RESUMEN
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.
RESUMEN
Apple stem pitting virus (ASPV) is an important latent virus of apple trees transmitted by grafting. In pear trees, ASPV is associated with pear vein yellows and pear necrotic spot diseases. Symptoms consist of chlorotic leaf banding and red mottling and flecking along the veins and necrotic spotting in some cultivars may also occur (4). During the spring of 2007, chlorotic leaf banding was observed in Bartlett pear (Pyrus communis L.) trees from one orchard in Bahía Blanca (Buenos Aires Province) and in Anjou, Packham, Abate Fetel, and Bartlett pears in another orchard in General Roca (Río Negro Province). The percentage of symptomatic plants was 10% in both cases. Pooled samples consisting of eight leaves per tree, 25 samples from Bahía Blanca and 25 samples from General Roca, were tested by double-antibody sandwich (DAS)-ELISA with a polyclonal antiserum from BIOREBA AG (Reinach, Switzerland). Five samples from Bahía Blanca and ten from General Roca were positive by DAS-ELISA. Only four positive samples by DAS-ELISA were also positive by immunocapture-reverse transcription (RT)-PCR using virions trapped in a microcentrifuge tube (3). A fragment of 370 bp was amplified with specific primers from each of these four samples. Amplicons were cloned and the nucleotide sequences were determined for one clone of each sample (GenBank Accession Nos. GQ356781, GQ356782, GQ356783, and GQ356784). All sequences had the highest identities with coat protein genes of ASPV. One of them was 94% identical with the coat protein gene of isolate PA66 isolate from Germany (GenBank Accession No. D21829.1) (1). Losses in pear by ASPV have not been demonstrated yet in Argentina. However, when the virus is present with other virus or virus-like diseases, a synergistic effect may occur and growth reduction may exceed 50% (2). Because of the mild symptoms in pear plants, nurserymen or growers must take care when they select material for propagation, in part because laws requiring virus-free propagation material do not exist in Argentina. To our knowledge, this is the first report of ASPV in pears in Argentina. References: (1) W. Jelkmann. J. Gen. Virol. 75:1535, 1994. (2) A. L. Jones and H. S. Aldwinckle. Compendium of Apple and Pear Diseases. The American Phytopathological Society, St. Paul, MN, 1990. (3) W. Menzel et al. J. Virol. Methods 99:81, 2002. (4) M. Németh. Virus, Mycoplasma and Rickettsia Disease of Fruit Trees. Martinus Nijhoff Publishers, Dordrecht, the Netherlands, 1986.