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National audience; Cultivated fabeae legumes (pea, fababean, lentil) develop root nodules resulting from the symbiotic interaction with Rhizobium leguminosarum sv. viciae (Rlv). Individual Rlv bacteria are able to associate with various potential hosts, but in soil they are in mixture and they display a wide range of competitiveness to form nodules (CFN). Because in Rlv, CFN and capacity to fix nitrogen are genetically independent, CFN limits the effectiveness of inoculation strategies as efficient bacteria are often outcompeted by poorly efficient Rlv bacteria of the soil community. We developed a strategy to identify bacterial genes controlling CFN. A worldwide collection of 240 Rlv isolates was obtained by combining bacteria described in GenBank with new isolates obtained worldwide by pea, fababean and lentil root nodule trapping. 100 genomes (22 already in GenBank) were sequenced. The extended Rlv complex species includes probably 16 genospecies and two main groups of symbiosis plasmids that can be horizontally transferred. We identified phylogenetic clades of Rlv displaying contrasted levels of CFN upon pea and fababean. A molecular barcode was designed on nodD gene to discriminate and quantify intraspecific variability of Rlv in root systems allowing estimate CFN in symbiotic pea, fababean or lentil associations with multiple Rlv potential partners. Several plasmid regions genetically associated with pea/fababean CFN phenotypes were identified. Candidate genes include specific nod genes as well as other genes with unknown function in symbiosis.

Soil is an heterogeneous and fluctuating environment. Water limitation in agronomic soils is often partial but lead locally to the suppression of symbiotic activity and therefore to reduction of N acquisition by the plant. A split-root system was developed in pea to evaluate the capacities of Pea-Rhizobium leguminosarum bv viciae (Rlv) symbiotic associations to compensate a local reduction of water availability. Root systems are split in two parts watered separately. Soil water content was monitored. Water limitation was exerted by stopping watering of a half root system. Arrest of nitrogen fixation is very rapidly observed in root directly exposed to water stress. This local and partial water limitation of the plant triggered a systemic signaling on well-watered roots of the same plant. The compensatory response occurs by increasing nodule biomass (and therefore nitrogen fixation capacities) of these roots not directly exposed to the water limitation. In preliminary experiments pea plants were inoculated with a reference Rlv strain. Response of and root and nodule development that compensate the local water limitation was confirmed. Interestingly, the compensatory response varies according to the Rlv strain as another Rlv strain, displaying similar nitrogen fixation efficiency in watered conditions, was not able to promote equivalent compensatory response. This experimental system has been used as a screen to select Rlv strains able to promote rapid compensatory responses to water stress.

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BAP GEAPSI; National audience; Les légumineuses sont des cultures de grand intérêt pour répondre aux enjeux de sécurité alimentaire et de développement durable. Dans un contexte de forte croissance démographique, ce sont des sources de protéines pour les alimentations humaine et animale. Un autre atout des légumineuses est qu’elles peuvent s’affranchir d’un apport d’engrais azoté grâce à leur capacité à former des associations symbiotiques au sein de nodosités racinaires avec des bactéries du sol (les rhizobia) qui fixent l’azote de l’air. Cependant, la fixation symbiotique n’est pas toujours optimale ; elle est très sensible aux stress abiotiques et dépend directement de l’efficacité symbiotique du partenaire bactérien. Les populations naturelles de rhizobia sont quantitativement et qualitativement hétérogènes et peuvent aboutir à des symbioses inefficaces. Une approche pluridisciplinaire a été menée afin d’évaluer l’impact du génotype de pois et des rhizobia qui nodulent cette espèce sur l’établissement et l’efficacité de la symbiose fixatrice d’azote. Des essais ont été réalisés en serre sur une collection de 104 accessions de pois avec des historiques de sélection et des origines géographiques diverses. Ces 104 accessions ont été inoculées par un mélange de cinq souches de Rhizobium leguminosarum sv. viciae (Rlv) choisies pour leur diversité. Une forte variation du choix entre les partenaires symbiotiques a été observée. Ceci a permis de mettre en évidence des liens entre la diversité génétique des accessions de pois et l’établissement préférentiel de la symbiose avec certaines souches de Rlv. Une expérimentation complémentaire sur un sous-ensemble de 18 génotypes de pois, inoculés chacun séparément avec chacune des cinq souches de Rlv, a révélé que la plante ne s’associe pas toujours préférentiellement avec la souche de rhizobium qui permet la fixation symbiotique la plus efficace. Des effets d’interaction pois x rhizobium ont été mis en évidence. Cette expérimentation a montré la grande variabilité de l’interaction symbiotique entre pois et Rlv et son importance pour l’établissement d’un rendement optimal. Des expérimentations supplémentaires permettront de préciser les déterminants génétiques de la plante et de la bactérie qui pilotent les capacités d’association des partenaires symbiotiques et/ou leurs efficacités. Des stratégies d’inoculation des semences de pois par des bactéries rhizobiacées, qui sont actuellement très peu pratiquées en Europe, combinées à une meilleure prise en compte de l’interaction symbiotique dans les processus de création de variétés sont des leviers à travailler pour accroître les performances des légumineuses dans des systèmes de cultures à bas intrants.

Plant symbioses are fundamental for ecosystem stability and sustainability. Introduction of legumes associated with microorganisms efficient in nitrogen fixation and promoting nutrient biodisponibility in limited-nutrient degraded sites is a key step for a successful plant establishment in stress situations such as those encountered in Mediterranean soils. A vegetation strategy based on microbial engineering of locally-adapted plants was investigated and tested in field conditions within the framework of calcareous quarry rehabilitation. Choice of symbiotic plants requires, first, a prospection of native legume species growing in relatively degraded sites in Mediterranean areas and easy to multiply, and second, an identification and production of efficient rhizobial partners. Plants produced in nursery and then transplanted into the quarry showed a good capacity for establishment after three years. Shrub species performed best with spontaneous mycorrhizal association and ability to enrich topsoil with organic matter, Les symbioses végétales sont une composante fondamentale de la stabilité et de la durabilité des écosystèmes. L’introduction de légumineuses associées à leurs auxiliaires microbiens fixateurs d’azote ou améliorant la biodisponibilité d’éléments nutritifs, dans des sites dégradés fortement appauvris en éléments nutritifs, est une condition primordiale pour réussir l’installation de plants en milieu particulièrement contraignant, comme le sont les sites de carrière de calcaire après exploitation sous climat méditerranéen. Une stratégie de végétalisation basée sur l’ingénierie microbiologique de plants adaptés aux conditions locales, a été élaborée puis testée sur le terrain en grandeur nature dans le cas particulier de carrières de granulats calcaires. Le choix des plants symbiotiques utilisables pour la végétalisation repose, d’une part, sur la prospection d’espèces de légumineuses repérées sur des sites assez dégradés en milieu méditerranéen, faciles à multiplier et, d’autre part, sur l’identification et la production de rhizobiums partenaires efficaces. Ces plants produits en pépinière puis transférés sur le terrain présentent après trois années une bonne capacité de croissance. Les espèces arbustives sont plus particulièrement performantes avec une capacité spontanée à s’associer aux mycorhizes et à favoriser l’enrichissement en matière organique de la surface du substrat.

Nitrogen-fixing Sinorhizobium sp. bacteria interact with Medicago truncatula plants, forming root nodules in this nitrogen fixation symbiosis. When rhizobia colonize rhizosphere’s legumes, symbiotic specificity is expressed at two levels, among-species for nodulation ability and within-species for nodulation competitivity. We investigated the genetic diversity of 223 Sinorhizobium sp. strains isolated from nodules sampled from four Medicago truncatula fixed lines, each issued from distinct geographic areas, and cultivated on the same French soil. The DNA molecular polymorphism of isolates was analyzed by CE-SSCP and RFLP on 5 loci chosen either for species characterization or for their involvement in symbiosis and effectors translocation pathways. Although more than 90% of the sample bacteria belonged to Sinorhizobium medicae (others belonging to S. meliloti), statistical tests showed a significant differentiation among M. truncatula lines for bacterial species proportion. Conversely, on the basis of the four other loci analysed, we could not detect any significant among-lines population differentiation, for each bacterial species. Nevertheless, several rare strains isolated were specific from a single plant line. Thus, we compared the nodulation ability and the fixation efficiency of these isolates on the different M. truncatula lines used for trapping. Moreover, to enlighten the role played by bacterial genetic diversity on nodulation competitivity, intraspecific competition assays were performed with genetically different strains on 80 plants. Hence, SNP molecular genotyping of 200 nodules allowed the choice of the most competitive strain for each of the two Sinorhizobium species. Therefore, those two strains were tested for their ability to compete in the formation of nodules on the four Medicago truncatula lines, with inoculation of strains at different ratios (90/10, 50/50 and 10/90 proportions) on 30 plants each. Results led us to formulate hypotheses explaining the S. medicae versus S. meliloti proportions found initially in this bacterial trapping. This work outlines the influence of the genetic diversity of the host plant on the distribution of symbiotic genotypes in rhizospheric bacterial populations, and allows a better understanding of bacterial selection in natural and cultivated ecosystems.

Anthropogenic degradation activities (overgrazing, deforestation, quarry exploitation¿) together with a long dry and hot summer, with scarce, erratic but torrential rainfalls, is a major threat to Mediterranean ecosystem sustainability. Quarry exploitation, in particular, removes native plants and soils, and generates an anthropogeomorphic soil material. Revegetation of such degraded ecosystems requires selection of suitable plant species adapted to unfavorable soil and climatic conditions. Therefore, nitrogen-fixing legumes are key components of plant successions because symbioses associated with rhizobium are major nitrogen input to poor ecosystems. Several Mediterranean native legumes were selected from different areas in the South of France: some of them, Coronilla glauca and Medicago arborea, belonged to the shrub community whereas others like Astragalus monspessulanus, Dorycnium hirsutum and Coronilla varia were herbaceous. Seeds were collected for plant multiplication. Rhizobial partners were isolated, characterized and the most N2-fixing ones were selected for inoculum's preparation. Seedlings were transferred into plastic containers according to usual forestry nursery practices, then were inoculated with the appropriate inoculum and allowed to grow for 6-9 months in an experimental nursery before vegetalisation. Symbiotic plants were transferred into a rock-spolic soil materiel of a calcareous quarry and experimental variables including survival rates, plant growth, endomycorrhizae, soil organic matter and total N accumulation were recorded after three years. Plant growth rates were major for C. glauca, M. arborea and D. hirsutum, despite adverse conditions. A thin (0-5 mm) and discontinuous O horizon was formed under plants of C. glauca, M. arborea and D. hirsutum. In this last case, an important amount of organic carbon carbon was accumulated in some points (11.9 % + 13). Total nitrogen (0.03-0.1 %) was slightly incorporated into top vegetalised spolic material (0-5 cm) in comparison with non-vegetalised material. Thus, C. glauca, M. arborea and D. hirsutum was the most suitable legume species for reclamation and rehabilitation of spolic soil material. (Texte intégral)

Heavy metal contamination of soils originating from industrial activities (e.g., metal mining and smelting) is a major environmental problem in many parts of the world. In addition to the high heavy metal levels, plants and microorganisms have to face to very low content in nitrogen and to limited water supply due to porous substrates (Requena et al., 2001 ; Escarré et al., 2000). Isolation and selection of both metal-resistant and symbiotic nitrogen-fixing plant-bacterium system will be a crucial aspect to overcome the main constraint of N input in mine tailings. We have identified in an ancient Zn-Pb mine in the South of France, a metalliferous Anthyllis vulneraria ecotype able to fix nitrogen in the presence of heavy metals. Our objective was therefore to isolate and to characterize rhizobium strains from nodules directly collected from root plant systems growing in metal-contaminated substrates. According to the 16S sequencing data, the bacterial isolates obtained belong to Mesorhizobium genus, with an unexpected diversity. Metal-resistant phenotypes of Mesorhizobium isolates were characterised by culture in media supplemented with Zn or Pb. Isolates exhibited variable tolerance degree to Zn and at least 4 different phenotypes were recorded. Pb resistance was 5-10 times lower and few phenotypes were observed. The presence of the resistance genes like czcD gene was confirmed by PCR and sequencing in some strains. The selection and the practical use of metalliferous efficient nitrogen-fixing Anthyllis vulneraria-Mesorhizobium associations will help to improve plant cover of heavy-metal-enriched spoils heaps and therefore could be proposed as a new tool for bioremediation of area affected by heavy-metal contamination. (Texte intégral)

International audience; To learn more about the diversity in the Caribbean basin of Xenorhabdus and Photorhabdus in relation with their nematode host and their environment, an extensive survey was conducted in this area. A total of 77 Caribbean isolates, recovered from 72 Heterorhabditis and 5 Steinernema, were studied. ln order to type faster these numerous bacterial isolates, a method previously assessed with reference strains (see Brunel et al., in this book), Restriction Fragment Length Polymorphism analysis of PCR-amplified 16S rRNA genes, was used. The ribosomal genotypic patterns were compared to those of 40 reference strains. Most of the EPN isolates had been previously identified and ecological data about the samples origin were available. The five Xenorhabdus isolates exhibited a high degree of genetic polymorphism and only one was genotypically identical to a reference strain. ln contrast, very little variability was evidenced within the 72 Photorhabdus isolates, as only four closely related genotypes, over the 12 already identified, were detected. When we compared these results with geographical and ecological data, no correlation could be drawn, excepted those directly associated to the environmental habitat of host nematodes. ln fact, a closely relatedness between bacterial genotypes and nematode species was found. This result was particularly obvious within the Photorhabdus-Heterorhabditis couple where two bacterial genotypes were exclusively associated to H. indica and two others to H. bacteriophora. Within the Xenorhabdus-Steinernema association, new symbiont genotypes corresponded to new nematode species. The availability on one hand of a fast molecular identification of bacterial symbionts, and on the other hand the characterization with satellite DNA probes of most EPNs, allowed to compare their respective taxonomie structure on a large scale. Thereby, a congruenee was evidenced which strongly supports an early co-evolution of the symbiotic partners of both couples.

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