies transmission of CHV‐1. First, host species occur sympatrically and therefore have the opportunity to come into physical contact. Second, we transmitted CHV‐1 between species experimentally in the laboratory. Third, phylogenetic analysis of 476 bp of the ORF B region of CHV‐1 showed that sequences from Cryphonectria sp. were more closely related to those from C. parasitica than to each other. Local geographical subdivision of virus sequences from both host species argues against the alternative hypothesis of independent evolution of CHV‐1 since speciation of their hosts. Based on these findings, we rule out the hypotheses that CHV‐1 diverged from viruses in a common ancestor of the hosts, or that ancestral polymorphisms in CHV‐1 persisted in the two host taxa. Estimating the direction and frequency of interspecies transmission in nature will require more extensive samples of CHV‐1 from both host species.

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.

The family narnaviridae: simplest of RNA viruses.

Members of the virus family Narnaviridae contain the simplest genomes of any RNA virus, ranging from 2.3 to 3.6 kb and encoding only a single polypeptide that has an RNA-dependent RNA polymerase domain. The family is subdivided into two genera based on subcellular location: members of the genus Narnavirus have been found in the yeast Saccharomyces cerevisiae and in the oomycete Phytophthora infestans and are confined to the cytosol, while members of the genus Mitovirus have been found only in filamentous fungi and are found in mitochondria. None identified thus far encodes a capsid protein; like several other RNA viruses of lower eukaryotes, their genomes are confined within lipid vesicles. As more family members are discovered, their importance as genetic elements is becoming evident. The unique association of the genus Mitovirus with mitochondria renders them potentially valuable tools to study biology of lower eukaryotes.

Phylogenetic and population genetic divergence correspond with habitat for the pathogen Colletotrichum cereale and allied taxa across diverse grass communities.

Over the past decade, the emergence of anthracnose disease has newly challenged the health of turfgrasses on North American golf courses, resulting in considerable economic loss. The fungus responsible for the outbreaks, Colletotrichum cereale, has also been identified from numerous natural grasses and cereal crops, although disease symptoms are generally absent. Here we utilize phylogenetic and population genetic analyses to determine the role of ecosystem in the advancement of turfgrass anthracnose and assess whether natural grass and/or cereal inhabitants are implicated in the epidemics. Using a four‐gene nucleotide data set to diagnose the limits of phylogenetic species and population boundaries, we find that the graminicolous Colletotrichum diverged from a common ancestor into distinct lineages correspondent with host physiology (C3 or C4 photosynthetic pathways). In the C4 lineage, which includes the important cereal pathogens Colletotrichum graminicola, C. sublineolum, C. falcatum, C. eleusines, C. caudatum and several novel species, host specialization predominates, with host‐associated lineages corresponding to isolated sibling species. Although the C3 lineage —C. cereale— is comprised of one wide host‐range species, it is divided into 10 highly specialized populations corresponding to ecosystem and/or host plant, along with a single generalist population spread across multiple habitat types. Extreme differentiation between the specialized C. cereale populations suggests that asymptomatic nonturfgrass hosts are unlikely reservoirs of infectious disease propagules, but gene flow between the generalist population and the specialized genotypes provides an indirect mechanism for genetic exchange between otherwise isolated populations and ecosystems.

Systematic analysis of the falcate-spored graminicolous Colletotrichum and a description of six new species from warm-season grasses

Species limits in the fungal genus Colletotrichum are traditionally distinguished by appressorial and/or conidial morphology or through host plant association, but both criteria are criticized for their inability to resolve distinct taxa. In previous research eight novel falcate-spored Colletotrichum species were identified from graminicolous hosts using multilocus molecular phylogenetic analysis. In the present work formal descriptions and illustrations are provided for six of the new taxa: C. hanaui sp. nov., C. nicholsonii sp. nov., C. paspali sp. nov., C. jacksonii sp. nov., C. miscanthi sp. nov. and C. axonopodi sp. nov.; and an emended description with epitypification is provided for C. eleusines. Comparison of hyphopodial appressoria and host association against phylogenetic species boundaries and evolutionary relationships in the graminicolous Colletotrichum group demonstrate that, while these characters can be useful in combination for the purpose of species diagnosis, erroneous identification is possible and species boundaries might be underestimated if these characters are used independently, as exemplified by the polyphyletic taxa C. falcatum. Appressoria have been subject to convergent evolution and were not predictive of phylogenetic relationships. Despite these limitations, the results of this work establish that in combination appressorial and host range characters could be used to generate informative dichotomous identification keys for Colletotrichum species groups when an underlying framework of evolutionary relationships, taxonomic criteria and nomenclature have been satisfactorily derived from molecular systematic treatments.

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.

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination.

Abstract Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polylmerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae.