UC Davis

Crown gall (CG) caused by a Gram-negative bacterium Agrobacterium tumefaciens is a serious threat to the California walnut industry. Understanding the classification and diversity of this pathogen is important for the development of sustainable management strategies for CG. In the present study, average nucleotide identity (ANI), phylogenetic, and pangenomic analyses of 311 Agrobacterium strains, including 29 newly isolated and sequenced strains from walnut orchards in California were performed. These analyses revealed 35 ANI groups (genomic species) and four main clades corresponding to three previously described biovars, i.e., Biovars 1, 2, and 3 and a mix of known and unknown Agrobacterium species. The Agrobacterium genus had an open pangenome, indicating great diversity among individuals in this group. Five Phenotypic traits (virulence, growth, antibiotic resistance, K84 sensitivity, motility) of the 29 strains were determined. Virulence testing on Datura stramonium and walnut hybrid genotypes (J. regia X J. hindsii) revealed considerable variation. The growth rates ranged from 0.15 min-1 to 0.65 min-1 in a nutrient-rich medium, whereas the growth rates in the nutrient-poor medium ranged from 0.05 min-1 to 0.07 min-1. Most strains were resistant to streptomycin and vancomycin, whereas they were sensitive to the other six tested antibiotics. Nearly half of the strains were resistant to the biocontrol Agrobacterium strain K84. Comparative genomic analysis of the 29 strains revealed a highly conserved T4SS, the T-DNA, and the virA/virG two-component regulatory system. The 29 strains were divided into agropine-, succinamopine-, and nopaline-types. The agropine and succinamopine opine types were related to the size of tumors induced on Datura stramonium, but not on walnut. The different hosts (D. stramonium and walnut) also exerted a significant influence on the tumor size induced by a given A. tumefaciens opine genotype. These data indicate that the opine type may influence a strain’s host preference. Interestingly, three virulent strains did not contain a T6SS. The remaining strains contained a classical T6SS in which the imp operon was conserved, whereas the hcp operons were found to be variable among the strains. Variability in the hcp operon may contribute to the ability of A. tumefaciens to adapt to a wide range of environments. Collectively, these data will be useful in the development of effective crown gall management strategies for the walnut industry in California.