Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

Abstract

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

Keywords: PCR; TaqMan PCR; detection; halo blight; oat.

Fig. 1

Gel electrophoresis of the polymerase chain reaction products formed with primer Pc-12-F/Pc-12-R and bacterial DNA of Pseudomonas coronafaciens strains. Lanes 1~13, P. coronafaciens LMG 5060, KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, LMG 2330; lane 14, water as a negative control.

Fig. 2

Gel electrophoresis of the polymerase cain reaction products formed with primer Pc-12-F/Pc-12-R and total DNA of lane 1, Pseudomonas coronafaciens LMG 5060, lanes 2–31, P. syringae pvs, actinidiae KACC 10582, antirrhini ICMP 4303, aptata DSM 50252, atrofaciens ICMP 4394, berberidis NCPPB 2724, ciccaronei NCPPB 2355, delphhinii ICMP 529, dysoxyli ICMP 545, eriobotyae NCPPB 2331, helianthi NCPPB 1229, japonica ICMP 6305, lachrymans ATCC 11965, lapsa ATCC 10859, maculicola ICMP 3935, mellea ICMP 5711, mori ICMP 4331, morsprunorum ICMP 5795, myricae ICMP 7118, panici NCPPB 1498, papulans ICMP 4040, passiflorae NCPPB 1386, persicae NCPPB 2761, pisi ICMP 4433, ribicola NCPPB 963, sesami NCPPB 1016, syringae NCPPB 388, tabaci ICMP 2835, tagetis ICMP 4091, tomato NCPPB 2683, ulmi NCPPB 632; lanes 32~34, P. savastanoi pvs. glycinea NCPPB 1134, pahseolicola KACC 10575, and savastanoi NCPPB 639; lane 35, Acidovorax avenae subsp. avenae NCPPB 1011; lanes 36–38, Clavibacter michiganensis subsp. insidiosus NCPPB 1020, michiganensis NCPPB 1064, sepedonicus NCPPB 2137; lane 39, Pectobacterium carotovorum subsp. carotovorum, NCPPB 312; lanes 40–43, Rhizobium radiobacter DSM 30205, R. rhizogenes ATCC 11325, R. rubi NCPPB 1854, R. vitis NCPPB 3554; lane 44, Rhodococcus fascians LMG 3601; lane 45, Ralstonia solanacearum NCPPB 339; lanes 46–47, Xanthomonas campestris pvs. campestris KACC 10377, vesicatoria KACC 11157; lane 48, X. oryzae pv. oryzae KACC 10331; lane 1, P. coronafaciens LMG 5060, as a positive control; lane 49, water as a negative control.

Fig. 3

Sensitivity and specificity of the TaqMan real-time PCR with primer, Pc-12-ne-F/Pc-12-ne-R and TaqMan probe, Pc-taqman. (A) The linear regression generated by ten-fold dilution of DNA of Pseudomonas coronafaciens LMG 5060 and (B) TaqMan PCR with DNAs (circle dot) of P. coronafaciens LMG 5060, and P. coronafaciens strains (square dot): P. coronafaciens KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, and LMG 2330.