A Rapid and Sensitive Method to DetectAgrobacterium vitisin Grapevine Cuttings Using the Polymerase Chain Reaction

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 pehA-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.