First Report of Tobacco ringspot virus Infecting an American Hybrid Grape Cultivar in Texas
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© 2017, American Phytopathological Society. All rights reserved. Statewide efforts to document the occurrence and distribution of viruses in Texas vineyards began during spring 2016. Plants of the interspecific hybrid bunch grape cultivar Blanc du Bois (Vitis spp.: ‘Florida D 6-148’ × ‘Cardinal’) showing symptoms of leaf deformation, chlorotic specks, stunted shoots, and general decline were observed in a vineyard block located in Hidalgo County of the Lower Rio Grande Valley region of South Texas. Due to the consistency of the symptoms with those described for grapevine-infecting nepoviruses, total nucleic acid extracts obtained from eight (four symptomatic and four nonsymptomatic) plants were subjected to a two-step reverse transcription (RT)-PCR using primers targeting Grapevine fanleaf virus (GFLV) (Rowhani et al. 1993) and Tobacco ringspot virus (TRSV) (Fuchs et al. 2010). The expected TRSV-specific 316 bp DNA was obtained from the four symptomatic plants while all eight vines were negative for GFLV. Symptomatic plants also tested positive for TRSV by DAS-ELISA using virus-specific antibodies (BIOREBA AG, Reinach, Switzerland) based on a threshold of >3× mean absorbances (OD405) over the manufacturer-supplied negative control. To enable the molecular typing of TRSV isolates in the positive samples, their cDNA aliquots were subjected to PCR with two gene-specific primer pairs targeting distinct genes encoded by the RNA1 and RNA2 segments of the virus. The primer pairs TRSVRdRpfwd6005 and TRSVRdRprev7067 amplified a 1,055 bp DNA band specific to the RNA-dependent RNA polymerase (RdRp) and primers TRSVCPfwd2442 and TRSVCPrev3452 amplified a 1,013 bp DNA band specific to the coat protein (CP) genes of TRSV (Fisher 2013) from each of the four samples. Gene-specific DNA bands from two representative vines were cloned individually into pCR2.1 (ThermoFisher Scientific, Carlsbad, CA) and two to three clones per amplicon were sequenced in both orientations. BLASTn analysis of the obtained nucleotide (nt) and derived amino acid (aa) sequences confirmed their relation to TRSV. In pairwise comparisons, the 1,002 nt (333 aa) long partial RdRp cistron obtained in this study (GenBank accession nos. KY304030-35) showed 92 to 100% nt and 97 to 100% aa identities among themselves. They also showed 88 to 96% nt and 94 to 97% aa identity levels with corresponding global sequences of TRSV obtained from GenBank. The 987 nt (328 aa) long partial CP cistron (KY304036-40) showed 100% nt and 99 to 100% aa identities among themselves. They also showed 92 to 97% nt and 98 to 100% aa identity levels with corresponding sequences of several TRSV isolates obtained from GenBank. To our knowledge, this is the first report of TRSV occurrence in grapevines in Texas and the first record of Blanc du Bois, an American hybrid grape, as host to the virus with implications for disease management in vineyards where the cultivar is grown primarily for its disease resistance attributes. Efforts are ongoing to assess the prevalence of TRSV across vineyards in the state and to investigate the presence of its nematode vector.
author list (cited authors)
McBride, S., Appel, D. N., Pontasch, F. M., Gregg, L., & Alabi, O. J.