The Identification And Multiplex RT-PCR Detection Of Pepper Virus Disease In Chongqing

Chongqing is one of the major pepper production and consumption areas in China.Viral diseases have caused serious damage to pepper by affecting the production and quality in the past few years. In order to investigate the major pathogens,278 pepper samples with virus-like infection symptoms were collected from twelve regions of Chongqing, such as Beibei, Pengshui, Wulong, Wanzhou, Shizhu,Wushan, Wuxi, Yongchuan, Jiulongpo, Fuling, Xiushan and Hechuan in 2013 and 2014. Using antibodies against Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV), Broad bean wilt virus 2 (BBWV-2), Tomato mosaic virus (ToMV) and Tomato spotted wilt virus (TSWV), the samples were detected by Dot-Enzyme Linked Immunosorbent Assay (Dot-ELISA). Resultes showed that the detection rate of CMV was the highest,56.47%. The detection rate of TMV was 33.45%. The detection rate of TSWV was the lowest,14.39%. Mixed-infection was common and diverse, three main types are included: TMV+CMV, TMV+TuMV and TMV+CMV+TuMV. Mixed-infection rate of TMV and CMV occupied 14.38% and was the highest. Among the twelve regions, pepper viral diseases occured most seriously in shizhu. The detection rates of six viruses were all over 30%.Specific primers were designed based on their conserved region obtained from multiple sequence alignment of the corresponding isolates in GenBank. Multiple RT-PCR detection system of (Pepper mild mottle virus,PMMoV), (Chilli vein mottle virus,ChiVMV), (Potato virus Y, PVY), TSWV and TuMV were established. The complete CP genes of PMMoV, PVY and TSWV and partical CP genes of ChiVMV, TuMV were amplified from these samples. The sequence comparison results showed that PMMoV-WL02 shared the highest identities (99.2%) with two Chinese isolates (PMMoV-BD and Guizhou), ChiVMV-WZ07 shared the highest identity (99.8%) with isolate (Pp3) from China, PVY-BB31 which belonged to PVYO shared the highest identity (98.5%) with Chinese isolate (HeB20) and TSWV-SZ21 shared the highest identity (99.6%) with two Chinese isolates (CG-1 and KM-T),geographical selection force has played a role in the evolution of the four viruses to some extent. At last, TuMV-WZ08 revealed the highest identity (99.5%) with two Chinese isolates (Baoji2 and Xianyang3).Peppers samples collected in Chongqing were commonly infected with TMV, CMV, TuMV, BBWV-2 and PMMoV by Dot-ELISA and molecular methods, mixed-infection was diverse. Thus, it is necessary to establish an optimized mulptiple RT-PCR system to identify the five main viruses. Comparison of multiple RT-PCR confirmed its accuracy, high efficiency and low cost with single RT-PCR.The main pathogens on pepper have been identified by Dot-ELISA and RT-PCR in Chognqing. Among these viruses, TuMV was detected on pepper for the first time in China, and PMMoV, ChiVMV, PVY and TSWV were firstly reported infecting pepper in Chongqing.