First Report of Grapevine leafroll-associated virus-2 and -3 in Texas Vineyards.

Abstract

Grapevine leafroll disease (GLD), caused by the grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae) is an important disease in all grapevine-growing regions of the world (2). It negatively affects vine vigor, fruit yield, and grape quality (e.g., sugar accumulation) (3). Typical disease symptoms include downward rolling of grape leaves accompanied by interveinal reddening in red-fruited varieties and interveinal chlorosis in white-fruited varieties (2). The state of Texas currently has over 275 commercial vineyards and acreage under grape production is expanding. Currently, there is limited information on the presence of either GLRaV-2 (genus Closterovirus) or GLRaV-3 (genus Ampelovirus) in this state. During the 2012 season, 19 individual, symptomatic grapevines (13 cv. Lenoir and 6 cv. Blanc du Bois) were sampled (14 petioles per vine) from one vineyard site in Richards, TX. Total nucleic acid was extracted from the samples as described before (5) and tested by RT-PCR using species specific primers to amplify a 334-bp fragment of the HSP70h gene of GLRaV-2 (L2 F: 5'-ATAATTCGGCGTACATCCCCACTT-3' and U2 R: 5'-GCCCTCCGCGCAACTAATGACAG-3') (1) and a 541-bp fragment of the HSP70h gene of GLRaV-3 (LC1 F: 5'-CGCTAGGGCTGTGGAAGTATT-3' and LC2 R: 5'-GTTGTCCCGGGTACCAGATAT-3') (4). Samples were also subjected to triple (TAS) and double (DAS) antibody sandwich ELISA for GLRaV-2 and GLRaV-3 using commercially available antibody test kits (AC Diagnostics, Fayetteville, AR). Five samples tested positive for GLRaV-2 and one for GLRaV-3, all from the variety Lenoir with no incidences of mixed infection. In addition to the RT-PCR and ELISA, the presence of GLRaV-2 and GLRaV-3 was confirmed by direct sequencing of select RT-PCR products, which was purified using the QIAquick PCR Purification kit (Qiagen Inc., CA). The sequencing took place at the Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA. GLRaV-2 isolate TX12 (GenBank Accession No. KF417612) and GLRaV-3 isolate TX9 (KJ545571) shared 85 to 100% and 94 to 100% nucleotide identity and 74 to 100% and 82 to 100% amino acid identity, respectively, with previously reported isolates from around the world. All samples tested negative for GLRaV-1, -4, -4 strain 5, and -4 strain 9 (4), suggesting that some of the symptomatic vines may have a different disease or abiotic disorder, such as a nutrient deficiency. To our knowledge, this is the first report of GLRaV-2 and GLRaV-3 in the state of Texas.