Identification Of Species Of Tobacco Aphid-transmitted Viruses In Sichuan Province And The Establishment Of LAMP

Tobacco aphid-transmitted virus is the important virus in tobacco planting areas all over the world, which could cause serious lost on the yield and quality of tobacco. Potato virus Y (PVY), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV), Tobacco vein banding mosaic virus (TVBMV) and Tobacco ring spot virus (TRSV) are the main tobacco aphid-transmitted viral pathogens. In order to develop effective prevention and control measures for tobacco aphid-transmitted virus, we indentify the aphid-transmitted viral pathogens in Sichuan region.The general survey of tobacco aphid-transmitted viral pathogens was carried out in tobacco production areas in Sichuan during 2012-2013.252 samples were collected in Guangyuan, Dazhou, Xichang, Panzhihua. Specific primers were designed and synthesized based on coat protein(CP) the nucleotide sequence of PVY, CMV, TEV, TVBMV, TRSV, respectively. The infection status of the two kind of virus(CMV, PVY) have been detected by RT-PCR. Among 252 tobacco samples,84 samples were infected by CMV and 87 samples were infected by PVY. The results showed that CMV detection rate is 33.33% and PVY is 34.52%. The complex infection of CMV and PVY is 16.17%.The target fragments about 657bp of CMV CP and 759bp of PVY CP were amplified by RT-PCR from the RNA template of infected samples. The amplified cDNA fragments were then cloned and sequenced. CMV is divided into two subgroups, subgroup Ⅰ and subgroup Ⅱ, subgroups Ⅰ can be further divided into IA and IB. According to the phylogenetic tree analyses of CP gene nucleotide sequences, CMV isolates of Sichuan belonged in CMV IB subgroup. The CMV isolates we obtained have a high homology with CMV isolates from vegetables and medicinal plants. The vegetables and other crops around tobacco fields may be the main source of CMV.Currently, PVY can be classified as three strains, tobacco ordinary strain(PVYO), tobacco vein necrosis strain(PVYN) and tobacco stipple streak strain(PVYc). The differentiation of PVY strains of Sichuan is obvious and there are three PVY stains including PVYN:O, PVYO and PVYN.Loop-mediated isothermal amplification(LAMP) detection of CMV and PVY were developed with SYBR Green I as a fluorescence indicator in this study. Four primers (F3 and B3, BIP and FIP) were designed according to CP gene of CMV and PVY, respectively. The reaction conditions of LAMP were optimized, the sensitivity of LAMP were testified. LAMP reaction was carried out at 63 ℃ for 40 min. The green color is regarded as positive while the orange is negative. The limit of LAMP detection was about 10"3 ng/μl, which was 100 times more sensitive than that of PCR. The detection of random samples collected in the field showed that LAMP we established is a accurate and reliable method.