Whole-Genome Resources and Species-Level Taxonomic Validation of 89 Plant-Pathogenic Xanthomonas Strains Isolated from Various Host Plants.

Bacterial spot disease caused by Xanthomonas spp. is a global threat to tomato and pepper plants. A recent classification of these pathogens indicated the need for a diverse dataset of whole-genome resources. We report whole-genome resources of 89 Xanthomonas strains isolated from Canada (n = 44), the United States (n = 29), Argentina (n = 4), Brazil (n = 3), Costa Rica (n = 3), New Zealand (n = 1), Australia (n = 1), Mexico (n = 1), Taiwan (n = 1), Thailand (n = 1), and unknown (n = 1). Of these strains, 48 were previously identified to species-level based on nongenome-based approaches while 41 strains were classified only at the genus level. The average coverage of the sequencing reads was 103×. The draft genome sizes ranged from 4.53 to 5.46 Mbp with a G + C content of 63.53 to 67.78% and comprised 4,233-5,178 protein-coding sequences. Using average nucleotide identity (ANI) and genome-based DNA-DNA hybridization (gDDH) values, the taxonomic classifications were validated for 38 of the 48 strains previously assigned to species level using other methods. Ten strains previously identified as Xanthomonas campestris, X. axonopodis, X. vasicola, and X. arboricola were incorrectly assigned, and new species-level delineations are proposed. Data from ANI, gDDH, and pangenome phylogeny of shared protein families were used to assign the 41 strains, previously identified only to genus level, into five distinct species: X. euvesicatoria (pv. euvesicatoria or pv. perforans), X. hortorum pv. gardneri, X. vesicatoria, X. campestris, and X. arboricola. These 89 whole-genome sequences of Xanthomonas strains, the majority (49.4%) of which are from Canada, could be useful resources in our understanding of the global population structure and evolution of these pathogens.

Development and Evaluation of PCR-Based Diagnostic Assays for the Bacterial Speck and Bacterial Spot Pathogens of Tomato.

Bacterial speck and bacterial spot lesions can easily be confused with each other and with those formed by other tomato pathogens. To facilitate disease diagnosis, we developed and evaluated polymerase chain reaction (PCR)-based lesion assays using crude DNA extracts and primer sets COR1/2 (bacterial speck) and BSX1/2 (bacterial spot). All 29 pathogenic Pseudomonas syringae pv. tomato strains tested produced a 689-bp amplicon with COR1/2; 28 of the 37 geographically diverse bacterial spot-causing xanthomonad (BSX) strains that were tested generated the 579-bp BSX1/2 amplicon. The detection limit with plant samples was 30 to 50 CFU/reaction. In a 6-year study, the COR1/2 PCR assay diverged from the culture-based classical assay for only 3 of 70 bacterial speck lesion samples collected from Ontario greenhouses and tomato fields; the BSX1/2 assay was positive for 112 of the 124 confirmed bacterial spot lesions sampled. The majority (66%) of the BSX strains isolated from these lesions belonged to group D; the 12 strains that were BSX1/2-negative belonged to group C. Group D strains produced a 425-bp PCR product with crude DNA extracts but a 579-bp product with purified DNA; the former was identical to the latter except that it was missing 150 bp from the middle of the 579-bp sequence.

Reduction of Bacterial Spot Disease Severity on Tomato and Pepper Plants with Foliar Applications of Ammonium Lignosulfonate and Potassium Phosphate.

Bacterial spot is a serious and persistent disease problem of tomato and bell pepper in both the United States and Canada. Current disease management practices, based primarily on fixed copper bactericides, do not give consistent, effective protection. Foliar applications of ammonium lignosulfonate (ALS), derived from the wood pulping process, and the fertilizer potassium phosphate (KP) were tested for their ability to control this disease under both greenhouse and field conditions. Acibenzolar-S-methyl was included as a control. Greenhouse-grown tomato transplants treated with acibenzolar-S-methyl, 2 or 4% (vol/vol) ALS, 25 mM KP, or 2% ALS plus 10 mM KP and then inoculated with Xanthomonas campestris pv. vesicatoria had significantly less disease than the unprotected controls. Weekly foliar applications of acibenzolar-S-methyl, ALS, or KP significantly reduced disease severity on the foliage of inoculated field-grown tomato and pepper plants; although less disease appeared on the fruit of these plants, the effect was not always statistically significant except for the acibenzolar-S-methyl treatment. Acibenzolar-S-methyl increased the yield of marketable tomato fruit in 2 of 3 years of the study and that of pepper fruit in 1 of 2 years. There was a marked increase in the yield of marketable fruit on all ALS-treated pepper plants in 2001. None of the treatments significantly increased total tomato or pepper yield. ALS and KP had no observable phytotoxic effect on tomato or pepper foliage. Our results indicate that future integrated disease management programs for bacterial spot may be enhanced by including foliar sprays of these two products.

Chemotaxis by Pseudomonas syringae pv. tomato.

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl(2) in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23 degrees C, which is 5 degrees C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 x 10 CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.