DIGOXIGENIN-LABELED RIBOPROBES APPLIED TO PHYTOSANITARY CERTIFICATIONOF TOMATO IN ITALY

A dot-blot hybridization system using digoxigenin-labeled riboprobes a nd chemiluminescent detection was developed for the diagnosis of infec tions by cucumber mosaic cucumovirus (CMV), tomato spotted wilt tospov irus (TSWV), potato Y potyvirus (PVY), tomato yellow leaf curl geminiv irus (TYLCV), and, occasionally, alfalfa mosaic alfamovirus (AMV) and tomato mosaic tobamovirus (ToMV) in tomato seedlings. This system was successfully applied to sanitary certification purposes. Tomato sample s were collected using a systematic sampling method. The optimal ratio of tissue sample to extraction solution was 1.2 g to 6 ml. Hybridizat ion reactions were done using a riboprobe mixture. This procedure save d time and cut costs without reducing sensitivity. TSWV was detected u p to a ratio of 21 mu g of infected tissue per spot; whereas the other viruses were detectable at a ratio of 17 mu g of infected tissue per spot. The method allowed the analysis of 400 to 500 samples (represent ative of approximately 1.15 million tomato seedlings) per day and fulf illed the requirements for virus detection in routine diagnosis.