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CHARACTERISATION AND DIAGNOSIS OF YAM POTYVIRUSES BY RT-PCR
CHARACTERISATION AND DIAGNOSIS OF YAM POTYVIRUSES BY RT-PCR E. CANNING and S.SEAL Natural Resources Institute, Central Avenue, Chatham Maritime, Kent ME4 4TB Background and Objectives Potyviruses are the most diverse of yam-infecting viruses [1],yam mosaic virus MV) and yam mild mosaic virus MMV) being especially important because of their high incidence and wide distribution amongst the tv most widely cultivated yams; Dioscorea rotundata-cayenensis and D. alata. A lack of knowledge on their variability, coupled with the recalcitrant nature of yam tissue, has resulted in diagnostic tests being unreliable. This situation severely hampers efforts to improve the yam crop by preventing the international exchange of breeding materials. In an effort to rectify this situation, an approach using PCR-based techniques has been employed and has resulted in an increased understanding of yam potyvirus variability and the development of reliable diagnostics. Materials and Methods Diagnosis of YMV and YMMV infection is performed by a modified method of the immunocapturereverse transcription-polymerase chain reaction (IC-RT-PCR) described by Mumford & Seal (in press). The RT-PCR is carried out in a single tube and includes positive controls to ensure that all stages of the RT-PCR function efficiently. Reagents are dried to allow storage at room temperature. Amplified viral products were cloned using the pGEM-T vector system (Promega) and sent for automated sequencing. Results and Conclusions By utilising a single-tube IC and combined RT-PCR, rapid, sensitive and reliable diagnosis of these viruses can be achieved, suitable for routine use in yam growing areas. Transfer of the test is undergoing trials in West Africa. Sequence obtained from yam potyvirus isolates from throughout Africa and Asia and from both domesticated and wild yams. The sequence indicates the existence of four distinct yam potyvirus groups. References Porth A, Lesemann DE, Vetten HJ, 1987. Journal of Phytopathology 120,188-183