Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut
L. E. YAKABE (1), S. R. Parker (1), D. A. Kluepfel (1)
(1) USDA, Agricultural Research Service, Crops Pathology/Genetics Research Unit, Davis, CA, U.S.A.
Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even with the use of semi-selective media. Many semi-selective media have been developed for A. tumefaciens and more recent studies of Agrobacterium species have relied on the medium1A. In this study, six previously published semi-selective media were evaluated using a panel of A. tumefaciens biovar 1 and closely related Rhizobiaceae species isolated from walnut orchards in California. Based on the antibiotic and carbon-utilization profiles of twelve A. tumefaciens isolates, D1 and 1A recipes were modified by antibiotic amendment or substitution of the primary carbon source to increase isolation efficiencies and/or selectiveness out of a complex environment. The antibiotics ampicillin, trimethoprim, and vancomycin were evaluated in addition to the carbon sources proline, serine, theonine, and β-hydroxybutyric acid. Results from these experiments revealed significant variation in isolation efficiency within a genetically diverse group of A. tumefaciens isolates and allowed development of a more selective medium for diagnostics and quantification from soil.
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