Molecular characterization of Hellenic variants of <i>Apple scar skin viroid</i> and <i>Pear blister canker viroid</i> in pome fruit trees

Abstract

Apple scar skin viroid (ASSVd) and Pear blister canker viroid (PBCVd) are members of the genus Apscaviroid (family Pospiviroidae). In order to study the nucleotide sequence and secondary structure of Hellenic variants of these viroids, a large number of collected samples were initially screened by imprint hybridization; then ASSVd and PBCVd positive samples were assayed for the viroids by RT-PCR. Total RNA extracts were reverse-transcribed and amplified by polymerase chain reaction using two different specific primer pairs for each viroid. Purified RT-PCR products were directly sequenced or cloned into the pGEM-T and pCR® II vectors and then sequenced. Fourteen Hellenic full length ASSVd variants from 3 apple, 3 wild apple (Malus sylvestris), 1 wild pear (Pyrus amygdaliformis) and 3 pear trees are 330-335 nucleotides long. They differ from the reference sequences of ASSVd (ASSCS and Y00435) at 15-29 and 3-36 sites, respectively. Fifteen nucleotide changes (differences from ASSCS) are common among all Hellenic variants. Hellenic ASSVd variants share high identity (97-100%) with ASSVd isolates from Asian apples. Three Hellenic variants, deriving from different hosts and areas, are identical with each other (wild apple and apple from Pella [Macedonia] and pear from Achaia [Peloponnesus]) and with another group of 3 apple variants from China (Liaoning, AM1 and B-9). Sixteen full length Hellenic PBCVd variants from 12 trees (4 apples, 1 wild apple, 5 pears, 1 wild pear and 1 quince) are 314-316 nucleotides long. There are 6-50 nucleotide changes among all Hellenic variants and the prototype PBCVd isolate (NC001830). Twenty-two (22) changes are identical among the majority of the Hellenic variants, regardless of origin, and 28-35 changes occur in PBCVd sequences obtained from apple and wild apple samples. In addition, 2 Hellenic PBCVd variants are 97-98% homologous to some Australian and European (Bosnian) PBCVd pear isolates, whereas the remaining 14 share 86-94% identity with Australian PBCVd isolates from pear, quince and Japanese pear (Pyrus pyrifolia). This is the first detailed molecular study of ASSVd and PBCVd in Hellenic cultivated and wild pome fruit trees.

Keywords: ASSVd, PBCVd, pome fruit, molecular characterization