Molecular Characterization Of Mycoviruses From Sclerotinia Sclerotiorum Hypovirulence Strain 8-2-3 And Their Influence On The Host

The fungus Sclerotinia sclerotiorum(Lib.) de Bary is one of the most widespread pathogens, which cause severe diseses in rapeseed, vegetables and other crops worldwide. Due to the complexity of the disease cycle, a broad range of hosts and a lack of resistant species, no safe and effective measures to control sclerotial disease. Mycoviruses are widely present in fungi, some of which can reduce the virulence of the pathogenic fungi. Hypovirulence-associated mycoviruses from S. sclerotiorum can provide new materials for the biological control of sclerotial disease. In the present research, a hypovirulent strain 8-2-3 of S. sclerotiorum was obtained from Sichuan province, and mycoviruses in strain 8-2-3 were characterizated both molecualar and biological level.Strain 8-2-3 was a hypovirulent strain characterizated by slow growth rate on PDA, thin hyphae and weak pathogenicity on detached leaves of rapeseed. Preliminary research showed that strain 8-2-3 was co-infected by two ds RNA viruses and one negative-stranded RNA virus. One of the ds RNA virus was a partitivirus, named as Sclerotinia sclerotiorum Partitivirus 3(Ss PV3), whose genome was composed of two segments(ds RNA1 and ds RNA2), with sizes of 1793 nt and 1566 nt respevtively. One ORF was found in ds RNA1, which encoded Rd RP, while one ORF was found in ds RNA2, which encoded a coat protein. The virions of Ss PV3 were spherical, 22-28 nm in diameter. The other ds RNA virus was Sclerotinia sclerotiorum botybirnavirus 3(Ss BRV3). Its genome was composed of two segments of ds RNA, with 96% and 83% sequence similarity of Bp RV1, respectively. Sequence homology analysis showed that Ss BRV3 and Bp RV1 belong to different strains of a same genus. The full-length sequence of Ss NSRV3 was expected to be larger than 10 Kb, and we have obtained 4672 nt and 2081 nt sequence in the 3 ’end and 5’ end, respectively. Ss NSRV3 comprised three conserved mononegaviral m RNA capping-associated domains, Virus-capping methyltransferase(Methyltrans_Mon, pfam14314, 8.50e-44), Mononegavirales m RNA- capping region V(Mononeg_m RNAcap, pfam14318, 1.68e-24), m RNA capping enzyme(Paramyx_RNAcap, TIGR04198, 4.30e-20). Phylogenetic analysis based on L protein indicated that Ss NSRV3 belongs to the family Rhabdoviridae and had a close relationship with unclassified rhabdovirus, indicating that Ss NSRV3 may represent a new genus in the family Rhabdoviridae.By protoplast regeneration, we obtained a strain 9-22 which was infected by Ss PV3 and Ss BRV3, and a strain P4 which was only infected by Ss PV3. The strain 9-22 had a normal colony morphology and was able to produce sclerotium; Strain P4 was hypovirulent with an abnormal colony morphology and lose the ability to produce sclerotium. Ss PV3 was very stable in S. sclerotiorum, and was difficult to be eliminated through protoplast regeneration. Both two ds RNA viruses could be horizontally transmitted through host’s hyphal contact regardless of the limitation of vegetative compatibility. Ss NSRV3 was associated with the hypovirulence, but it could not be horizontally transmitted between different vegetative-incompatibility strains and could be eliminated easily.