Mycoviromic Analysis Unveils Complex Virus Composition in a Hypovirulent Strain of Sclerotinia sclerotiorum

Mycoviruses are ubiquitous in pathogenic fungi including Sclerotinia sclerotiorum. Using RNA sequencing, more mycoviruses have been identified in individual strains, which were previously reported to be infected by a single mycovirus. A hypovirulent strain of S. sclerotiorum, HC025, was previously thought to harbor a single mitovirus, Sclerotinia sclerotiorum mitovirus 1 (SsMV1), based on the analysis of the conventional dsRNA extraction method. We found HC025 to be co-infected by five mycoviruses. In addition to SsMV1, four mycoviruses were identified: Sclerotinia sclerotiorum narnavirus 4 (SsNV4), Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV1), Sclerotinia sclerotiorum ourmia-like virus 14 (SsOLV14), and SsOLV22. Three mycoviruses including SsNV4, SsNSRV1, and SsOLV14 share high replicase identities (more than 95%) with the previously reported corresponding mycoviruses, and SsOLV22 shows lower identity to the known viruses. The complete genome of SsOLV22 is 3987 nt long and contains a single ORF-encoded RdRp, which shares 24.84% identity with the RNA-dependent RNA polymerase (RdRp) of Hubei narna-like virus 10 (query coverage: 26%; e-value: 8 × 10(−19)). The phylogenetic tree of RdRp suggests that SsOLV22 is a new member within the family Botourmiaviridae. All of the mycoviruses except for SsNSRV1 could horizontally co-transfer from HC025 to the virulent strain Ep-1PNA367 with hypovirulent phenotypes, and converted a later strain into a hypovirulent strain. In summary, we molecularly characterized the hypovirulent strain HC025 and identified five RNA mycoviruses including a new member within Botourmiaviridae.