| Mycoviruses are viruses that infect fungi,and some of them could confer hypovirulence,thus they have a potential to control fungal diseases.Sclerotinia sclerotiorum,a widespread ascomycetous fungus,can attack more than 450 plant species and subspecies;It causes stem rot on rapeseed(Brassica napus)and leads to huge economic loss in China each year.Previously,several hypovirulence-associated mycoviruses have been identified from S.sclerotiorum,suggesting that this fungus could host various mycoviruses.In this study,we isolated 2000 strains of S.sclerotiorum from Hubei province,Jiangxi Province,and Anhui Province,and then we selected 173 strains which showed abnormal phenotypes to screen novel mycoviruses.We extracted RNA samples from the mycelial mass of these 173 strains,and then mixed them equally for RNA_Seq analysis.After carefully splicing,we acquired 247 contigs which showed the properties of viral genomes,this result suggested that various viruses could be existed in the 173 strains.We then searched the NCBI database using the putative proteins encoded by 247 contigs,and we found that these contigs possibly represented 48 mycoviruses,among them,35 were(+)ss RNA viruses,7 were(-)ss RNA viruses,5 were ds RNA viruses,and one was ss DNA viruses,with mitoviruses were major group of these viruses.We confirmed four novel mycoviruses represented by four contigs(contig3,contig50,contig71 and ontig1178),the viruses represented by contig3 and contig50 were(-)ss RNA viruses,and viruses represented by contig71 and contig1178 were(+)ss RNA viruses.The virus represented by contig3 was belonged to the Family Bunyaviridae and was named Sclerotinia sclerotiorum bunya-like virus 1(Ss Bl V1);the virus represented by contig 50 was named Sclerotinia sclerotiorum negative-stranded RNA virus 9(Ss Ns RV9),it was closely related to unclassified viruses.Both these viruses represented by contig71 and contig 1178 were belonged to the Order Tymovirales,and were named Sclerotinia sclerotiorum mycotymovirus 1(Ss MTV1)and Sclerotinia sclerotiorum deltaflexivirus 2(Ss DFV2).We selected Ss DFV2 for further study,we found that Ss DFV2 was in a strain named 228,and found that this strain was also infected by Botrytis porri RNA virus 1(Bp RV1),a virus originally isolated from a hypovirulent strain of Botrytis porri.Strain 228 grew on PDA slowly and produced few sclerotia at very late stage of growth,this strain also could not induce lesion on detacted rapeseed leaves.Based on the sequence of contig,we used 5’-and 3’-RACE to obtain the 5’-and 3’-termini of virual genome of Ss DFV2.The full-length sequence of viral genome was 6711 nt excluding a Poly(A)tail.The genome of Ss DFV2 has a large open reading frame(ORF),and encodes a putative viral RNA replicase,in which three domains,Methyltransferase,vrial Helicase and Rd Rp were identified.Ss DFV2 was closely phylogenetically related to previously identified virus,Sclerotinia sclerotiorum deltaflexivirus 1(Ss DFV1)from S.sclerotiorum belonging to the newly suggested Family Deltaflexiviridae.The amino acid identity of RNA replicase of Ss DFV2 with Ss DFV1 was 446/1185(38%)and similarity was 625/1185(52%),thus Ss DFV2 was also suggested to be a member of this new family.Intrestingly,comparing to Ss DFV1 which had been reported to contain 4 genes,Ss DFV2 have only one large ORF(single gene)and it is assumed that this virus lost other genes during its evolution.Vegetative incompatiblity restricts mycoviruses transmission among host’s different vegative compatibility groups,and extremely limits the efficacy of biological control with hypovirulence-associated mycoviruses.In this study,we found that Ss DFV2 had strong infectivity,when strain 228 was dual cultured with its vegetative incompatible strain Ep-1PNA367hyg/ Ep-1PNA367 or strain 1980hyg/1980,Ss DFV2 could transmit to the colonies of these vegetative imcompatible strains,while Bp RV1 in strain 228 could not transmit.When Ss DFV2-infected strain Ep-1PNA367 was dual cultured with strain strain 1980 hyg,virus still could be detected in the colony of the later,further suggested that the transmission to vegetative incompatible strain was not strain-dependent.Our finding suggested that some RNA mycoviruses may overcome the limitation caused by vegetative incompatibility reaction to spread among host’s individuals;it will provide novel ideas for exploring RNA mycoviruses to control crop fungal diseases. |
