Molecular characterization of a single-negative-stranded RNA virus from the rice blast fungus Magnaporthe oryzae isolate NJ39.

A novel negative-sense single-stranded RNA mycovirus, designated as "Magnaporthe oryzae mymonavirus 1" (MoMNV1), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ39. MoMNV1 has a single genomic RNA segment consisting of 10,515 nucleotides, which contains six open reading frames. The largest open reading frame contains 5837 bases and encodes an RNA replicase. The six open reading frames have no overlap and are arranged linearly on the genome, but the spacing of the genes is small, with a maximum of 315 bases and a minimum of 80 bases. Genome comparison and phylogenetic analysis indicated that MoMNV1 is a new member of the genus Penicillimonavirus of the family Mymonaviridae.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Fusarium oxysporum f. sp. melonis strain T-SD3.

A novel mitovirus was identified in Fusarium oxysporum f. sp. melonis strain T-SD3 and designated as "Fusarium oxysporum mitovirus 3" (FoMV3). The virus was isolated from diseased muskmelon plants with the typical symptom of fusarium wilt. The complete genome of FoMV3 is 2269 nt in length with a predicted AU content of 61.40% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a polypeptide of 679 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain with a molecular mass of 77.39 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5'-untranslated region (UTR) and 3'-UTR of FoMV3 were predicted to fold into stem-loop structures. BLASTp analysis revealed that the RdRp of FoMV3 shared the highest aa sequence identity (83.85%) with that of Fusarium asiaticum mitovirus 5 (FaMV5, a member of the family Mitoviridae) infecting F. asiaticum, the causal agent of wheat fusarium head blight. Phylogenetic analysis further suggested that FoMV3 is a new member of the genus Unuamitovirus within the family Mitoviridae. This is the first report of a new mitovirus associated with F. oxysporum f. sp. melonis.

Discovery of novel RNA viruses through analysis of fungi-associated next-generation sequencing data.

BACKGROUND: Like all other species, fungi are susceptible to infection by viruses. The diversity of fungal viruses has been rapidly expanding in recent years due to the availability of advanced sequencing technologies. However, compared to other virome studies, the research on fungi-associated viruses remains limited. RESULTS: In this study, we downloaded and analyzed over 200 public datasets from approximately 40 different Bioprojects to explore potential fungal-associated viral dark matter. A total of 12 novel viral sequences were identified, all of which are RNA viruses, with lengths ranging from 1,769 to 9,516 nucleotides. The amino acid sequence identity of all these viruses with any known virus is below 70%. Through phylogenetic analysis, these RNA viruses were classified into different orders or families, such as Mitoviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Mymonaviridae, Bunyavirales, and Partitiviridae. It is possible that these sequences represent new taxa at the level of family, genus, or species. Furthermore, a co-evolution analysis indicated that the evolutionary history of these viruses within their groups is largely driven by cross-species transmission events. CONCLUSIONS: These findings are of significant importance for understanding the diversity, evolution, and relationships between genome structure and function of fungal viruses. However, further investigation is needed to study their interactions.

Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Comparative Analysis of Viromes Identified in Multiple Macrofungi.

Macrofungi play important roles in the soil elemental cycle of terrestrial ecosystems. Fungal viruses are common in filamentous fungi, and some of them can affect the growth and development of hosts. However, the composition and evolution of macrofungal viruses are understudied. In this study, ninety strains of Trametes versicolor, Coprinellus micaceus, Amanita strobiliformis, and Trametes hirsuta were collected in China. Four mixed pools were generated by combining equal quantities of total RNA from each strain, according to the fungal species, and then subjected to RNA sequencing. The sequences were assembled, annotated, and then used for phylogenetic analysis. Twenty novel viruses or viral fragments were characterized from the four species of macrofungi. Based on the phylogenetic analysis, most of the viral contigs were classified into ten viral families or orders: Barnaviridae, Benyviridae, Botourmiaviridae, Deltaflexiviridae, Fusariviridae, Hypoviridae, Totiviridae, Mitoviridae, Mymonaviridae, and Bunyavirales. Of these, ambi-like viruses with circular genomes were widely distributed among the studied species. Furthermore, the number and overall abundance of viruses in these four species of macrofungi (Basidiomycota) were found to be much lower than those in broad-host phytopathogenic fungi (Ascomycota: Sclerotinia sclerotiorum, and Botrytis cinerea). By employing metatranscriptomic analysis in this study, for the first time, we demonstrated the presence of multiple mycoviruses in Amanita strobiliformis, Coprinellus micaceus, Trametes hirsute, and Trametes versicolor, significantly contributing to research on mycoviruses in macrofungi.

Fusarium sacchari hypovirus 1, a Member of Hypoviridae with Virulence Attenuation Capacity in Phytopathogenic Fusarium Species.

In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.

Characterization of Mycoviruses in Armillaria ostoyae and A. cepistipes in the Czech Republic.

Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described "ambivirus" group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.

Virome analysis of an ectomycorrhizal fungus Suillus luteus revealing potential evolutionary implications.

Suillus luteus is a widespread edible ectomycorrhizal fungus that holds significant importance in both ecological and economic value. Mycoviruses are ubiquitous infectious agents hosted in different fungi, with some known to exert beneficial or detrimental effects on their hosts. However, mycoviruses hosted in ectomycorrhizal fungi remain poorly studied. To address this gap in knowledge, we employed next-generation sequencing (NGS) to investigate the virome of S. luteus. Using BLASTp analysis and phylogenetic tree construction, we identified 33 mycovirus species, with over half of them belonging to the phylum Lenarviricota, and 29 of these viruses were novel. These mycoviruses were further grouped into 11 lineages, with the discovery of a new negative-sense single-stranded RNA viral family in the order Bunyavirales. In addition, our findings suggest the occurrence of cross-species transmission (CST) between the fungus and ticks, shedding light on potential evolutionary events that have shaped the viral community in different hosts. This study is not only the first study to characterize mycoviruses in S. luteus but highlights the enormous diversity of mycoviruses and their implications for virus evolution.

Mycovirus Hunting Revealed the Presence of Diverse Viruses in a Single Isolate of the Phytopathogenic Fungus Diplodia seriata From Pakistan.

Diplodia seriata in the family Botryosphaeriaceae is a cosmopolitan phytopathogenic fungus and is responsible for causing cankers, fruit rot and leaf spots on economically important plants. In this study, we characterized the virome of a single Pakistani strain (L3) of D. seriata. Several viral-like contig sequences were obtained via a previously conducted next-generation sequencing analysis. Multiple infection of the L3 strain by eight RNA mycoviruses was confirmed through RT-PCR using total RNA samples extracted from this strain; the entire genomes were determined via Sanger sequencing of RT-PCR and RACE clones. A BLAST search and phylogenetic analyses indicated that these eight mycoviruses belong to seven different viral families. Four identified mycoviruses belong to double-stranded RNA viral families, including Polymycoviridae, Chrysoviridae, Totiviridae and Partitiviridae, and the remaining four identified mycoviruses belong to single-stranded RNA viral families, i.e., Botourmiaviridae, and two previously proposed families "Ambiguiviridae" and "Splipalmiviridae". Of the eight, five mycoviruses appear to represent new virus species. A morphological comparison of L3 and partially cured strain L3ht1 suggested that one or more of the three viruses belonging to Polymycoviridae, "Splipalmiviridae" and "Ambiguiviridae" are involved in the irregular colony phenotype of L3. To our knowledge, this is the first report of diverse virome characterization from D. seriata.

A Capsidless Virus Is trans-Encapsidated by a Bisegmented Botybirnavirus.

RNA viruses usually have linear genomes and are encapsidated by their own capsids. Here, we newly identified four mycoviruses and two previously reported mycoviruses (a fungal reovirus and a botybirnavirus) in the hypovirulent strain SCH941 of Sclerotinia sclerotiorum. One of the newly discovered mycoviruses, Sclerotinia sclerotiorum yadokarivirus 1 (SsYkV1), with a nonsegmented positive-sense single-stranded RNA (+ssRNA) genome, was molecularly characterized. SsYkV1 is 5,256 nucleotides (nt) in length, excluding the poly(A) structure, and has a large open reading frame that putatively encodes a polyprotein with the RNA-dependent RNA polymerase (RdRp) domain and a 2A-like motif. SsYkV1 was phylogenetically positioned into the family Yadokariviridae and was most closely related to Rosellinia necatrix yadokarivirus 2 (RnYkV2), with 40.55% identity (78% coverage). Although SsYkV1 does not encode its own capsid protein, the RNA and RdRp of SsYkV1 are trans-encapsidated in virions of Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3), a bisegmented double-stranded RNA (dsRNA) mycovirus within the genus Botybirnavirus. In this way, SsYkV1 likely replicates inside the heterocapsid comprised of the SsBV3 capsid protein, like a dsRNA virus. SsYkV1 has a limited impact on the biological features of S. sclerotiorum. This study represents an example of a yadokarivirus trans-encapsidated by an unrelated dsRNA virus, which greatly deepens our knowledge and understanding of the unique life cycles of RNA viruses. IMPORTANCE RNA viruses typically encase their linear genomes in their own capsids. However, a capsidless +ssRNA virus (RnYkV1) highjacks the capsid of a nonsegmented dsRNA virus for the trans-encapsidation of its own RNA and RdRp. RnYkV1 belongs to the family Yadokariviridae, which already contains more than a dozen mycoviruses. However, it is unknown whether other yadokariviruses except RnYkV1 are also hosted by a heterocapsid, although dsRNA viruses with capsid proteins were detected in fungi harboring yadokarivirus. It is noteworthy that almost all presumed partner dsRNA viruses of yadokariviruses belong to the order Ghabrivirales (most probably a totivirus or toti-like virus). Here, we found a capsidless +ssRNA mycovirus, SsYkV1, from hypovirulent strain SCH941 of S. sclerotiorum, and the RNA and RdRp of this mycovirus are trans-encapsidated in virions of a bisegmented dsRNA virus within the free-floating genus Botybirnavirus. Our results greatly expand our knowledge of the unique life cycles of RNA viruses.

A Novel Heptasegmented Positive-Sense Single-Stranded RNA Virus from the Phytopathogenic Fungus Colletotrichum fructicola.

In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1-3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

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.

Metagenomics Analysis of the Wheat Virome Identifies Novel Plant and Fungal-Associated Viral Sequences.

Wheat viruses including wheat streak mosaic virus, Triticum mosaic virus, and barley yellow dwarf virus cost substantial losses in crop yields every year. Although there have been extensive studies conducted on these known wheat viruses, currently, there is limited knowledge about all components of the wheat (Triticum aestivum L.) virome. Here, we determined the composition of the wheat virome through total RNA deep sequencing of field-collected leaf samples. Sequences were de novo assembled after removing the host reads, and BLASTx searches were conducted. In addition to the documented wheat viruses, novel plant and fungal-associated viral sequences were identified. We obtained the full genome sequence of the first umbra-like associated RNA virus tentatively named wheat umbra-like virus in cereals. Moreover, a novel bi-segmented putative virus tentatively named wheat-associated vipovirus sharing low but significant similarity with both plant and fungal-associated viruses was identified. Additionally, a new putative fungal-associated tobamo-like virus and novel putative Mitovirus were discovered in wheat samples. The discovery and characterization of novel viral sequences associated with wheat is important to determine if these putative viruses may pose a threat to the wheat industry or have the potential to be used as new biological control agents for wheat pathogens either as wild-type or recombinant viruses.

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.

Genome Organizations and Functional Analyses of a Novel Gammapartitivirus from Rhizoctonia solani AG-1 IA Strain D122.

Here, we describe a novel double-stranded (ds) RNA mycovirus designated Rhizoctonia solani dsRNA virus 5 (RsRV5) from strain D122 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. The RsRV5 genome consists of two segments of dsRNA (dsRNA-1, 1894 bp and dsRNA-2, 1755 bp), each possessing a single open reading frame (ORF). Sequence alignments and phylogenetic analyses showed that RsRV5 is a new member of the genus Gammapartitivirus in the family Partitiviridae. Transmission electron microscope (TEM) images revealed that RsRV5 has isometric viral particles with a diameter of approximately 20 nm. The mycovirus RsRV5 was successfully removed from strain D122 by using the protoplast regeneration technique, thus resulting in derivative isogenic RsRV5-cured strain D122-P being obtained. RsRV5-cured strain D122-P possessed the traits of accelerated mycelial growth rate, increased sclerotia production and enhanced pathogenicity to rice leaves compared with wild type RsRV5-infection strain D122. Transcriptome analysis showed that three genes were differentially expressed between two isogenic strains, D122 and D122-P. These findings provided new insights into the molecular mechanism of the interaction between RsRV5 and its host, D122 of R. solani AG-1 IA.

Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand.

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

Characterization of the Mycovirome from the Plant-Pathogenic Fungus Cercospora beticola.

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (-)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.

Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum.

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family "Alternaviridae". Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family "Alternaviridae" members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1-and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family "Alternaviridae". Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family "Alternaviridae", and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.