Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences.
Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.
Cho
Min Seok
MS
National Academy of Agricultural Science, Rural Development Administration, 441-707 Suwon, Republic of Korea.
Jeon
Yong Ho
YH
Kang
Man Jung
MJ
Ahn
Hong Il
HI
Baek
Hyung-Jin
HJ
Na
Young Wang
YW
Choi
Yu Mi
YM
Kim
Tae San
TS
Park
Dong Suk
DS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D007887:Fabaceae / Q000382:microbiology*; D009952:Ornithine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D044224:Pseudomonas syringae / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; D045522:Recombinases; C021234:phaseolotoxin; D009952:Ornithine; D019914:Taq Polymerase