Molecular detection of whitefly-transmissible geminiviruses (Geminiviridae: Begomovirus) from selected areas in the Philippines

This study was conducted to detect whitefly transmissible geminiviruses (Geminiviridae: Begomovirus) from selected areas in the Philippines by amplifying Begomovirus DNA fragments by with the polymerase chain reaction (PCR), to confirm the identity of the amplified DNA fragments Southern hybrization, and to differentiate the amplicons by digestion with five restriction endonucleases. Leaf curls samples of tomato and squash were obtained from different areas of the country and each sample was labeled as a collection. Begomovirus DNA fragments were amplified by PCR from total DNA extracts of different leaf curl samples using three sets of degenerate primers that amplify different regions of the degenerate primers genomic A DNA component of Begomoviruses. Not all samples produced amplicons/PCR products of the expected sizes in PCR. Some samples produced bands in all three primer sets, some with only two of the three sets of primers, while some produced PCR fragments with only one of the three primer pairs, which suggests variation in the virus DNA sequence in terms of the presence or absence of primer annealing sites. Southern blot analysis using as probe the PCR product amplified from top half clone of the A DNA component of the Philippine isolate of tomato leaf curl geminivirus (TYLCV-Ph) confirmed the Begomovirus identity of the top half primer pair-generated amplicons. Digestion of the top halfamplicons with five different restriction endonucleases (Alul, Hhal, Hpa2, Hind3, and Sau3AI) revealed different banding profiles of agarose gel-resolved DNA digests. Total size of the cut fragments exceeded that of the original uncut fragment which suggests the presence of more than a single Begomovirus isolate in a sample, which have not been discriminated by PCR. In some instances, the presence of uncut fragments was observed and this could be attributed to incomplete enzyme digestion or it could represent a DNA sequence that lacks the restriction site for a particular endonuclease or both. Results of this study detected and confirmed the presence of begomoviruses in leaf curl diseased samples from selected areas in the Philippines