First Report of Pear blister canker viroid in Pear Trees in Argentina.

During December 2009 and January 2010, pear trees (Pyrus communis L.) from five orchards located in Allen (Alto Valle) and one in Rio Colorado, both in Rio Negro Province in Argentina, were randomly sampled for Pear blister canker viroid (PBCVd). Ninety-six trees were tested, 20 of cv. Williams, 4 of Abate Fetel, 30 of D'Anjou, and 38 of Packhamn, that showed no symptoms of PBCVd, plus four trees of cv. Red Bartlett that exhibited symptoms of bark pustules and rounded, scaly cankers varying from 2 to 6 cm in diameter on the stems. Purified dsRNA from leaves of 96 trees were analyzed by dot-blot hybridization with a specific probe for PBCVd (2). Of the plants tested, 18 were positive for PBCVd. Three of the positive dsRNAs, with a higher dot-blot signal, were analyzed by electrophoresis in 5% nondenaturing polyacrylamide and a second denaturing polyacrylamide gel. A band, in the portion of viroids migration, was detected with ethidium bromide. The segment corresponding to the three bands was excised and electroeluted. Two-step reverse transcription (RT)-PCR was performed using Moloney-murine leukemia virus (M-MLV) reverse-transcriptase (Promega Corporation, Madison, WI), retrotranscriptase, and PBCVd primers F-5'-GCGGGACAGAAGACGAGGCTCAGGCAGGAAGCAAC-3' and R-5'-TATAAAAGAAAAAAGCGCTTCGGCGGTGCTCGGG-3' (3). The product was legated into pUC 19 vector (Fermentas Inc., Glen Burnie, MD) and cloned following the manufacturer's instructions. Four clones were sequenced by the Unidad de Genómica, Instituto de Biotecnología (Argentina) and the sequences were analyzed with the Lasergene Biocomputing Software for Windows (version 8.0.2; DNASTAR, Madison, WI). The four partial sequences of 296 nucleotides from the local sequences were identical to each other and had the highest nucleotide identity (99.7%) with the Spanish PBCVd (GenBank Accession No. D12823). The local sequence was submitted to GenBank (Accession No. HQ606079). PBCVd is a member of the genus Apscaviroid within the family Pospiviroidae. PBCVd is a 316-nucleotide viroid responsible for pear blister canker disease. It causes pustules, cankers, and/or bark symptoms on the pear indicators A 20 and Fieud 37, whereas infections on most commercial pear cultivars remain symptomless (1). These lesions are entry points for other pathogens to infect the plant. This research indicates the need to test pear propagation material in Argentina, since this is the primary way of spreading this pathogen. To our knowledge, this is the first report of PBCVd in Argentina. References: (1) J. Desvignes et al. Plant Dis. 83:419, 1999. (2) R. Flores et al. J. Gen. Virol. 72:1199, 1991. (3) C. Hernandez et al. J. Gen. Virol. 73:2503, 1992.