Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium.