| Botrytis cinerea is an important necrotrophic phytopathogen and cause grey mold in numerous crops.B.cinerea leads to serious economic losses worldwide due to its wide host range,fast spread,easy variation and strong resistance to different fungicides.Grey mold is difficult to control and new control methods are urgently needed.Mycoviruses are natural resources for biological control of crop fungal diseases.In our Lab,the virome of406 B.cinerea strains from Israel were investigated and 79 species of mycoviruses have been determined.In this study,two pathogenicity-defective abnormal strains,IBc-352 and IBc-114 which could not produce any spore or sclerotia,were chosen from the 406 strains to identify novel mycoviruses and evaluate their potential use to control grey mold.Thirteen mycoviruses were detected in strain IBc-352,including Botrytis cinerea mitovirus 1(Bc MV1-IBc-352),Botrytis cinerea mitovirus 4(Bc MV4-IBc-352),Botrytis cinerea mitovirus 9(Bc MV9-IBc-352)and Sclerotinia sclerotiorum mitovirus 3(Ss MV3-IBc-352)in Mitoviridae,Botrytis cinerea binarnavirus 5(Bcb NV5-IBc-352)in Narnaviridae,Botrytis cinerea hypovirus virus 1(Bc HV1-IBc-352)in Hypoviridae,Botrytis cinerea alpha-like virus 1(Bc ALV1-IBc-352)in Togaviridae,Botrytis cinerea betaendornavirus 2(Bc BEV2)in Endornaviridae,Botrytis cinerea umbra-like virus 2(Bc ULV2)in Tombusviridae,Botrytis cinerea victorivirus 4(Bc VV4)in Totiviridae,and2 unclassified viruses Sclerotinia sclerotiorum ds RNA mycovirus-L(Ssd ML-IBc-352)and Botrytis cinerea mycovirus 5.Among them,Bc BEV2,Bc ULV2 and Bc VV4 are novel viruses.Bc BEV2 is a novel virus of the Endornaviridae.Bc BEV2 has a +ss RNA genome,the obtained genome sequence is 11097 nt,it has a complete ORF encoding a polyprotein of 3583 aa.The polyprotein has conserved domains including MTR domain(338-561 aa),DEADc domain(1269-1405 aa),Hel domain(1843-2076 aa)and Rd Rp(3241-3406 aa).Bc BEV2 is closely phylogenetically related to Botrytis cinerea betaendornavirus 1,they share 67.68% aa identity of polyproetin.Bc ULV2 is a novel virus of Tombusviridae.Bc ULV2 has a +ss RNA genome,the obtained genome is 4279 nt with two complete ORFs.ORF1 encodes a 331 aa polyprotein of unknown function while ORF2 encodes a polyprotein of 513 aa including Rd Rp domain.Bc ULV2 is closely phylogenetically related to Botrytis cinerea umbra-like virus 1,they shares 72.99% aa identity of polyprotein encoded by ORF2.Bc VV4 is a novel virus of Totiviridae.Bc VV4 is a ds RNA virus,its genome 5284 bp,including a 303 bp of 5?-UTR and a 68 bp of 3?-UTR,and two ORFs,ORF1(304-2679 bp)encodes CP and ORF2(2709-5214 bp)encodes Rd Rp,and there is a small intergenic region of 30 bp between two genes.Bc VV4 adopts a novelty expression strategy different from three known expression strategies of Totiviridae.Bc VV4 is closely phylogenetically related to Fusarium sambucinum victorivirus 1,they share 56.05% aa identity of Rd RP.Based on the phylogenetic analysis of Rd Rp and CP,Bc VV4 is a member of the genus Victorivirus.Virions of Bc VV4 are icosahedral spherical with 23-25 nm in diameter,which is smaller than these,usually 30-40 nm,of other members of Totiviridae.Bc VV4 can be easily removed by protoplast regeneration technique.Bc VV4 was proved to have no significant effects on the phenotype of B.cinerea.The mycoviruses in the strain IBc-352 can be transfected by RNA/ds RNA,and four mycoviruses,including Bc ALV1-IBc-352,Bc MV4-IBc-352,Bc MV9-IBc-352 and Bc HV1-IBc-352,can be transferred into strain B05.10.The growth rate and virulence of protoplast regenerants of the strain IBc-352 partially recovered though they still carried some viruses.The removal of Bc ALV1-IBc-352 may be the cause of the phenotypic change,and the weak virulence is related to the Bc HV1-IBc-352 related.Spraying the homogenized hyphae containing fermentation broth of strain IBc-352 on tomato leaves might induce severe hypersensitiv reaction,and high concentration spraying could lead to leaf death;while,B.cinerea could not induce any lesion on the leaves treated with 100-fold diluted fermentation broth.Interestingly,hyphae-free filtrate when diluted 5-100 folds could inhibit the lesion expansion induced by B.cinerea on leaves.These results indicate that strain IBc-352 can also synthesize antifungal substance.Strain IBc-114 was identified as s atrain of Schizophyllum commune with molecular identification,but not B.cinerea.It was co-infected by two mycoviruses,one is Bc MV9-IBc-352,another is novel virus.The novel virus has +ss RNA genome og 7370 nt in length,and contains two ORF,ORF1(130-6036 nt),ORF2(6102-6828 nt),a 303 bp of5?-UTR and a 68 bp of 3?-UTR.ORF1 encodes a 1968 aa protein with Hel and Rd Rp;ORF2 encodes a 242 aa protein with no predicted conserved domain.Phylogenetic analysis based on Rd Rp and Hel indicated that the virus was related to genus Benyvirus of family Benyviridae,and might represent a new genus of this family,thus was named Schizophyllum commune beny-like virus 1(Sc BLV1).Sc BLV1 could inhibit the hyphal growth of Sc commune.Strain IBc-114 could not inhibit the hyphal growth of B.cinerea or lesion expansion induced by B.cinerea on tomato leaves.In conclusion,we investigated the mycoviruses that co-infect strains IBc-352 and IBc-114 from Israel,we found three novel viruses from these two strains,found that a mitovirus(Bc MV9)can infect both B.cinerea belonging to ascomycetes and Sc.commune belonging to basidiomycetes,and preliminarily found that IBc-352 has a potential to control grey mold.Our findings may enrich the understanding of virus diversity,ecology and evolution;at the same time,it provides novel mycoviruses and hypovirulent strains for the control of diseases caused by B.cinerea. |
