Grapevine is the natural host of at least five viroid species, i.e. Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd). Some species (HSVd, GYSVd-1, GYSVd-2) are of worldwide occurrence, whereas others (AGVd and CEVd) seem to be more restricted geographically. The lack of fast and reliable laboratory procedures for the contemporary detection of multiple viroidal infections is an important limitation for studying the distribution and relative incidence of these viroids in grapevine-growing areas. To fill this gap, a multiplex RT-PCR (mRT-PCR) assay has been developed for the contemporary detection of the five viroids and the simultaneous amplification of the cDNA fragment of a host-derived mRNA (actine mRNA), as an internal positive control. The method was validated testing 57 grapevine plants from Iran infected by one or more viroid species. Concordant results were obtained by our mRT-PCR protocol and single RT-PCR (sRT-PCR) tests conducted using primers previously reported for amplifying separately cDNAs of the monomeric full-length genomic RNA of each targeted viroid. Negative samples were further assayed by Northern-blot hybridization. Except for CEVd, in this survey HSVd, AGVd, GYSVd-1, and GYSVd-2 were detected in 100, 95, 93 and 65% of the tested samples, respectively, confirming their wide distribution in Iran. The present mRT-PCR protocol has the potential to be used routinely for large-scale surveys and certification programs.
