First Report of Bois Noir Phytoplasma in Grapevine in Canada

During the summer and fall of 2006, a survey was done to detect European phytoplasmas of quarantine significance in Canadian vineyards. This survey was developed as one of the 2006 import requirements for grapevine nursery stock from Europe. This addresses the increased concerns regarding inadvertent phytoplasma introductions. Grapevines imported in 2006 and established grapevines were observed for symptoms typical of those associated with diseases caused by phytoplasmas on grapevine. Samples were tested from 155 grapevines. One plant, located in the lower Okanagan Valley, British Columbia, tested positive by a modified real-time PCR assay and TaqMan probe targeting the 16S region of the ribosomal RNA gene (1), which detects a wide variety of known phytoplasmas. The sample was further analyzed and found to be positive by conventional PCR with the phytoplasma-specific primers, P1/P7 (3), and Stolbur specific primers, STOL11f2/r1 (2). Additional PCR tests with primers specific to flavescence doree (FD9f/r) (2) and western X disease (P1/W INT) (3) were negative. These phytoplasmas are also known to infect grapevine. The approximate 1,800-bp fragment obtained with P1/P7 was sequenced (GenBank Accession No. EU086529) and found to have 99.7% nucleotide sequence identity to the Stolbur STOL #11 isolate (GenBank Accession No. AF248959) originally isolated from eastern Europe. This was the highest match to any available phytoplasma sequence obtained and indicates that the phytoplasma in the British Columbian sample is an isolate of bois noir, a pest of quarantine significance to Canada. Additional phylogenetic analysis using CLUSTAL W (Lasergene; DNASTAR, Madison, WI) confirmed this result. The presence and identity of the phytoplasma was confirmed from a second tissue sample that was analyzed by PCR and sequenced using the same test procedures as for the first sample, with identical results. The bois noir phytoplasma belongs to the stolbur group (16SrVII) with the principal vector being a cixiid planthopper. Stolbur phytoplasmas cause diseases in other crops, but bois noir disease is caused by a specific member of that group and is the only stolbur phytoplasma known to infect grapevines in Europe. The infected grapevine was from a lot of 1,965 plants of Grenache clone 70 on rootstock 3309 clone 143 that was imported from Europe in 2006. All plants in this importation have been destroyed. This phytoplasma has not been detected in any other grapevines in Canada. Additional import conditions requiring hot water treatment of European vines have been implemented for 2007. Further survey work for phytoplasma in grapevine will continue. References: (1) N. M. Christensen et al. Mol. Plant-Microbe Interact. 17:1175, 2004. (2) X. D. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.