Spinach latent virus Infecting Tomato in Virginia, United States

Plants in a single field of commercial tomato (Solanum lycopersicum) of unidentified cultivars in Virginia in July, 2012, were observed showing stunting, leaf distortion, twisting and thickening, discoloration, and color streaking and ringspots on fruits. Serological tests were negative for Cucumber mosaic virus, Groundnut ringspot virus, Tomato spotted wilt virus, Tomato chlorotic spot virus, Impatiens necrotic spot virus, Tobacco mosaic virus, and Tomato bushy stunt virus (Agdia, Inc., Elkhart, IN). Using a membrane-based macroarray (3), hybridization was observed to 8 of 9 70-mer oligonucleotide probes of Spinach latent virus (SpLV; genus Ilarvirus, family Bromoviridae). To confirm the hybridization results, complementary DNA (cDNA) was synthesized using random hexamers and MMLV reverse transcriptase (Promega, Madison, WI), followed by PCR amplification using ilarvirus degenerate primers (4). Fragments of approximately 380 bp were amplified and directly sequenced (GenBank Accession KC_466090); a BLAST search showed a 99% identity to the SpLV RNA 2 reference genome (NC_003809). Primers for SpLV RNA1 (SpLVRNA1f-GGTGTCACCATGCAAACTGG, SpLVRNA1r-AGCTCTTCGTAATAGGCCTGC) and SpLV RNA3 (SpLVCPf-GAAGTCTTTCCCAGGTGAGCA, SpLVCPr-AGGTGGGCATATGGACTTGG) were designed and cDNA was amplified using the IQ supermix (Biorad, Hercules, CA) with thermocycling of 94°C for 4 min, 35× (94°C 45 s, 55°C 45 s, 72°C 45 s), and 72°C for 10 min. The resulting fragments of 538 bp for RNA1 (KC_466088) and 661 bp for RNA3 (KC_466089) showed 100% identity to reference genome sequences for SpLV (NC_003808 and NC_003810, respectively). To demonstrate virus transmissibility, Chenopodium quinoa plants were mechanically inoculated using tomato leaf material (same source described above) ground in 30 mM Na₂HPO₄ buffer, pH 7.0. Necrotic spots developed on the inoculated leaves 10 dpi. Younger, non-inoculated leaves showed yellow mottling and tested positive for SpLV by RT-PCR (two of two plants tested). The detection of SpLV is rarely reported, with only one record from the United States (2). Although SpLV is described as a latent virus, it has been found associated with tomato fruit symptoms in New Zealand (1). It is not known if the fruit ringspot and other symptoms on the Virginia samples were due to virus infection. Since SpLV is seed-transmissible and seed production takes place in different parts of the world, it has the potential to spread with germplasm and become more widespread in North America. References: (1) B. S. M. Lebas et al. Plant Dis. 91:228, 2007. (2) H. Y. Liu and J. E. Duffus. Phytopathology 76:1087, 1986. (3) K. L. Perry and X. Lu. Phytopathology 100:S100, 2010. (4) M. Untiveros, et al. J. Virol. Methods 165:97, 2010.