Detection by tissue printing hybridization of Pome fruit viroids in the mediterranean basin

Available data on the incidence and biodiversity of pome fruit viroids in the Mediterranean basin are limited. Before starting a research survey to fill this gap, a tissue-printing hydridization (TPH) method to detect Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd) and Apple dimple fruit viroid (ADFVd) has been developed and validated. Afterward, TPH was used in large-scale indexing of pome fruit viroids in Bosnia and Herzegovina, Malta, Lebanon and Turkey. A total of about 1,000 trees was randomly collected and tested. Positive results obtained by TPH were confirmed by at least one additional detection method (RT-PCR and/or Northern-blot hybridization) and viroids were finally identified by sequencing full-length cDNA clones. PBCVd was detected in 13%, 12.4% and 5.4% of the tested pear trees in Bosnia and Herzegovina, Malta and Turkey, respectively, showing a wider diffusion of this viroid than expected. In contrast, ASSVd was never detected and ADFVd was only found in symptomatic trees (cv. Starking Delicious) in Lebanon, confirming a restricted presence of these viroids in the Mediterranean basin. Altogether, these data support the use of TPH as an easy and valuable tool for exploring pome fruit viroid spread.