Detection of tobamoviruses by RT-PCR using a novel pair of degenerate primers

Authors: YUEYUE, LI - Yunnan Agricultural University; TAN, GUANLIN - Yunnan Agricultural University; LAN, PINGXIU - Southwest University; ZHANG, ANSHENG - Yunnan Agricultural University; LIU, YONG - Yunnan Agricultural University; Li, Ruhui; LI, FAN - Yunnan Agricultural University

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2018
Publication Date: 6/23/2018
Citation: Yueyue, L., Tan, G., Lan, P., Zhang, A., Liu, Y., Li, R., Li, F. 2018. Detection of tobamoviruses by RT-PCR using a novel pair of degenerate primers. Journal of Virological Methods. 259:122-128.

Interpretive Summary: Tobamoviruses are a large group of plant viruses that infect many economically important vegetable (e.g. melons, pepper, potato, tomato, squash), ornamental (e.g. orchids) and other (e.g. tobacco) crops. They usually cause severe diseases and losses. Their impact is increased because the same tobamovirus often infects numerous crops, and many crops can be infected by more than one related, but distinct, tobamovirus. In this study, a generic RT-PCR assay using universal primers was developed and optimized to detect a wide array of tobamoviruses. The assay detected nine different viruses infecting orchid plants, solanaceous crops and cucurbitaceous vegetables. The assay was further validated using pepper and tomato samples collected from field locations, from which five additional tobamoviruses were detected. Generic or broad spectrum diagnostic tests use resources efficiently and are especially beneficial for certification, quarantine, and resistance breeding programs.

Technical Abstract: A generic RT-PCR assay was developed for the universal detection of viruses of the genus Tobamovirus using a novel pair of degenerate primers designed based on conserved regions on replicase genes of 32 tobamoviruses. The assay detected nine tobamoviruses, including six Solanaceae-infecting subgroup tobamoviruses of Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus (ToMMV), Tobacco mottle green mosaic virus (TMGMV), Pepper mild mottle virus (PMMoV), Paprika mild mottle virus (PaMMV), one Orchidaceae-infecting tobamovirus of Odontoglossum ringspot virus (ORSV) and two Cucurbitaceae-infecting subgroup tobamoviruses of Cucumber green mottle mosaic virus (CGMMV) and Zucchini green mottle mosaic virus (ZGMMV), with high amplification efficiency, specificity and sensitivity. The assay was applied to detect tobamoviruses in pepper and tomato fields. Five viruses, PMMoV, TMV, ToMV, ToMMV and TMGMV, were detected from the pepper fields in single and mixed infections. Single infections of PMMoV, ToMV and ToMMV and mix-infection of ToMV+PMMoV were detected from the tomato fields. Among these viruses, PMMoV was first detected from tomato worldwide, while ToMMV was first detected from tomato plants in China. This generic assay is simple and cost-effective, and has potential to detect known and unknown tobamoviruses.