A Study On Methods For Detection Of Pathogens Of Rice Black-streaked Dwarf Disease

Rice is one of most important food crop in China, however, its production is often threatened by a variety of infectious diseases. Rice black streaked dwarf disease is one of most serious viral diseases and mainly distributed in Japan, Korea, Vietnam, and China. Until now, it has been found that the disease can be caused by single or mixture infection of two viruses. One is Rice black streaked dwarf virus (RBSDV), a recognized member of Genus Fijivirus, Family Reoviridae, mainly transmitted by small brown planthopper (SBPH), Laodelphax striatellus Fallen, in a persistent manner. Another is Southern rice black streaked dwarf virus (SRBSDV) or Rice black streaked dwarf virus 2 (RBSDV-2), a novel putative member of Genus Fijivirus, Family Reoviridae, mainly transmitted by white-backed planthopper (WBPH), Sogatella furcifera Horvath. The similarity of symptoms, the overlapping patterns of distribution, and the complexity of infection in fields make detection and diagnosis more difficult but rapid and accurate measurement of diseased plant and viruliferous planthoppers is pivotal for predicting and monitoring epidemics of the disease. Therefore, the methods for detection of these two viruses were developed in this study and the main results were listed as follows.1、Serological method:Based on the cloning of viral gene encoding RBSDV P8, the viral protein were expressed in prokaryotic system, purified, and used for preparation of antibody aginst viral P8. ELISA and Western blotting assays showed the antibody can specifically and strongly reacted with the extract of RBSDV- or SRBSDV-infected plants. Based on the titre of the antibody and the optimized reaction conditions, both ELISA and Dot-Blot methods were established and used to detect the proportion of viruliferous planthoppers, which provided supports for the disease control.2、High Resolution Melting Curve (HRM) method:Based on plenty of real-time quantity RT-PCR and multiple RT-PCR assays, a pair of primers were designed and the reaction conditions were optimized according to the properties of HRM system. Then a HRM protocol for simultaneous detection of RBSDV and SRBSDV was developed and used to detect these two viruses in field samples. The results showed that the method proved to be simple, rapid, sensitive and suitable for routine and effective detection and diagnosis of RBSDV and SRBSDV.