Evaluation of Agrobacterium tumefaciens genetic diversity in CA walnut growing regions and resistance to the biocontrol agent, Agrobacterium rhizogenes K84

Crown gall of walnut (Juglans sp.), caused by the bacterium Agrobacterium tumefaciens, is of great concern to the California walnut growers and nurseries due to significant yield and tree losses. To determine the genetic diversity of A. tumefaciens throughout the California Central Valley, we collected isolates from ten counties, which encompass the CA walnut growing regions. A total of 340 A. tumefaciens biovar 1 isolates were isolated from both soil and gall tissue using the Agrobaeterium semi-selective medium 1A amended with 80 ppm potassium tellurite. A PCR-based approach was used to detect the presence of the Ti plasmid, the virulence determinant of A. tumefaciens. One-hundred ninety isolates were Ti-plasmid positive or shown to be virulent in bioassays. To assess genetic diversity, BOX PCR profiles were generated for the confirmed virulent isolates and analyzed using GelCompar II. Several distinct genotypes clustered as a function of geography. Due to the level of genotypic diversity observed among the A. tumefaciens isolates we hypothesized that control of crown gall by the biocontrol agent A. rhizogenes K84 would be variable as well. To this end, we tested the ability of the biocontrol agent Agrobaeterium rhizogenes K84 to inhibit the growth of a subset of virulent A. tumefaciens biovar 1 isolates displaying distinct genetic fingerprints. Analysis of the genetic heterogeneity of California A. tumefaciens isolates will facilitate the design of effective crown gall management strategies, including judicious application of A. rhizogenes K84 depending on the A. tumefaciens genotype present.