Genomic Structure And Biological Function Of The RNA Fungal Virus Of Colletotrichum Gloeosporioides

Colletotrichum gloeosporioides is an important plant pathogenic fungus,which has a wide host range and geographical distribution,causing a variety of plant anthracnose and causing great losses in agricultural production.At present,the control of diseases caused by the pathogen mainly depends on chemicals,but it is easy to pollute the environment and form drug resistance.Therefore,it is of great theoretical and practical significance to seek environmentally friendly methods for the prevention and control of anthrax.Mycoviruses(fungal viruses)exists widely in various fungi.Most fungal viruses have no obvious symptoms in the host and present latent infection.However,some fungal viruses can cause phenotypic changes in the host,such as slower growth rate,reduced spore production,reduced pathogenicity,etc.Such fungal viruses are called weakly virus-related fungal viruses.Attenuated fungal viruses can reduce the pathogenicity of host fungi and can be used as potential biocontrol factors to control plant fungal diseases.Therefore,screening and functional study of fungal viruses will provide a new way and idea for biological control of plant diseases.In this study,using the strain LS-1,HJ1-2 of Colletotrichum gloeosporioides as the research material,the molecular biological characteristics of ds RNA fungal virus carried by the two strains were studied,the relationship between the genome structure and evolutionary classification of the virus was analyzed,and the function of the virus to the host in strain LS-1 was discussed.The results are as follows:(1)Strain HJ1-2 contains a new type of RNA virus,named Colletotrichum gloeosporioides Polymycovirises-1(Cg Pm V1).Cg Pm V1 has seven genomic fragments(S1to S7),each containing one ORF.Sequence analysis showed that the virus belongs to a new type of RNA virus,which has a genomic structure similar to that of Beauveria bassiana polymycovirus3(Bb Pm V-3),Aspergillus fumigatus tetramycovirus-1(Afu Tm V-1)and Colletotrichum camelliae filamentous virus1(Cc FV-1).The Rd Rp encoded by Ds RNA-1has amino acid similarity from 31.92% to 47.06% with Bb Pm V-3,Afu Tm V-1 and Cc FV-1.In classification,Cg Pm V1 is a new member of the provisional family Polymycoviridae.(2)Strain LS-1 contained six ds RNA fragments.According to sequence analysis,it was found that the virus in the strain was mixed infection and belonged to three viruses,named Colletotrichum gloeosporioides Virus1(Cg V1),Colletotrichum gloeosporioides RNA Virus1(Cg RV1)and Colletotrichum gloeosporioides Partitivirus1(Cg PV1)respectively.Cg V1 is composed of a ds RNA,full-length 4912 bp(classified as ds RNA-L).Based on the phylogenetic analysis of Rd Rp,it was found that Cg RV1 was similar to(Tombusviridae)virus of tomato rosette stunt virus,and Cg RV1 contained a ds RNA fragment,the size of which was 297 5bp(classified as ds RNA-M).It is predicted to contain two ORF(ORF1 and ORF2),encoding a functional unknown protein and Rd Rp,respectively.Sequence homology search showed that Cg RV1 had the greatest similarity with Beauveria bassiana non-segmented RNAvirus1(Bb NRV1),with 71.11% Rd Rp amino acid similarity.Evolutionary analysis showed that the virus was a kind of unclassified virus which was distantly related to Partitiviradae.This part of the study has been published in Archives of Virology;Cg PV1 contains four ds RNA fragments(classified as ds RNA-S),each containing one ORF.The Rd Rp,encoded by ORF1 is similar to the virus in Partitiviradae family.ORF2 encodes a protein with unknown function,which is similar to the unknown protein of virus in Partitiviradae family.ORF3 and ORF4 have no homology with known virus sequences.Virions about 36-40 nm in diameter could be extracted from strain LS-1,and the nucleic acid extracted from virions showed that they contained four bands of ds RNA-S.Therefore,it is inferred that only viral Cg PV1 forms virions in LS-1,and wraps ds RNA-S,and further proves that the four bands of ds RNA-S belong to the genome of Cg PV1.Evolutionary analysis shows that Cg PV1 belongs to Partitiviradae family and is a new atypical bivirus.(3)The function of virus in LS-1 was preliminarily verified by virus elimination and horizontal transfer.The derived strains LS-1-Y41(containing virus Cg RV1 and Cg PV1),LS-1-Y42(containing Cg PV1)and LS-1-Y42-26(virus-free)were obtained by protoplast regeneration.Through biological comparison,it was found that compared with LS-1-Y41 and LS-1-Y42-26,LS-1 and LS-1-Y42 showed a declining phenotype,and microscopic observation showed that the mycelium was distorted slightly and the pathogenicity was obviously weaker;Cg PV1 was transferred horizontally to another avirulent Colletotrichum anthracis strain SYGJ3-1 by plate confrontation.It was found that the infection of Cg PV1 could slow down the mycelium growth of SYGJ3-1.To sum up,Cg PV1 can affect the growth of host and other Colletotrichum gloeosporioides,and make the host show declining phenotype.It was inferred that there was interaction between viruses in LS-1.When Cg V1,Cg RV1 and Cg PV1 coexisted and Cg PV1 existed alone,the host phenotype declined,but when Cg RV1 and Cg PV1 coexisted,the phenotype of the strain was normal.This study expands our understanding of virus diversity in Colletotrichum gloeosporioides and provides important materials for the exploitation of fungal virus biocontrol resources.