Hypovirulence of phytopathogenic fungi are often conferred by mycovirus(es) infections and for this reason many mycoviruses have been characterized, contributing to a better understanding of virus diversity. In this study, three strains of Fusarium head blight fungus (Fusarium boothii) were isolated from Ethiopian wheats as dsRNA-carrying strains: hypovirulent Ep-BL13 (>10, 3 and 2.5 kbp dsRNAs), and virulent Ep-BL14 and Ep-N28 (3 kbp dsRNA each) strains. The 3 kbp-dsRNAs shared 98% nucleotide identity and have single ORFs encoding a replicase when applied to mitochondrial codon usage. Phylogenetic analysis revealed these were strains of a new species termed Fusarium boothii mitovirus 1 in the genus Mitovirus. The largest and smallest dsRNAs in Ep-BL13 appeared to possess single ORFs and the smaller was originated from the larger by removal of its most middle part. The large dsRNA encoded a replicase sharing the highest amino acid identity (35%) with that of Botrytis virus F, the sole member of the family Gammaflexiviridae. Given that the phylogenetic placement, large genome size, simple genomic and unusual 3′-terminal RNA structures were far different from members in the order Tymovirales, the virus termed Fusarium boothii large flexivirus 1 may form a novel genus and family under the order.

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature.