Diversity Analysis Of Mycoviruses In Sclerotinia Sclerotiorum And Molecular Characteristics Of Two Novel RNA Mycoviruses

Sclerotinia sclerotiorum a important phytopathogenic fungus, has a wide range of hosts. Mycoviruses can cause hypovirulence on plant pathogenic fungi and are potential biological method to control of plant fungal diseases. Previous researches have been suggested that S. sclerotiorum habors rich diversity mycoviruses and some of the characterizated mycoviruses are responsible for hypovirulence on S. sclerotiorum. Thus, the hypovirulence-associated mycoviruses have the potential for development as biocontrol agents for combating for Sclerotinia diseases and for exploitation as tools to explore the basic biology of S. sclerotiorum. In the present research, we successfully screened 125 S. sclerotiorum strains with abornal colony morphology and analysized the diversity of mycovirus via high-throughput sequencing technique. The main results were shown as following.1800 sclerotium of S. sclerotiorum were collected from three provinces of Hunan, Heilongjiang, and Inner Mongolia. All sclerotium were re-isolated and observed their colony morphology on PDA plate. In total 125 strains were screened with abnormal colony morphology. Moreover, these obtained strains slowed slower growth rate, and lower virulence on the detached leaves of rapeseed.High-throughput sequencing analyses showed that S. sclerotiorum was infected by a diverse mycoviruses. Most of mycoviruses detected in S. sclerotiorum have not reported and characterizated. The classification for some of novel mycoviruses was not still uncertained. They possess diverse genomes of mostly ssRNA and dsRNA and circular ssDNA. All detected RNA mycoviruses possess diverse genomes of double-stranded RNA(dsRNA), positive-sense single-stranded RNA(+ssRNA), negative-sense single-stranded RNA(-ssRNA), and circular single-stranded(ssDNA). 80% mycoviruses are belonging to ssRNA viruses, and 18% mycoviruses are assigned into dsRNA viruses. In total, the sequence information of 78 mycoviruses, with exception of members within Narnaviridae, was obtained and those mycoviruses distribute in ninteen virus families and other to be established families.Strain 230 showed developed aerial hyphae, lower sclerotium production, and furthermore it could not cause disease lesion on the detached rape leaf. A new negative- stranded RNA virus with sveral segments was detected in strain 230 and tentatively designated this mycovirus as Sclerotinia sclerotiorum Mirafiore lettuce virus–like(SsMiLV). The full-length cDNA of SsMiLV-RNA1 genome was 7832 nt, and contained two presumed open reading frames(ORF1 and ORF2). ORF2 encoded a putative protein with molecular weight of 269 KD, which contains a typical conserved domain of RNA dependent RNA polymerase(RdRp). Phylogenetic analysis showed that SsMiLV is closely related to the members within the family Ophioviridae. However, according to the genetic evolution, there is a significant difference between SsMi LV and all reported phioviruses. SsMiLV with other two mycovirus discovered in in Rhizoctonia solani phylogenetically formed an independent clade that was distant from all reported viruses. Therefor we proposed that family Ophioviridae was divided into two genera based on their hosts range and phylogenetic analysis.Strain 767 showed typical hypovirulent features including slower growth rate, yellow pigment production, and lower sclerotium production. More important, strain 767 could not cause lesion formation, and no diseased lesion on the detached rapeseed leaves. Preliminary study found that strain 767 was infected by a new RNA virus, named Sclerotinia sclerotiorum Norovirus(SsNorV). SsNorV is a a novel virus in S. sclerotiorum, which has the similarity(24% identity) with Bat norovirus.The full-length cDNA of SsNorV was 11024 bp, and encoded two ORF which contained a conserved domain of RdRp. Phylogenetic analysis showed that SsNorV is closely to the the members within family Astroviridae, but SsNorV may present a new virus genus.SsMiLV and SsNorV could horizontally transmit between difference S. sclerotiorum strains via hyphae fusion. SsMiLV can only transmitted from strain 230 to the strain 1980, and the transmission efficiency is low(5%); While SsNorV can horizontally transmitted from strain 767 to strains Ep-1PNA367 and 1980 with higher transmission efficiency of 75%.