Hop stunt viroid (HSVd), Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are known to infect the grapevine in nature (Little and Rezaian, 2003). Except for CEVd, the other viroids have been previously detected in vines grown in southern Iran (zaki-Aghl and Izadpanah, 2009). However, little information is available regarding their combination in mixed infections as well as their presence in other Iranian regions. During summer 2010, leaf samples were collected from 57 grapevine vines grown in north-west Iran and tested for viroid infections by RT-PCR using primers specific for each viroid species. Viroid identity was conclusively established by sequencing the amplified cDNAs. The large majority (96,2%) of the 53 positive samples had mixed infections, whereas single infections by HSVd or AGVd were detected in only two samples. HSVd, AGVd, GYSVd-1, and GYSVd-2 occurred in various combinations in 87.7%, 78.9%, 71.9% and 45.6% of the samples, respectively. HSVd was present in most double- and triple-infected vines, representing 15.8% and 38.5% of the samples respectively. Specifically, HSVd/GYSVd-1 and HSVd/AGVd were found in 7% of the samples, whereas the AGVd/GYSVd-2 combination was less frequent (1.8%). Vines contemporarily infected by HSVd, GYSVd-1 and AGVd were 29.8%, whereas the combinations HSVd/GYSVd-1/GYSVd-2 (3.5%) and HSVd/AGVd/GYSVd2 (5.3%) were less frequent. In contrast, double infections by AGVd/GYSVd-1 or GYSVd-1/GYSVd-2 and triple infections by AGVd/GYSVd-1/GYSVd.2 were not detected. Interestingly, 18 the 57 tested grapevines (31.6%) contained four different viroids species, so that ca. 70% of the tested grapevines were infected by three and four viroids. It was not possible to associate any of the viroid combinations with a specific symptomatology. Altogether, these data show that most grape-infecting viroids are widespread in north-west Iran.
