Research On The Molecular Characterization Of Two Mycoviruses In Hypovirulent Strain SX247of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is an important plant fungal pathogen and causes stem rot disease of crops, which is responsible for yield loss and the quality drop of crops. Since its life cycle is complicated, the effective control method is still not available. The hypovirulence-associated mycoviruses have a potential for exploitation as alternative biological control agents.In the present study, a hypovirulent strain SX247was isolated from the sclerotium of the diseased rapeseed plant stem in Shanxi Province. Compared to normal strain Ep-1PNA367, strain SX247showed abnormal colony morphology, slower growth rate, and no sclerotia production. Furthermore, SX247was incapable of causing diseased lesion on the detached leaves of rapeseed plant. Thus strain SX247is a hypovirulent strain.Two dsRNA segments, approximately15Kb and5.4Kb, were extracted from strain SX247. Analysis of cDNA sequence revealed that those two dsRNA segments are the genomes of two mycoviruses, which were named as Sclerotinia sclerotiorum hypovirus2(SsHV2/SX247), and Sclerotinia sclerotiorum Debilitation-Associated RNA Virus strain SX247(SsDRV/SX247), respectively.SsHV2/SX247shared sequence similarity (23%-28%) to mycoviruses belonging to the family Hypoviridae, and the full-length cDNA of SsHV2/SX247was15219nts, excluding Poly (A) and contained a large putative open reading frame (ORF) which encoded a putative ployprotein with three conserved domains of papain-like protease (Prot), RNA-dependent RNA polymerase (RdRp) and viral RNA helicase (Hel).The phylogenetic tree based on three conserved domains and ployprotein showed that SsHV2/SX247is most closely to the Alphahypovirus in the family Hypoviridae. However, there are also some differences between SsHV2/SX247and mycoviruses of Alphahypovirus based on genome size, genetic organization and phylogenetic analysis. Thus we proposed to establish a new genus Gammahypovirus.The other mycovirus SsDRV/SX247was5493nts in length excluding Poly (A), and it contained a single ORF which included three conserved domains, of Methyl transferase, RNA Helicase and RdRp. SsDRV/SX247shared a high identity (81%) to the previously reported a positive ssRNA virus SsDRV.The relationship between SsHV2/SX247, SsDRV/SX247and their host was determined via the horizontal transmission and protoplast regeneration assays. Both results revealed that SsHV2/SX247was associated with hypovirulent traits, such as the abnormal colony morphology, slower growth rate, no sclerotia production and lower virulence. However, whether SsDRV/SX247was responsible for hypovirulence of strain SX247is still not understood.In summary, SX247and SsHV2/SX247would be a potential as agent to control Sclerotinia disease. This mycovirus also provides excellent viral material for studying on viral evolution and ecology.