Identification of traits shared by rhizosphere-competent strains of fluorescent pseudomonads.

Rhizosphere competence of fluorescent pseudomonads is a prerequisite for the expression of their beneficial effects on plant growth and health. To date, knowledge on bacterial traits involved in rhizosphere competence is fragmented and derived mostly from studies with model strains. Here, a population approach was taken by investigating a representative collection of 23 Pseudomonas species and strains from different origins for their ability to colonize the rhizosphere of tomato plants grown in natural soil. Rhizosphere competence of these strains was related to phenotypic traits including: (1) their carbon and energetic metabolism represented by the ability to use a wide range of organic compounds, as electron donors, and iron and nitrogen oxides, as electron acceptors, and (2) their ability to produce antibiotic compounds and N-acylhomoserine lactones (N-AHSL). All these data including origin of the strains (soil/rhizosphere), taxonomic identification, phenotypic cluster based on catabolic profiles, nitrogen dissimilating ability, siderovars, susceptibility to iron starvation, antibiotic and N-AHSL production, and rhizosphere competence were submitted to multiple correspondence analyses. Colonization assays revealed a significant diversity in rhizosphere competence with survival rates ranging from approximately 0.1 % to 61 %. Multiple correspondence analyses indicated that rhizosphere competence was associated with siderophore-mediated iron acquisition, substrate utilization, and denitrification. However, the catabolic profile of one rhizosphere-competent strain differed from the others and its competence was associated with its ability to produce antibiotics phenazines and N-AHSL. Taken together, these data suggest that competitive strains have developed two types of strategies to survive in the rhizosphere.

Fluorescent pseudomonads harboring type III secretion genes are enriched in the mycorrhizosphere of Medicago truncatula.

Type III secretion systems (T3SSs) of Gram-negative bacteria mediate direct interactions with eukaryotic cells. Pseudomonas spp. harboring T3SS genes (T3SS+) were previously shown to be more abundant in the rhizosphere than in bulk soil. To discriminate the contribution of roots and associated arbuscular mycorrhizal fungi (AMF) on the enrichment of T3SS+ fluorescent pseudomonads in the rhizosphere of Medicago truncatula, their frequency was assessed among pseudomonads isolated from mycorrhizal and nonmycorrhizal roots and from bulk soil. T3SS genes were identified by PCR targeting a conserved hrcRST DNA fragment. Polymorphism of hrcRST in T3SS+ isolates was assessed by PCR-restriction fragment length polymorphism and sequencing. Genotypic diversity of all pseudomonads isolated, whether or not harboring T3SS, was described by BOX-PCR. T3SS+ pseudomonads were significantly more abundant in mycorrhizal than in nonmycorrhizal roots and in bulk soil, and all were shown to belong to the phylogenetic group of Pseudomonas fluorescens on the basis of 16S rRNA gene identity. Four hrcRST genotypes were described; two only included isolates from mycorrhizal roots. T3SS+ and T3SS- pseudomonads showed different genetic backgrounds as indicated by their different BOX-PCR types. Taken together, these data suggest that T3SSs are implicated in interactions between fluorescent pseudomonads and AM in medic rhizosphere.

Phenazine antibiotics produced by fluorescent pseudomonads contribute to natural soil suppressiveness to Fusarium wilt.

Natural disease-suppressive soils provide an untapped resource for the discovery of novel beneficial microorganisms and traits. For most suppressive soils, however, the consortia of microorganisms and mechanisms involved in pathogen control are unknown. To date, soil suppressiveness to Fusarium wilt disease has been ascribed to carbon and iron competition between pathogenic Fusarium oxysporum and resident non-pathogenic F. oxysporum and fluorescent pseudomonads. In this study, the role of bacterial antibiosis in Fusarium wilt suppressiveness was assessed by comparing the densities, diversity and activity of fluorescent Pseudomonas species producing 2,4-diacetylphloroglucinol (DAPG) (phlD+) or phenazine (phzC+) antibiotics. The frequencies of phlD+ populations were similar in the suppressive and conducive soils but their genotypic diversity differed significantly. However, phlD genotypes from the two soils were equally effective in suppressing Fusarium wilt, either alone or in combination with non-pathogenic F. oxysporum strain Fo47. A mutant deficient in DAPG production provided a similar level of control as its parental strain, suggesting that this antibiotic does not play a major role. In contrast, phzC+ pseudomonads were only detected in the suppressive soil. Representative phzC+ isolates of five distinct genotypes did not suppress Fusarium wilt on their own, but acted synergistically in combination with strain Fo47. This increased level of disease suppression was ascribed to phenazine production as the phenazine-deficient mutant was not effective. These results suggest, for the first time, that redox-active phenazines produced by fluorescent pseudomonads contribute to the natural soil suppressiveness to Fusarium wilt disease and may act in synergy with carbon competition by resident non-pathogenic F. oxysporum.

Diversity of root-associated fluorescent pseudomonads as affected by ferritin overexpression in tobacco.

A transgenic tobacco overexpressing ferritin (P6) was recently shown to accumulate more iron than the wild type (WT), leading to a reduced availability of iron in the rhizosphere and shifts in the pseudomonad community. The impact of the transgenic line on the community of fluorescent pseudomonads was assessed. The diversity of 635 isolates from rhizosphere soils, rhizoplane + root tissues, and root tissues of WT and P6, and that of 98 isolates from uncultivated soil was characterized. Their ability to grow under iron stress conditions was assessed by identifying their minimal inhibitory concentrations of 8-hydroxyquinoline for each isolate, pyoverdine diversity by isoelectrofocusing and genotypic diversity by random amplified polymorphism DNA. The antagonistic activity of representative isolates and of some purified pyoverdines against a plant pathogen (Pythium aphanidermatum Op4) was tested in vitro. In overall, isolates taken from P6 tobacco showed a greater ability to grow in iron stress conditions than WT isolates. The antagonism by some of the representative isolates was only expressed under iron stress conditions promoting siderophore synthesis and their pyoverdines appeared to have a specific structure as assessed by mass spectrometry. For other isolates, antagonism was still expressed in the presence of iron, suggesting the involvement of metabolites other than siderophores. Altogether, these data indicate that the transgenic tobacco that over-accumulates iron selected fluorescent pseudomonads, less susceptible to iron depletion and more antagonistic to the tested plant pathogen than those selected by the tobacco WT.

Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils.

The taxonomic position of eight fluorescent Pseudomonas strains isolated from bulk and rhizospheric soils, and from water was examined. These eight strains clustered in one phenon together with Pseudomomas mandelii (CFBP 4844T), but could still be differentiated from this type strain by four phenotypic features. The eight stains exhibited internal DNA-DNA hybridization values ranging from 60 to 100%, with deltaTm below 5 degrees C (3.9 and 4.3 degrees C) for the lowest values (60 and 66%). The percentages of hybridization with type or reference strains of other Pseudomonas species tested ranged from 12 to 60% (deltaTm = 5.5 degrees C), indicating that the eight isolates studied constituted a discrete DNA homology group. Comparison of the 16S rDNA sequence of the strain representing this group (CFBP 5737T) with the sequences of other strains belonging to the genus Pseudomonas revealed that strain CFBP 5737T was a member of this genus and that these bacteria did not cluster with any previously described species of the genus Pseudomonas. The eight isolates belonged to two siderovars different from those described so far. On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, and of siderotyping, a new species, Pseudomonas lini sp. nov. (type strain CFBP 5737T) is proposed.