The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut.

BACKGROUND: The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome. RESULTS: The genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors. CONCLUSIONS: This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen's life cycle and a solid foundation for studying EFB.

Genetic Diversity Analysis of Anisogramma anomala in the Pacific Northwest and New Jersey.

Anisogramma anomala, a biotrophic ascomycete, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). EFB is endemic in eastern North America, preventing the commercial production of European hazelnut (C. avellana L.). In contrast, the historic absence of A. anomala in the Pacific Northwest (PNW) supported the development of a robust hazelnut industry. Circa 1960, A. anomala was inadvertently introduced into southwestern Washington, causing orchard devastation. Distribution of the pathogen in the PNW has been hypothesized to be the result of a single-point introduction. This study aimed to investigate the single-point introduction hypothesis of A. anomala by comparing the genetic diversity of A. anomala samples from the PNW and New Jersey (NJ). Specimens from the main PNW production region (n = 60) and an area within the pathogen's native range, NJ (n = 151), were genotyped using 15 simple sequence repeat (SSR) markers. The following were used to assess genetic diversity and population structure: allelic summary statistics, discriminant analysis of principal components, network median-joining tree, analysis of multilocus genotypes, and allelic population diversity analysis. Analyses separated the samples into one cluster containing all the PNW isolates, and five clusters of NJ isolates. The PNW samples were nearly genetically uniform, and the NJ isolates were diverse. These findings support the hypothesis that A. anomala in the PNW was derived from a single-point introduction and corroborate previous studies that have shown A. anomala is very diverse in NJ. This indicates that maintaining restrictions on the movement of Corylus into the PNW is important to prevent the introduction of new populations of A. anomala, thus protecting the PNW hazelnut industry.

In-Tree Behavior of Diverse Viruses Harbored in the Chestnut Blight Fungus, Cryphonectria parasitica.

The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

ICTV Virus Taxonomy Profile: Chrysoviridae.

Members of the family Chrysoviridae are isometric, non-enveloped viruses with segmented, linear, dsRNA genomes. There are 3-7 genomic segments, each of which is individually encapsidated. Chrysoviruses infect fungi, plants and possibly insects, and may cause hypovirulence in their fungal hosts. Chrysoviruses have no known vectors and lack an extracellular phase to their replication cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Chrysoviridae, which is available at ictv.global/report/chrysoviridae.

Investigation of Host Range of and Host Defense against a Mitochondrially Replicating Mitovirus.

Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses.IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen.

Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

ICTV Virus Taxonomy Profile: Megabirnaviridae.

Megabirnaviridae is a family of non-enveloped spherical viruses with dsRNA genomes of two linear segments, each of 7.2-8.9 kbp, comprising 16.1 kbp in total. The genus Megabirnavirus includes the species Rosellinia necatrix megabirnavirus 1, the exemplar isolate of which infects the white root rot fungus (Rosellinia necatrix) to which it confers hypovirulence. Megabirnaviruses are characterized by their bisegmented genome with large 5'-untranslated regions (1.6 kb) upstream of both 5'-proximal coding strand ORFs, and large protrusions on the particle surface. This is a summary of the ICTV Report on the family Megabirnaviridae, which is available at ictv.global/report/megabirnaviridae.This Profile is dedicated to the memory of our valued colleague Professor Said A. Ghabrial.

Phytophthora infestans RNA virus 2, a novel RNA virus from Phytophthora infestans, does not belong to any known virus group.

Phytophthora infestans is the causal agent of potato and tomato late blight. In this study, we obtained the complete genome sequence of a novel RNA virus from this plant pathogen, tentatively named "Phytophthora infestans RNA virus 2" (PiRV-2). The PiRV-2 genome is 11,170 nt in length and lacks a polyA tail. It contains a single large open reading frame (ORF) with short 5' and 3' untranslated regions. The ORF is predicted to encode a polyprotein of 3710 aa (calculated molecular weight, 410.94 kDa). This virus lacks significant similarity to any other known viruses, even in the conserved RNA-dependent RNA polymerase region. Phylogenetic analysis demonstrated that it did not cluster with any known virus group. We conclude that PiRV-2 belongs to a new virus family yet to be described. This virus was found to be faithfully transmitted through asexual reproduction.

Population Differentiation Within Anisogramma anomala in North America.

Anisogramma anomala, a biotrophic ascomycete in the order Diaporthales, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). Until recently, little has been documented on its genetic diversity and population structure. In this study, 18 simple sequence repeat markers were used to fingerprint 182 accessions of the fungus originating from across North America. Our results, based on summary statistics of the allelic data, a discriminant analysis of principal components (DAPC) scatterplot, an unweighted pair group method with arithmetic mean (UPGMA) dendrogram, and analysis of multilocus genotypes, show that A. anomala exhibits considerable genetic diversity across multiple populations. Eleven clusters were resolved from the DAPC scatterplot, five of which were validated by statistically supported clusters in the UPGMA dendrogram. The 11 DAPC clusters were statistically significant via an analysis of molecular variance. Dendrogram topology and DAPC scatterplot groups showed some correlation with collection origin; samples collected in proximity tended to cluster together and be genetically similar. However, some locations held populations that were diverse and some populations with a high degree of similarity had disparate origins, suggesting movement by humans. Overall, the results demonstrate the presence of multiple, genetically distinct populations of A. anomala in North America and serve as a reference to assist in understanding and managing EFB.

ICTV Virus Taxonomy Profile: Chrysoviridae.

The Chrysoviridae is a family of small, isometric, non-enveloped viruses (40 nm in diameter) with segmented dsRNA genomes (typically four segments). The genome segments are individually encapsidated and together comprise 11.5-12.8 kbp. The single genus Chrysovirus includes nine species. Chrysoviruses lack an extracellular phase to their life cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. There are no known natural vectors for chrysoviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Chrysoviridae, which is available at www.ictv.global/report/chrysoviridae.

ICTV Virus Taxonomy Profile: Hypoviridae.

The Hypoviridae, comprising one genus, Hypovirus, is a family of capsidless viruses with positive-sense, ssRNA genomes of 9.1-12.7 kb that possess either a single large ORF or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e. internal ribosome entry site-mediated and stop/restart translation. Hypoviruses have been detected in ascomycetous or basidiomycetous filamentous fungi, and are considered to be replicated in host Golgi-derived, lipid vesicles that contain their dsRNA as a replicative form. Some hypoviruses induce hypovirulence to host fungi, while others do not. This is a summary of the current ICTV report on the taxonomy of the Hypoviridae, which is available at www.ictv.global/report/hypoviridae.

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica.

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.

A member of the virus family Narnaviridae from the plant pathogenic oomycete Phytophthora infestans.

A virus that has properties consistent with inclusion in the virus family Narnaviridae was described in Phytophthora infestans, the oomycete that caused the Irish potato famine. The genome of phytophthora infestans RNA virus 4 (PiRV-4) is 2,984 nt with short complementary terminal sequences and a single open reading frame predicted to encode an RNA-dependent RNA polymerase (RdRp) most closely related to saccharomyces cerevisiae narnavirus 20S (ScNV-20S) and ScNV-23S, the members of the genus Narnavirus, family Narnaviridae. This report constitutes the first description of a member of the family Narnaviridae from a host taxon outside of the kingdom Fungi.

Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4.

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)-likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

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.

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 associated publication. 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.

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.

PiRV-2 stimulates sporulation in Phytophthora infestans.

Phytophthora infestans is the causal agent of potato and tomato late blight. This pathogen, which caused the Irish potato famine, is of profound historical significance and still poses a major threat in today's agroecosystems. Research on late blight epidemics usually focuses on pathogen virulence, host resistance, environmental factors and fungicide resistance. In this study, we examined the effect of PiRV-2, an RNA virus harbored by some P. infestans isolates, on its host. Comparing isogenic isolates with or without the virus demonstrated that the virus stimulated sporangia production in P. infestans. Transcriptome analysis suggested that it achieved sporulation stimulation likely through down-regulation of ammonium and amino acid intake in P. infestans. Survey of a limited P. infestans collection found PiRV-2 presence in most strains in the US-8 lineage, a very successful clonal lineage of P. infestans in North America. We suggest that PiRV-2 may affect the ecological fitness of P. infestans and thus could contribute to late blight epidemiology.

A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing.

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection.