Novel T-RFLP method to investigate six main groups of 2,4-diacetylphloroglucinol-producing pseudomonads in environmental samples

von Felten A., Meyer JB., Défago G. and Maurhofer M.

Journal of Microbiological Methods 84: 379-387, 2011

Strains offluorescent 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-RFLPmethodwas determined for the two P. fluorescens strains F113 (group B) and CHA0 (group F) in rhizosphere samples and was found to be 5×103 CFU/g and 5×104 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 Fwere detected in greenhouse samples but only by phlD-T-RFLP and not by phlD-DGGE. In conclusion, the newphlD-T-RFLPmethod proved to be a fast and reliablemethod to detect strains of the six main groups of DAPG-producers in environmental samples with an improved detection limit compared to phlD-DGGE.

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