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Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun
Published on: June 12, 2010
First Report of Iris yellow spot virus on Onion in Uruguay
P Colnago1, R Achigar2, P H González3
1Plant Production Department, Centro Regional Sur (CRS), Facultad de Agronomía, Universidad de la República, Camino Folle km 36, Progreso, Canelones, Uruguay.
Abstract:
From October to December 2005, onion (Allium cepa) plants in seed-production fields in south Uruguay (Canelones) had symptoms suggestive of those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae). Symptoms included diamond-shaped lesions on seed stalks (scapes), each 1 to 5 cm long with a necrotic border, green center, and sometimes a second necrotic area in the center of the diamond (2,3). Necrotic lesions with more irregular shape were also associated with diseased plants. In 2006, scape samples with these symptoms were collected from four onion seed crops and assayed for IYSV using an IYSV-specific antiserum (Agdia Inc., Elkhart, IN) in a double-antibody sandwich-ELISA. IYSV was detected in all four onion seed crops monitored in 2006. IYSV incidence, expressed as the number of plants with symptoms, ranged from <1% (1 of 120 plants evaluated) to 20% (24 of 120 plants). Two fields were monitored in 2007, in which IYSV incidence increased from 2 and 3% in October to 7% (198 of 2,768 plants) and 40% (253 of 638 plants) in December, respectively. The highest incidence was observed in the same farm in 2006 and 2007. Scapes were sampled from the field with the highest incidence of symptoms in 2007 and tested for IYSV with IYSV-specific primers (3). Total RNA was extracted from 100 mg of symptomatic tissue, with green tissue adjacent to typical lesions, following a Trizol-based protocol (1). A reverse transcriptase-PCR assay with nucleocapsid gene-specific primers was used (3). A PCR product of approximately 26 bp was obtained, coincident with the expected length for IYSV. The PCR product was cloned and sequenced. The tospovirus N sequence of the isolate in Uruguay (Accession No. GU550518) had maximum identity (97%) with an Australian IYSV isolate (Accession No. AY345227), and >87% identity only with IYSV N protein sequences in GenBank. Because of the presence of IYSV in Brazil, Chile, and Peru, this first documentation, to our knowledge, of IYSV in onion crops in Uruguay suggests that the threat of IYSV to onion is increasing in South America. References: (1) P. Chomczynski and K. Mackey. Biotechniques 19:942, 1995. (2) D. H. Gent et al. Plant Dis. 90:1468, 2006. (3) H. R. Pappu et al. Plant Dis. 92:588, 2008.
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