Developing a multiplex real-time RT-PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid

Authors: SOMBART, SUKHONTIP - Kasetsart University; REANWARAKORN, KANUNGNIT - Kasetsart University; Ling, Kai-Shu

Submitted to: Australasian Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2018
Publication Date: 12/1/2018
Citation: Sombart, S., Reanwarakorn, K., Ling, K. 2018. Developing a multiplex real-time RT-PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid. Australasian Plant Pathology. 47:615-621. https://doi.org/10.1007/s13313-018-0597-1.
DOI: https://doi.org/10.1007/s13313-018-0597-1

Interpretive Summary: Tomato is one of the most important vegetable crops in the world. In recent years, severe disease outbreaks caused by a diverse group of pospiviroids in tomato production have caused serious concerns from growers. With the seed-borne nature of pospiviroids in tomato seeds, demand for developing a sensitive and reliable seed health assay is extremely high. Scientist at the U.S. Vegetable Laboratory, in collaboration with researchers at the Kasetsart University in Thailand, developed a multiplex real-time RT-PCR that would allow simultaneous detection of two emerging viroids, Pepper chat fruit viroid (PCFVd) and Columnea latent viroid (CLVd), which would save time and money. It is expected that broader adaptation of this seed health testing system would ensure that only those clean seed lots are in the international seed trade and in grower fields, thus to control these emerging viroids from further spreading.

Technical Abstract: Tomato (Solanum lycopersicum L.) is one of the most important vegetables in the world and Asian countries are the center for hybrid tomato seed production. Seed-borne diseases, if not managed well through effective seed health assay, could seriously hamper the global seed trade. Pepper chat fruit viroid (PCFVd) and Columnea latent viroid (CLVd) are two emerging and economically important pospiviroid species infecting tomato and other solanaceous crops. With their greater sequence diversity from those of other common tomato-infecting pospiviroids, it is necessary to develop a new detection method that could effectively detect PCFVd and CLVd, useful for seed health assay. In the present study, we developed a multiplex real time (quantitative) reverse transcription-polymerase chain reaction (qRT-PCR) to detect PCFVd and CLVd in a single tube, thus to save time and money. Using known viroid-contaminated seeds, this multiplex qRT-PCR could detect CLVd and PCFVd from a single contaminated seed, suggesting its sensitivity for seed health assay. The effectiveness of this newly developed multiplex qRT-PCR for detection of PCFVd and CLVd was validated using field-produced tomato plant tissue or commercial tomato seeds.