USE OF DEGENERATE PRIMERS IN THE POLYMERASE CHAIN-REACTION TO DETECT WHITEFLY-TRANSMITTED GEMINIVIRUSES

Geminiviruses are widely recognized as a serious threat to vegetable production in many tropical and subtropical regions. This has increased the need for accurate identification of these viruses. Geminiviruses are well suited to polymerase chain reaction (PCR) methods because they replicate via a double-stranded, circular DNA form. Degenerate PCR primers were designed to anneal to highly conserved nucleotide sequences identified in the genomes of 10 whitefly-transmitted geminiviruses. The PCR primers were tested for their effectiveness in the amplification of viral DNA fragments from the DNA-A and/ or DNA-B components of 15 previously uncharacterized geminiviruses from the Americas, the Caribbean Basin, and Africa. Symptomatic plant samples tested included tomato, bean, pepper, soybean, cassava, and four weed species. Two primer pairs designed to anneal to DNA-A amplified viral DNA from the monopartite, leafhopper-transmitted geminivirus, beet curly top geminivirus. PCR-amplified viral fragments were further characterized by Southern blot DNA-DNA hybridization analysis with geminivirus DNA probes, by restriction fragment length polymorphism analysis, and/or by cloning and sequencing. These methods also detected mixed geminivirus infections in two cases. PCR using these degenerate geminivirus primers offers a rapid means of geminivirus detection.