Six viroids have been reported to infect grapevine and among them only Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2) are able to induce yellow speckle (YS) disease, characterized by yellowish flecks scattered on the leaf blade or massed along the veins. The mixed infection of GYSVd-1 or GYSVd-2 with Grapevine fanleaf virus (GFLV) may elicit vein banding (VB), a syndrome characterized by chrome-yellow flecks along the main veins of mature leaves and expanding through the interveinal tissues of infected leaves. In the present study, we have developed a multiplex RT-PCR (mRT-PCR) for simultaneous detection of GYSVd-1, GYSVd-2 and GFLV. Hop stunt viroid (HSVd), another viroid that infecting most grapevines worldwide is used as a positive control instead of a host- derived internal control. The test developed here proved to be reliable for simultaneous detection of three pathogens, i.e. GYSVd-1, GYSVd-2 and GFLV, in grapevine; hence it is useful for detection of YS and VB in large-scale surveys and certification programs.
