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In this study, we identified a new mycotombus-like mycovirus from the phytopathogenic fungus Nigrospora oryzae, which was tentatively designated as "Nigrospora oryzae umbra-like virus 1" (NoULV1). The complete genome of NoULV1 is 3,381 nt long, containing two open reading frames (ORF1 and ORF2). ORF1 encodes a hypothetical protein with an unknown function, while ORF2 encodes an RNA-dependent RNA polymerase (RdRp) with a conserved RdRp domain containing a metal-binding 'GDN' triplet in motif C, which is distinct from the 'GDD' motif found in most + ssRNA mycoviruses. A homology search revealed that the RdRp encoded by ORF2 was similar to the RdRp of umbra-like mycoviruses. Phylogenetic analysis based on the RdRp indicated that NoULV1 was grouped into a clade together with umbra-like mycoviruses belonging to the proposed family "Mycotombusviridae"., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Here, we describe a novel mycovirus, tentatively designated as "Nigrospora sphaerica fusarivirus 2" (NsFV2), which was isolated from the phytopathogenic fungus Nigrospora sphaerica strain HNXX-Ns20. NsFV2 has a single-stranded positive-sense RNA (+ ssRNA) genome of 6,156 nucleotides, excluding the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,509 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a viral helicase domain. The ORF1-encoded polypeptide shares 29.40-68.48% sequence identity with other fusariviruses and shares the highest sequence identity (68.48%) with Nigrospora sphaerica fusarivirus 1 (NsFV1). The small ORF2 encodes a polypeptide of 483 aa that contains a conserved chromosome segregation ATPase (Smc) domain. Multiple sequence alignments and phylogenetic analysis based on the ORF1-encoded polypeptide indicated that NsFV2 should be considered a new member of the genus Alphafusarivirus of the family Fusariviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Mycoviruses pervade the fungal kingdom, yet their diversity within various fungal families and genera remains largely unexplored. In this study, 10 publicly available fungal transcriptomes from Ceratocystidaceae were analyzed for the presence of mycoviruses. Despite mycovirus associations being known in only four members of this family, our investigation unveiled the discovery of six novel mycoviruses. The majority of these mycoviruses are composed of positive sense single stranded RNA and are putatively assigned to the viral family Mitoviridae (with tentative classification into the genera Unuamitovirus and Duamitovirus). The double stranded RNA viruses, however, were associated with the family Totiviridae (with tentative classification into the genus Victorivirus). This study also revealed the discovery of an identical unuamitovirus in the fungal species Thielaviopsis ethacetica and Thielaviopsis paradoxa. This discovery was notable as these fungal isolates originated from distinct geographical locations, highlighting potential implications for the transmission of this mitovirus. Moreover, this investigation significantly expands the known host range for mycoviruses in this family, marking the initial identification of mycoviruses within Ceratocystis platani, Thielaviopsis paradoxa, Thielaviopsis ethacetica, and Huntiella omanensis. Future research should focus on determining the effects that these mycoviruses might have on their fungal hosts., Competing Interests: Declarations Conflict of interest The authors declare no competing interests. Ethical approval The manuscripts constitutes original research and has not been previously published. All authors are in agreement with manuscript submission, which complies with the ethical standards of the journal., (© 2024. The Author(s).)

Mycoviruses can alter the biological characteristics of host fungi, including change virulence or pathogenicity of phytopathogens and entomopathogenic fungi (EPF). However, most studies on the mycoviruses found in EPF have focused on the effects of the viruses on the virulence of host fungi towards insect pests, with relatively few reports on the effects to the host fungi with regard to plant disease resistance in hosts. The present study investigated the effects of the mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) virus infection on host biological characteristics, evaluated antagonistic activity of BbCV2 against two phytopathogenic fungi ( Sclerotinia sclerotiorum and Botrytis cinerea ), and transcriptome analysis was used to reveal the interactions between viruses and hosts. Our results showed that BbCV2 virus infection increased B. bassiana 's growth rate, spore production, and biomass, it also enhanced the capacity of host fungi and their metabolic products to inhibit phytopathogenic fungi. BbCV2 virus infection reduced the contents of the two pathogens in tomato plants significantly, and transcriptome analysis revealed that the genes related to competition for ecological niches and nutrition, mycoparasitism and secondary metabolites in B. bassiana were significantly up-regulated after viral infection. These findings indicated that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. We suggest that mycovirus infection causes a positive effect on B. bassiana against phytopathogens, which should be considered as a potential strategy to promote the plant disease resistance of EPF.

Here, we identified a new mycovirus infecting the phytopathogenic fungus Nigrospora oryzae, which we have designated "Nigrospora oryzae partitivirus 2" (NoPV2). The genome of NoPV2 consists of two dsRNA segments (dsRNA 1 and dsRNA 2), measuring 1771 and 1440 bp in length, respectively. dsRNA 1 and dsRNA 2 each contain a single open reading frame (ORF) that encodes the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. A BLASTp search showed that the RdRp of NoPV2 had significant sequence similarity to the RdRps of other partitiviruses, including Nigrospora sphaerica partitivirus 1 (75.61% identity) and Magnaporthe oryzae partitivirus 1 (67.53% identity). Phylogenetic analysis revealed that NoPV2 is a new member of the genus Gammapartitivirus in the family Partitiviridae. This study provides important information for understanding the diversity of mycoviruses in N. oryzae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named "Nigrospora oryzae mitovirus 3" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Some mycoviruses can be considered as effective biocontrol agents, mitigating the impact of phytopathogenic fungi and consequently reducing disease outbreaks while promoting plant health. Cryphonectria parasitica , the causal agent of chestnut blight and a highly destructive pathogen, experienced a notable decrease in its virulence with the identification of cryphonectria hypovirus 1 (CHV1), a naturally occurring biocontrol agent. In this study, two innovative diagnostic protocols designed for the accurate and efficient detection of CHV1 are introduced. The ORF A and ORF B regions of CHV1 are targeted by these techniques, which employ colorimetric loop-mediated isothermal amplification (LAMP) with 2 Colorimetric LAMP Master Mix and real-time quantitative PCR (qPCR) with SYBR Green chemistry, respectively. The LAMP assay presents a discernible color transition, changing from pink to yellow after a 35 min incubation period. Comparative analysis, when assessed against two established reverse transcription-PCR (RT-PCR) techniques, reveals a significant enhancement in sensitivity for both the LAMP approach, which offers a tenfold increase, and the qPCR method, which showcases a remarkable 100-fold sensitivity improvement. Throughout the comparison phase, it was evident that the RT-PCR, LAMP, and qPCR procedures displayed superior performance compared to the Bavendamm test, relying on phenol oxidase activity, effectively distinguishing hypovirulent strains. Consequently, this study introduces two pioneer diagnostic assays for highly sensitive CHV1 detection, representing a substantial advancement in the realm of CHV1 surveillance techniques. These methodologies hold significant promise for enhancing research endeavors in the domain of the biological control of C . parasitica .

Monilinia fructicola is one of the most devastating fungal diseases of rosaceous fruit crops, both in the field and postharvest, causing significant yield losses. Here, we report the discovery of a novel positive single-stranded RNA virus, Monilinia fructicola hypovirus 3 (MfHV3), in a strain (hf-1) of the phytopathogenic fungus Monilinia fructicola. The complete genome of MfHV3 is 9259 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt position 462 to 8411. This ORF encodes a polyprotein with three conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), and DEAD-like helicase. The MfHV3 polyprotein shares the highest similarity with Colletotrichum camelliae hypovirus 1. Phylogenetic analysis indicated that MfHV3 clustered with members of the genus Betahypovirus within the family Hypoviridae. Taken together, the results of genomic organization comparisons, amino acid sequence alignments, and phylogenetic analysis convincingly show that MfHV3 is a new member of the genus Betahypovirus, family Hypoviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission., (© 2024. The Author(s).)

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1-P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As the application of fungicides is often unfeasible and not a sustainable solution, only a few other control options are available, including biological control. In this context, the use of parasitic mycoviruses as biocontrol agents of fungal pathogens has recently gained particular attention. Since the 1990s, the Asian fungus Hymenoscyphus fraxineus has been causing lethal ash dieback across Europe. In the present study, we investigated the biocontrol potential of the mitovirus Hymenoscyphus fraxineus mitovirus 2 (HfMV2) previously identified in Japanese populations of the pathogen. HfMV2 could be successfully introduced via co-culturing into 16 of 105 HfMV2-free isolates. Infection with HfMV2 had contrasting effects on fungal growth in vitro, from cryptic to detrimental or beneficial. Virus-infected H. fraxineus isolates whose growth was reduced by HfMV2 showed overall a lower virulence on ash ( Fraxinus excelsior ) saplings as compared with their isogenic HfMV2-free lines. The results suggest that mycoviruses exist in the native populations of H. fraxineus in Asia that have the potential for biological control of ash dieback in Europe. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

We have previously discovered a virus neo-lifestyle exhibited by a capsidless positive-sense (+), single-stranded (ss) RNA virus YkV1 (family Yadokariviridae ) and an unrelated double-stranded (ds) RNA virus YnV1 (proposed family " Yadonushiviridae ") in a phytopathogenic ascomycete, Rosellinia necatrix . YkV1 has been proposed to replicate in the capsid provided by YnV1 as if it were a dsRNA virus and enhance YnV1 replication in return. Recently, viruses related to YkV1 (yadokariviruses) have been isolated from diverse ascomycetous fungi. However, it remains obscure whether such viruses generally show the YkV1-like lifestyle. Here, we identified partner viruses for three distinct yadokariviruses, YkV3, YkV4a, and YkV4b, isolated from R. necatrix that were coinfected with multiple dsRNA viruses phylogenetically distantly related to YnV1. We first established transformants of R. necatrix carrying single yadokarivirus cDNAs and fused them with infectants by single partner candidate dsRNA viruses. Consequently, YkV3 and YkV4s replicated only in the presence of RnMBV3 (family Megabirnaviridae ) and RnMTV1 (proposed family " Megatotiviridae "), respectively. The partners were mutually interchangeable between the two YkV4 strains and three RnMTV1 strains but not between other combinations involving YkV1 or YkV3. In contrast to YkV1 enhancing YnV1 accumulation, YkV4s reduced RnMTV1 accumulation to different degrees according to strains. Interestingly, YkV4 rescued the host R. necatrix from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3) or host fungus. Overall, we revealed that while yadokariviruses generally require partner dsRNA viruses for replication, each yadokarivirus partners with a different dsRNA virus species in the three diverse families and shows a distinct symbiotic relation in a fungus. IMPORTANCE A capsidless (+)ssRNA virus YkV1 (family Yadokariviridae ) highjacks the capsid of an unrelated dsRNA virus YnV1 (proposed family " Yadonushiviridae ") in a phytopathogenic ascomycete, while YkV1 trans -enhances YnV1 replication. Herein, we identified the dsRNA virus partners of three yadokariviruses (YkV3, YkV4a, and YkV4b) with genome organization different from YkV1 as being different from YnV1 at the suborder level. Their partners were mutually interchangeable between the two YkV4 strains and three strains of the partner virus RnMTV1 (proposed family " Megatotiviridae ") but not between other combinations involving YkV1 or YkV3. Unlike YkV1, YkV4s reduced RnMTV1 accumulation and rescued the host fungus from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3, family Megabirnaviridae ) or host fungus. These revealed that while each yadokarivirus has a species-specific partnership with a dsRNA virus, yadokariviruses collectively partner extremely diverse dsRNA viruses and show three-layered complex mutualistic/antagonistic interactions in a fungus.

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khan, Telengech, Kondo, Bhatti and Suzuki.)

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.

Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in the presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analysed genome-wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at an interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency-dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e., inbreeding) and genetic drift are probably at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1-mediated biocontrol was probably retained., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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 .

Sclerotinia sclerotiorum ourmiavirus 17 (SsOV17) was isolated from the hypovirulent strain GF3 of Sclerotinia sclerotiorum. The genome of SsOV17 is 2,802 nt in length and contains a single long open reading frame (ORF) flanked by a short structured 5'-untranslated region (5'-UTR) (28 nt) and a long 3'-UTR (788 nt), respectively. The ORF encodes a protein with 663 amino acids and a predicted molecular mass of 75.0 kDa. A BLASTp search indicated that the protein encoded by SsOV17 is closely related to the putative RNA-dependent RNA polymerase (RdRp) of Sclerotinia sclerotiorum ourmiavirus 13 (71% identity). A multiple sequence alignment indicated that eight conserved amino acid motifs were present in the RdRp conserved region of SsOV17. Phylogenetic analysis demonstrated that SsOV17 clustered with members of the genus Botoulivirus., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Here, we describe a novel mycovirus, tentatively designated as "Botryosphaeria dothidea mitovirus 3" (BdMV3), isolated from Botryosphaeria dothidea strain FJ, which causes pear ring rot disease in Fujian Province, China. The complete genome nucleotide sequence of BdMV3 is 2538 nt in length and contains a single 2070-nt open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 689 amino acids (aa) using the fungal mitochondrial genetic code. BLASTp analysis revealed that the RdRp of BdMV3 shares 28.91%-69.36% sequence identity (query sequence coverage more than 90%) with those of members of the genus Mitovirus, with the highest sequence identity of 69.36% and 68.79% to the corresponding RdRp aa sequences of Rhizoctonia solani mitovirus 10 and Macrophomina phaseolina mitovirus 4, respectively. Phylogenetic analysis based on RdRp aa sequences indicated that BdMV3 is a new member of the genus Mitovirus in the family Mitoviridae., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation., Competing Interests: The authors have declared that no competing interests exist.

We previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense single-stranded RNA (ssRNA) virus, yadokari virus 1 (YkV1), and an unrelated double-stranded RNA (dsRNA) virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix. We have proposed that YkV1 diverts the YnV1 capsid to trans -encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp and whether YnV1 capsid copackages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs in the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein cotranslationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsids solely packaging YkV1 dsRNA and RdRp. These results provide proof of concept that a capsidless positive-sense ssRNA [(+)ssRNA] virus is hosted by an unrelated dsRNA virus. IMPORTANCE Viruses typically encode their own capsids that encase their genomes. However, a capsidless positive-sense single-stranded RNA [(+)ssRNA] virus, YkV1, depends on an unrelated double-stranded RNA (dsRNA) virus, YnV1, for encapsidation and replication. We previously showed that YkV1 highjacks the capsid of YnV1 for trans -encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the heterocapsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains of the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsids solely packaging YkV1 dsRNA and RdRp. This study provides proof of concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae . Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea , and M. fructicola . SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm ( S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea , and Monilinia fructicola . The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

In this report, we describe the molecular characterization of two novel mycoviruses coinfecting the plant pathogenic fungus Nigrospora sphaerica, which were designated "Nigrospora sphaerica fusarivirus 1" (NsFV1) and "Nigrospora sphaerica partitivirus 1" (NsPV1). NsFV1 has an undivided genome measuring 6,147 nt, excluding the polyA tail, and was predicted to contain two nonoverlapping open reading frames (ORF1 and 2). The larger ORF1 encodes a polyprotein containing a conserved RNA-dependent RNA polymerase (RdRp) and a helicase domain that has functions related to RNA replication, and the smaller ORF2 encodes a putative protein with an unknown function. NsPV1 consists of two genome segments, which measure 1,796 bp and 1,455 bp in length. Each of the two dsRNAs has a single ORF, and they are predicted to encode proteins with homology to viral RdRps and coat proteins of members of the family Partitiviridae. Phylogenetic analysis indicated that NsFV1 is a member of the recently proposed family "Fusariviridae", while NsPV1 was determined to belong to the genus Gammapartitivirus in the family Partitiviridae. To the best of our knowledge, this report is the first to describe mycoviruses infecting N. sphaerica., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A putative polymycovirus tentatively named "Setosphaeria turcica polymycovirus 1" (StPmV1) was discovered in the phytopathogenic fungus Setosphaeria turcica. StPmV1 has a genome comprising five double-stranded RNAs (dsRNAs). dsRNA1, 2, and 3 each encode a protein sharing significant similarity but lower than 64% sequence identity to the corresponding proteins of other polymycoviruses. dsRNA4 and 5 each encode a protein with a sequence that is not conserved among polymycoviruses. However, the protein encoded by dsRNA4 is rich in proline (P), alanine (A), and serine (S) residues, which is a feature shared by the so-called PAS-rich proteins encoded by all polymycoviruses. Phylogeny reconstruction using the RNA-dependent RNA polymerase (RdRp) sequences of accepted or putative polymycoviruses revealed that StPmV1 is most closely related to Plasmopara viticola lesion associated polymycovirus 1 (PvaPolymyco1), a putative polymycovirus recovered from the phytopathogenic oomycetes Plasmopara viticola. These data suggest that StPmV1 may represent a novel species of the genus Polymycovirus, family Polymycoviridae. To our knowledge, this is the first polymycovirus reported from S. turcica., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A novel mycovirus with the proposed name "Magnaporthe oryzae botourmiavirus 9" (MoBV9) was found in the rice blast fungus Magnaporthe oryzae isolate SH05. The virus has a positive single-stranded RNA genome of 2,812 nucleotides and contains a single open reading frame predicted to encode an RNA-dependent RNA polymerase that is closely related to those of some unclassified viruses of the family Botourmiaviridae, including Plasmopara viticola lesion associated ourmia-like virus 44, Plasmopara viticola lesion associated ourmia-like virus 47, and Cladosporium uredinicola ourmiavirus 1. Genome sequence comparisons and phylogenetic analysis supported the notion that MoBV9 is a new member of the family Botourmiaviridae.

In this study, we isolated and determined the complete genome sequence of a novel mitovirus, "Botryosphaeria dothidea mitovirus 2" (BdMV2), from the phytopathogenic fungus Botryosphaeria dothidea isolate DT-5. BdMV2 has a genome 2,482 nt in length with an A+U content of 67%. The genome of BdMV2 contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 717 amino acids (aa) with a molecular mass of 81.86 kDa. A BLASTp comparison of the RdRp sequence showed the highest identity (66.67%) with that of Alternaria arborescens mitovirus 1 (AbMV1). Sequence comparisons and phylogenetic analysis revealed that BdMV2 is a new member of the genus Mitovirus of the family Mitoviridae.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Aplosporella javeedii is a pathogenic fungus that causes canker and dieback of jujube in China. In this study, we report a new mycovirus, Aplosporella javeedii partitivirus 1 (AjPV1), isolated from A. javeedii strain NX55-3. The AjPV1 genome contains two double-stranded RNA elements (dsRNA1 and dsRNA2). The size of dsRNA1 is 2,360 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp), while dsRNA2 is 2,301 bp in length and encodes a putative capsid protein (CP). The sequences of RdRp and CP have significant similarity to those of members of the family Partitiviridae. Sequence alignment and phylogenetic analysis showed that AjPV1 is a new member of the family Partitiviridae that is related to members of the genus Betapartitivirus. To our knowledge, AjPV1 is the first mycovirus reported from A. javeedii.

A novel virus, Botryosphaeria dothidea bipartite mycovirus 1 (BdBMV1), was isolated from the plant-pathogenic fungus Botryosphaeria dothidea strain HNDT1, and the complete nucleotide sequence of its genome was determined. BdBMV1 consists of two genomic segments. The first segment is 1,976 bp in length and contains a single open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) (68.95 kDa). The second segment is 1,786 bp in length and also contains a single ORF encoding a hypothetical protein of 35.19 kDa of unknown function. Based on the sequence of its RdRp, BdBMV1 is phylogenetically related to several other unclassified dsRNA mycoviruses, including Cryphonectria parasitica bipartite mycovirus 1 (CpBV1), and has a distant relationship to members of the family Partitiviridae.

Understanding the dynamics of white-nose syndrome spread in time and space is an important component for the disease epidemiology and control. We reported earlier that a novel partitivirus, Pseudogymnoascus destructans partitivirus-pa, had infected the North American isolates of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome in bats. We showed that the diversity of the viral coat protein sequences is correlated to their geographical origin. Here we hypothesize that the geographical adaptation of the virus could be used as a proxy to characterize the spread of white-nose syndrome. We used over 100 virus isolates from diverse locations in North America and applied the phylogeographic analysis tool BEAST to characterize the spread of the disease. The strict clock phylogeographic analysis under the coalescent model in BEAST showed a patchy spread pattern of white-nose syndrome driven from a few source locations including Connecticut, New York, West Virginia, and Kentucky. The source states had significant support in the maximum clade credibility tree and Bayesian stochastic search variable selection analysis. Although the geographic origin of the virus is not definite, it is likely the virus infected the fungus prior to the spread of white-nose syndrome in North America. We also inferred from the BEAST analysis that the recent long-distance spread of the fungus to Washington had its root in Kentucky, likely from the Mammoth cave area and most probably mediated by a human. The time to the most recent common ancestor of the virus is estimated somewhere between the late 1990s to early 2000s. We found the mean substitution rate of 2 X 10-3 substitutions per site per year for the virus which is higher than expected given the persistent lifestyle of the virus, and the stamping-machine mode of replication. Our approach of using the virus as a proxy to understand the spread of white-nose syndrome could be an important tool for the study and management of other infectious diseases., Competing Interests: The authors have declared that no competing interests exist.

Neofusicoccum parvum is a fungal plant-pathogen belonging to the family Botryosphaeriaceae , and is considered one of the most aggressive causal agents of the grapevine trunk disease (GTD) Botryosphaeria dieback. In this study, the mycovirome of a single strain of N. parvum (COLB) was characterized by high throughput sequencing analysis of total RNA and subsequent bioinformatic analyses. Contig annotations, genome completions, and phylogenetic analyses allowed us to describe six novel mycoviruses belonging to four different viral families. The virome is composed of two victoriviruses in the family Totiviridae , one alphaendornavirus in the family Endornaviridae , two mitoviruses in the family Mitoviridae , and one narnavirus belonging to the family Narnaviridae. The presence of the co-infecting viruses was confirmed by sequencing the RT-PCR products generated from total nucleic acids extracted from COLB. This study shows that the mycovirome of a single N. parvum strain is highly diverse and distinct from that previously described in N. parvum strains isolated from grapevines.

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., (Copyright © 2021 American Society for Microbiology.)

Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2 , which are orthologous to rid and dim-2 of Neurospora crassa , respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1 , not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1 -null mutant is hypervirulent, while the CpDmt2 -null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations. IMPORTANCE Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus C. parasitica We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance., (Copyright © 2021 Ko et al.)

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition., Competing Interests: The authors have declared that no competing interests exist.

Here, a novel mycovirus, Botryosphaeria dothidea mitovirus 1 (BdMV1), was isolated from a phytopathogenic fungus, Botryosphaeria dothidea, and its molecular characteristics were determined. BdMV1 has a genome of 2,667 nt that contains a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 727 amino acids with a molecular mass of 81.64 kDa. BLASTp analysis revealed that the RdRp domain of BdMV1 has 39.59% and 39.18% sequence identity to Plasmopara viticola associated mitovirus 43 and Setosphaeria turcica mitovirus 1, respectively. Phylogenetic analysis further suggested that BdMV1 is a new member of the genus Mitovirus within the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in B. dothidea.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A putative mycovirus belonging to the proposed family "Fusariviridae" was discovered in Setosphaeria turcica by sequencing a double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named "Setosphaeria turcica fusarivirus 1" (StFV1). StFV1 has a genome comprising 6685 nucleotides. The genome contains three open reading frames (ORF). The largest ORF, ORF1, is preceded by an untranslated region (UTR) of 16 nucleotides and separated from ORF2 by an intergenic region of 63 nucleotides. The smallest ORF, ORF3, overlaps ORF2 by 16 nucleotides and is followed by a 3'-UTR of 82 nucleotides. The protein encoded by ORF1 is 71.8%, 67.4% and 68.1% identical to the RNA-dependent RNA polymerases (RdRps) of Pleospora typhicola fusarivirus 1 (PtFV1), Plasmopara viticola lesion-associated fusarivirus 1 (PvlaFV1), and Plasmopara viticola lesion-associated fusarivirus 3 (PvlaFV3), respectively, but has less than 47% amino acid sequence identity to the RdRps of other fusariviruses. To our knowledge, this is the first fusarivirus discovered in S. turcica and the first virus to be identified in this fungus using conventional cloning methods.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868 bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22 bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907 bp and 1918 bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Cryphonectria parasitica, the causal agent of chestnut blight is controlled in Europe through natural spread of Cryphonectria hypovirus 1 (CHV1), a mycovirus able to induce hypovirulence to the host. In recent years C. parasitica was reported infecting Azerbaijani population of chestnut, but the presence of CHV1 still needs to be confirmed. Aim of this work was to investigate fifty-five C. parasitica isolates collected in Azerbaijan to describe the associated viruses. Our work found i) the first negative-sense ssRNA virus known to infect C. parasitica naturally for which we propose the name Cryphonectria parasitica sclerotimonavirus 1 (CpSV1) and ii) an RNA sequence showing peculiar features suggesting a viral nature for which we propose the name Cryphonectria parasitica ambivirus 1 (CpaV1). The discovery of CpaV1 expands our knowledge of the RNA virosphere suggesting the existence of a new lineage that cannot presently be reliably associated to the monophyletic Riboviria., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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"., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The complete genome sequence of a novel mycovirus, Phoma matteucciicola RNA virus 1 (PmRV1), derived from Phoma matteucciicola strain LG-01, was sequenced and analyzed. The complete cDNA sequence of PmRV1 is 3432 bp in length with a GC content of 57.17%. The genome of PmRV1 contains two putative open reading frames (ORFs): ORF1 and ORF2. ORF1 encodes a hypothetical protein with significant similarity to a protein encoded by Periconia macrospinosa ambiguivirus 1 (PmAV1). ORF2 encodes a protein of 491 amino acids with a conserved RNA-dependent RNA polymerase (RdRp) domain. Additionally, the triad within domain III has an asparagine (GDN) instead of the nearly universally conserved aspartic acid (GDD). RdRp phylogeny showed that PmRV1 grouped together with PmAV1 as a sister branch of a new member of the recently proposed family of mycotombus-like viruses. This is first report of the complete sequence of a novel mycovirus, PmRV1, infecting Phoma matteucciicola strain LG-01, the causal agent of leaf blight of Curcuma wenyujin.

Two isolates of Rosellinia necatrix (Rn118-8 and Rn480) have previously obtained from diseased avocado trees in commercial orchards of the coastal area in southern Spain. Rn118-8 and Rn480 have weak virulence on avocado plants, and are infected by R. necatrix hypovirus 2 (RnHV2). In this work, the possible biological effects of the hypovirus on R. necatrix were tested. First, RnHV2 was transmitted from each of Rn118-8 and Rn480 to a highly virulent, RnHV2-free isolate of R. necatrix (Rn400) through hyphal anastomosis, using zinc compounds which attenuate the mycelial incompatibility reactions and allow for horizontal virus transfer between vegetatively incompatible fungal strains. Next, we carried out an analysis of growth rate in vitro and a virulence test of these newly infected strains in avocado plants. We obtained five strains of Rn400 infected by RnHV2 after horizontal transmission, and showed some of them to have lower colony growth in vitro and lower virulence on avocado plants compared with virus-free Rn400. These results suggest that R. necatrix isolates infected by RnHV2 could be used as novel virocontrol agents to combat avocado white root rot., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae , one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae . Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3' untranslated region (3'-UTR) and 5'-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae . Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae , and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae .

A novel mycovirus, named "Corynespora cassiicola bipartite mycovirus 1" (CcBV1), was isolated from a phytopathogenic fungus, Corynespora cassiicola, the causal agent of rubber leaf fall disease. The nucleotide sequence of the complete genome of CcBV1, which consists of two double-stranded RNA (dsRNA) segments, was determined. The first dsRNA is 2,002 bp in length and contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) (69 kDa), while the second is 1,738 bp in length and contains a single ORF encoding a hypothetical protein of unknown function, with an approximately molecular weight of 36 kDa. The amino acid sequences of the both deduced proteins are most similar (58.9% and 45.1% identity, respectively) to those of Cryphonectria parasitica bipartite mycovirus 1 (CpBV1). Phylogenetic analysis indicated that CcBV1 clusters together with CpBV1 and other unassigned dsRNA mycoviruses. To the best of our knowledge, this represents the first report of a mycovirus infecting C. cassiicola.

Members of the family Partitiviridae are reported from a variety of fungal and plant taxa. After dsRNA-preparation, deep sequencing, and bioinformatics, we here reveal the existence of various divergent partitiviruses co-infecting the ectomycorrhizal fungus Sarcosphaera coronaria, symbiotically associated with the pine species Pinus brutia in Turkey. A total of 75 complete or nearly complete sequences related to the members of Alphapartitivirus and Betapartitivirus, were detected from the ascocarp sample of the fungal isolate. Two of the identified partitivirus genome segments encoding for partitiviral capsid protein represent evolutionarily distinct members of Alphapartitivirus, indicating that they may have diverged in the presence of long spatial isolation. In an attempt to match the two genome segments of the identified partitiviruses and distinguish individual species co-inhabiting a single host, nine possible genome segment pairs were identified., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host-pathogen interactions in bat WNS.

Rosellinia necatrix is responsible for the white rot root disease of avocado in Southern Spain. Entoleuca sp. is a fungus isolated from roots of these same trees, but it is not pathogenic in avocado. Here, we describe two new species of partitiviruses detected in isolates of the avocado sympatric fungi Entoleuca sp. and R. necatrix, termed Entoleuca partitivirus 1 (EnPV1), genus Alphapartitivirus, and Entoleuca partitivirus 2 (EnPV2), genus Betapartitivirus. For both R. necatrix and Entoleuca sp., the dsRNA of the RdRp genomic segment of EnPV1 accumulates at a higher rate than the CP dsRNA, except for a set of Entoleuca sp. isolates where titers of the CP dsRNA are 35-50 times higher than those of the RdRp dsRNA and between 250-380 times higher than the CP dsRNA titers found in the rest of Entoleuca sp. and R. necatrix isolates. For EnPV2, the accumulation rates of the RdRp dsRNA in Entoleuca sp., is in most of the cases, higher than the CP dsRNA. In contrast, in R. necatrix isolates, EnPV2 dsRNA2 generally accumulates at a higher rate. Genetic analysis of the partitiviruses revealed that there is no apparent variation in the nucleotide sequences among the strains. RNA silencing of the partitiviruses appears to be limited in Entoleuca sp., as shown by small RNA sequencing. Finally, the investigation of the presence of these partitiviruses in a fungal collection revealed that they have no role in the pathogenicity of R. necatrix in avocado or in the avirulence of Entoleuca sp. in this host., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this study, we report the molecular characterization of a novel positive-sense single-stranded RNA (+ssRNA) mycovirus from Botryosphaeria dothidea strain G91, which we name "Botryosphaeria dothidea botourmiavirus 1" (BdBOV-1). The complete genome of BdBOV-1 is 2547 nucleotides (nt) long, contains one open reading frame (ORF) potentially encoding an RNA-dependent RNA polymerase (RdRp), and shows a close relationship to mycoviruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp sequences confirmed that BdBOV-1 clustered together with the members of the family Botourmiaviridae. To the best of our knowledge, this is the first report of a +ssRNA mycovirus infecting B. dothidea.

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (-) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum . In this study, functional analysis of Open Reading Frame Ι (ORF Ι) of SsNSRV-1 was performed. The integration and expression of ORF Ι led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF Ι were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF Ι were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF Ι could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF Ι may be a potential gene used for the prevention of S. sclerotiorum ., Competing Interests: The authors declare no conflict of interest.