Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on King’s medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.
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.
In 1978 and 1979, Pseudomonas syringae pv. maculicola strains DAR 33362, DAR 33363, and DAR 33406 were isolated from diseased Brassica hirta, B. nigra, and B. napus var. napus, respectively, in Wagga Wagga and Armatree, NSW, Australia (2). Peters et al. (2) demonstrated that these strains were similar to P. syringae pv. maculicola ICMP 4326 (CFBP 1637), which was recently transferred to Pseudomonas cannabina pv. alisalensis (1). We evaluated these Australian strains to determine if they might also be P. cannabina pv. alisalensis. Amplification of DNA using the BOXA1R primer and PCR resulted in identical DNA fragment banding patterns for Australian strains DAR 33362 and DAR 33363 and P. cannabina pv. alisalensis ICMP 4326 and CFBP 6875. The third Australian strain, DAR 33406, was 90% similar to P. cannabina pv. alisalensis; in contrast, it was only 77% similar to P. syringae pv. maculicola. All strains of P. cannabina pv. alisalensis, including the pathotype strain (CFBP 6866) and all three Australian strains, were lysed by bacteriophage PBS1, which is specific for P. cannabina pv. alisalensis strains (1). To complete Koch's postulates, pathogenicity was evaluated on B. hirta, B. nigra, and B. napus var. napus. In two independent experiments, two plants of each species were inoculated with each Australian strain or a phosphate buffer control treatment. In separate experiments, pathogenicity was evaluated on the differential hosts radish (Raphanus sativus cv. Comet) and broccoli raab (Brassica rapa cv. Sorrento), and plants inoculated with the pathotypes of P. cannabina pv. alisalensis and P. syringae pv. maculicola served as additional control treatments. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h (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 108 CFU/ml). Treatments were applied by spraying until runoff. DAR 33362, DAR 33363, and DAR 33406 caused typical bacterial blight symptoms on B. hirta, B. nigra, and B. napus var. napus. Infected leaves became yellow, followed by the development of small (<2 mm in diameter), angular, water-soaked, and eventually, shot-holed spots. Bacteria isolated from symptomatic tissue following surface disinfestation of tissue with sodium hypochlorite (0.525%) had identical characteristics (rep-PCR DNA fragment banding patterns and phage sensitivity) to the strains used to inoculate the plants. Additionally, DAR 33362, DAR 33363, and DAR 33406, as well as P. cannabina pv. alisalensis, caused symptoms on radish and broccoli raab while P. syringae pv. maculicola and the buffer control did not. These data support the transfer of the Australian crucifer strains, originally identified as P. syringae pv. maculicola, to P. cannabina pv. alisalensis. To our knowledge, this is the first report of a bacterial disease of crucifers caused by P. cannabina pv. alisalensis in Australia. Differentiation of these pathogens will inform crop rotation strategies for disease management. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) B. J. Peters et al. Plant Pathol. 53:3, 2004.
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