Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products

Abstract

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies 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-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-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 coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length 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 different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related. In conclusion, the region amplified by PCR was highly effective for the detection of coronatine-producing P. syringae strains, and the sequence analysis of PCR products proved valuable in showing relatedness between strains and pathovars.

Evaluation of cherry cultivar susceptibility to bacterial canker and leaf spot disease

“…Obtained isolates were tested for different biochemical characters: Gram reaction; presence of catalase; production of fluorescent pigment on King B medium; oxidative-fermentative metabolism of glucose; LOPAT and GATTa tests (Latorre & Jones, 1979;Lelliott & Stead, 1987;Lelliott et al, 1966). Molecular identification was based on the detection of genes for discriminative bacterial toxin production or synthesis, namely: coronatine production gene (cfl) using Primer1/2 primer set (Bereswill et al, 1994) and genes for syringomycin synthesis (syrB) and syringomycin secretion (syrD) by two sets of primers, B1/B2 and SyD1/SyD2 (Iličič, Balaž, et al, 2016). The reference strains KFB0120 (pv.…”

Current situation and characterization ofPseudomonas syringaepv.actinidiaeon kiwifruit inGalicia (northwestSpain)

“…The ability of isolates to produce phytotoxins was determined by PCR amplification of the cfl gene coding for coronatine (with primers CFLF/CFLR) and of fragments of the tox-argK gene cluster coding for phaseolotoxin (with primers ArgKF3/ArgKR) under the conditions established by Templeton et al (2005) and Bereswill et al (1994), respectively.…”

Curtobacterium flaccumfaciens pv. flaccumfaciens on Soybean in Germany – A Threat for Farming

“…According to our expectations, a population size of Psg of c. 10 6 -10 7 cfu⁄cm 2 was found on the soybean cvs Lissabon, Alma ATA, Maple Arrow, Primus and Capnor. The pathogen Psg was identified as described by Vo¨lksch et al (1992) and Bereswill et al (1994). However, the isolation procedure revealed the occurrence of a high number of yellow colonies, especially in samples of cv.…”