IDENTIFICATION AND RELATEDNESS OF CORONATINE-PRODUCING PSEUDOMONAS-SYRINGAE PATHOVARS BY PCR ANALYSIS AND SEQUENCE DETERMINATION OF THE AMPLIFICATION PRODUCTS

Production of the chlorosis-inaucing phytotoxin coronatine in the Pseu domonas syringae pathovars atropurpurea, glycinea, maculicola, morspru norum, and tomato has been previously reported. DNA hybridization stud ies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin . In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine produc ing P. syringae strains by PCR analysis. The primer set amplified diag nostic 0.65-kb PCR products from genomic DNAs of five different corona tine-producing pathovars of P. syringae. The 0.65 kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for cor onatine production. When the 0.65-kb PCR products were digested with C laI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment lengt h polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65 -kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two di fferent P. syringae pv. glycinea strains contained identical DNA seque nces, and these showed relatedness to the sequence obtained for the pa thovar morsprunorum. The PCR products obtained from the pathovars macu licola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related. In conclu sion, the region amplified by PCR was highly effective for the detecti on of coronatine-producing P. syringae strains, and the sequence analy sis of PCR products proved valuable in showing relatedness between str ains and pathovars.