Development of a protocol for the identification of tospoviruses and thrips species in individual thrips

A protocol for identifying tospovirus and thrips species in an individual thrips sample was successfully developed. First, an individual thrips was soaked in an RNA stabilization solution to preserve protein and nucleic acids and ground in a carbonate buffer containing 0.2% sodium diethyldithiocarbamate. Initially, the thrips extracts were screened for tospovirus infection by dot blot analysis using antibodies to nucleocapsid (N) proteins of tospoviruses. Thrips extracts with positive results by dot blot analysis were further subjected to RNA extraction. Next, tospovirus species were identified by reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers for the N genes of four tospoviruses known to occur in Thailand, including Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV) and Watermelon silver mottle virus (WSMoV). The residual genomic DNA in the thrips RNA extract was used as a template to identify thrips species by PCR with species-specific primers to the internal transcribed spacer 2 regions of the rRNA of Ceratothripoides claratris, Frankliniella intonsa, Scirtothrips dorsalis and Thrips palmi. This protocol was initially validated against laboratory-reared thrips and then used to determine the occurrence of viruliferous thrips species collected from tomato, pepper, watermelon and cucumber fields in Thailand.