First report of Persimmon virus a in Italy

Extensive necrosis of the veinlets were observed in early summer of 2011 on both sides of the leaf blades of a Japanese persimmon (Diospyros kaki) tree growing in a private garden in the vicinity of Bari (Apulia, southern Italy). This tree (accession SSPI) hosted a previously uncharacterized cryptovirus whose presence was apparently unrelated to the vein necrosis condition as it occurred also in a large number of symptomless trees (Morelli et al., 2012). Highthroughput sequencing analysis of the SSPI virome, performed on dsRNA extracts with an Illumina platform, allowed the identification of four contigs which, upon BLAST analysis (Altschul et al., 1997) showed a 97% sequence identity at the nucleotide level with sequences of the putative cytorhabdovirus Persimmon virus A (PeVA, GenBank AB735628), recently described in Japan (Ito et al., 2013). The sequence of the largest contig (983 bp), spanning part of the polymerase gene and the 3'-UTR, was deposited in GenBank under the accession No. KM407515. To confirm deep-sequencing findings, the PCR primer set PeVAfor/Pe- VArev (5'-AGGATCATTACAAAATCCGTGAGG-3'/ 5'- TTCCCGAAAGACAATCTGTCCC-3'), intended to amplify a 250 bp product, was designed on the KM407515 sequence. An amplicon of the expected size was repeatedly obtained from the symptomatic (SSPI) but not from 10 symptomless trees. This product was cloned into pSC-A-amp/kan and custom-sequenced (Macrogen Europe, The Netherlands). BLAST analysis matched previous identification, as cloned sequences shared ca. 98% identity with those of the Japanese PeVA isolate. There was no amplification when RT-PCR assays were extended to the 10 symptomless persimmon trees. Whether or not PeVA is involved in the induction of vein necrosis remains to be ascertained. To our knowledge, this is the first report of PeVA in a country other than Japan.