EVALUATION OF PCR AND NONRADIOACTIVE MOLECULAR HYBRIDIZATION TECHNIQUES FOR THE ROUTINE DIAGNOSIS OF TOMATO LEAF CURL NEW DELHI VIRUS TOMATO YELLOW LEAF CURL VIRUS AND TOMATO YELLOW LEAF CURL SARDINIA VIRUS
A. Alfaro-Fernández, J.A. Sánchez-Navarro, M. Landeira, M.I. Font, D. Hernández-Llópis, V. Pallás
The begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) has been reported as a causal agent of leaf curl disease in tomato and other solanaceous crops and, more recently, affecting different cucurbitaceous crops. ToLCNDV was first detected in Asia and recently in Europe, in 2013. In the present analysis, we have evaluated the PCR and the non-radioactive nucleic acids spot hybridization (NASH) techniques together with two nucleic acids extraction protocols, for the routine diagnosis of ToLCNDV and its discrimination from the closely related Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV). A protocol, designed to extract only the DNA, gave the best results with the PCR technique meanwhile the use of silica, which favors total nucleic acids extraction, was the best extraction protocol for the NASH. All allowed the specific detection of ToLCNDV but only some of the general begomovirus primers allowed the detection of all three viruses. The two ToLCNDV riboprobes analyzed by NASH, targeting the replicase and the coat protein genes, respectively, detected the virus with no cross-reaction with the TYLCV and TYLCSV infected extracts, obtaining a better detection limit with the replicase riboprobe. Direct comparison between the PCR and NASH techniques by the analysis of 42 field samples, revealed a good correlation between the two techniques. In addition, some samples were detected only by NASH due the presence of PCR inhibitors. The use of the PCR and NASH for the routine diagnosis of ToLCNDV is discussed.