Identification and characterization of Apple dimple fruit viroid in Lebanon

Apple trees of cv. Starking Delicious producing fruits with depressed green spots 3-4 mm in diameter, scattered on the whole fruit surface and merging around the calyx, were observed in the Bekaa valley (Lebanon). These symptoms resembled those of apple dimple fruit and dapple apple diseases induced by Apple dimple fruit viroid (ADFVd) and Apple scar skin viroid (ASSVd), respectively. Dot-blot hybridization using digoxigenin-labelled riboprobes, multiplex RT-PCR amplification assay specific for detecting these viroids in both single and mixed infections and sequencing of the generated amplicons showed that the symptoms were associated exclusively with the presence of ADFVd, whereas ASSVd was never detected. Full length ADFVd cDNAs from one symptomatic apple tree were also amplified and cloned. Sequencing of four independent clones identified ADFVd sequence variants composed by 307 nt with limited variability. Multiple sequence alignment of these variants with those reported previously in databank allowed the identification of one additional polymorphic position in the viroid genome. To our knowledge, this is the first report of ADFVd in Lebanon and the first in a country other than Italy. Tissue-printing hybridization was successfully used for a large-scale survey for the presence of ADFVd and ASSVd in Lebanon, the results of which are presented and discussed.