Application of Multiplex RT-PCR for Simultaneous Identification of Tomato Spotted Wilt Virus and Thrips Species in an Individual Thrips on Chrysanthemum

Abstract

We have developed a simultaneous diagnostic method that can identify both the species of thrips and tomato spotted wilt virus (TSWV) that are problematic in chrysanthemum plants. This is a method of amplifying DNA by performing reverse transcription-polymerase chain reaction by simultaneously adding primers specific to TSWV coat protein (N) gene and primers specific to the internal transcribed spacer 2 region of Frankliniella occidentalis and F. intonsa using total nucleic acid extracted from one thrips. The sizes of DNA fragments for TSWV, F. occidentalis, and F. intonsa were 777, 287, and 367 bp, respectively. These results showed species identification of thrips and whether thrips carrying TSWV can be simultaneously confirmed. Further usefulness of the simultaneous diagnostic method was made from greenhouse survey at chrysanthemum greenhouses in Taean (Chungcheongnam-do) and Changwon (Gyeongsangnam-do) to investigate the identification of thrips species and the rate of thrips carrying TSWV. Of thrips collected from the greenhouses, 83.7% thrips was F. occidentalis and 72.9% F. occidentalis carried TSWV in Taean. Similarly, the diagnostic method showed that 92.2% thrips was F. occidentalis and 84.0% F. occidentalis carried TSWV in Changwon. These results confirm that F. occidentalis is a dominant thrips species and the thrips species plays a crucial role in the transmission of TSWV in chrysanthemum plants in the greenhouses. Taken together, this study showed a simple diagnostic method for thrips identification and epidemiological studies of the timing and spread of TSWV through thrips in chrysanthemum greenhouses in South Korea.