The Identification Of Pathogens Causing Common Vegetable Virus Diseases And Establishment Of Multiplex RT-PCR Detection System

Vegetable viral disease is one of the most important diseases in our country’s vegetable industry. Viruses infection resulted a great reduction of quality and production of many vegetables. Professor Wei Ningsheng had identified the vegetable viral diseases in Shaanxi Province from 1950 s to 1960 s. In the 1980 s, Professor Wu Yunfeng(Northwest A&F University) indentified several viral pathogens which were associated with viral diseases of vegetables from Cruciferae and Solanaceae. Since then, there were few studies on vegetable virus disease. At present, the field incidence of main vegetable(Brassicaceae, Solanaceae, Leguminosae, Cucurbitaceae 4 large families) viral diseases in Shaanxi and Gansu Province is still not very clear. On the other hand, development of modern agriculture has changed the cultivation patterns of many vegetables. This provides a possibility for viruses escaping from the overwintering stage and starting a new infection cycle, accelerating the spread of viral diseases. Therefore, it is necessary to do more research on vegetable viral diseases, in order to provide reliable guidelines for vegetable production.We investigated vegetable viral diseases in seven vegetable growing regions of Shaanxi and six of Gansu from 2014 to 2015. A total of more than 2,000 vegetable samples were collected, including cabbage and carrot in Cruciferae, white gourd, sponge gourd and cucumber in Cucurbit, tomato, pepper and eggplant in Solanaceae, bean and cowpea in Leguminous. The results of Dot-ELISA showed that TMV occurred in almost every vegetable growing area and TMV widely distributed in the Shaanxi and Gansu. CMV mainly infected cucumber plants but was occasionally detected in Solanaceous vegetables. TuMV was only found in cabbage samples in this research. TSWV had high incidence in Solanaceae vegetables and BBWV primarily damaged cowpea, occasionally infect tomato but causing few symptoms. CGMMV could be detected in Cucumber.Results of RT-PCR indicated that RMV, CaMV, ORMV, ToMV and TZSV were not detected in major vegetable growing regions of Shaanxi. Additionally, PVX widely occurred in potato, tomato and pepper and PVY mainly infected potato. Both of ZYMV and SqMV had high incidences of infection in Cucurbit crops while the incidence of WMV was very low. ToCV widely occurred in greenhouse-grown tomato plants.Mixed infection of viral pathogens is very general on vegetables. In order to ensure the yield and quality of vegetables, it is imperative to develop a sensitive, accurate and rapid method for identification of numerous viruses causing vegetable diseases. In this study, according to symptoms of viral diseases in the main growing area in Shaanxi and Gansu Province, vegetable viral pathogens were first determined using Dot-ELISA and single RT-PCR(sRT-PCR). According to the viral species, two multiplex RT-PCR(mRT-PCR) systems for simultaneous detection of three Cruciferae vegetable viruses TMV, CMV and TuMV and four Solanaceae vegetable viruses TMV, CMV, TSWV and PVX were established, respectively. The mRT-PCR system was optimized in terms of the concentrations of the main ingredients such as Taq DNA polymerase, dNTPs, primers, Mg2+and PCR conditions including annealing temperature, extension times and amplification cycles successively. In order to verify the accuracy of mRT-PCR results, the PCR products of two multiplex RT-PCR system were purified and then sequenced. The sensitivity of each mRT-PCR system was very close to corresponding sRT-PCR method. Overall, two less time-consuming, more efficient and type-specific mRT-PCR systems were established and could detect a variety of vegetable viruses more efficiently.In this study, through investigation of vegetable virus diseases, viral pathogens causing common vegetable viral diseases were first indentified in Shaanxi and Gansu Province.. Moreover, two rapid detection system of 3 and 4 kinds of vegetable viral diseases were established, respectively. The mRT-PCR systems are able to detect a variety of viral diseases simultaneously, and has been successfully applied to detect vegetable samples in the field.