Molecular Characterizations Of Two Novel RNA Viruses Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.) de Bary is a serious necrotrophic pathogenic fungus, which may infect more than 450 species of plants belonging to 75 families. The diseases caused by S. sclerotiorum cause great losses of yield to plants including soybean, rapeseed, sunflower and other vegetable crops, thus bring huge economic losses to agricultural production.Strain JMTJ14 from Jingmen and strain JZJL2 from Jingzhou, Hubei were identified to be sclerotia deficient screeed from above 3,000 strains collected from Hubei, Shanxi and Sichuan province, China 3,600 sclerotia. There were no significant difference in colony morphology, growth rate and virulence among strain JMTJ14, JZJL2, virus-free strain Ep-1PNA367 and 1980. The genes involved in sclerotium-forming and oxalic acid-degradating expressed in the same pattern as well. Now, many mycovirus are found in fungi, a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum, and many of them are virulence-associated viruses. Using of virulence-associated virus or antagonistic and / or parasitic microorganisms such as Coniothyrium minitans is one of the most important ways of biological control of disease caused by S. scleotiorum. In this paper, we were verify that there existed one individual ds RNA fragment in the two strains and cloned the ds RNA segments.Sclerotinia sclerotiorum fusarivirus1(Ss FV1) was isolated from the strain JMTJ14. Excluding a poly(A) tail, the genome of Ss FV1 comprises 7,754 nts in length with 83 and 418 nts for 5’- and 3’- untranslated regions, respectively. Ss FV1 has four non-overlapping open reading frames(ORFs): ORF1 encodes a 191 k Da polyprotein(1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase(Rd Rp) and helicase domains; the other three ORFs encode three putative hypothetical proteins with unknown function. Phylogenetic analysis based on Rd Rp and Helicase domains indicated that Ss FV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1(Rn FV1), Fusarium graminearum virus-DK21(Fg V1), and Penicillium roqueforti RNA mycovirus 1(Pr RV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, Ss FV1 is markedly different from Fg V1 and Rn FV1 in genome organization and nucleotide sequence. Ss FV1 was transmitted successfully to strains Ep-PNA367 and 1980. The results suggested that Ss FV1 was not responsible for colony morphology, growth rate and sclerotium-forming.Sclerotinia sclerotiorum endornavirus 1(Ss EV1) was isolated from the strain JZJL2. The genome of Ss EV1 is 10,770 nucleotides(nt) in length and has 5’ and 3’ untranslated regions(UTRs) that are 140 and 54 nt, respectively. Ss EV1 has a single open reading frame encoding a large protein that consists of 3,491 amino acids. This protein contains a cysteine-rich region and four conserved domains, including the putative viral methyltransferase, DEXDc box helicase, viral RNA helicase 1 and RNA-dependent RNA polymerase domains. The genome organization and sequence of Ss EV1 were similar to the previously described Gremmeniella abietina type B RNA virus(Ga BRV). A phylogenetic analysis indicated that the Rd Rp domain in Ss EV1 has significant sequence similarity with viruses from the Endornaviridae family. However, the viral methyltransferase and DEXDc box helicase domains of Ss EV1 and Ga BRV were clustered with ss RNA viruses of the “alpha-like” supergroup. It is noteworthy that these two conserved domains are absent in typical endornaviruses. Ss EV1 was transmitted successfully to two vegetatively incompatible virus-free strains Ep-PNA367 and 1980. The results suggested that Ss EV1 was not responsible for on colony morphology, growth rate and sclerotium-forming..In this study, two novel mycoviruses have been reported, and the influences to their host fungus were studied. The results provide important references for studying the evolution of fungal virus.