Research And Application Of Methods For Detection Of Three Rice Viruses

Rice is one of the most important food crops in China, but its production is often affected by the viral diseases. So far it has been reported that more than10viral diseases occurred on rice plants in our country. Rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), and southern rice black-streaked dwarf virus (SRBSDV) have been epidemic in Jiangsu and Zhejiang provinces and caused ones of the most serious viral diseases in recent years. Detection and diagnosis of pathogen always is the basis of accurate forecasting and efficient controlling of crop diseases. Effective control of the3viral diseases also depends on rapid and accurate detection of3viruses. Therefore, here we established and developed the indirect ELISA, multiplex RT-PCR, RT-LAMP methods for detection of the3viruses.We firstly developed an indirect ELISA method for detection of RBSDV and SRBSDV using P8antibody, which was previously prepared in our laboratory. Experimental results showed that we get the best results of ELISA test when P8antibody was diluted at3000times, antigen was reacted with antibody for2hours, and color reaction was done for40minutes. We used these optimized condition for ELISA detection of white-backed planthopper (WBPH) samples, which collected from different region in Zhejiang Province. The results showed that the viruliferous rate of WBPH from Pujiang County was up to14.6%, more than those from any other counties in Zhejiang Province.Then we have designed three pairs of specific primers, which respectively matched the conserved sequences within the genomes of RSV, RBSDV and SRBSDV, and developed multiplex RT-PCR technology for simultaneous detection of the rice viruses. Gradient PCR experiments showed that the optimal annealing temperature is63℃. Specificity and sensitivity analysis showed that this method was simple, rapid, accurate, and sensitive for simultaneous detection of the three important rice viruses. The method was applied to detection of single planthopper and plant samples collected from fields. The results showed that, SRBSDV was detected in samples from Vietnam, Shanghai, Guangdong, Hainan, Hunan, Hubei and Zhejiang provinces. Only RBSDV was detected in maize plant samples from21counties and cities in Shandong Province and Lin’an city in Zhejiang Province, Only RBSDV was detected in maize plant samples from21counties and cities in Shandong Province and Lin’an city in Zhejiang Province. All three viruses were detected in rice plant and planthopper samples from Zhejiang Province, indicating there were the diversity and complex infection of rice viral pathogens in Zhejiang Province.Reverse transcription loop-mediated isothermal amplification technology (RT-LAMP) is a new technology for amplification of nucleic acids. Here we designed8specific primers according to the genomic sequences of RBSDV and SRBSDV S10, and developed a RT-LAMP method for detection of the two viruses. Experimental results showed that the technology was sensitive and specific for distinguishing between the two viruses and can be effectively used for detection of the viruses in single planthopper and diseased plant samples.In addition, we based on serological and RT-PCR detection methods and analyzed the transmission properties of RSV, RBSDV and SRBSDV by SBPH and WBPH.