DETECTION OF GEMINIVIRUSES IN SWEET POTATO POLYMERASE CHAIN REACTION

Authors: Li, Ruhui; Salih, Sarbagh; Hurtt, Suzanne

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2004
Publication Date: 7/2/2004
Citation: Li, R., Salih, S.S., Hurtt, S.S. 2004. Detection of geminiviruses in sweet potato polymerase chain reaction. Plant Disease. 88(12):1347-1351

Interpretive Summary: Several viruses cause important diseases in sweet potato, and one of them is Sweet potato leaf curl virus (SPLCV), a geminivirus. To prevent the introduction of viral pathogens during the importation of germplasm, sweet potato materials imported into the United States must be tested for viruses by the Plant Germplasm Quarantine Office (PGQO) of USDA-ARS. The current method to detect geminiviruses is to graft the material onto a susceptible indicator plant, Ipomoea setosa, in summer. The geminiviruses induce leaf curl in the indicator plant two to four weeks after grafting. To ensure the accuracy, this biological assay has to be repeated. This process is laborious, and takes three months. Thus, the current method is laborious and expensive, and requires a long time period, thus slowing the introduction of germplasm. Subsequently, any virus-infected material undergoes meristem tip culture, normally requiring six months to a year, to eliminate the virus. To get a virus-free plant, many plantlets generated from meristem tip culture need to be grown out and tested by the biological assay. Thus it is necessary to develop a rapid and sensitive virus detection assay suitable for testing large numbers of plants. We have developed a molecular technique to detect the geminiviruses in sweet potato plantlets and greenhouse-grown plants. This method is sensitive and reliable, and is completed in two days instead of three months. The method has a broad detection range, and can detect not only SPLCV but also other geminiviruses. Moreover, it could be used year-round, thereby speeding up dissemination of healthy plant material to the intended users and greatly reducing costs. This method should be of value to other scientists interested in detection of geminiviruses.

Technical Abstract: Geminivirus infection of sweet potato (Ipomea setosa) germplasm acquired from foreign regions is common. Graft-inoculation of the indicator host, I. setosa, is the accepted method of detection for these viruses, but the assay is laborious and takes up to eight weeks. When infected sweet potato material undergoes meristem tip culture to eliminate the viruses, the eradication rate is low. In this study, a polymerase chain reaction (PCR)-based detection system was developed for detection of geminiviruses in a variety of sweet potato cultivars. Different methods were evaluated to extract suitable nucleic acids for PCR assays, and a reliable and simple extraction procedure was developed for large scale sample preparation. PCR assays using degenerate primers and virus-specific primers were tested for the virus detection. PCR products of predicted sizes were amplified from infected plants using these primers, but not from healthy plants. Viral identities of three amplicons from a Taiwan isolate were confirmed by sequencing. PCR assays using three primer pairs detected a range of nine uncharacterized isolates of the geminiviruses in sweet potato from Asia and America. However, the best PCR result was obtained with degenerate primers SPG1/SPG2, which detected Sweet potato leaf curl virus in a sample diluted to 10-9. The PCR assay using the degenerate primers also had a broader detection range than those using the virus-specific primers in detection of the geminiviruses and it was used to detect geminiviruses in in vitro and greenhouse-grown sweet potato plants, as well as in tissue from several Ipomoea hosts. The PCR assay was found to be as reliable as the biological assay. Moreover, it could be used year-round, and required no greenhouse space for growing the indicator plants. This detection system will accelerate detection of the geminiviruses and subsequent selection of virus-free plants after in vitro therapy. It can also facilitate the development of more efficient therapy for eradication of the geminiviruses in sweet potato.