A MULTIPLEX RT-PCR FOR SIMULTANEOUS DETECTION OF THE AGENTS OF YELLOW SPECKLE AND VEIN BANDING DISEASES IN GRAPEVINE

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.