Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses.

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Dicer functions transcriptionally and posttranscriptionally in a multilayer antiviral defense.

In antiviral RNA interference (RNAi), Dicer plays a primary role in processing double-stranded RNA (dsRNA) molecules into small-interfering RNAs (siRNAs) that guide Argonaute effectors to posttranscriptional suppression of target viral genes. Here, we show a distinct role for Dicer in the siRNA-independent transcriptional induction of certain host genes upon viral infection in a filamentous fungus. Previous studies have shown that the two key players, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), of antiviral RNAi in a phytopathogenic ascomycete, Cryphonectria parasitica, are highly transcriptionally induced upon infection with certain RNA mycoviruses, including the positive-stranded RNA hypovirus mutant lacking the RNAi suppressor (Cryphonectria hypovirus 1-Δp69, CHV1-Δp69). This induction is regulated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, a well-known transcriptional coactivator. The present study shows that diverse host genes, in addition to dcl2 and agl2, were up-regulated more than 10-fold by SAGA upon infection with CHV1-Δp69. Interestingly, DCL2, but not AGL2, was essential for SAGA-mediated global gene up-regulation. Moreover, deletion of certain virus-induced genes enhanced a CHV1-Δp69 symptom (growth rate) but not its accumulation. Constitutive, modest levels of dcl2 expression drastically reduced viral siRNA accumulation but were sufficient for full-scale up-regulation of host genes, suggesting that high induction of dcl2 and siRNA production are not essential for the transcriptional up-regulation function of DCL2. These data clearly demonstrate the dual functionality of DCL2: as a dsRNA-specific nuclease in posttranscriptional antiviral RNA silencing and as a key player in SAGA-mediated host gene induction, which independently represses viral replication and alleviates virus-induced symptom expression.

Viruses shuttle between fungi and plants.

The intimate relationships between plants and fungi provide an opportunity for the shuttling of viruses. Dai et al. recently discovered that a virus undergoes cross-kingdom transmission, and naturally spreads to both plant and fungal populations. This finding expands our understanding of viral host range, evolution, transmission, and disease management.

Cross-Kingdom Interactions Between Plant and Fungal Viruses.

The large genetic and structural divergences between plants and fungi may hinder the transmission of viruses between these two kingdoms to some extent. However, recent accumulating evidence from virus phylogenetic analyses and the discovery of naturally occurring virus cross-infection suggest the occurrence of past and current transmissions of viruses between plants and plant-associated fungi. Moreover, artificial virus inoculation experiments showed that diverse plant viruses can multiply in fungi and vice versa. Thus, virus cross-infection between plants and fungi may play an important role in the spread, emergence, and evolution of both plant and fungal viruses and facilitate the interaction between them. In this review, we summarize current knowledge related to cross-kingdom virus infection in plants and fungi and further discuss the relevance of this new virological topic in the context of understanding virus spread and transmission in nature as well as developing control strategies for crop plant diseases.

A Transfectable Fusagravirus from a Japanese Strain of Cryphonectria carpinicola with Spherical Particles.

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5'-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.

A new tetra-segmented splipalmivirus with divided RdRP domains from Cryphonectria naterciae, a fungus found on chestnut and cork oak trees in Europe.

Positive-sense (+), single-stranded (ss) RNA viruses with divided RNA-dependent RNA polymerase (RdRP) domains have been reported from diverse filamentous ascomycetes since 2020. These viruses are termed splipalmiviruses or polynarnaviruses and have been characterized largely at the sequence level, but ill-defined biologically. Cryphonectria naterciae, from which only one virus has been reported, is an ascomycetous fungus potentially plant-pathogenic to chestnut and oak trees. We molecularly characterized multiple viruses in a single Portuguese isolate (C0614) of C. naterciae, taking a metatranscriptomic and conventional double-stranded RNA approach. Among them are a novel splipalmivirus (Cryphonectria naterciae splipalmivirus 1, CnSpV1) and a novel fusagravirus (Cryphonectria naterciae fusagravirus 1, CnFGV1). This study focused on the former virus. CnSpV1 has a tetra-segmented, (+)ssRNA genome (RNA1 to RNA4). As observed for other splipalmiviruses reported in 2020 and 2021, the RdRP domain is separately encoded by RNA1 (motifs F, A and B) and RNA2 (motifs C and D). A hypothetical protein encoded by the 5'-proximal open reading frame of RNA3 shows similarity to a counterpart conserved in some splipalmiviruses. The other RNA3-encoded protein and RNA4-encoded protein show no similarity with known proteins in a blastp search. The tetra-segment nature was confirmed by the conserved terminal sequences of the four CnSpV1 segments (RNA1 to RNA4) and their 100% coexistence in over 100 single conidial isolates tested. The experimental introduction of CnSpV1 along with CnFGV1 into a virus free strain C0754 of C. naterciae vegetatively incompatible with C0614 resulted in no phenotypic alteration, suggesting asymptomatic infection. The protoplast fusion assay indicates a considerably narrow host range of CnSpV1, restricted to the species C. naterciae and C. carpinicola. This study contributes to better understanding of the molecular and biological properties of this unique group of viruses.

Fungal Virus, FgHV1-Encoded p20 Suppresses RNA Silencing through Single-Strand Small RNA Binding.

Fungal viruses are widespread in fungi infecting plants, insects and animals. High-throughput sequencing has rapidly led to the discovery of fungal viruses. However, the interactive exploration between fungi and viruses is relatively limited. RNA silencing is the fundamental antivirus pathway in fungi. Fusarium graminearum small RNA (sRNA) pattern was regulated by Fusarium graminearum hypovirus 1 (FgHV1) infection, indicating the activation of RNA silencing in virus defense. In this study, we focused on the function of an uncharacterized protein sized at 20 kD (p20) encoded by FgHV1. In the agro-infiltration assay, p20 was identified as a novel fungal RNA silencing suppressor. p20 can block systemic RNA silencing signals besides local RNA silencing suppression. We further elucidated the RNA silencing suppression mechanism of p20. The single-strand sRNA, instead of double-strand sRNA, can be incorporated by p20 in electrophoretic mobility shift assay. p20 binds sRNA originating from virus and non-virus sources in a non-sequence-specific manner. In addition, The F. graminearum 22 and 23-nt sRNA abundance and pathways related to RNA processing and redox regulation were regulated by p20. Our study revealed the first fungal virus-encoded RNA silencing suppressor with sRNA binding capability.

Virus Infection of Aspergillus fumigatus Compromises the Fungus in Intermicrobial Competition.

Aspergillus and Pseudomonas compete in nature, and are the commonest bacterial and fungal pathogens in some clinical settings, such as the cystic fibrosis lung. Virus infections of fungi occur naturally. Effects on fungal physiology need delineation. A common reference Aspergillus fumigatus strain, long studied in two (of many) laboratories, was found infected with the AfuPmV-1 virus. One isolate was cured of virus, producing a virus-free strain. Virus from the infected strain was purified and used to re-infect three subcultures of the virus-free fungus, producing six fungal strains, otherwise isogenic. They were studied in intermicrobial competition with Pseudomonasaeruginosa. Pseudomonas culture filtrates inhibited forming or preformed Aspergillus biofilm from infected strains to a greater extent, also seen when Pseudomonas volatiles were assayed on Aspergillus. Purified iron-chelating Pseudomonas molecules, known inhibitors of Aspergillus biofilm, reproduced these differences. Iron, a stimulus of Aspergillus, enhanced the virus-free fungus, compared to infected. All infected fungal strains behaved similarly in assays. We show an important consequence of virus infection, a weakening in intermicrobial competition. Viral infection may affect the outcome of bacterial-fungal competition in nature and patients. We suggest that this occurs via alteration in fungal stress responses, the mechanism best delineated here is a result of virus-induced altered Aspergillus iron metabolism.

Novel and diverse mycoviruses co-inhabiting the hypogeous ectomycorrhizal fungus Picoa juniperi.

Viruses hosted by ectomycorrhizal fungi remain poorly studied. In this study, we detected eight new fungal viruses co-infecting a single isolate of the hypogeous ectomycorrhizal fungus Picoa juniperi using high-throughput sequencing. Phylogenetic analysis of one identified virus abbreviated as PjMTV1 revealed its closest relatives as members of the newly proposed family "Megatotiviridae". Phylogenetic analyses of two identified viruses abbreviated as PjV1 and PjV2 showed that these viruses are associated with members of the proposed family "Fusagraviridae". Phylogenetic analysis of the identified one another virus abbreviated as PjYV1 demonstrated that this virus is related to the members of the proposed family Yadokariviridae. The remaining four identified virus-like contigs were determined as segments of the bipartite dsRNA mycoviruses from the family Partitiviridae. The mycoviruses reported in this study are the first viruses described in Picoa juniperi, and PjMTV1 characterized herein is the secondly reported member of the newly proposed family "Megatotiviridae".

Cryphonectria nitschkei chrysovirus 1 with unique molecular features and a very narrow host range.

Cryphonectria nitschkei chrysovirus 1 (CnCV1), was described earlier from an ascomycetous fungus, Cryphonectria nitschkei strain OB5/11, collected in Japan; its partial sequence was reported a decade ago. Complete sequencing of the four genomic dsRNA segments revealed molecular features similar to but distinct from previously reported members of the family Chrysoviridae. Unique features include the presence of a mini-cistron preceding the major large open reading frame in each genomic segment. Common features include the presence of CAA repeats in the 5'-untranslated regions and conserved terminal sequences. CnCV1-OB5/11 could be laterally transferred to C. nitschkei and its relatives C. radicalis and C. naterciae via coculturing, virion transfection and protoplast fusion, but not to fungal species other than the three species mentioned above, even within the genus Cryphonectria, suggesting a very narrow host range. Phenotypic comparison of a few sets of CnCV1-infected and -free isogenic strains showed symptomless infection in new hosts.

Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum.

The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms.IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.

The True Host/s of Picobirnaviruses.

Picobirnaviruses (PBVs) are bisegmented double-stranded RNA viruses that have been detected in a wide variety of animal species including invertebrates and in environmental samples. Since PBVs are ubiquitous in feces/gut contents of humans and other animals with or without diarrhea, they were considered as opportunistic enteric pathogens of mammals and avian species. However, the virus remains to be propagated in animal cell cultures, or in gnotobiotic animals. Recently, the classically defined prokaryotic motif, the ribosomal binding site sequence, has been identified upstream of putative open reading frame/s in PBV and PBV-like sequences from humans, various animals, and environmental samples, suggesting that PBVs might be prokaryotic viruses. On the other hand, based on the detection of some novel PBV-like RNA-dependent RNA polymerase sequences that use the alternative mitochondrial genetic code (that of mold or invertebrates) for translation, and principal component analysis of codon usage bias for these sequences, it has been proposed that PBVs might be fungal viruses with a lifestyle reminiscent of mitoviruses. These contradicting observations warrant further studies to ascertain the true host/s of PBVs, which still remains controversial. In this minireview, we have focused on the various findings that have raised a debate on the true host/s of PBVs.

Molecular Characterization of a Novel Polymycovirus From Penicillium janthinellum With a Focus on Its Genome-Associated PASrp.

The genus Polymycovirus of the family Polymycoviridae accommodates fungal RNA viruses with different genomic segment numbers (four, five, or eight). It is suggested that four members form no true capsids and one forms filamentous virus particles enclosing double-stranded RNA (dsRNA). In both cases, viral dsRNA is associated with a viral protein termed "proline-alanine-serine-rich protein" (PASrp). These forms are assumed to be the infectious entity. However, the detailed molecular characteristics of PASrps remain unclear. Here, we identified a novel five-segmented polymycovirus, Penicillium janthinellum polymycovirus 1 (PjPmV1), and characterized its purified fraction form in detail. The PjPmV1 had five dsRNA segments associated with PASrp. Density gradient ultracentrifugation of the PASrp-associated PjPmV1 dsRNA revealed its uneven structure and a broad fractionation profile distinct from that of typical encapsidated viruses. Moreover, PjPmV1-PASrp interacted in vitro with various nucleic acids in a sequence-non-specific manner. These PjPmV1 features are discussed in view of the diversification of genomic segment numbers of the genus Polymycovirus.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

Viruses infecting macrofungi.

Ever since their discovery just about 56 years ago in the cultivated mushroom Agaricus bisporus, many more viruses infecting fungi have been identified in a wide range of fungal taxa. With mostly being asymptomatic, especially the ones that are detrimental to their phytopathogenic hosts are intensively studied due to their considerable importance in developing novel plant protection measures. Contrary to the rapid accumulation of notable data on viruses of plant pathogenic microfungi, much less information have hitherto been obtained in regards to the viruses whose hosts are macrofungi. According to the current literature, only more than 80 distinct viruses bearing either linear dsRNA or linear positive sense ssRNA genome and infecting a total number of 34 macrofungal species represented with four Ascomycota and 30 Basidiomycota have been identified so far. Among these 34 macrofungal species, 14 are cultivated edible and wild edible mushroom species. According to the 10th ICTV (International Committee on Taxonomy of Viruses) Report, macrofungal viruses with linear dsRNA genome are classified into five families (Partitiviridae, Totiviridae, Chrysoviridae, Endornaviridae and Hypoviridae) and macrofungal viruses with linear positive sense ssRNA genome are classified into seven families (Betaflexiviridae, Gammaflexiviridae, Barnaviridae, Narnaviridae, Virgaviridae, Benyviridae and Tymoviridae). In this review, following a brief overview of some general characteristics of fungal viruses, an up to date knowledge on viruses infecting macrofungal hosts were presented by summarizing the previous, recent and prospective studies of the field.

Viruses of Plant-Interacting Fungi.

Plant-associated fungi are infected by viruses at the incidence rates from a few % to over 90%. Multiple viruses often coinfect fungal hosts, and occasionally alter their phenotypes, but most of the infections are asymptomatic. Phenotypic alterations are grouped into two types: harmful or beneficial to the host fungi. Harmful interactions between viruses and hosts include hypovirulence and/or debilitation that are documented in a number of phytopathogenic fungi, exemplified by the chestnut blight, white root rot, and rapeseed rot fungi. Beneficial interactions are observed in a limited number of plant endophytic and pathogenic fungi where heat tolerance and virulence are enhanced, respectively. Coinfections of fungi provided a platform for discoveries of interesting virus/virus interactions that include synergistic, as in the case for those in plants, and unique antagonistic and mutualistic interactions between unrelated RNA viruses. Also discussed here are coinfection-induced genome rearrangements and frequently observed coinfections by the simplest positive-strand RNA virus, the mitoviruses.

Evidence for contemporary plant mitoviruses.

Mitoviruses have small RNA(+) genomes, replicate in mitochondria, and have been shown to infect only fungi to date. For this report, sequences that appear to represent nearly complete plant mitovirus genomes were recovered from publicly available transcriptome data. Twenty of the refined sequences, 2684-2898 nt long and derived from 10 different species of land plants, appear to encompass the complete coding regions of contemporary plant mitoviruses, which furthermore constitute a monophyletic cluster within genus Mitovirus. Complete coding sequences of several of these viruses were recovered from multiple transcriptome (but not genome) studies of the same plant species and also from multiple plant tissues. Crop plants among implicated hosts include beet and hemp. Other new results suggest that such genuine plant mitoviruses were immediate ancestors to endogenized mitovirus elements now widespread in land plant genomes. Whether these mitoviruses are wholly cryptic with regard to plant health remains to be investigated.

Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae.

Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed.

Natural infection of the soil-borne fungus Rosellinia necatrix with novel mycoviruses under greenhouse conditions.

Fungi are an important component of the soil ecosystem. Mycoviruses have numerous potential impacts on soil fungi, including phytopathogenic fungal species. However, the diversity and ecology of mycoviruses in soil fungi is largely unexplored. Our previous work has shown that the soil-borne phytopathogenic fungus Rosellinia necatrix was infected with several novel mycoviruses after growing for 2-3 years in an apple orchard. In this study, we investigated whether natural infection of R. necatrix with mycoviruses occurs under limited conditions. Virus-free R. necatrix isolates were grown in a small bucket containing soil samples for a short time (1.5-4.5 months) under greenhouse conditions. Screening of dsRNA mycoviruses among 365 retrieved isolates showed that four, including 6-31, 6-33, 6-35, and 7-11, harbored virus-like dsRNAs. Molecular characterization of the dsRNAs revealed that three retrieved isolates, 6-31, 6-33, and 6-35 were infected with a novel endornavirus and isolate 7-11 is infected with a novel partitivirus belonging to the genus Alphapartitivirus. These novel mycoviruses had no overt biological impact on R. necatrix. Overall, this study indicates that natural infections of R. necatrix with new mycoviruses can occur under experimental soil conditions.