To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which "Rosellinia necatrix partitivirus 11-Rosellinia necatrix partitivirus 25" were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.

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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.

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