title: Isolation and identification of Pseudomonas syringae facilitated by a PCR targeting the whole P. syringae group.
abstract: We present a reliable PCR-based method to avoid the biases related to identification based on the conventional phenotypes currently used in the identification of Pseudomonas syringae sensu lato, a ubiquitous environmental bacterium including plant pathogens. We identified a DNA target suitable for this purpose by applying a comparative genomic pipeline to Pseudomonas genomes. We designed primers and developed PCR conditions that led to a clean and strong PCR product from 97% of the 185 strains of P. syringae strains tested and gave a clear negative result for the 31 non-P. syringae strains tested. The sensitivity of standard PCR was determined with pure strains to be 10(6) bacteria mL(-1) or 0.4 ng of DNA μL(-1). Sensitivity could be improved with the touchdown method. The new PCR-assisted isolation of P. syringae was efficient when deployed on an environmental sample of river water as compared to the isolation based on phenotypes. This innovation eliminates the need for extensive expertise in isolating P. syringae colonies, was simpler, faster and very reliable. It will facilitate discovery of more diversity of P. syringae and research on emergence, dispersion and evolution to understand the varied functions of this environmental bacterium.
authors.affiliation: INRA, UR0407 Pathologie Végétale, F-84143 Montfavet cedex, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Guilbaud
authors.affiliation: INRA, UR0407 Pathologie Végétale, F-84143 Montfavet cedex, France.
authors.forename: Cindy E
authors.initials: CE
authors.lastname: Morris
authors.affiliation: CEA, IBEB, Lab Ecol Microb Rhizosphere and Environ Extrem, Saint-Paul-lez-Durance, F-13108, France CNRS, UMR 7265 Biol Veget and Microbiol Environ, Saint-Paul-lez-Durance, F-13108, France Aix Marseille Université, BVME UMR7265, Marseille, F-13284, France.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Barakat
authors.affiliation: CEA, IBEB, Lab Ecol Microb Rhizosphere and Environ Extrem, Saint-Paul-lez-Durance, F-13108, France CNRS, UMR 7265 Biol Veget and Microbiol Environ, Saint-Paul-lez-Durance, F-13108, France Aix Marseille Université, BVME UMR7265, Marseille, F-13284, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Ortet
authors.affiliation: INRA, UR0407 Pathologie Végétale, F-84143 Montfavet cedex, France odile.berge@avignon.inra.fr.
authors.forename: Odile
authors.initials: O
authors.lastname: Berge
mesh_terms: D001483:Base Sequence; D005075:Biological Evolution; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D058889:Molecular Typing / Q000379:methods; D010641:Phenotype; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D044224:Pseudomonas syringae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D045483:Rivers / Q000382:microbiology*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: P. syringae diversity; P. syringae identification; PCR-assisted detection; environmental pathogen; population structure; specific PCR