Sclerotinia sclerotiorum is a devastating plant pathogen that attacks numerous economically important broad acre and vegetable crops worldwide. Mycoviruses are widespread viruses that infect fungi, including S. sclerotiorum. As there were no previous reports of the presence of mycoviruses in this pathogen in Australia, studies were undertaken using RNA_Seq analysis to determine the diversity of mycoviruses in 84 Australian S. sclerotiorum isolates collected from various hosts. After RNA sequences were subjected to BLASTp analysis using NCBI database, 285 contigs representing partial or complete genomes of 57 mycoviruses were obtained, and 34 of these (59.6%) were novel viruses. These 57 viruses were grouped into 10 distinct lineages, namely Endornaviridae (four novel mycoviruses), Genomoviridae (isolate of SsHADV-1), Hypoviridae (two novel mycoviruses), Mononegavirales (four novel mycovirusess), Narnaviridae (10 novel mycoviruses), Partitiviridae (two novel mycoviruses), Ourmiavirus (two novel mycovirus), Tombusviridae (two novel mycoviruses), Totiviridae (one novel mycovirus), Tymovirales (five novel mycoviruses), and two non-classified mycoviruses lineages (one Botrytis porri RNA virus 1, one distantly related to Aspergillus fumigatus tetramycovirus-1). Twenty-five mitoviruses were determined and mitoviruses were dominant in the isolates tested. This is not only the first study to show existence of mycoviruses in S. sclerotiorum in Australia, but highlights how they are widespread and that many novel mycoviruses occur there. Further characterization of these mycoviruses is warranted, both in terms of exploring these novel mycoviruses for innovative biocontrol of Sclerotinia diseases and in enhancing our overall knowledge on viral diversity, taxonomy, ecology, and evolution.

Mycovirus diversity is generally analyzed from isolates of fungal culture isolates at a single point in time as a snapshot. The stability of mycovirus composition within the same geographical location over time remains unclear. Not knowing how the population fluctuates in the field can be a source of unpredictability in the successful application of virocontrol. To better understand the changes over time, we monitored the interannual dynamics and abundance of mycoviruses infecting Sclerotinia sclerotiorum at a rapeseed-growing field for three years. We found that the virome in S. sclerotiorum harbors unique mycovirus compositions each year. In total, 68 mycoviruses were identified, among which 24 were detected in all three successive years. These 24 mycoviruses can be classified as the members of the core virome in this S. sclerotiorum population, which show persistence and relatively high transmissibility under field conditions. Nearly two-thirds of the mycoviruses have positive-sense, single-stranded RNA genomes and were found consistently across all three years. Moreover, 28 mycoviruses are newly described, including four novel, multi-segmented narnaviruses, and four unique bunyaviruses. Overall, the newly discovered mycoviruses in this study belong to as many as 20 families, into which eight were first identified in S. sclerotiorum, demonstrating evolutionarily diverse viromes. Our findings not only shed light on the annual variation of mycovirus diversity, but also provide important virus evolutionary clues.

Recently, an increasing number of ourmia-like viruses have been found in fungi; however, the features of these viruses remain unknown. Here, we report a novel ourmia-like virus isolated from Sclerotinia sclerotiorum. This virus, named S. sclerotiorum ourmia-like virus 4 (SsOLV4), has a genome 2,982 nt in length with a G-pentamer (GGGGG) at the 5terminus and a C-pentamer (CCCCC) at the 3-terminus. The SsOLV4 genome has only one large putative open reading frame (ORF) predicted with both standard codes and mitochondrial codes and encodes an RNA-dependent RNA polymerase (RdRp). SsOLV4 is closely phylogenetically related to Pyricularia oryzae ourmia-like virus 1, with 42% identity between the RdRp amino acid sequences. We constructed full-length cDNA of SsOLV4 and synthesized RNA in vitro using the T7 RNA polymerase. The synthesized RNA could transfect S. sclerotiorum protoplasts efficiently. We further found that viral RNA could infect mycelia when mixed with PEG buffer. Our study suggests that a novel genus in family Botourmiaviridae should be established for SsOLV4 and other related viruses and demonstrates that one single-stranded RNA segment encoding RdRp is sufficient for ourmia-like viruses in fungi.

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.