Identification of a novel mitovirus in grapevine through high-throughput sequencing.

BACKGROUND: Transcriptome data from a plant sample frequently include numerous reads originating from RNA virus genomes that were concurrently isolated during RNA preparation. These high-throughput sequencing reads from the virus can be assembled to form a new sequence for the plant RNA genome. METHODS AND RESULTS: Here, we identify putative novel mitovirus, grapevine mitovirus 1 (GMV1) through high-throughput sequencing (HTS) of grapevine rootstocks (Vitis spp.), and the identified virus was confirmed using virus-specific primers in RT-PCR assay. The genomic RNA of GMV1 encodes complete open reading frame (ORF) of 2,496 nucleotides (nts) in length. RNA-dependent RNA polymerase (RdRp) encoded by the viral genome contained one RdRp conserved domain. BLASTx analysis of GMV1 genome showed sequence identity of 33.18-56.75% with the existing mitovirus sequences. Phylogenetic analysis based on genome sequences showed that GMV1 clustered in a distinct clade to other mitoviruses. CONCLUSION: Grapevine mitovirus 1 represents a newly discovered species within the Unuamitovirus genus of the Mitoviridae family, targeting fungal mitochondria. While the majority of recognized mitoviruses typically lack a functional RdRp as per the plant mitochondrial genetic code, GMV1 encodes a complete RdRp in accordance with both fungal and plant mitochondrial genetic codes.

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.

Complete genome sequence of a novel mitovirus isolated from the fungus Fusarium equiseti causing potato dry rot.

In this study, a novel mitovirus was isolated from the fungus Fusarium equiseti causing potato dry rot and tentatively designated as "Fusarium equiseti mitovirus 1" (FeMV1). The full-length genome sequence of FeMV1 consists of 2,459 nucleotides with a predicted A + U content of 69.5%. Using the mold mitochondrial genetic code, an open reading frame (ORF) of 725 amino acids (aa) was predicted to encode an RNA-dependent RNA polymerase (RdRp). The RdRp protein contains six conserved motifs, with the highly conserved GDD in motif IV, and the 5'-untranslated region (UTR) and 3'-UTR of FeMV1 have the potential to fold into stem-loop secondary structures and a panhandle structure, both of which are typical characteristics of members of the family Mitoviridae. Results of a BLASTp search showed that the RdRp aa sequence of FeMV1 shared the highest sequence similarity with that of Fusarium poae mitovirus 2 (FpMV2) (76.84% identity, E-value = 0.0). Phylogenetic analysis based on the complete aa sequence of RdRp further suggested that FeMV1 is a new member of the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus associated with F. equiseti.

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa.

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

Biological and Molecular Characterization of Chenopodium quinoa Mitovirus 1 Reveals a Distinct Small RNA Response Compared to Those of Cytoplasmic RNA Viruses.

Indirect evidence of mitochondrial viruses in plants comes from discovery of genomic fragments integrated into the nuclear and mitochondrial DNA of a number of plant species. Here, we report the existence of replicating mitochondrial virus in plants: from transcriptome sequencing (RNA-seq) data of infected Chenopodium quinoa, a plant species commonly used as a test plant in virus host range experiments, among other virus contigs, we could assemble a 2.7-kb contig that had highest similarity to mitoviruses found in plant genomes. Northern blot analyses confirmed the existence of plus- and minus-strand RNA corresponding to the mitovirus genome. No DNA corresponding to the genomic RNA was detected, excluding the endogenization of such virus. We have tested a number of C. quinoa accessions, and the virus was present in a number of commercial varieties but absent from a large collection of Bolivian and Peruvian accessions. The virus could not be transmitted mechanically or by grafting, but it is transmitted vertically through seeds at a 100% rate. Small RNA analysis of a C. quinoa line carrying the mitovirus and infected by alfalfa mosaic virus showed that the typical antiviral silencing response active against cytoplasmic viruses (21- to 22-nucleotide [nt] vsRNA peaks) is not active against CqMV1, since in this specific case the longest accumulating vsRNA length is 16 nt, which is the same as that corresponding to RNA from mitochondrial genes. This is evidence of a distinct viral RNA degradation mechanism active inside mitochondria that also may have an antiviral effect.IMPORTANCE This paper reports the first biological characterization of a bona fide plant mitovirus in an important crop, Chenopodium quinoa, providing data supporting that mitoviruses have the typical features of cryptic (persistent) plant viruses. We, for the first time, demonstrate that plant mitoviruses are associated with mitochondria in plants. In contrast to fungal mitoviruses, plant mitoviruses are not substantially affected by the antiviral silencing pathway, and the most abundant mitovirus small RNA length is 16 nt.

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.

Novel Divavirus (the family Betaflexiviridae) and Mitovirus (the family Narnaviridae) species identified in basil (Ocimum basilicum).

Transcriptome data obtained from a plant sample often contain a large number of reads that are derived from associated RNA virus genomes that were co-isolated during RNA preparation. These virus-derived reads can be assembled into a novel plant RNA genome sequence. Here, a basil (Ocimum basilicum) transcriptome dataset was analyzed to identify two new RNA viruses, which were named Ocimum basilicum RNA virus 1 (ObRV1) and Ocimum basilicum RNA virus 2 (ObRV2). A phylogenetic analysis of the ObRV1 RNA-dependent RNA polymerase (RdRp) motif indicated that ObRV1 is a novel species of the genus Divavirus of the family Betaflexiviridae. ObRV1 is the fourth divavirus species to be identified. The ObRV2 RdRp motif showed sequence similarity to viruses of the genus Mitovirus of the family Narnaviridae, which infect fungal mitochondria. Although most of the known mitoviruses do not produce a functional RdRp using the plant mitochondrial genetic code, the ObRV2 encodes a full-length RdRp using both the fungal and plant mitochondrial genetic codes.

Co-infection of a hypovirulent isolate of Sclerotinia sclerotiorum with a new botybirnavirus and a strain of a mitovirus.

BACKGROUND: Sclerotinia sclerotiorum, a notorious plant fungal pathogen, causes yield loss of many crops and vegetables, and is a natural host of a diverse viruses with positive-sense RNA (+ssRNA), negative-sense RNA (-ssRNA), double-stranded RNA (dsRNA), or DNA genomes. Mixed-infection with multiple related or unrelated mycoviruses is a common phenomenon in S. sclerotiorum. However, a single strain co-infected with dsRNA and + ssRNA viruses has not been reported in S. sclerotiorum. RESULTS: We report two unrelated viruses, Sclerotinia sclerotiorum botybirnavirus 2 (SsBRV2) with a bipartite dsRNA genome and Sclerotinia sclerotiorum mitovirus 4 (SsMV4/AH16) with a + ssRNA genome, which were originally detected in a single hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 has a typical genome of mitovirus and is a strain of mitovirus SsMV4. The genome of SsBRV2 consists of two separated dsRNA segments. The large dsRNA segment is 6159 bp in length and only has a single open reading frame (ORF) encoding a putative 1868-aa polyprotein with a conserved RNA dependent RNA polymerase (RdRp) domain. The small dsRNA segment is 5872 bp in length and encodes a putative 1778-aa protein. Phylogenetic analysis using RdRp conserved domain sequences revealed that SsBRV2 is phylogenetically related to the previously reported three bipartite viruses SsBRV1, Botrytis porri RNA virus 1 (BpRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1). Electron microscopy demonstrated that SsBRV2 forms rigid spherical virions with a diameter of approximately 40 nm in infected mycelia. The virion of SsBRV2 was successfully introduced into a virus-free strain, which provides conclusive evidence that SsBRV2 confers hypovirulence on phytopathogenic fungus S. sclerotiorum. CONCLUSIONS: A bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented + ssRNA virus (SsMV4/AH16) were characterized in a hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 is a strain of a reported mitovirus, whereas SsBRV2 is a new botybirnavirus. SsBRV2 is the causal agent of hypovirulence on S. sclerotiorum. Our findings supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and + ssRNA mycoviruses.

Characterization of Two Novel Single-Stranded RNA Viruses from Agroathelia rolfsii, the Causal Agent of Peanut Stem Rot.

Peanut stem rot is a soil-borne disease caused by Agroathelia rolfsii. It occurs widely and seriously affects the peanut yield in most peanut-producing areas. The mycoviruses that induce the hypovirulence of some plant pathogenic fungi are potential resources for the biological control of fungal diseases in plants. Thus far, few mycoviruses have been found in A. rolfsii. In this study, two mitoviruses, namely, Agroathelia rolfsii mitovirus 1 (ArMV1) and Agroathelia rolfsii mitovirus 2 (ArMV2), were identified from the weakly virulent A. rolfsii strain GP3-1, and they were also found in other A. rolfsii isolates. High amounts of ArMV1 and ArMV2in the mycelium could reduce the virulence of A. rolfsii strains. This is the first report on the existence of mitoviruses in A. rolfsii. The results of this study may provide insights into the classification and evolution of mitoviruses in A. rolfsii and enable the exploration of the use of mycoviruses as biocontrol agents for the control of peanut stem rot.

First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

Corrigendum: Characterization of a novel mitovirus infecting Melanconiella theae isolated from tea plants.

[This corrects the article DOI: 10.3389/fmicb.2021.757556.].

A Novel Mitovirus PsMV2 Facilitates the Virulence of Wheat Stripe Rust Fungus.

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as "Puccinia striiformis mitovirus 2" (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.

Metatranscriptomic analysis uncovers prevalent viral ORFs compatible with mitochondrial translation.

RNA viruses are ubiquitous components of the global virosphere, yet relatively little is known about their genetic diversity or the cellular mechanisms by which they exploit the biology of their diverse eukaryotic hosts. A hallmark of (+)ssRNA (positive single-stranded RNA) viruses is the ability to remodel host endomembranes for their own replication. However, the subcellular interplay between RNA viruses and host organelles that harbor gene expression systems, such as mitochondria, is complex and poorly understood. Here we report the discovery of 763 new virus sequences belonging to the family Mitoviridae by metatranscriptomic analysis, the identification of previously uncharacterized mitovirus clades, and a putative new viral class. With this expanded understanding of the diversity of mitovirus and encoded RNA-dependent RNA polymerases (RdRps), we annotate mitovirus-specific protein motifs and identify hallmarks of mitochondrial translation, including mitochondrion-specific codons. This study expands the known diversity of mitochondrial viruses and provides additional evidence that they co-opt mitochondrial biology for their survival. IMPORTANCE Metatranscriptomic studies have rapidly expanded the cadre of known RNA viruses, yet our understanding of how these viruses navigate the cytoplasmic milieu of their hosts to survive remains poorly characterized. In this study, we identify and assemble 763 new viral sequences belonging to the Mitoviridae, a family of (+)ssRNA viruses thought to interact with and remodel host mitochondria. We exploit this genetic diversity to identify new clades of Mitoviridae, annotate clade-specific sequence motifs that distinguish the mitoviral RdRp, and reveal patterns of RdRp codon usage consistent with translation on host cell mitoribosomes. These results serve as a foundation for understanding how mitoviruses co-opt mitochondrial biology for their proliferation.

Expanding the knowledge frontier of mitoviruses in Cannabis sativa.

Mitoviruses were initially known for their presence in the mitochondria of fungi and were considered exclusive to these organisms. However, recent studies have shown that they are also present in a large number of plant species. Despite the potential impact that mitoviruses might have on the mitochondria of plant cells, there is a lack of information about these ancient RNA viruses, especially within the Cannabaceae family. Cannabis sativa has been in the spotlight in recent years due to the growing industrial applications of plant derivatives, such as fiber and secondary metabolites. Given the importance of Cannabis in today's agriculture, our study aimed to expand the knowledge frontier of Mitoviruses in C. sativa by increasing the number of reference genomes of CasaMV1 available in public databases and representing a larger number of crops in countries where its industrial-scale growth is legalized. To achieve this goal, we used transcriptomics to sequence the first mitoviral genomes of Colombian crops and analyzed RNA-seq datasets available in the SRA databank. Additionally, the evolutionary analysis performed using the mitovirus genomes revealed two main lineages of CasaMV1, termed CasaMV1_L1 and CasaMV1_L2. These mitoviral lineages showed strong clustering based on the geographic location of the crops and differential expression intensities.

Four Novel Mycoviruses from the Hypovirulent Botrytis cinerea SZ-2-3y Isolate from Paris polyphylla: Molecular Characterisation and Mitoviral Sequence Transboundary Entry into Plants.

A hypovirulent SZ-2-3y strain isolated from diseased Paris polyphylla was identified as Botrytis cinerea. Interestingly, SZ-2-3y was coinfected with a mitovirus, two botouliviruses, and a 3074 nt fusarivirus, designated Botrytis cinerea fusarivirus 8 (BcFV8); it shares an 87.2% sequence identity with the previously identified Botrytis cinerea fusarivirus 6 (BcFV6). The full-length 2945 nt genome sequence of the mitovirus, termed Botrytis cinerea mitovirus 10 (BcMV10), shares a 54% sequence identity with Fusarium boothii mitovirus 1 (FbMV1), and clusters with fungus mitoviruses, plant mitoviruses and plant mitochondria; hence BcMV10 is a new Mitoviridae member. The full-length 2759 nt and 2812 nt genome sequences of the other two botouliviruses, named Botrytis cinerea botoulivirus 18 and 19 (BcBoV18 and 19), share a 40% amino acid sequence identity with RNA-dependent RNA polymerase protein (RdRp), and these are new members of the Botoulivirus genus of Botourmiaviridae. Horizontal transmission analysis showed that BcBoV18, BcBoV19 and BcFV8 are not related to hypovirulence, suggesting that BcMV10 may induce hypovirulence. Intriguingly, a partial BcMV10 sequence was detected in cucumber plants inoculated with SZ-2-3y mycelium or pXT1/BcMV10 agrobacterium. In conclusion, we identified a hypovirulent SZ-2-3y fungal strain from P. polyphylla, coinfected with four novel mycoviruses that could serve as potential biocontrol agents. Our findings provide evidence of cross-kingdom mycoviral sequence transmission.

Novel RNA viruses from the native range of Hymenoscyphus fraxineus, the causal fungal agent of ash dieback.

The native Japanese population of the fungus Hymenoscyphus fraxineus, the causal agent of ash dieback in Europe, was screened for viruses using a high-throughput sequencing method. Five RNA viruses were detected in 116 fungal isolates sequenced via Illumina RNA-seq platform, with an overall virus prevalence of 11.2%. The viruses were completely sequenced by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) followed by Sanger sequencing. The sequences appear to represent new species from three established families (Mito-, Endorna- and Partitiviridae), one recognized genus (Botybirnavirus) and a negative-sense single-stranded RNA virus in the order Bunyavirales from the proposed family "Mybuviridae". The highest prevalence was found for the mitovirus (7.8%), that had two genomic forms (linear and circular), while the other viruses were detected each in one isolate. Co-infection of a mitovirus and an endornavirus was also observed in one of the infected isolates. Here we describe the molecular characterization of the identified viruses. This study expands the diversity of viruses in H. fraxineus and provides the basis for investigating the virus-mediated control of ash dieback in Europe.

The Mycovirome in a Worldwide Collection of the Brown Rot Fungus Monilinia fructicola.

The fungus Monilinia fructicola is responsible for brown rot on stone and pome fruit and causes heavy yield losses both pre- and post-harvest. Several mycoviruses are known to infect fungal plant pathogens. In this study, a metagenomic approach was applied to obtain a comprehensive characterization of the mycovirome in a worldwide collection of 58 M. fructicola strains. Deep sequencing of double-stranded (ds)RNA extracts revealed a great abundance and variety of mycoviruses. A total of 32 phylogenetically distinct positive-sense (+) single-stranded (ss)RNA viruses were identified. They included twelve mitoviruses, one in the proposed family Splipalmiviridae, and twelve botourmiaviruses (phylum Lenarviricota), eleven of which were novel viral species; two hypoviruses, three in the proposed family Fusariviridae, and one barnavirus (phylum Pisuviricota); as well as one novel beny-like virus (phylum Kitrinoviricota), the first one identified in Ascomycetes. A partial sequence of a new putative ssDNA mycovirus related to viruses within the Parvoviridae family was detected in a M. fructicola isolate from Serbia. The availability of genomic sequences of mycoviruses will serve as a solid basis for further research aimed at deepening the knowledge on virus-host and virus-virus interactions and to explore their potential as biocontrol agents against brown rot disease.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

Characterization of a Novel Mitovirus Infecting Melanconiella theae Isolated From Tea Plants.

A dsRNA segment was identified in the fungus Melanconiella theae isolated from tea plants. The complete dsRNA sequence, determined by random cloning together with RACE protocol, is 2,461 bp in length with an AU-rich content (62.37%) and comprises a single ORF of 2,265-nucleotides encoding an RNA-dependent RNA-polymerase (RdRp, 754 amino acids in size). The terminus sequences can fold into predicted stable stem-loop structures. A BLASTX and phylogenetic analysis revealed the dsRNA genome shows similarities with the RdRp sequences of mitoviruses, with the highest identity of 48% with those of grapevine-associated mitovirus 20 and Colletotrichum fructicola mitovirus 1. Our results reveal a novel member, tentatively named Melanconiella theae mitovirus 1 (MtMV1), belongs to the family Mitoviridae. MtMV1 is capsidless as examined by transmission electron microscope, efficiently transmitted through conidia as 100 conidium-generated colonies were analyzed, and easily eliminated by hyphal tipping method combined with green-leaf tea powder. MtMV1 has a genomic sequence obviously divergent from those of most members in the family Mitoviridae and some unique characteristics unreported in known members. This is the first report of a mycovirus infecting Melanconiella fungi to date.