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. Keywords: Viroid disease, viroid spread, pome fruit trees, detection methods, molecular hybridizatio

Molecular characterization of Turkish isolates of Pear blister canker viroid and assessment of the sequence variability of this viroid

A survey of the presence of Pear blister canker viroid (PBCVd) in pear trees in Turkey was carried out by tissue print hybridization. Four out of 74 trees (5.4%) from a pear germplasm collection tested positive and this result was confirmed by RT-PCR and sequencing. Molecular characterization of the Turkish PBCVd isolates from four different local pear cultivars, together with multiple sequence alignments and phylogenetic analyses including all PBCVd sequences reported previously, provided an insight in the sequence variability of this viroid and showed that: (i) a total of 98 polymorphic positions distributed throughout the molecule are present in PBCVd genomic RNA; (ii) some regions with presumed relevant functions are strictly preserved and, (iii) the geographic origin of the isolates and the host species apparently have a bearing on the sequence variability of the viroid. This study records the presence of PBCVd in Turkey and confirms that its geographic distribution has been underestimated, most likely because the majority of infected pear cultivars are symptomless