| Sclerotinia sclerotiorum is a notorious plant fungal pathogen with a broad host range.Agricultural production suffers great losses because of sclerotiorum disease every year in the world. Hypovirulence-associated mycoviruses have attracted much attention for their potential as biological control agents to combate plant fungal diseases. Therefore, it is imperative to screen hypovirulence-associated mycoviruses in S. sclerotiorum. Moreover,those hypovirulence-associated mycoviruses will not only supply some potential biological resources to control Sclerotinia disease, but also facilitate to research on viral diversity, evolution and interaction between mycovirus and its hosts.1603 sclerotium of S. sclerotiorum were collected from Shanxi Province and 100 strains with abnormal colony morphology were screened for deep sequencing. In total, 49 viruses with different features were obtained, and a novel virus of 49 viruses, named as Sclerotinia sclerotiorum Rhabdovirus 1(SsRhV1), was further reasearched.RT-PCRconfirmed that strain SX276 was infected by SsRhV1. Strain SX276 showed abnormal colony morphology with lower growth rate and no pathogenicity on rapeseed plant. Thus, strain SX276 is a typical hypovirulent strain. DsRNA extraction and sequence clone further confirmed that strain SX276 is co-infected by other three mycovirus of Sclerotinia sclerotiorum endornavirus 2(SsEV2), Sclerotinia sclerotiorum hypovirus 1-like(SsHV1-like) and Sclerotinia sclerotiorum botytivirus 2(SsBRV2) with exception of SsRhV1.SsRhV1 as a member of Rhabdoviridae is firstly characterizated in fungi. The genome of SsRhV1 is consists of 11356 nucleotides(nts) in length with reverse complement of the 5,and 3,terminus sequence, but structures of cap and poly A is lack.SsRhV1 contains five nonoverlapping and linearly arranged ORFs. ORF1/4/5 encodes N/G/L proteins which show conserved sequence with members of Rhabdoviridae, but ORF2/3-encoded proteins have no sequence similarity to any known proteins in the protein database. Termination codon UAA, termination sequence AUAUAA, termination signal sequence, initiation codon AUG, initiation sequence UCA(U/C/A)(U/C/A) AUG,and initiation signal sequence are found among the conserved gene-junction sequence with A/U rich. The conserved overlap sequence between termination signal sequence and initiation signal sequence contains SsRhV1 TTP sequence AAA(A/G) A and TI sequence AACAGA. The phylogenetic analysis indicated that SsRhV1 has a closed relationshipwith rhabdoviruses that infect animals, but formed an independent phylogenetical branch.Thus, we proposed to be created a new genus to accommodate SsRhV1. The virions of SsRhV1 are filamentous with 30 nm in diameter and 500-1200 nm in length. Experiment result prove that SsRhV1 has some effect on biological properties of host S. sclerotiorum.The partial cDNA sequence of SsEV2 was obtained with 11586 nts and two complete ORFs were predicted from the obtained sequence of SsEV2. ORF1-encoded protein contains 5 conserved domains of MTR, CRR, DExH, HEL, and S7, whereas ORF2-encoded protein contains a conserved RdRp domain. This similar phenomenon has not been previously reported in all known endornaviruses. Multiply alignment and phylogenetic analysis indicated that SsEV2 contains 8 conserved amino acid sites or motifs of RdRp, and SsRhV1 may belong to a new endornavirus species, which has a closed relationship with previously reported endornaviruses. Using mixed culture methods, the virulent strain Ep-1PNA367 GFP was successfully obtained SsEV2 from strain SX276 via hyhal fusion. The newly SsEV2-infected isolates displayed debilitation symptoms that were similar to that of strain SX276. Therefore, SsEV2 has great influence on biological properties of host S. Sclerotiorum, which is singnificant different with all previously reported fungal endornavirus. |
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