First Report of Tomato blistering mosaic virus Infecting Tomato in Argentina

Tomato (Solanum lycopersicum) ranks as the most important crop among greenhouse-planted crops in terms of cultivated areas (17,800 ha) and production in Argentina. In September 2014, farmers reported that tomato plants of cv. Elpida (Enza Zaden) showed mosaic and blistering on leaves in La Plata (Buenos Aires green-belt). The diseased plants occurred together in the same line, showing a typical pattern of mechanically transmitted pathogens. Although incidence did not exceed 1.5%, it was distributed in several distant locations within the district, posting a serious threat to the tomato production. Five samples were collected from symptomatic plants and tested for Tospovirus, Begomovirus, and Cucumber mosaic virus by reverse transcription polymerase chain reaction (RT-PCR) (Dewey et al. 1996), PCR (Rojas et al. 1993), and enzyme-linked immunosorbent assay (ELISA), respectively. All results were negative. The samples were then examined under transmission electron microscopy. Isometric virus-like particles of 25 to 30 nm in diameter were seen in leaf-dip preparations, and isometric virus-like particles aggregated in the cytoplasm of mesophyll cells and alterations of chloroplast membrane in association to virus-like particles were observed in ultrathin sections suggesting a Tymovirus. To identify this virus, leaf and stem samples were dot blotted on a nitrocellulose membrane and incubated with Tomato blistering mosaic virus(ToBMV) antiserum, kindly provided by Dr. Alice K. Inoue-Nagata (Embrapa-Horticulture, Brazil). The samples reacted to the ToBMV antiserum, indicating the presence of ToBMV or a ToBMV-related virus. Sap of the diseased samples was used to inoculate tomato cv. Elpida and several indicator plants (Datura stramonium, Nicotiana benthamiana, Nicotiana tabacum cv. TNN, Capsicum annuum cv. Almuden, and Chenopodium quinoa). The inoculated tomato plants showed foliar chlorosis, mosaic, necrosis, and blistering similar to those observed in the greenhouses, and the indicator plants reproduced the symptoms reported by de Oliveira et al. (2013). Furthermore, two samples were subjected to RT-PCR using primers specifically designed to amplify the complete capsid protein (CP) from ToBMV: TymoF3RaceFor, 5'-TCCAATCCATGCCTTTTGAT-3'; and ToBMV3EndNdeIRev, 5'-TTCATATGGTTTCCGATACCCTCGG-3'. DNA fragments of expected size (1000 bp) were amplified, cloned in pGEM-T easy (Promega), and sequenced. Sequence analysis showed the nucleotide sequences of the two isolates