Preliminary Studies On The 6.4kb DsRNA Genome Of The Mycovirus Infecting The Hypovirulent Isolate Ep-1PN Of Sclerotinia Sclerotiorum

In this dissertation, the 6.4kb dsRNA genome of the mycovirus infecting the hypovirulent isolate Ep-lPN of Sclerotinia sclerotiorum (SsHV) was studied using techniques of cDNA synthesis, cloning, and sequencing. The relationship between hypovirulence of Sclerotinia sclerotiorum Ep-lPN and the dsRNA mycovirus was further confirmed at the molecular level. Results achieved so far include:1 Positive cDNA clones of the 6.4kb dsRNA of Ep-lPN were obtained. After sequencing the insertial cDNA fragments, in the plasmid partial cDNA sequence of the 6.4kb dsRNA genome of 4629 nucleotides was aligned with DNAMAN program. The sequence (4629bp) was submitted to the GenBank with the accession number AY147260.2 A incomplete open reading frame (ORF) encoding a fusion protein of 1440 ammo acid residues of helicase and RNA dependent RNA Polymerase(RdRp) of RNA virus was revealed. The gene coat protein amino acids sequence was not included in this sequence. Based on the characteristics of putative fusion protein, the 6.4 kb dsRNA element could be regarded as mycovirus, and named as Sclerotinia sclerotiorum hypovirulence-associated virus(SsHV).3 Both the nucleotide sequence and putative amino acid sequence were subjected to do homology search with Blast Program on http://www.ddbj.nig.ac.jpT the result revealed that the obtained nucleotide sequence had the characteristic of plant Potexvirus, subsequently the putative protein coded by the 6.4kb dsRNA was highly similar to those proteins coded by Potexvirus, such as BaMV(Bamboo mosaic virus), PVX(Potato virus X), ClYMV(Clover yellow mosaic virus) and TuVX(Tulip virus X) et al. The result suggested SsHV might derive from plant virus or showed the same ancestor with plant virus.4 Based on the cDNA sequence of SsHV, specific primers was designed to detect dsRNA in Ep-lPN and its derivatives using RT-PCR. the recovered cultures includ thee ascospore progenies (Ep-1PNAa, Ep-1PNAb and Ep-1PNAe), thee protoplast-regeneration cultures (Ep-1PNP14, Ep-1PNP94 and Ep-lPN131) and one single-sclerotium-isolationculture (Ep-1PNSSB9). The PCR product of 871bp in size occurred in Ep-lPNSSB9 and Ep-1PNP131, both cultures exhibited some atypical colony morphology, compared to health strains of S. sclerotiorum. This result suggests that S. sclerotiorum may activate some unknown mechanism to eliminate the hypovirulence associated mycovirus during sexual reproduction.