| Sclerotinia sclerotiorum is a destructive pathogen with worldwide distribution,and can infect over 700 plant species including many important crops,such as beans,rapeseed and sunflower.The white rot of beans infected by S.sclerotiorum results in substantial yield losses every year worldwide.Mycoviruses are viruses that replicate and proliferate in fungal cells and have a great diversity with wide distribution;among which the hypovirulence-associated mycoviruses have good potential for biological control of plant disease.Hence,mycoviruses discovery from S.sclerotiorum is of great potential to control white rot disease,and provides new ideas and insights for the study of viral ttaxonomy,evolution and ecology.More than 2000 sclerotia of S.sclerotiorum were collected from Pinto bean(Phaseolus vulgaris cv.Pinto)and Garden bean(Phaseolus vulgaris L.)in Washington State,USA,and mycoviruses were screened from 280 strains which have abnormal colony morphology using RNA＿Seq analysis.111 contigs representing partial or complete genomes of 33 mycoviruses were identified,and comfirmed by RT-PCR,among them,14 viruses were as novel viruses.According to the current Virus Taxonomy,these 33 viruses were classified into 9 families: Alphaflexiviridae(one virus),Botourmiaviridae(six viruses),Deltaflexiviridae(one virus),Hypoviridae(three viruses),Mitoviridae(seventeen viruses),Narnaviridae(one virus),Tombusviridae(one virus),Tymoviridae(one virus),and Mymonaviridae(two viruses),and mitoviruses were up to 51.5%.Interestingly,some viruses(12.6%)that show more than 90% amino acid sequence identity to these of viruses that infect Botrytis cinerea and Macrophomina phaseolina and that of Plasmopara viticola associated virus,suggesting that these viruses may transmit among fungal genera or fungal classes,even among kingdoms.Among 14 of those novel viruses,Sclerotinia sclerotiorum hypovirus 9(Ss HV9),Sclerotinia sclerotiorum ourmia-like virus 19(Ss OLV19),Sclerotinia sclerotiorum ourmia-like virus 20(Ss OLV20),and Sclerotinia sclerotiorum narnavirus 4-U(Ss NV4-U)were selected for research.Ss HV9 and Ss NV4-U coinfected in strain vGB375,Ss OLV19 infected strain PB821,and Ss OLV20 infected strain GB334.The full-length genomes of these four viruses were obtained by terminal cloning,and were analyzed.The genome of Ss HV9 is 14067 nt in length,including a Poly(A)tail and contained a large putative open reading frame(ORF)which encoded a putative 4196 aa ployprotein with four conserved domains of papain-like protease(Prot),protein of unknown function(DUF3525),RNA-dependent RNA polymerase(Rd Rp),and viral RNA helicase(Hel).The phylogenetic analysis based on ployprotein sequences showed that Ss HV9 is more closely to the Trichoderma asperellum hypovirus 1,is a member of Alphahypovirus of Hypoviridae.The full length genomes of Ss OLV19 and Ss OLV20 were 2697 nt and 3890 nt,respectively,and both contained one ORF which encoded putative Rd Rp with the size of 705 aa and 917 aa.The 5’ termini and 3’termini of them included consecutive guanines and consecutive cytosines,respectively,which are similar to that of the recently reported Sclerotinia sclerotiorum ourmia-like virus 4.The phylogenetic tree based on Rd Rp showed that Ss OLV19 belongs to the genus Magoulivirus and Ss OLV20 belongs to the recently proposed genus of Penoulivirus of Botourmiaviridae.Moreover,Ss OLV20 has a largest genome among Botourmiaviruses that infect S.sclerotiorum.The full length of Ss NV4-U was 3106 nt and contained a complete ORF putatively encoding Rd Rp of 987 aa.The phylogenetic tree based on Rd Rp showed that Ss NV4-U belongs to the family Narnaviridae.The biological characteristics of viruses-infected strains GB334,GB375 and PB821 were determined and the results demonstrated that strains GB334 and GB375 showed abnormal colony morphology with lower growth rate,twisted hyphal tip,reduction population of acidic substances,and decrease of pathogenicity;while strain PB821 was not significantly different from the virus-free strain 1980.Strains GB334,GB375 and PB821 carried 10 species,13 species,and 7 species of viruses,respectively.The viruses that infected strain GB375 were classified into Botourmiaviridae,Hypoviridae,Mitoviridae,Narnaviridae and Mymonaviridae;the viruses that infected strain GB334 were classified into Botourmiaviridae,Hypoviridae,Mitoviridae and Mymonaviridae and the viruses that infected strain PB821 were classified into Botourmiaviridae,Hypoviridae and Mitoviridae.All these three strains were infected by Sclerotinia sclerotiorum hypovirus 7,Sclerotinia sclerotiorum hypovirus 7 satellite-like RNA,Sclerotinia sclerotiorum mitovirus 9-U and Sclerotinia sclerotiorum mitovirus 11-U.To determine the relationship between viruses and the abnormal traits of virus-infected strains,a method combined by protoplast regeneration and ribavirin was used to dismiss viruses.However,only Ss HV9 in strain GB375 were possibly dismissed by two round treatment,and two regenerants GB375/19 a and GB375/19 b in which Ss HV9 could not detected via RT-PCR amplication.The virulence,growth and colony morphology of GB375/19 a and GB375/19 b were not significantly different form original strain GB375,suggesting that Ss HV9 was not affect host’virulence and abnormal morphology.The viruses that co-infected regenerants GB375/19 a and GB375/19 b were further determined,and the results showed that that strains GB375/19 a and GB375/19 b carried 10 species and 11 species of viruses,respectively,including members of Botourmiaviridae,Hypoviridae,Mitoviridae,Narnaviridae and Mymonaviridae.According to current reports,only Sclerotinia sclerotiorum hypovirus7 may be related to the hypovirulence of strain GB375.Briefly,in this study,the virome of S.sclerotiorum on beans from Washington State,USA was analyzed,and 14 novel mycoviruses were discovered.The full-length genomes of four novel viruses were sequenced.Our findings could enrich the knowledge on virus diversity and provide new materials for the research of the interaction between viruses and their hosts. |
