Molecular Identification And Pathogenicity Research Of Cotton Leaf Curl Multan Virus

Geminiviruses are characterized by their geminate virion particles and circular single-stranded DNA(ssDNA) genome.Geminiviruses infect both monocots and dicots and have caused significant losses in many economically important crops.In China,many begomoviruses were identified in tomato,tobacco,squash,ageratum and malvastrum.Virus isolates GD37 was obtained from Hibiscus rosa-sinensis showing leaf curl and enation symptoms in Guangdong Province of China.Sequencing the total DNA-A of GD37 shows that it is most closely related to some isolates of Cotton leaf curl Multan virus(CLCuMV),such as G6,Nanning and Okra06,with more than 99%of sequence identity.Satellite DNA was also obtained from GD37 by special primers, which contains 1347 nts and shows the high sequence identity(99%) with G6, Nanning and Okra06.Therefore,GD37 was an isolate of CLCuMV.The infectious clones of CLCuMV GD37 and CLCuMV GD37 DNAβwere constructed and their infectivity was tested in host plants.Infectivity assay shows that CLCuMV GD37 alone could systemically infect Nicotiana benthamiana,N.tabacum, N.tabacum Samsun,and N.glutinosa without typical viral symptoms or mild.While co-inoculated with CLCuMV GD37 DNAβ,the virus can induce severe leaf curling, stunt phenotype in these host plants.Southern blot analysis shows that CLCuMV GD37 DNAβdepends on the helper virus for its replication and,in turn,is required for helper virus accumulation.In order to establish a silencing system in plants by using CLCuMV,we co-inoculate CLCuMV with heterogeneous satellite Tomato yellow leaf curl China virus(TYLCCNV) DNAβin N.benthamiana,the inoculated plants show leaf curling, enations and stunt,however,the efficiency is not as highas that with CLCuMV DNAβ.It means that CLCuMV can maintain the replication of heterogeneous satellite TYLCCNV DNAβ.When CLCuMV is co-inoculated in N.benthamiana with the silencing vector 2mDNA1-NbSu351,a virus induced gene silencing(VIGS) vector derived from heterogeneous satellite Tobacco curly shoot virus(TbCSV) DNA 1,the system leaves and stems result in white,showing the phenotype of Su silencing.It indicates that CLCuMV can maintain the replication of heterogeneous DNA 1 silencing vector and efficiently induce endogenous genes silencing.