Rapid Detection of Rice Tungro Spherical Virus by RT-PCR and Dot Blot Hybridization

Abstract

Tungro disease of rice is very difficult to identify in the field, as characteristic symptoms are not expressed in all varieties. Moreover, physiological disorders mimic the disease symptoms quite often and make it more complex for diagnosis. In the present work, Rice tungro spherical virus (RTSV) was detected in diseased samples using sequence specific primers and probes. The disease symptoms observed were stunting, yellow to orange discolouration and twisting of leaf tips. A two step reverse transcription polymerase chain reaction (RT-PCR) and RNA dot blot hybridization methods were used to detect RTSV in rice plants. Total RNA was extracted from symptomatic and non-symptomatic rice leaf samples and was used to amplify the specific sequences of RTSV. Primers were designed based on the flanking regions of the coat protein gene sequences available in NCBI database. The same amplicon was cloned and further used as a probe to detect the virus through RNA-dot blot hybridization. Both the methods of diagnosis were demonstrated to be very specific in identifying the virus in diseased samples. As eight of the 26 tungro suspected but non-symptomatic leaf samples were diagnosed positive for RTSV, it confirmed further that viral symptoms do not correspond to the viral titer always.