Comparative analysis of techniques for detection of grapevine and citrus viroids in Tunisia

Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of Citrus exocortis viroid (CEVd) in total nucleic acids from infected citrus indicator hosts or directly from field trees. Grapevine yellow speckle viroid-1 (GYSVd-1) and Australian grapevine viroid (AGVd) were also detected by this technique in total nucleic acids from infected grapevine plants. Three protocols for nucleic acid extraction were tested: a phenol-based method, a protocol using a commercial reagent (Trizol) and a method in which extraction with a high-salt buffer was combined with partial purification using non-ionic cellulose. The third protocol, coupled to RT-PCR, seems to be the most suitable, allowing detection of the three viroids in Tunisian citrus and grapevine samples. This method should be used in a testing programme to produce certified viroid-free planting material.