title: Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining.
abstract: The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.
authors.affiliation: DLO Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
authors.forename: J M
authors.initials: JM
authors.lastname: van der Wolf
authors.forename: J R
authors.initials: JR
authors.lastname: van Beckhoven
authors.forename: P M
authors.initials: PM
authors.lastname: de Vries
authors.forename: J M
authors.initials: JM
authors.lastname: Raaijmakers
authors.forename: P A
authors.initials: PA
authors.lastname: Bakker
authors.forename: Y
authors.initials: Y
authors.lastname: Bertheau
authors.forename: J W
authors.initials: JW
authors.lastname: van Vuurde
mesh_terms: D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D016974:Dickeya chrysanthemi / Q000235:genetics* / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique / Q000379:methods*; D010944:Plants / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial