Résultat de la recherche
How elevated pCO2 modifies total and metabolically active bacterial communities in the rhizosphere of two perennial grasses grown under field conditions
The response of total (DNA-based analysis) and active (RNA-based analysis) bacterial communities to a pCO2 increase under field conditions was assessed using two perennial grasses: the nitrophilic Lolium perenne and the oligonitrophilic Molinia coerulea. PCR- and reverse transcriptase-PCR denaturing gradient gel electrophoresis analysis of 16S rRNA genes generated contrasting profiles. The pCO2 increase influenced mainly the active and root-associated component of the bacterial community. Bacterial groups responsive to the pCO2 increase were identified by sequencing of corresponding denaturing gradient gel electrophoresis bands. About 50% of retrieved sequences were affiliated to Proteobacteria. Our data suggest that Actinobacteria in soil and Myxococcales (Deltaproteobacteria) in root are stimulated under elevated pCO2.
Nitrogen fertiliser rate affects the frequency of nitrate-dissimilating Pseudomonas spp. in the rhizosphere of Lolium perenne grown under elevated pCO2 (Swiss FACE)
The effect of elevated pCO2 (60 Pa) on the frequency of nitrate-dissimilating Pseudomonas (NDP) was investigated in the rhizosphere of fertilised Lolium perenne swards in the Swiss Free Air Carbon dioxide Enrichment (FACE) experiment. Numbers of cultivable root-associated Pseudomonas were greater under elevated (60 Pa) than under ambient (36 Pa) pCO2 in both high and low N-fertilised swards. For both pCO2 conditions, the NDP frequency decreased with closer root proximity to L. perenne roots in low fertilised swards. Anyway, in high N swards the NDP frequency was similar in root and soil fractions. Thus, N availability may be a major factor influencing NDP populations under elevated pCO2, most likely due to increased competition for N between plant and nitrate-dissimilating bacteria.
Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns
Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities. However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation. A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process. The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles. Applying statistical method make the DGE fingerprinting technique a promising tool. Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter. As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.
Phenotypic structure of Pseudomonas populations is altered under elevated pCO2 in the rhizosphere of perennial grasses
The increasing atmospheric CO2 content (pCO2) is likely to modify the ecosystem functioning including rhizosphere bacteria that are directly dependent on rhizodeposition. This may include alteration of Pseudomonas populations that display phenotypic traits in relation with plant fitness. In the present study, 1228 Pseudomonas strains were isolated from the non-rhizosphere soil, rhizosphere soil and root fractions of perennial grassland systems: Lolium perenne and Molinia coerulea. Both plants were grown under ambient (36 Pa) and elevated (60 Pa) pCO2 in the Swiss Free Air CO2 Enrichment (FACE) system. Pseudomonas spp. were tested for their ability to produce auxin, siderophores and hydrogen cyanide, and to dissimilate nitrate. No effect of root proximity and elevated pCO2 was observed on the proportions of auxin producers. For L. perenne and M. coerulea, siderophore and hydrogen cyanide Pseudomonas producers were stimulated in the root fraction. In contrast lower frequencies of nitrate reducers were observed in the root fraction compared to non-rhizosphere soil. The frequencies of siderophore producers and nitrate dissimilating strains were higher, and those of hydrogen cyanide producers lower, under elevated pCO2 for L. perenne. This alteration of the phenotypic structure of Pseudomonas guild in the root fraction is discussed in relation with the physico-biochemical modifications of the rhizosphere condition via rhizodeposition and environmental changes occurring under elevated pCO2.
Examination of Gould’s modified S1 (mS1) selective medium and Angle’s non-selective medium for describing the diversity of Pseudomonas spp. in soil and root environments
Studies on the diversity of environmental culturable Pseudomonas populations are dependent on the isolation procedure. This procedure includes the use of selective media which may influence the recovery of strains and thus the diversity described. In this study, we assessed the use of two agar isolation media for describing the diversity of soil- and root-inhabiting Pseudomonas associated with the perennial grass Molinia coerulea. A total of 382 Pseudomonas strains were recovered on either non-selective Angle’s medium, or on Gould’s modified S1 (mS1) Pseudomonas-selective medium. Their diversity was assessed by restriction analysis of PCR (polymerase chain reaction)-amplified 16S–23S rDNA internal transcript spacer sequences. The comparison of mS1- and Angle-recovered populations showed that the use of mS1 selective medium led to an underestimation of both Pseudomonas counts and diversity, especially in the soil environment.
nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass
Rhizosphere associative dinitrogen fixation could be a valuable source of nitrogen in many nitrogen limited natural ecosystems, such as the rhizosphere of Molinia coerulea, a hemicryptophytic perennial grass naturally occurring in contrasted oligonitrophilic soils. The diversity of the dinitrogen-fixing bacteria associated with this environment was assessed by a cloning–sequencing approach on the nifH gene directly amplified from environmental DNA extracts. Seventy-seven randomly picked clones were analysed. One type of NifH sequence was dominant in both roots and surrounding soil, and represented 56% of all retrieved sequences. This cluster included previously described environmental clones and did not contain any NifH sequences similar to cultivated diazotrophs. The predominance of few NifH sequence types in the roots and the rhizosphere of Molinia coerulea indicate that the plant environment mediates a favourable niche for such dinitrogen-fixing bacteria.
Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions
A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO2 content (pCO2) at the Swiss FACE (Free Air CO2 Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO2 in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO2 treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO2. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO2 treatment.
Influence d'une augmentation en pCO2 atmosphérique sur les communautés bactériennes associées à Molinia coerulea
La thèse traite de l'influence de l'augmentation de la teneur en dioxide de carbone de l'atmosphère observée depuis un siècle sur terre. Nous avons postulé que l'augmentation en CO2 atmosphérique modifierait en premier lieu les microorganismes du sol situés à proximité des racines, car cela altérerait la quantité et la qualité des exsudats racinaires. Nous nous sommes plus particulièrement intéressés aux communautés bactériennes associées au système racinaire des graminées pérennes. En effet, ces plantes sont particulièrement intéressantes dans ce contexte car d'une part, elles poussent au même endroit d'une année à l'autre et d'autre part, les graminées sont connues pour allouer une grande part des produits de la photosynthèse dans l'interface sol-racine, nommée rhizosphère. La plante modèle choisie est Molinia coerulea (la canche bleue), une graminée pérenne hémicryptophyte qui pousse naturellement dans des sols ayant très peu d'azote disponible (sols à nappe battante, tourbières). Nous pensons que l'écologie et la physiologie même de cette plante en fait une candidate idéale pour réagir à une augmentation en CO2 atmosphérique. Le manuscrit suit trois axes principaux correspondant aux trois grands thèmes menés en parallèle: La première partie s'intéresse aux communautés microbiennes dans leur ensemble. Nous avons particulièrement orienté nos efforts sur l'interprétation statistique des empreintes moléculaires (fingerprint). Ces fingerprints sont basés sur la séparation éléctrophorétique en gel avec gradient de dénaturation (DGGE) du gène ribosomique 16S. Des indices de diversité spécialement adaptés aux fingerprints ont été mis au point pour permettre une meilleure interprétation des profils. Les méthodes d'ordination (AFC et ACC) appliquées à ces profils moléculaires ont permis de mettre en exergue des populations de d-protéobactéries plus abondantes près des racines lorsque les plantes étaient cultivées sous une atmosphère enrichie en CO2. De même, on a repéré des populations d'actinomycètes peu abondantes mais actives dans le sol dans ces mêmes conditions de culture. La technique Biolog nous a permis de montrer que les communautés bactériennes de la rhizosphère, et non celles du sol distant des racines, réagissaient à une augmentation de la teneur en CO2 atmosphérique. Les hydrates de carbones sont en particulier plus facilement consommés sont par les communautés racinaires sous atmosphère enrichie. La deuxième partie de mon doctorat a concerné les populations du genre Pseudomonas. Nous avons d'abord mis au point un outil de caractérisation de la diversité spécifique à ce genre, puis nous avons caractérisé cette diversité dans la prairie naturelle. Enfin, nous avons comparé les Pseudomonas réducteurs de nitrate (première étape de la dénitrification) sous atmosphère enrichie et sous atmosphère ambiante. La troisième partie du travail est consacrée aux bactéries fixatrices d'azote (BFN). Nous avons choisi comme marqueur moléculaire le gène nifH (codant pour la nitrogénase réductase, l'enzyme clé de la nitrogénase) pour suivre la guilde des BFN dans l'environnement racinaire. Le résultat le plus frappant a été la mise en évidence d'un groupe de BFN non-cultivables qui représentaient 58% des BFN dans l'environnement naturel de Molinia coerulea. Les BFN semblent présentes aussi bien dans les racines que dans le sol environnant, mais la fixation d'azote semble s'effectuer uniquement dans la racine (détection des transcrits des gènes nifH). Nous en avons conclu que le sol pouvait servir d'habitat et de réservoir aux BFN mais que c'était la racine qui constituait le véritable siège de la fixation d'azote dans notre système., The thesis deals with the influence of the rising of the atmospheric carbon dioxide content observed on earth since one century. We postulated that the global changes would first alter the soil organisms located near the roots because of a modification of the quantity and quality of root exudates with an elevation of CO2 content. We focused on the microbial communities living near the roots of perennial grasses. They are particularly interesting because they grow at the same place from one year to an other and grasses are known to allocate an especially huge amount of photosynthesis derived carbon in the soil-root interface, called the rhizosphere. The model plant studied is Molinia coerulea, an hemicryptophytic perennial grass, naturally occurring in available nitrogen poor soil. We postulated that its physiology allow to react to an increase of atmospheric CO2 content. The manuscript followed three main axes. The first part deals with the microbial communities taken as a whole. A special effort has been intended in the statistical interpretation of 16S rDNA DGGE fingerprinting profiles. New diversity indices were designed to better interpret the profiles. Ordination methods (CA and CCA) were successfully applied: active delta-proteobacteria populations were highlighted near the roots under elevated CO2 conditions and non-dominant but active actinobacteria were characteristic of the soil environment. The physiological status of the bacterial communities has been studied using BIOLOG plates. As expected, the bacterial root community under elevated CO2 better metabolized the carbohydrates than the control one. The second part of the manuscript focused on the Pseudomonas genus. We first described the diversity of the Pseudomonas present in the natural conditions were Molinia coerulea is found. We showed an increase of the nitrate reducing Pseudomonas under elevated CO2 content. The third part of the manuscript targeted the nitrogen-fixing bacteria (NFB) in the rhizospheric environment. We used the nifH gene (encoding for the catabolic unit of the dinitrogenase reductase) as molecular marker to follow the guild of NFB. The most striking result is the dominance of an uncultured group of NFB in the natural condition detected by molecular methods. Working on nifH mRNA leads to an other important result. The NFB seems to be either present in soil and root environments but the NFB were only active near the roots. We concluded that the soil could be a reservoir of NFB and the root itself could be considered as the niche for NFB.
Specific PCR Amplification for the Genus Pseudomonas Targeting the 3′ Half of 16S rDNA and the Whole 16S–23S rDNA Spacer
A PCR protocol was developed for the selective amplification of a segment of the ribosomal RNA operon in Pseudomonas strains. Two specific conserved sequences suitable for PCR priming were identified in the middle of the 16S rDNA and at the very beginning of the 23S rDNA respectively. As a result, amplified region includes the 3′ half of the 16S rDNA with the whole 16S–23S rRNA Internal Transcripted Spacer (ITS1) sequence. The specificity of the primer set was checked on sequence databases and validated on collection strains and on one hundred soil bacterial isolates. Our results showed that both collection, soil-inhabiting Pseudomonas and some Pseudomonas-related Azotobacter DNAs could be amplified. This specific PCR for the detection of Pseudomonas strains was in good agreement with colony hybridisation using a Pseudomonas-specific probe. The targeted segment is relevant for a characterisation at the species (16S rDNA) as well as at the infraspecific (ITS1) levels. This PCR-based approach offers promising potential for the characterisation of environmental Pseudomonas populations.