Grapevine red blotch-associated virus Is Widespread in the United States.

Grapevine red blotch disease has been recognized since 2008 as affecting North American grape production. The presence of the newly described Grapevine red blotch-associated virus (GRBaV) is highly correlated with the disease. To more effectively detect and monitor the presence of the virus, a sample processing strategy and multiplex polymerase chain reaction assay were developed. A total of 42 of 113 vine samples collected in or received from seven of the United States were shown to harbor the virus, demonstrating the virus is widely distributed across North America. Phylogenetic analyses of a viral replication-associated protein (Rep) gene fragment from the 42 isolates of GRBaV demonstrated distinct clades of the virus (1 and 2), with clade 1 showing the greatest variability. The full-length genome of six virus isolates was sequenced, and phylogenetic analyses of 14 whole genomes recapitulated results seen for the Rep gene. A comparison of GRBaV genomes revealed evidence of recombination underlying some of the variation seen among GRBaV genomes within clade 1. Phylogenetic analyses of coat and replicase-associated protein sequences among single-stranded DNA viruses showed GRBaV to group within the family Geminiviridae. This grouping is distinct from members of the families Nanoviridae and Circoviridae, with limited significant affinities to both recognized genera and novel plant-infecting, gemini-like viruses.

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.

Potato virus M in Bittersweet Nightshade (Solanum dulcamara) in New York State.

Potato virus M (PVM) was detected in upstate New York in two plants of the widely naturalized, weedy perennial Solanum dulcamara. The virus was detected with a macroarray assay for potato viruses (1). Amplified, complimentary DNAs from the two isolates hybridized to 5 and 7 of the 15 oligonucleotide probes for PVM. Testing of the samples by double-antibody sandwich-ELISA using PVM-specific antibodies (Agdia, Elkhart, IN) showed a clear positive result. Sequence information for a 118-bp genomic region was obtained by amplification using carlavirus-specific primers (2) (GenBank Accession No. HQ446853). Comparison with a reference PVM genome (GenBank Accession No. NC_001361) showed that the sequence corresponded to nucleotide positions 8418 to 8533 with 86% identity. The infected plants were symptomless and collected from two sites, 50 miles apart. One site was a weedy roadside location in Tompkins County in 2009, while the second was from a hedgerow in a (non-potato) vegetable production area of Ontario County in 2010. The virus could be detected throughout the growing season in this perennial host. PVM was reported from S. dulcamara L. in Hungary and described as being found frequently from a diversity of habitats (3). Importantly, the virus was transmitted via tubers and by Myzus persicae with low efficiency (3). These results suggest that the virus may be endemic in S. dulcamara in the northeastern United States and this host may serve as a reservoir for the virus from which it could move into potato. To our knowledge, PVM has not been reported in this host in North America. References: (1) B. Agindotan and K. L. Perry. Plant Dis. 92:730, 2008. (2) J. Badge et al. Eur. J. Plant Pathol. 102:305, 1996. (3) P. Salamon. Eur. Assoc. Pot. Res. Virol. Sect. Meet. 42:121, 2006.