Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings

Abstract

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.

FIG. 1

Molecular analysis of P. fluorescens F113Rif and the 498 isolates of RCFP by ARDRA. The DNA fragments obtained by ARDRA were separated by electrophoresis using an automated laser fluorescent sequencer. The resulting traces were then incorporated into image analysis software (WinCam2.2) and converted into the banding patterns shown. Four ARDRA groups were identified for 496 of the 498 RCFP isolates when restriction analysis of amplified 16S rDNA was done using AluI and TaqI as follows. With AluI, the isolates yielded profile AluI-2 or AluI-13 (A). With TaqI, isolates with profile AluI-13 displayed profile TaqI-1, TaqI-7, or TaqI-12, and those with profile AluI-2 yielded profile TaqI-1 (B). The biocontrol inoculant F113Rif displayed profiles AluI-13 and TaqI-1.

FIG. 2

Diversity of RAPD profiles for 16 isolates randomly chosen from the 498 RCFP isolates (lanes 3 to 23) and for strain F113Rif (lane 1). PCR fragments generated by the RAPD technique were separated by electrophoresis, and the photograph was taken after silver staining of the gel. The size marker ΦX174-RF-DNA (HaeIII digest; Amersham Pharmacia Biotech) is included in lane 2 and shows bands (from top to bottom) of 1,358, 1,078, 872, 603, 310 to 271 (in fact, three bands too close to each other to be distinguished), 234, 194, and 118 bp (a 72-bp band is too faint to be seen). Isolates X109, X116, and X117 (lanes 14, 21, and 22, respectively) exhibited the same RAPD profile, as did isolates X113 and X115 (lanes 18 and 20, respectively). The reproducibility of the RAPD procedure is illustrated by duplicate assays of isolates X103 (in lanes 8 and 9) and X110 (in lanes 15 and 16).