Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 10 3 CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation.

Pseudomonas syringae pv. tomato produces the chlorosis-inducing phytotoxin coronatine. Five of 3700 (0.13%) kanamycin-resistant mutants generated by random Tn5 mutagenesis were unable to synthesize this toxin. Clone pEC18, isolated from a cosmid pLAFR1 library of wild-type P. syringae pv. tomato DC3000 genomic DNA, complemented four of the mutants. Restriction enzyme analysis of pEC18 and corresponding clones from the four mutants indicated that the Tn5 insertion sites of these four mutants spanned a 19-kb region of DC3000 genomic DNA. Complementation tests with subclones of pEC18 confirmed the relative locations of the Tn5 insertions. Because pEC18 did not complement all five mutants or confer the ability to produce toxin on nonproducing P. syringae pathovars, sequences outside this cloned region must also be involved in toxin synthesis. As demonstrated by Southern blot analysis, this cloned region was not on the 68-kb indigenous plasmid of DC3000. The only P. syringae pathovars with DNA homologous to sequences within the coronatine gene cluster were the coronatine producers P. syringae pv. tomato, P. syringae pv. atropurpurea, and P. syringae pv. glycinea. Since the hybridization patterns of these toxin producers were identical, this locus is highly conserved and appears crucial to the synthesis of coronatine.Key words: coronatine, phytotoxin mutants, Pseudomonas syringae pv. tomato.