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

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 phID 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 phID-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 x 10(3) CFU/g and 5 x 10(4) CFU/g respectively. PhID-T-RFLP and phID-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 phID-T-RFLP. Additionally, groups C and F were detected in greenhouse samples but only by phID-T-RFLP and not by phID-DGGE. In conclusion, the new phID-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 phID-DGGE. (C) 2010 Elsevier B.V. All rights reserved.