Detection of subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction

The DNA of several monopartite and bipartite whitefly-transmitted (WFT) geminiviruses was amplified from a viral template present in infected leaves after either direct addition of clarified plant extracts to an otherwise complete polymerase chain reaction (PCR) mix or after immobilization of template to microfuge tubes. A degenerate primer pair was designed to specifically target the middle or 'core' region of the capsid protein gene of subgroup III geminivirus isolates and amplify a viral DNA fragment of approximately 550 bp. Using this method, a single PCR product of the expected size (550 bp), as estimated by agarose gel electrophoresis, was amplifiable from plants infected with a representative set of subgroup III geminivirus isolates with a broad biogeographic base. That the 550-bp PCR product had a geminiviral gene origin was demonstrated by direct sequencing of the 550-bp fragments (yielding approximately 470 to 490 bases of informative sequence) and was validated through comparison (alignment) of the sequences with the published DNA sequences of several well-characterized WFT geminiviruses. Analogous viral fragments were not detectable by PCR with the subgroup III core coat protein primers and extracts of plants infected with either subgroup I or II geminivirus isolates. The demonstrated exclusive specificity of the assay for subgroup III geminiviruses offers a highly simplified PCR- based assay that permits the detection of a geographically diverse collection of WFT geminiviruses infecting cultivated crops, ornamentals, and weed hosts with minimal sample preparation. This approach is highly useful for the amplification of subgroup III geminiviral DNA templates from total nucleic acid extracts from infected plants and partially purified virion preparations.