Biodiversity of rhizosphere bacteria associated with perennial grasses under elevated CO2

 

Keywords: rhizosphere, biodiversity, perennial grasses, Molinia coerulea, Lolium perenne, elevated atmospheric CO2 content, FACE, plant – bacteria interactions, molecular ecology.

Introduction

The rhizosphere environment, at the interface between root and soil, is a major habitat for soil processes. A significant part of the Carbon fixed by plants during photosynthesis is released into the soil during rhizodeposition. Root exudates, the soluble part of rhizodeposition, are used for the growth of microorganisms naturally occurring in the soil. Rhizodeposition (its quality and composition) has therefore a great influence on the development of rhizosphere microflora, which contributes to soil and plant health.
Our project is focusing on bacterial communities associated to the rhizosphere of two perennial hemicryptophytic grasses with different trophic requirements, Molinia coerulea and Lolium perenne. We are investigating the diversity of rhizobacteria in relation to the plant and its growth conditions with a particular interest for elevation of atmospheric CO2 content.


Keywords: rhizosphere, perennial grasses Molinia coerulea, Lolium perenne, elevated CO2, FACE, plant-bacteria interactions

Research areas

Rhizosphere bacterial communities: molecular profiling

Keywords: bacterial community, DGGE, fingerprinting, diversity, statistical analysis

The characterisation and monitoring of total and active bacterial communities is achieved by a DGGE (Denaturing Gradient Gel Electrophoresis) analysis of 16S rDNA gene fragments directly amplified from soil and root DNA or RNA extracts (after RT step). The dominant bacterial populations are displayed on the DGGE profiles and can be used for the monitoring of the bacterial community structure in relation to the growth conditions of the plants: proximity to the plant root, elevated atmospheric CO2 content, and sampling date (which reflects the growth stage and other environmental conditions). ‘Indicator’ populations (for which occurrence is affected by these parameters) can then be selected by using ordination methods (PCA, CA, CAA) on DGGE patterns. The use of statistical tools is helpful for an ecological interpretation of DGGE patterns in terms of community structure and ecosystem functioning. Such approaches allow to test the statistical meaning of changes in the patterns and to correlate these changes with other environmental parameters.

  • Fromin et al. (2002) Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environmental Microbiology 4(11): 634-643.
  • Hamelin et al. From microbial community barcode to genetic heterogeneity indices (in prep)
In the FACE experiment, the plant roots highly influence the bacterial community structure. Moreover, changes related to CO2 treatment have been evidenced. Both root and CO2 influenced more strongly the active communities (RNA patterns). The sequencing of ‘indicator’ bands revealed that Proteobacteria (delta and alpha mainly) responded to these environmental changes.

Rhizosphere bacterial communities: metabolic profiling

Keywords: bacterial community, Biolog ECOPlates, metabolic profiling, diversity

Under elevated atmospheric CO2 content, a stimulation of rhizodeposition is generally described (more Carbon released in the rhizosphere, qualitative changes of exudate composition). In order to assess the influence of this altered rhizodeposition on bacterial communities, we have measured the ability of these communities to respire on 31 different Carbon sources using ECOPlates (Biolog®).
Statistical analyses of these metabolic profiles allowed to identify substrates that are differentially metabolised by the communities of plants grown under ambient and elevated CO2 content. Statistical analyses were performed with the help of Florian Kohler (department of Plant Ecology, University of Neuchâtel).

Contact : Jérôme Hamelin

Nitrogen fixing rhizobacteria

Keywords: nifH, nitrogen fixation, ARA, hybridisation, cloning, sequencing

The aim of our team is to understand to what extent Nitrogen limitation in the rhizosphere can influence the microbial communities associated to the plant. Two model plants are currently used in our laboratory: Lolium perenne, that needs high Nitrogen supplies for its growth, and Molinia coerulea, that is naturally occurring on low-Nitrogen soils. Both plants grown under ambient and CO2-enriched atmosphere (FACE experiments in Eschikon). Under elevated CO2, more Carbon is released in the rhizosphere by plant exudation, we thus expect a greater Nitrogen limitation (augmentation of the C:N ratio).
A molecular approach (DNA/RNA extraction, PCR, cloning, and sequencing of nifH genes) allowed to evidence a major bacterial group which nifH sequence is different of already culturable nitrogen-fixer sequences to date described. The relevance of this group in the nitrogen-fixing guild as well as its activity is currently assessed (RNA extraction, RT-PCR on nifH mRNA) and first results suggest that nitrogenase is active in the root fraction of M. coerulea.
At the same time, we are trying to isolate some representatives of this group. The Nitrogen fixation activity is measured by Acetylene Reduction Assay (ARA) on pure cultures, young plants and surrounding soil.

  • Hamelin et al. (2001) Inventaire moléculaire des bactéries fixatrices d’azote associées à la rhizosphère de Molinia coerulea. Bulletin de la Société Suisse de Pédologie 25: 35-40.
  • Hamelin et al. (2002) nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass. Environmental Microbiology 4(8):477-481.

Contact : Jérôme Hamelin

Diversity of Pseudomonas spp. populations associated with Lolium perenne and Molinia coerulea : effect of an atmospheric CO2 increase

Keywords : Pseudomonas, population structure, 16S-23S rDNA spacer, nitrate reduction, PGPR, rhizosphere competence.

Pseudomonas populations are frequently found in the rhizosphere of annual as well as perennial grasses. A previous study has shown that their dominance increased in the rhizosphere of Lolium perenne grown under elevated CO2 content :

  • Marilley et al. (1999) Influence of an elevated atmospheric CO2 content on soil and rhizosphere bacterial communities beneath Lolium perenne and Trifolium repens under field (FACE) conditions. Microbial Ecology 38:39-49.

In the present project, we compared the structure of Pseudomonas populations in the rhizosphere of L. perenne and M. coerulea grown under ambient and elevated pCO2.

A protocol of PCR amplification of 16S-23S rDNA spacer (ITS1) specific to the genus Pseudomonas was developed in order to check the affiliation of environmental isolates to Pseudomonas.

  • Locatelli et al. (2002) Specific PCR amplification for the genus Pseudomonas targeting the 3' half of 16S rDNA and the whole 16S-23S rDNA spacer. Systematic and Applied Microbiology 25(2):220-227.

The use of a selective (Gould’s modified mS1 medium) versus non-selective (Angle’s medium) for the isolation of environmental Pseudomonas strains was assessed. Our results showed that the use of the mS1 selective medium led to underestimate both Pseudomonas counts and diversity, especially in the soil environment.

  • Tarnawski et al. (2003) Examination of Gould’s modified S1 (mS1) selective medium and Angle’s non-selective medium for collecting diversity of Pseudomonas spp. in soil and root environments. FEMS Microbiology and Ecology 45: 97-104.

The influence of elevated pCO2 on the phenotypic structure of Pseudomonas was assessed by characterizing about 1200 Pseudomonas strains isolated from the rhizosphere of L. perenne and M. coerulea. The strains were characterized for potential traits related to plant fitness and rhizosphere competence (production of auxin, HCN, or sidérophores, nitrate dissimilation).

Our results showed that elevated pCO2 modified the phenotypic structure of Pseudomonas, especially for root-associated strains, probably via an alteration of rhizodeposition and, consequently, of the physico-biochemical conditions prevailing in the rhizosphere under high pCO2.

  • Tarnawski et al. Phenotypic structure of Pseudomonas populations is altered under elevated pCO2 in the rhizosphere of perennial grasses. Submitted to Soil Biology & Biochemistry.

Finally, the diversity of rhizosphere Pseudomonas was assessed by direct amplification of ITS1 sequences from environmental samples (root and soil suspensions). In that sense, we are investigating the use of ITS1 sequences as taxonomic markers.

Contact : Sonia Tarnawski

Nitrate-dissimilating Pseudomonas

In rhizosphere environment, the oxygen partial pressure is lower than in soil, because of root and rhizobacteria cell respiration. Some Pseudomonas can use nitrate as electron acceptor when oxygen tension is low.

In the present study, the response of nitrate-dissimilating Pseudomonas to an elevated atmospheric pCO2 was investigated.

The frequencies of nitrate-dissimilating and denitrifying Pseudomonas strains differed according to both root proximity and pCO2 content. The type of nitrate reductase enzyme (membrane-bound NAR versus periplasmic NAP) present in rhizosphere nitrate-dissimilating strains was characterized by a narG-napA multiplex PCR. A large diversity of narG and napA genes (encoding the catalytic subunits of NAR and NAP respectively) was described.

  • Roussel-Delif et al. (2004) Frequency and diversity of nitrate reductase genes among nitrate-dissimilating Pseudomonas in the rhizosphere of perennial grasses grown in field conditions. Microbial Ecology (in press).

The diversity of nitrate-dissimilating Pseudomonas is being evaluated using narG, napA as well as 16S-23S rDNA sequences. Finally, further research is dealing with the influence of nitrate fertilization on nitrate-dissimilating Pseudomonas.

Contact : Sonia Tarnawski

Collaborations

 

last update : 07.08.2004