Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Abstract

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.

FIG. 1

Cluster analysis of genomic fingerprint patterns of phlD-containing Pseudomonas strains generated by PCR amplification of whole-cell template with the BOXA1R primer. Only patterns of strains representing unique genotypes isolated from individual soils are shown. Two independent amplifications were used for each strain. Using GelCompar 4.0, the UPGMA algorithm was applied to the similarity matrix generated from the tracks of the whole patterns by using Pearson's correlation coefficient. The similarity coefficient used to define distinct groups (see Materials and Methods) is noted (∗). Distinct groups of genotypes, labeled alphabetically A through N, are discussed more fully in the text.

FIG. 2

Cluster analysis of genomic fingerprint patterns of phlD-containing Pseudomonas strains generated by PCR amplification of whole-cell template with the ERIC primer set. Only patterns of strains representing unique genotypes isolated from individual soils are shown. Two independent amplifications were used for each strain. Using GelCompar 4.0, the UPGMA algorithm was applied to the similarity matrix generated from the tracks of the whole patterns by using Pearson's correlation coefficient. The similarity coefficient used to define distinct groups (see Materials and Methods) is noted (∗). Distinct groups of genotypes, labeled alphabetically A through N, are discussed more fully in the text.

FIG. 3

Cluster analysis of carbon source utilization patterns of phlD-containing Pseudomonas strains cultured for 3 days. Only strains representing unique genotypes isolated from individual soils were assayed. Two independent assays were performed on each strain. Using MULTIV 1.2.1, the simple linkage (a) and complete linkage (b) algorithms were applied to the similarity matrix generated by using Pearson's correlation coefficient. The similarity coefficient used to define distinct groups (see Materials and Methods) is noted (∗). Distinct groups of phenotypes, labeled alphabetically A through G, are discussed more fully in the text.