Identification of Tobacco streak virus Associated With a Virus-like Mottle Symptom on Hosta

Tobacco streak virus (TSV) is the type species of the Ilarvirus genus of the family Bromoviridae (4), and is transmitted mechanically through thrips feeding, by pollen (5), and by seed (3). The virus has a tripartite genome of single-stranded messenger-sense RNA which encodes four non-structural proteins and a single structural capsid protein (4). The movement protein and capsid protein encoding genes are located on the 5’ half and 3’ half, respectively, of RNA 3 (4). In the spring of 2010, a Hosta sp. ‘Fried Green Tomatoes’ plant displaying a virus-like mottle symptom (Fig. 1) was submitted to the Ohio Plant Diagnostic Network for analysis. Hosta sp. ‘Fried Green Tomatoes’ is a solid green sport of the variegated Hosta sp. ‘Guacamole’ (7), and the submitted sample was collected from a nursery block of approximately 100 plants grown from bare root plugs imported from Europe, a majority of which showed the symptom. The sample tested positive for TSV and negative for the Potyvirus group, Alfalfa mosaic, Arabis mosaic, Cucumber mosaic, Hosta virus X, Impatiens necrotic spot, Lily symptomless, Tobacco mosaic, Tobacco ringspot, Tomato mosaic, Tomato ringspot, and Tomato spotted wilt viruses by enzyme-linked immunosorbent assays (ELISA) using commercially available antibodies (Agdia Inc., Elkhart, IN). The sample also tested negative for Tobacco rattle virus by RT-PCR. Double-stranded ribonucleic acid (dsRNA) was purified from symptomatic tissue as previously described (6) with no visible dsRNAs detected using agarose gel electrophoresis. However, cDNAs were synthesized from purified dsRNA template as previously described (1). For immunocapture reverse transcription (IC-RT), magnetic beads conjugated with sheep anti-rabbit IgG were incubated with polyclonal rabbit anti-TSV IgG (Agdia, Inc.) (2). Leaf tissue samples were extracted, incubated with TSV-conjugated beads, washed, and cDNAs synthesized from bound virions (2). Three full length TSV RNA 3 sequences (accession no. FJ655173.1, FJ403377.1, X00435.1), one movement protein (MP) sequence (accession no. DQ141601.1), and five coat protein (CP) sequences (accession no. AY606066.1, DQ864458.1, EU375481.1, GQ370526.1, HM622157.1) were aligned and used to design two sets of TSV-specific primers to amplify the MP (TSV fwd69: 5‘-CCTACAAGTY GAGACCATTGGTC-3’; TSV rev1111: 5’-CCATGTCTTACWCAACCSAACARTC-3’) and CP (TSV fwd1190: 5GCTTCTCGGACTTACCTGRGATG-3’; TSV rev2160: 5’GGTTTYCCAYGGAAATCGTCCGATTC-3’; Integrated DNA Technologies Inc., Coralville, IA) open reading frames (ORFs). Five μL of cDNA or sterile water was used as template for separate PCR reactions. Amplification was done in 25 μL reactions (1.5 mM MgCl , 0.2 mM dNTP mix, 0.2 μM primer pair, 0.625 units GoTaq Flexi polymerase (Promega Inc., Madison, WI) with the cycling parameters of 94°C (2 min), 40 cycles of 94°C (45 sec), 55°C (30 sec), 72°C (60 sec), and final extension of 72°C (10 min). 2