title: Novel T-RFLP method to investigate six main groups of 2,4-diacetylphloroglucinol-producing pseudomonads in environmental samples.
abstract: Strains of fluorescent pseudomonads producing 2,4-diacetylphloroglucinol (DAPG) are involved in the protection of plant roots against soil-borne plant pathogens. Recently, a multilocus sequence analysis of a world wide collection of DAPG-producers led to the identification of six main groups (A-F). In this study a T-RFLP method based on the phlD gene was developed to efficiently identify the members of these six groups in environmental samples. A combination of six restriction enzymes was identified which leads to group specific terminal fragments (T-RF). The detection limit of the phlD-T-RFLP method was determined for the two P. fluorescens strains F113 (group B) and CHA0 (group F) in rhizosphere samples and was found to be 5×10(3)CFU/g and 5×10(4)CFU/g respectively. PhlD-T-RFLP and phlD-DGGE analysis of wheat and maize root samples from greenhouse and field revealed similarly the presence of multilocus groups A, B and D. However, they were more frequently detected with phlD-T-RFLP. Additionally, groups C and F were detected in greenhouse samples but only by phlD-T-RFLP and not by phlD-DGGE. In conclusion, the new phlD-T-RFLP method proved to be a fast and reliable method to detect strains of the six main groups of DAPG-producers in environmental samples with an improved detection limit compared to phlD-DGGE.
authors.affiliation: Institute of Integrative Biology, Plant Pathology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: von Felten
authors.forename: Joana Beatrice
authors.initials: JB
authors.lastname: Meyer
authors.forename: Geneviève
authors.initials: G
authors.lastname: Défago
authors.forename: Monika
authors.initials: M
authors.lastname: Maurhofer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004783:Environmental Microbiology; D009691:Nucleic Acid Denaturation; D010696:Phloroglucinol / Q000031:analogs & derivatives / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D011549:Pseudomonas / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D058441:Rhizosphere; D014908:Triticum; D003313:Zea mays
chemical_list: D001426:Bacterial Proteins; C096183:PhlD protein, Pseudomonas; C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol