I. Rubio, California State University, Monterey Bay, Undergraduate Research Opportunities Center, Seaside, CA 93955;
G. Hiddink, Enza Zaden Seed Operations B. V., The Netherlands;
M. Asma, Bejo Zaden B. V., The Netherlands; and
C. T. Bull, USDA-ARS Agricultural Research Station, Salinas, CA 93905
In 2008, a bacterial blight was observed on Raphanus sativus in the Pfalz region in Germany. Disease was sporadic but severe when present within R. sativus fields, which resulted in unmarketable crops. Symptoms consisted of small, angular, water-soaked flecks that often were surrounded by chlorotic haloes. Lesions were visible from adaxial and abaxial leaf surfaces and generally retained chlorotic borders. A gram-negative, bluefluorescing bacterium was isolated from surface-disinfested leaf tissue on King's medium B agar. The radish isolate was levan positive, oxidase negative, and arginine dihydrolase negative. The isolate did not rot potato slices but induced a hypersensitive reaction in tobacco. These reactions corresponded to Lelliot's LOPAT group 1 (2). Repetitive extragenic palindromic sequence (rep)-PCR assays using the BOXA1R primer resulted in different DNA fragment banding patterns between the radish isolate and the pathotype strain of Pseudomonas syringae pv. maculicola (CFBP 1657), but identical DNA fragment banding patterns between the radish isolate and the pathotype strain of P. cannabina pv. alisalensis (CFBP 6866). Unlike P. syringae pv. maculicola, P. cannabina pv. alisalensis and the radish isolate were lysed by bacteriophage PBS1 (1). Pathogenicity was evaluated on two hosts, radish (R. sativus cv. Comet) and broccoli raab (Brassica rapa cv. Sorrento). In each of two independent experiments, 3-week-old radish and broccoli raab plants were inoculated with either the radish isolate, P. cannabina pv. alisalensis, or P. syringae pv. maculicola. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h at 27°C, suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), and adjusting each suspension to 0.6 OD at 600 nm (approximately 1 × 108 CFU/ml). All plants were inoculated by spraying until runoff, incubated in a humidity chamber for 48 h, then placed in a greenhouse at 20 to 25°C for symptom development. Plants inoculated with P. cannabina pv. alisalensis or sprayed with buffer served as positive and negative control treatments, respectively. Seven to ten days postinoculation, the development of symptoms similar to those originally observed in the field were observed on plants inoculated with the radish isolate. In addition, symptoms on radish and broccoli raab plants caused by the radish isolate were similar to symptoms caused by P. cannabina pv. alisalensis in contrast to the lack of symptoms on plants inoculated with P. syringae pv. maculicola. Bacteria isolated from symptomatic tissue and surface-disinfested with sodium hypochlorite (0.525%) had identical characteristics to the radish isolate used to inoculate plants and to the P. cannabina pv. alisalensis pathotype for LOPAT reactions, rep-PCR DNA fragment banding pattern analysis, and sensitivity to phage PBS1, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. cannabina pv. alisalensis isolated from diseased crucifers in Germany. Verification of P. cannabina pv. alisalensis in Germany indicates that German crucifer growers should differentiate between outbreaks caused by P. cannabina pv. alisalensis and P. syringae pv. maculicola and apply appropriate, specific management strategies.
References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.