Molecular Identification And Pathogenicity Of Begomovirus Infecting Tomato In China

26 tomato samples showing leaf curl symptoms were collected in Zhejiang, Shanghai,Jiangsu,Shand0ng,Henan and Guangxi provinces of China.500-bp fragments were amplified from all the 26 samples by PCR using universal primer pair PA/PB specific for the genus Begomovirus.These isolates were grouped into three types according to their partial viral DNA sequences:Tomato yellow leaf curl virus (TYLCV),Papaya leaf curl China virus(PaLCuCNV) and Tomato leaf curl Taiwan virus(ToLCTWV),respectively.In addition,all isolates were not found to be associated with DNA B or DNAβmolecules.The full-length genomes of TYLCV isolates from Zhejiang,Jiangsu and Shandon provinces were sequenced and they shared high sequence identity(＞98%) and had more than 97%sequence identity with TYLCV-IL[IL:Reo](X15656).Phylogenetic relationship analyses reveal that although with little genetic variability,TYLCV can be clutered into two groups and all the TYLCV isolates from China belong to groupⅠ.The complete nucleotide sequences of PaLCuCNV isolates from Henan and Guangxi provinces were determined to be 2748(HeNZM1,FN256260) and 2738(GX4, FN297834) nt long,respectively.They shared 94.6%and 89.3%sequence identity with Papaya leaf curl China virus-[CN:G2](AJ558123).The full-length genome of ToLCTWV isolate ZJWZ5 was determined to be 2748 nt(FN256292) and it shared 99%sequence identity with ToLCTWV-[CN:ZJ16](AM698111).Infectious clone of TYLCV-[CN:SH2],PaLCuCNV-[CN:HeNZM1]and ToLCTWV-[CN:ZJ16]were constructed and agro-inoculated into Nicotiana benthamiana,N.tabacum Samsun,N.glutinosa,Solanum lycopersicum,Petunia hybrida,Cucumis sativus L.,Gossypium spp.,S.melongena and Capsicum annuum. TYLCV-[CN:SH2]and PaLCuCNV-[CN:HeNZM1]could induce severe leaf curling and stunting symptoms in these plants except C.sativus L.,Gossypium.spp.,S. melongena and C.annuum.Moreover,we verified that TYLCV could trans-replicate DNAβin N.benthamiana and S.lycopersicum plants and induced more severe symptoms with distortion and yellow vein.ToLCTWV-[CN:ZJ16]induced severe leaf curling symptoms only in S.lycopersicum but could not infect N.benthamiana,N. tabacum Samsun,N.glutinosa and P.hybridas.But,when co-inoculated with TYLCCNVDNAβ,it induced distortion and yellow vein symptoms in S.lycopersicum, severe symptoms in N.benthamiana,N.glutinosa and P.hybridas.20 tomato varieties were collected and resistance locus Ty-1,Ty-2 and Ty-3 are associated with these tomato varieties,respectively.When these tomato varieties were agroinoculated with TYLCV,different resistant phenotypes were found,and no co-relationship was found between infectivity and virus concentration.Based on alignment of the complete sequences of tomato infecting begomoviruses,three conserved fragments were selected.Primers were designed to amplify three conserved fragments and linked them by Overlap-PCR.Then the fragments were reverse-repeated and inserted into the binary vector pBin438 and then transformed into tomato by A.tumefaciens-mediated method via leaf disc,34 kanamycin-resistant transgenic plants were obtained and their resistance to viruses is being evaluated.