First report of tomato mottle mosaic virus infecting tomato in the United States.

In June 2010, eight tomato samples were collected from a commercial tomato field in southwest Florida (Collier County) with virus-like symptoms of necrotic spots on leaves and necrotic areas on petioles and stems, typical of Groundnut ringspot virus (GRSV) infection (5). Although six samples tested positive for GRSV using previously described methods (5), systemic symptoms of leaf chlorosis and deformation developed on Nicotiana benthamiana and necrotic local lesions developed on N. glutinosa four days postinoculation with one of these samples. As symptoms developed more quickly than expected for a tospovirus, but more typical of a tobamovirus, the N. benthamiana plant was tested using commercially available lateral flow immunoassay reagents for Tobacco mosaic virus (TMV; Agdia, Inc., Elkhart, IN). A positive result indicated tobamovirus infection. In November 2012, nine tomato samples with virus-like symptoms of foliar mosaic of light and dark green and deformation of leaves (Fig. 1) were collected from a commercial greenhouse in southeast Florida (St. Lucie County). Six samples tested positive for TMV using commercially available enzymelinked immunosorbent assay reagents (ELISA; Agdia). No Cucumber mosaic virus, Tomato spotted wilt virus, or potyviruses were detected using ELISA (Agdia). To identify the specific tobamovirus detected in both 2010 and 2012 samples, total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from symptomatic tomato leaves and tested by reverse transcription-polymerase chain reaction (RTPCR). Since Tomato mosaic virus (ToMV) commonly infects tomato in Florida (1), previously described ToMV coat protein (CP) gene primers (3) were initially used. When no ToMV RTPCR products were obtained, previously described degenerate solanaceous tobamovirus primers SolACPv and SolACPvc (2) were used to amplify a portion of the movement protein (MP) and the entire CP genes from two 2010 and two 2012 samples. RT-PCR products from one sample from each year were cloned (pGEM-T, Promega, Madison, WI), sequenced and submitted to GenBank (KM000122 and KM000123). These 662 nt clones (encompassing 180 nt of the MP gene, a 2 nt intergenic region, and the entire 480 nt CP gene) shared 99% nucleotide identity with each other and with the type isolate of a novel tobamovirus, provisionally named Tomato mottle mosaic virus (ToMMV; GenBank accession KF477193). ToMMV was isolated from a greenhouse tomato sample collected in 2009 in Mexico (4) and is closely related to, but distinct from, ToMV. The partial MP gene of the 2010 isolate was 100% identical to the ToMMV type isolate, while the 2012 isolate had 98% nucleotide and 96% deduced amino acid identity. The CP genes of 2010 and 2012 isolates shared 99% nucleotide and 100% deduced amino acid identity with the ToMMV type isolate. A N. tabacum ‘Xanthi nc’ local lesion-passaged ToMMV isolate from one 2010 sample was purified and examined by electron microscopy and indicator host inoculation. Typical tobamovirus virions were observed by electron microscopy and induced a mild mottle on upper, noninoculated leaves when used to mechanically inoculate tomato. Necrotic local lesions were induced on inoculated leaves of N. glutinosa, N. sylvestris, N. tabacum ‘Xanthi nc’, and Chenopodium quinoa. Systemic mosaic symptoms and systemic chlorosis and leaf deformation were induced on upper, noninoculated leaves of N. tabacum ‘Xanthi’ and N. benthamiana, respectively. No symptoms were induced in Datura stramonium or Solanum americanum although TMV ELISA testing revealed both species were systemically infected. To the best of our knowledge this is the first report of ToMMV in the United States. Comparison of several partial ToMV CP gene sequences in GenBank from pepper in Brazil (AM411425, AM411430), and tomato in China (JX025564) and Iran (JX121576), with the type ToMMV isolate revealed 98–100% nucleotide and deduced amino acid identity, suggesting that ToMMV may be more widespread than currently reported. Our current results provide further characterization of the emerging ToMMV and highlight the continued importance of tobamovirus management in solanaceous crop production.