new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

• Published in: FEMS microbiology ecology 2008-06, Vol.64 (3), p.468-481
• Main Authors: Frapolli, Michele, Moënne-Loccoz, Yvan, Meyer, Joana, Défago, Geneviève
• Format: Article
• Language: English
• Subjects: 2,4-diacetylphloroglucinol; Animal, plant and microbial ecology; Bacterial Proteins - genetics; Bacteriology; biocontrol; Biological and medical sciences; Biological control; Black root; Consortia; Deoxyribonucleic acid; DGGE; DNA; DNA Fingerprinting - methods; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Ecology; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Life Sciences; Microbial ecology; Microbiology; Microbiology and Parasitology; Miscellaneous; Molecular Sequence Data; Nicotiana - microbiology; Nucleic Acid Denaturation; Phloroglucinol - analogs & derivatives; Phloroglucinol - metabolism; Phylogeny; Plant protection; Plant Roots - microbiology; Plants (botany); Protocol; Pseudomonas; Pseudomonas - classification; Pseudomonas - genetics; Pseudomonas - growth & development; Pseudomonas - metabolism; Pseudomonas fluorescens; Rhizosphere; Root rot; Sequence Analysis, DNA; Sequence Homology; Soil Microbiology; Soils; Strains (organisms); suppressive soil; suppressive soils; Tobacco
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1111/j.1574-6941.2008.00471.x

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD⁺ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD⁺colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD⁺ pseudomonads.