A Rapid and Sensitive Method to Detect Agrobacterium vitis in Grapevine Cuttings Using the Polymerase Chain Reaction. Kenneth C. Eastwell, Research Scientist, Agriculture and Agri-Food Canada, Research Centre, Summerland, British Columbia, Canada VOH 1Z0. Leslie G. Willis, Research Technician, Association of B.C. Grape Growers, #5-1864 Spall Road, Kelowna, British Columbia, Canada V1Y 4R1; and Timothy D. Cavileer, Visiting Fellow, Agriculture and Agri-Food Canada, Research Centre, Summerland, British Columbia, Canada VOH 1Z0. Plant Dis. 79:822-827. Accepted for publication 31 March 1995. Copyright 1995 Department of Agriculture and Agri-Food, Government of Canada. DOI: 10.1094/PD-79-0822.

Oligonucleotide primers were designed for amplification of the virA, pehA, and 6a genes of Agrobacterium species and evaluated for their potential use in the identification of Agrobacterium vitis. In conjunction with the polymerase chain reaction, the peA-specific primer pair readily identified all strains of A. vitis that were tested. Four different extraction/purification methods were tested for the recovery of A. vitis DNA from grapevine cuttings. A protocol which relied on exposure to microwaves for cell lysis, followed by purification of DNA by ion exchange chromatography was the most effective in removing, from grapevine samples, inhibitors of the polymerase chain reaction. The reported method was both sensitive and rapid, making it amenable to routine diagnostic testing. It was demonstrated that the lysis in planta of A. vitis and analysis of the recovered DNA by polymerase chain reaction provided a more reliable indication of the occurrence of the bacteria in actively growing grapevines than did recovery of viable bacteria and growth on selective media.