Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (−ssRNA) viruses. In these viruses, five +ssRNA and four −ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel −ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses.

A novel double-stranded RNA (dsRNA) virus was isolated and described from strain ZZZ210557 of plant endophyte Allocryptovalsa sichuanensis. The dsRNA virus contains four dsRNA segments, dsRNA1 to dsRNA4, with a size range of 3.8 to 5.1 kbp. Each possesses a single large ORF and is encapsulated in isometric particles approximately 42–47 nm in diameter. Notably, the dsRNA3 encoded sequence revealed modest similarities to the amino acid sequences of RdRps predicted from the nucleotide sequences of known and suspected members of the family Quadriviridae. Phylogenetic analysis of the putative RdRp with the corresponding proteins of other quadriviruses revealed that the dsRNA virus is a new member belonging to the family Quadriviridae, tentatively named Allocryptovalsa sichuanensis quadrivirus 1 (AsQV1). All four segments of AsQV1 could be successfully cured through ribavirin treatment, whereas it likely has no apparent impact on the morphologies or virulence of the host fungus. This study is the first report of a quadrivirus isolated from the fungus A. sichuanensis, and our results enhance the diversity of the quadrivirus.