Detection of Geminiviruses in Sweetpotato by Polymerase Chain Reaction

Abstract

Geminivirus infection of sweetpotato (Ipomoea spp.) germplasm acquired from foreign regions is common. Graft inoculation of the indicator host, Ipomoea setosa, is the accepted detection method for these viruses, but the assay is laborious and requires up to 8 weeks. When infected sweetpotato is subjected to meristem tip culture to eliminate these viruses, the eradication rate is low. In this study, a polymerase chain reaction (PCR) detection assay was developed for the detection of geminiviruses in a variety of sweetpotato cultivars. Different methods were evaluated to extract nucleic acids suitable for PCR from Ipomoea spp., and a reliable and simple extraction method was developed for large-scale sample preparation. PCR products of the expected sizes were amplified from infected plants using degenerate and virus-specific primers, but not from noninoculated indicator plants. PCR assays using three primer pairs detected nine uncharacterized isolates of the geminiviruses in sweetpotato from Asia and America. However, the best PCR result was obtained with degenerate primers SPG1/SPG2, which detected a Taiwan isolate of Sweet potato leaf curl virus (SPLCV-Taiwan) in a sample diluted to 10-9. Viral identities of three amplicons from SPLCV-Taiwan were confirmed by sequencing. The degenerate primers had a broader detection range than virus-specific primers; therefore, they were used to detect geminiviruses in in vitro plantlets and greenhouse-grown sweetpotato plants, and in several Ipomoea hosts. PCR was shown to be as reliable for virus detection as grafting.