Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Abstract

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.

FIG. 1.

Mean population densities of different genotypes of indigenous phlD⁺ Pseudomonas spp. isolates on the roots of pea cvs. Little Marvel (○) and WSU 31 (▪) grown in Mt. Vernon FWS soil for eight successive cycles of 3 weeks each. Mean values and standard deviations are presented. Bars indicate the percentage of phlD⁺ isolates (n = 148) characterized as genotype L (white bars), D (black bars), P (grey bars), and Q (hatched bars) by BOX-PCR.

FIG. 2.

Cluster analysis of genomic fingerprint patterns generated by BOX-PCR amplification of whole-cell genomic DNA from phlD⁺ reference strains and representative fluorescent Pseudomonas spp. isolates indigenous to Mt. Vernon FWS and Quincy TAD soils (indicated by asterisks) (see Table 1). Two independent amplifications were used for each isolate. The UPGMA algorithm was applied to the similarity matrix generated from the tracks of the whole patterns by using the pairwise Pearson's product-moment correlation coefficient (r value). The similarity coefficient used to define distinct groups (see Materials and Methods) is noted (∗).

FIG. 3.

Cluster analysis of combined HaeIII, TaqI, and MspI RFLP patterns of the 629-bp phlD fragment amplified from isolates of fluorescent Pseudomonas spp. Patterns include those of reference strains (BOX-PCR genotypes A to N) and isolates used in the cycling experiment (Fig. 4), which are indicated by an asterisks. Two independent amplifications were used for each reference strain. The UPGMA algorithm was applied to the similarity matrix generated with the Dice coefficient. A 100-bp DNA size standard ladder is shown at the top.

FIG. 4.

Population dynamics of P. fluorescens Pf-5 (∗), Q2-1 (○), Q2-87 (•), MVP1-6 (⊞), MVW1-2 (□), Q8r1-96 (▪), 5MR2 (⋄), QT1-6 (♦), W4-4 (▴), 1MA1 (▵), 7MA12 (⋆), MVW1-1 (▿), MVP1-4 (▾), and MVW4-2 (×) belonging to different genotypes on the roots of pea cv. Rex grown in Quincy virgin soil for five successive cycles of 3 weeks each. Each isolate was introduced into the soil to give a final density of approximately log 4 CFU per g of soil (cycle 0). Mean values and standard deviations are presented. The arrow in the treatment with isolate MVW4-2 (Q genotype) indicates the point at which contamination with the P genotype isolate MVP1-4 occurred during the repotting of the soil (see Results).