A single-tube PCR assay for the detecting viruses and their recombinants that cause tomato yellow leaf curl disease in the mediterranean basin.

Tomato yellow leaf curl disease (TYLCD) is well known in Mediterranean countries, where it has been causing severe losses in tomato crops for decades. Until recently, two viruses (with several isolates) in the genus Begomovirus, family Geminiviridae, have been associated with the epidemics. Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). However, recombinants between these, such as Tomato yellow leaf curl Malaga virus (TYLCMalV), are spreading, and new methods for detecting all viruses present in the region are needed. By considering all DNA sequences available of viruses causing TYLCD in the Mediterranean basin,a PCR/RFLP protocol was developed that amplifies the intergenic region in a multiplex reaction, followed by digestion with AClI (=Psp1406I) restriction enzyme. This procedure generates an easily recognizable pattern on gels, with DNA fragments of specific size for each virus species and each recombinant. 800bp for TYLCSV, 410bp for TYLCV, 570bp for TYLCMalV and the order detected recombinants, 640bp for hypothetical recombinants of different type. This new methods gives, with a single reaction, an overview of the species present in the sample and will be usefull for screening the casual agents of TYLCD, as well as in breeding programs for resistance.