植物病理学および微生物学ジャーナル

概要

Development of Molecular based Method for Specific Detection of Begomovirus Species Causing Tomato Leaf Curl in the Philippines

Jeremy Roy Augustus I Gomez*

Tomato is one of the most important vegetable crops due to its economic and nutritional value but due to a disease caused by a Begomovirus, tomato production declined over the years. Begomoviruses causing tomato yellow leaf curl or tomato leaf curl affect tomato production in many tropical and subtropical areas of the world. Limited detection on these plant viruses made them so difficult to detect and control. The study aims to develop molecular based method for specific detection of ToLCPV and ToLCCeV. This was done by: 1) Validating whether the published primers could anneal to a specific Begomovirus species using multiple sequence alignment, 2) Designing a primers for specific detection of ToLCPV or ToLCCeV and optimized by PCR, 3) Validating the designed primer for specific detection of ToLCPV and ToLCCeV using field collected samples, 4) To identify RFLP pattern using full length genome DNA in silico. Published primers were able to anneal to ToLCPV strains and ToLCCeV, ToLCMiV species and thus these primers need to be validated in an actual experiment to determine their specificity. Primers ToLCPV19 and ToLCCeV19 were designed to specifically detect ToLCPV and ToLCCeV species respectively and PCR was optimized to a different annealing temperatures: 53℃, 55℃, and 57℃. A ~ 200 bp amplicons were observed on tomato samples infected with ToLCPV and also tomato samples infected with ToLCCeV when using ToLCPV19 and ToLCCeV19 primers. Also, there was no difference in the band size and the intensity of the bands at different annealing temperatures. Validation of the designed primers were done but to only a few selected tomato samples and thus further validation should be done to determine the specificity of the designed primers. RFLP analysis showed that restriction enzyme EcoRI could distinguish ToLCPV, ToLCCeV, and ToLCMiV as it produced different number of bands per Begomovirus species.