The Composition of Fluorescent Pseudomonad Populations Associated with Roots Is Influenced by Plant and Soil Type

Populations of fluorescent pseudomonads isolated from an uncultivated soil and from the roots of two plant species were previously shown to differ (P. Lemanceau, T. Corberand, L. Gardan, X. Latour, G. Laguerre, J.-M. Boeufgras, and C. Alabouvette, Appl. Environ. Microbiol. 61:1004-1012, 1995). The diversities of fluorescent pseudomonads, from two uncultivated soils and from the roots of two plant species cultivated in these two soils, were compared. The phenotypic diversity of the bacterial isolates was characterized on the basis of biochemical and physiological tests and on the basis of their ability to utilize 147 different organic compounds. The genotypic diversity of the isolates was characterized on the basis of the types of 16S genes coding for rRNA (rDNA), their repetitive extragenic palindromic patterns by PCR, and plasmid profiles. Taxonomic identification of the isolates was achieved with both biochemical and physiological tests and by comparing their 16S rDNA types to those of reference and type strains of fluorescent Pseudomonas spp. Numerical analysis of phenotypic characteristics allowed the clustering of isolates that showed high levels of similarity. This analysis indicated that both soil type and host plant had an effect on the diversity of fluorescent pseudomonads. However, of the two factors studied, the soil was clearly the dominating one. Indeed, the populations associated with the roots of each plant species varied from one soil to the other. This variation could possibly be ascribed to the differences recorded between the phenotypically diverse populations of fluorescent pseudomonads from the two uncultivated soils. The plant selection was, at least partly, plant specific. It was not related to bacterial species and biovars or to the presence of plasmid DNA. The phenotypic clustering of isolates was well correlated with genotypic characterization by repetitive extragenic palindrome-PCR fingerprinting.     

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.     

The Soil Type Affects Both the Differential Accumulation of Iron between Wild-type and Ferritin Over-expressor Tobacco Plants and the Sensitivity of their Rhizosphere Bacterioflora to Iron Stress

Transgenic tobacco P6 over-expressing ferritin is known to activate iron transport systems and to have increased iron content. Iron phytoextraction by this transgene is then expected to be higher than that of the wild-type (WT). In the present study, the possibility to modify iron availability for bacteria via the cultivation of the transgene P6 was explored by comparing the sensitivity to iron stress of bacteria isolated from the rhizosphere of the two plant genotypes (WT and P6). This sensitivity was evaluated by measuring the bacterial density when plated on a solid media depleted (supplemented with 8-hydroxiquinoline) or not (supplemented with Fe-8-hydroxyquinoline) in iron. The experimental conditions favorable to the differential iron accumulation between the wild-type and transgenic tobacco were identified. The two plant genotypes were grown in three soils (Hervau, Thory and Oudun) chosen for their differences in iron content, and the plants were yielded at three stages (vegetative, floral bud and flowering). The highest differential accumulation of iron in favor of the over-expressing transgene was found in the plants at the floral bud stage when cultivated in the Oudun and Thory soils. Since at that stage, the plant growth was significantly higher in the Oudun soil, the phytoextraction of iron was the highest in this soil. At the floral bud stage, bacteria isolated from the rhizosphere of the transgene cultivated in the Oudun and Thory soils appeared to be less susceptible to iron stress than those from the wild-type. Bacterial density recovered on agar medium depleted in iron was significantly the highest in the rhizosphere of the transgene cultivated in the Oudun soil. Altogether, these data indicate that the over-expressing ferritin transgenic plants, that accumulate and extract more iron from the rhizosphere than the wild-type plants, select in their rhizosphere bacteria less susceptible to iron stress compared to those selected by the wild-type plants.     

Meta‐barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO‐11063, GnS‐GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico‐chemical characteristics and land use. A meta‐barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS‐GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO‐11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO‐11063 standard.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Suppression of fusarium wilts by fluorescent pseudomonads: Mechanisms and applications

Fluorescent pseudomonads are involved in the natural suppressiveness of some soils to fusarium wilts. These bacteria have been applied successfully to suppress fusarium wilts of various plant species grown in conducive soils and growing substrates. Suppression of fusarium wilts by fluorescent pseudomonads can be ascribed to direct and indirect effects against pathogenic Fusarium oxysporum. Direct effects are expressed by a reduction of the saprophytic growth of the pathogen leading to delay and reduction of root infections. This antagonism was demonstrated to be related to siderophore‐mediated iron competition. Iron competition was also shown to enhance the antagonistic effect of non‐pathogenic F. oxysporum by making the pathogen more susceptible to fungal competition for carbon. Indirect effects of fluorescent pseudomonads against pathogenic F. oxysporum are mainly related to the induction of host plant resistance which can be associated with the bacterial lipopolysaccharides. Suppression of fusarium wilts by some fluorescent pseudomonads could also be related to their ability to detoxify fungal metabolites such as fusaric acid. Association of different mechanisms of suppression increases the efficacy and consistency of the biological control under various experimental conditions. Increased knowledge of mechanisms of suppression now enables management of the environment, to some extent, to favour expression of the beneficial activities. These activities are only expressed if the bacterial density is sufficiently high.     

Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp

A total of 137 soilborne and plant-associated bacterial strains belonging to different Pseudomonas species were tested for their ability to synthesize N-acyl-homoserine lactones (NAHL). Fifty-four strains synthesized NAHL. Interestingly, NAHL production appears to be more common among plant-associated than among soilborne Pseudomonas spp. Indeed, 40% of the analyzed Pseudomonas syringae strains produced NAHL which were identified most often as the short-chain NAHL, N-hexanoyl-L-homoserine lactone, N-(3-oxo-hexanoyl)-homoserine lactone, and N-(3-oxo-octanoyl)-L-homoserine lactone (no absolute correlation between genomospecies of P. syringae and their ability to produce NAHL could be found). Six strains of fluorescent pseudomonads, belonging to the species P. chlororaphis, P. fluorescens, and P. putida, isolated from the plant rhizosphere produced different types of NAHL. In contrast, none of the strains isolated from soil samples were shown to produce NAHL. The gene encoding the NAHL synthase in P. syringae pv. maculicola was isolated by complementation of an NAHL-deficient Chromobacterium mutant. Sequence analysis revealed the existence of a luxI homologue that we named psmI. This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to psyI and ahlI, two genes involved in NAHL production in P. syringae pv. tabaci and P. syringae pv. syringae, respectively. We identified another open reading frame that we termed psmR, transcribed convergently in relation to psmI and partly overlapping psmI; this gene encodes a putative LuxR regulatory protein. This gene organization, with luxI and luxR homologues facing each other and overlapping, has been found so far only in the enteric bacteria Erwinia and Pantoea and in the related species P. syringae pv. tabaci.     

Interaction between Medicago truncatula and Pseudomonas fluorescens: Evaluation of Costs and Benefits across an Elevated Atmospheric CO2

Soil microorganisms play a key role in both plants nutrition and health. Their relation with plant varies from mutualism to parasitism, according to the balance of costs and benefits for the two partners of the interaction. These interactions involved the liberation of plant organic compounds via rhizodeposition. Modification of atmospheric CO₂ concentration may affect rhizodeposition and as a consequence trophic interactions that bind plants and microorganisms. Positive effect of elevated CO₂ on plants are rather well known but consequences for micoorganisms and their interactions with plants are still poorly understood. A gnotobiotic system has been developed to study the interaction between Medicago truncatula Jemalong J5 and the mutualistic bacteria Pseudomonas fluorescens strain C7R12 under two atmospheric CO₂ concentrations: ambient (365 ppm) versus enriched (750 ppm). Costs and benefits for each partner have been determined over time by measuring plant development and growth, the C and N contents of the various plant parts and the density of the bacteria in rhizosphere compartments. Following the increase in CO₂, there was a beneficial effect of P. fluorescens C7R12 on development, vegetative growth, and C/N content of M. truncatula. Concerning plant reproduction, an early seed production was noticed in presence of the bacterial strain combined with increased atmospheric CO₂ conditions. Paradoxically, this transient increase in seed production was correlated with a decrease in bacterial density in the rhizosphere soil, revealing a cost of increased CO₂ for the bacterial strain. This shift of costs-benefits ratio disappeared later during the plant growth. In conclusion, the increase in CO₂ concentration modifies transiently the cost-benefit balance in favor of the plant. These results may be explained either by a competition between the two partners or a change in bacterial physiology. The ecosystem functioning depends on the stability of many plant-microbe associations that abiotic factors can disrupt.