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Many Aeschynomene sp. belonging to the Dalbergioids family, like Arachis hypogea, can develop a nitrogen-fixing root symbiosis with photosynthetic bradyrhizobia. Among them, certain strains are lacking the nod genes operon which is essential for Nod Factor biosynthesis. First genetic analysis in Aeschynomene evenia revealed that many genes involved in bacterial perception in model legumes are not functionals in the Nod-independent symbiosis whereas the genes involved in signal transduction are mainly conserved.In the aim to better understand how Nod-independent nodulation has been set up, we searched to identify new genes using forward genetics on A. evenia. An EMS mutagenized population has been generated leading to the isolation of 250 stable mutant lineages affected in the nodulation process. Among these mutants, half of them are totally unable to nodulate and are qualified by « Nod- ».A Targeted-Sequence-Capture analysis has been realized on the Nod- mutants and allows the identification of mutations in many known genes of the « Nod signaling pathway » This kind of analysis is possible thanks to a reference genome which has been sequenced and annotated recently. Mutations have been found in AePOLLUX, AeCCaMK, AeCYCLOPS, AeNIN, and AeNSP2 genes whereas no mutations have been found in any LysM-RLK (Nod factor receptors). Gene expression analysis by Q-PCR on different mutants have been realized to observe eventual deregulation of other symbiotic genes.Concerning Nod- mutants for which no known symbiotic gene has been associated, we performed a Mapping-by-Sequencing approach that is combining cartography and whole-genome sequencing to localize homozygous mutations. For some mutants with the same phenotype, we identify mutations on a gene coding a Receptor-Like Cytoplasmic Kinase (RLCK). This gene underwent a specific duplication event that is only observed in Nod-independent Aeschynomene species, suggesting a role of this cytoplasmic kinase during the Nod-independent symbiosis. This kind of protein devoid of the extracellular domain and belonging to a family known for associates with Receptor-Like Kinase (RLK) to transduce the signal and activation of genetic programs of development, reproduction, growth, and immunity. However, this is one of the first times that a protein of this family is shown to be involved in nitrogen-fixing symbiosis (except for LjNiCK4 in 2019).Furthermore, the Mapping-by-sequencing approach allows the identification of 3 other new symbiotic genes (AeCRK, AeRINRK, AeMLRR-RLP) that are not described in model legumes (except LjRINRK1 in 2019). In this manuscript, the results of these analyses will be presented and discussed in order to understand the molecular mechanisms governing the nodulation in the absence of Nod factors.; Plusieurs espèces appartenant au genre tropical Aeschynomene développent une symbiose fixatrice d’azote originale avec des Bradyrhizobium photosynthétiques, qui ne passe pas par la reconnaissance des facteurs Nod rhizobiens et la formation de cordons d’infection. Pour mettre à jour les mécanismes moléculaires de cette symbiose dite Nod-indépendante, une approche de génétique directe est mise en œuvre chez Aeschynomene evenia. Elle combine le crible d’une population de mutagenèse EMS, ayant permis d’isoler 250 mutants de nodulation, au séquençage du génome d’A. evenia pour faciliter l’identification de gènes symbiotiques à partir de ces mutants. Au cours de ma thèse, j’ai mis à profit le génome nouvellement disponible d’A. evenia en menant une analyse comparative de gènes symbiotiques afin d’obtenir une vue d’ensemble des mécanismes potentiellement conservés ou divergents entre A. evenia et les légumineuses modèles M. truncatula et L. japonicus. Ce travail a mis en exergue des différences génétiques importantes, notamment au niveau des étapes de reconnaissance des rhizobiums et d’infection. Ensuite, pour déterminer quels gènes connus chez les légumineuses modèles sont aussi impliqués dans la symbiose Nod-indépendante, j’ai recherché des mutations par Targeted Sequence Capture de ces gènes sur les 250 mutants de nodulation d’A. evenia. Cette approche a révélé que nombreux mutants Nod-, qui sont bloqués dans les étapes les plus précoces de l’interaction, sont altérés dans plusieurs gènes de la voie de signalisation symbiotique activée par les rhizobiums : AePOLLUX, AeCCaMK, AeCYCLOPS, AeNIN et AeNSP2. En revanche, aucune mutation n’a été trouvée dans les gènes codant des récepteurs symbiotiques.Pour identifier chez A. evenia de nouveaux gènes clé de la symbiose Nod-indépendante, j’ai appliqué une approche sans a priori, le Mapping-by-Sequencing, sur les mutants Nod- n’ayant pas de gène connu associé. Cette stratégie a été fructueuse pour identifier deux gènes : AeRLCK, qui code un Receptor-Like Cytoplasmic Kinase (RLCK), et AeCRK, codant une Cysteine-rich Receptor-like Kinase (CRK). Il est notable que ces deux gènes ne sont pas retrouvés chez les légumineuses modèles et qu’ils appartiennent à deux familles d’acteurs de signalisation précoce pouvant participer à la formation de complexe récepteurs avec des Receptor-Like Kinases (RLK) pour assurer la transduction d’un signal extracellulaire. Une caractérisation de ces deux nouveaux acteurs symbiotiques a révélé qu’AeRLCK est issu d’une duplication en tandem propre aux Aeschynomene Nod-indépendantes et qu’AeCRK est soumis à une sélection purifiante chez ces mêmes espèces, ce qui suggère qu’ils pourraient fonctionner spécifiquement dans la symbiose Nod-indépendante. Pour préciser leur rôle et les positionner par rapport à la voie de signalisation symbiotique conservée avec les légumineuses modèles, j’ai également initié une analyse comparative fine au niveau phénotypique et moléculaire. La découverte d’AeRLCK et AeCRK représente une avancée importante dans notre compréhension des mécanismes moléculaires qui contrôlent l’établissement de la symbiose Nod-indépendante. Elle ouvre maintenant la voie à une analyse fonctionnelle de ces deux acteurs symbiotiques et à une recherche des récepteurs symbiotiques avec qui ils peuvent interagir.

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium–legume symbiosis., The establishment of symbiotic interaction between Aeschynomene evenia and photosynthetic bradyrhizobia doesn’t involve the canonical Nod factors and infection threads. Here, the authors assemble the draft genome of A. evenia and identify a receptor-like kinase in mediating the symbiotic interaction.

Root hairs are elongated epidermal cells essential for the acquisition of mineral nutrients by the plant. In legumes, they are also the seat of initiation of symbiotic interactions with N2‐fixing rhizobia. In these plants, early ionic signals initiated at the root hair plasma membrane, involving changes in H+ , Ca2+ , anions and K + ion fluxes and resulting in transient depolarization of the cell membrane, are triggered as initial responses to perception of the signaling Nod factors secreted by the rhizobial partners present in the rhizosphere. Here, we aimed at building a repertoire of ion channel activities present at root hair plasma membrane of the model legume M. truncatula , which could be implicated in these early signaling events leading to symbiosis establishment. Apical spheroplasts of plasma membrane of young growing root hairs, recovered through cell‐wall laser ablation, were used in patch‐clamp experiments. Six ionic conductances selective for Ca2+ , K+ or anions, involved in ion influx and/or effluxes, were identified and their sensitivity to NF checked. This provides a first sketch of the set of transport systems involved in the Nod‐factor‐triggered electrical signals, paving the way to future confirmation through reverse genetics on corresponding candidate genes in this sequenced model legume.

Eight mesorhizobial symbiotic strains isolated from Anthyllis vulneraria root-nodules were studied and compared taxonomically with defined Mesorhizobium species. All strains presented identical 165 rDNA sequences but can be differentiated by multilocus sequence analysis of housekeeping genes (recA, atpD, glnll and dnaK). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses separate these strains in two groups and a separate strain. Levels of DNA-DNA relatedness were less than 55% between representative strains and their closest Mesorhizobium reference relatives. The two groups containing four and three strains, respectively, originating from border mine and non-mining areas in Cevennes, were further phenotypically characterized. Groupings were further supported by average nucleotide identity values based on genome sequencing, which ranged from 80 to 92% with their close relatives and with each other, confirming these groups represent new Mesorhizobium species. Therefore, two novel species Mesorhizobium delmotii sp. nov. (type strain STM4623(T) = LMG 296401(T)= CFBP 84361(T) ) and Mesorhizobium prunaredense sp. nov. (type strain STM4891(T) = LMG 29641(T) = CFBP 8437(T)) are proposed. Type strains of the two proposed species share accessory common nodulation genes within the new symbiovar anthyllidis as found in the Mesorhizobium metallidurans type strain.

International audience; Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

L'article original est publié par The American Society of Plant Biologists; International audience; LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogenfixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box–dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant–microbe interactions and development through interacting with receptor-like kinases is discussed.

Photosynthetic Bradyrhizobium strain ORS285 forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. Depending on the Aeschynomene species, this symbiotic interaction does or does not rely on the synthesis of Nod-factors (NFs). However, whether during the interaction of Bradyrhizobium ORS285 with NF-independent Aeschynomene species the nod genes are expressed and if the general regulator NodD plays a symbiotic role is unknown. Expression studies showed that in contrast to the interaction with the NF-dependent Aeschynomene species, A. afraspera, the Bradyrhizobium ORS285 nod genes are not induced upon contact with the NF-independent host plant A. indica. Mutational analysis of the two nodD genes present in ORS285, showed that deletion of nodD1 and nodD2 did not affect the symbiotic interaction between Bradyrhizobium ORS285 and A. indica whereas the deletions had an effect on the symbiotic interaction with A. afraspera plants. In addition, when the expression of nod genes was artificially induced by adding naringenin to the plant growth medium, the nodulation of A. indica by Bradyrhizobium ORS285 is delayed and resulted in lower nodule numbers.

In Morocco, the use of seed legumes is limited because significant water deficits and the low availability of soil phosphorus (P) limit nitrogen fixation. However, little is known about the ability of faba bean-rhizobium symbiosis to fix nitrogen in P-deficient soils and to transfer fixed nitrogen (N) to intercropped wheat. Arbuscular mycorrhizal fungi (AM) and their extraradical hyphae networks play an important role in the facilitation process by promoting interconnectivity and the transfer of nutrients, such as N and P, between associated plants. The aim of this study was to analyse the impact of AM inoculation on N-2 fixation and the transfer of fixed N from faba bean to intercropped wheat. Germinated faba bean and wheat seeds were transferred into 1-l pots filled with a P-deficient soil that was collected from the Haouz valley near Marrakech (Morocco). Plants from the two species were grown in pots in either pure or mixed stands under greenhouse conditions, and each cropping system was subjected to three mycorrhizal inoculation treatments with a non-inoculated (AM0) and two concentrations of Rhizophagus irregularis inoculants containing 1000 (AM1) or 2000 (AM2) spores pot(-1). The N-15 isotope dilution method was used to determine the amount and proportion of atmospheric N fixed by faba bean (Ndfa%) and the fixed N that was transferred to wheat. Mycorrhizal inoculation had a significantly positive effect on the shoot dry weights and total shoot N in faba bean, but not in wheat. The cropping system had no significant effect on the plant growth and total shoot N in both faba bean and wheat. The Ndfa percentage was very high in all of the treatments, varying from 86 to 91%. The total N fixed by faba bean was 27% significantly higher in the AM2 treatment compared with the AM1 and AM0 treatments for both cropping systems combined. The estimated proportions of fixed N that were transferred from faba bean to wheat were far higher in AM1 (50%) and AM2 (32%) treatments than in AM0 (15%) treatment as well as for the total transferred fixed N. As corroborated by a parallel observation of root mycorrhizal colonization, these results suggest that the development of mycorrhizal networks stimulates the transfer of fixed N from faba bean to wheat, which could significantly contribute to the facilitation process under intercropping conditions.

Pour les chercheurs en biologie, les légumineuses arborées présentent de nombreuses qualités pour l'agronomie, en particulier la capacité à fixer l'azote atmosphérique. Celle-ci permet la production de parties végétales riches en matière azotée qui, lorsqu'elles se décomposent dans le sol, sont supposées en améliorer la " fertilité ". D'autre part, on met souvent en avant leur " richesse " pour la nutrition des hommes et des animaux et la qualité de leur bois. Les spécialistes du reboisement soulignent leur capacité de multiplication sexuée et végétative ainsi que leur aptitude à se développer en milieu peu fertile et ouvert. Les agro-sylvo-pastoralistes travaillant en terrains tropicaux constatent bien ces vérités scientifiques, parfois devenues de véritables paradigmes, mais en éprouvent au quotidien les limites. Il est bien connu que toutes les légumineuses ne fixent pas l'azote et aussi que cette fixation dépend des espèces, de leur association rhizobienne et des conditions environnementales, mais ceci n'est pas toujours dit dans les messages destinés à la " vulgarisation ". Si des transferts " directs " d'azote entre plantes ont été mis en évidence, l'importance du transfert d'azote et d'autres nutriments au bénéfice de plantes associées ou en rotation dépend du recyclage des organes des plantes fixatrices. A l'inverse, la récolte de leurs bois, fruits ou feuilles, peut aboutir à un appauvrissement de la fertilité du sol qui les porte. Leur formidable capacité à se multiplier et à s'ancrer durablement dans un milieu, en particulier lorsqu'il est dégradé, a aussi son " revers de médaille ", à savoir qu'elles peuvent souvent devenir envahissantes. Par ailleurs, les écologistes dogmatiques regretteront que des légumineuses provenant d'autres continents soient maintenant présentes dans la plupart des forêts. D'autres, plus pragmatiques, y verront l'émergence de " forêts nouvelles " dont il faut étudier l'écosystème mais aussi valoriser les biens et services, comme le font souvent les paysans. Il reste cependant que ces légumineuses arborées occupent une place de choix dans la palette des possibilités disponibles pour le praticien en charge de la restauration de milieux dégradés ou de la création de systèmes agro-sylvo-pastoraux productifs et résilients face aux changements globaux, à condition de manier avec prudence la lancinante tentation de l'introduction de la " plante miracle " qui peut parfois, si on n'y prend pas garde, devenir une " plante cauchemar ".

International audience; The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.

In this review, we discuss the recent progress in research on symbiotic association of rhizobia, Frankia and fungi with plant roots. We compare infection processes of symbiotic establishment; structure, functioning and molecular biology of the symbiotic organ including the regulation of genes implicated in rhizobial, actinorhizial and arbuscular mycorhizal symbioses. (African Journal of Biotechnology: 2003 2(1): 1-7)

Highlight Persistence of intracellular rhizobia in legumes involves plant and bacterial genes. Here we show that DNF2, bacA, SymCRK/RSD and nifA/nifH successively prevent bacterial death during the symbiotic process., Medicago truncatula belongs to the legume family and forms symbiotic associations with nitrogen fixing bacteria, the rhizobia. During these interactions, the plants develop root nodules in which bacteria invade the plant cells and fix nitrogen for the benefit of the plant. Despite massive infection, legume nodules do not develop visible defence reactions, suggesting a special immune status of these organs. Some factors influencing rhizobium maintenance within the plant cells have been previously identified, such as the M. truncatula NCR peptides whose toxic effects are reduced by the bacterial protein BacA. In addition, DNF2, SymCRK, and RSD are M. truncatula genes required to avoid rhizobial death within the symbiotic cells. DNF2 and SymCRK are essential to prevent defence-like reactions in nodules after bacteria internalization into the symbiotic cells. Herein, we used a combination of genetics, histology and molecular biology approaches to investigate the relationship between the factors preventing bacterial death in the nodule cells. We show that the RSD gene is also required to repress plant defences in nodules. Upon inoculation with the bacA mutant, defence responses are observed only in the dnf2 mutant and not in the symCRK and rsd mutants. In addition, our data suggest that lack of nitrogen fixation by the bacterial partner triggers bacterial death in nodule cells after bacteroid differentiation. Together our data indicate that, after internalization, at least four independent mechanisms prevent bacterial death in the plant cell. These mechanisms involve successively: DNF2, BacA, SymCRK/RSD and bacterial ability to fix nitrogen.

Plant-plant facilitation is an ecological process occurring in most terrestrial ecosystems. Plant-plant facilitation is considered as a positive interaction between both plant partners in which one plant species promotes the growth, survival or reproduction of the neighbouring plant. Recent studies have underlined the role of mycorrhizal fungi, i.e. arbuscular mycorrhizal fungi (AMF) and particularly extra radical hyphae of AMF, in interconnecting plants and consequently their importance in plant-plant facilitation process. Networks of AMF impact soils both physically and biologically and are considered as an important pathway for the transference of nutrients such as nitrogen and phosphorus. In parallel, AMF increase plant growth and nutrient uptake and decrease the deleterious effects of pathogens and drought. The aims of this chapter are to describe how the AMF are involved in the plant facilitation process and to assess the main mycorrhizal effects on the plant nutrition (P and N) and plant health. Also the contribution of AMF in cropping systems as well as agricultural strategies which improve AM associations in arid agrosystems will be reviewed and illustrated by experimental results from field studies in Mediterranean environment.

International audience; Rhizobia and legumes are able to interact in a symbiotic way leading to the development of root nodules. Within nodules, rhizobia fix nitrogen for the benefit of the plant. These interactions are efficient because spectacularly high densities of nitrogen fixing rhizobia are maintained in the plant cells. DNF2, a Medicago truncatula gene has been described as required for nitrogen fixation, bacteroid's persistence and to prevent defense-like reactions in the nodules. This manuscript shows that a Rhizobium mutant unable to differentiate is not sufficient to trigger defense-like reactions in this organ. Furthermore, we show that the requirement of DNF2 for effective symbiosis can be overcome by permissive growth conditions. The dnf2 knockout mutants grown in vitro on agarose or Phytagel as gelling agents are able to produce nodules fixing nitrogen with the same efficiency as the wild-type. However, when agarose medium is supplemented with the plant defense elicitor ulvan, the dnf2 mutant recovers the fix- phenotype. Together, our data show that plant growth conditions impact the gene requirement for symbiotic nitrogen fixation and suggest that they influence the symbiotic suppression of defense reactions in nodules.

Some leguminous species of the genus Aeschynomene are specifically stem-nodulated by photosynthetic bradyrhizobia. To study the effect of bacterial photosynthesis during symbiosis, we generated a photosynthesis-negative mutant of the Bradyrhizobium sp. strain ORS278 symbiont of Aeschynomene sensitiva . The presence of a functional photosynthetic unit in bacteroids and the high expression of the photosynthetic genes observed in stem nodules demonstrate that the bacteria are photosynthetically active during stem symbiosis. Stem inoculation by the photosynthetic mutant gave a 50% decrease in stem-nodule number, which reduced nitrogen fixation activity and plant growth in the same proportion. These results indicate an important role of bacterial photosynthesis in the efficiency of stem nodulation.

Mesorhizobium metallidurans STM 2683 T is a nitrogen-fixing bacterium that nodulates Anthyllis vulneraria in mine tailings highly contaminated in zinc, lead and cadmium. To study the mechanisms whereby this bacterium copes with metals, we functionally screened a cosmid genomic library of M. metallidurans for zinc or cadmium tolerance. A cosmid clone harbored a gene encoding P IB -type ATPase homologous to CadA that leads to cadmium and zinc resistance in Escherichia coli . The CadA protein structure presents one duplication of the two N-terminal metal binding domains (i.e. a heavy metal-associated domain followed by a histidine-rich domain) which allows specific binding to zinc and cadmium cations. A cadA -deleted strain of M. metallidurans failed to grow at high zinc concentrations (2 mM) and its growth was delayed at lower zinc concentrations. Expression studies using a transcriptional fusion of cadA promoter to gfp showed that cadA is specifically induced in a dose-dependent manner by zinc and cadmium in M. metallidurans in vitro conditions and into A. vulneraria nodules after Zn stress. Metal induction sensitivity was increased in the strain where cadA gene was deleted. This study identified cadA as a first mesorhizobial resistance determinant involved in detoxification of cadmium and zinc and which confers upon M. metallidurans greater capacity for coping with high zinc concentrations. This improves the knowledge of this bacterium for potential use as a symbiotic inoculant of Anthyllis in phytostabilization strategies of metal-rich sites.

International audience; The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules.

EA SPE Pôle GEAPSI Pôle EcolDur CT1 (EA) CT2 (EA) EJ1 (EA) EJ2 (EA) Ce numéro de la revue Innovations Agronomiques est dédié aux articles issus des programmes Casdar 2008-2010.; National audience; L'intensification agricole amorcée depuis les années 1950 a globalement conduit au déclin des cultures légumineuses, qui représentent aujourd'hui moins de 3 % des grandes culture françaises et européennes. Pourtant, les légumineuses présentent plusieurs atouts : (i) en tant que source de protéines végétales pour l'alimentation humaine ou animale et (ii) en tant que plantes fixatrices de l'azote de l'air, économes en intrants azotés, et (iii) en tant que cultures de diversification dans les systèmes de culture. Une analyse des systèmes de production des légumineuses à graines et fourragères en France montre que les différents facteurs freinant l'insertion de ces cultures relèvent d'un processus de " verrouillage " du régime socio-technique agricole dominant. Pourtant les légumineuses pourraient jouer un rôle majeur dans la transition agroécologique vers des systèmes agricoles, alimentaires et énergétiques plus durables. Une approche économique mobilisant l'approche multi-niveaux de la théorie des transitions a permis d'identifier des voies de déverrouillage de la production de légumineuses. A la lumière de cette analyse, quatre grands groupes de questions de recherche ont été identifiés, concernant (i) l'analyse des verrouillages et l'identification des leviers de transition vers des systèmes intégrant plus de légumineuses (ii) l'identification de débouchés nouveaux et diversifiés pour les légumineuses dans une chaine alimentaire durable, (iii) l'évaluation et l'amélioration des services écosystémiques rendus par les légumineuses à l'échelle des systèmes de culture et des territoires, et (iv) l'optimisation de la conduite des cultures de légumineuses dans une démarche globale d'intensification écologique.

L’intensification agricole amorcée depuis les années 1950 a globalement conduit au déclin des cultures légumineuses, qui représentent aujourd’hui moins de 3 % des grandes culture françaises et européennes. Pourtant, les légumineuses présentent plusieurs atouts : (i) en tant que source de protéines végétales pour l’alimentation humaine ou animale et (ii) en tant que plantes fixatrices de l’azote de l’air, économes en intrants azotés, et (iii) en tant que cultures de diversification dans les systèmes de culture. Une analyse des systèmes de production des légumineuses à graines et fourragères en France montre que les différents facteurs freinant l’insertion de ces cultures relèvent d'un processus de « verrouillage » du régime socio-technique agricole dominant. Pourtant les légumineuses pourraient jouer un rôle majeur dans la transition agroécologique vers des systèmes agricoles, alimentaires et énergétiques plus durables. Une approche économique mobilisant l'approche multi-niveaux de la théorie des transitions a permis d’identifier des voies de déverrouillage de la production de légumineuses. A la lumière de cette analyse, quatre grands groupes de questions de recherche ont été identifiés, concernant (i) l’analyse des verrouillages et l’identification des leviers de transition vers des systèmes intégrant plus de légumineuses (ii) l’identification de débouchés nouveaux et diversifiés pour les légumineuses dans une chaine alimentaire durable, (iii) l’évaluation et l’amélioration des services écosystémiques rendus par les légumineuses à l'échelle des systèmes de culture et des territoires, et (iv) l’optimisation de la conduite des cultures de légumineuses dans une démarche globale d'intensification écologique., Intensification of agriculture since 1950 has globally led to the decline of grain legume crops, which currently represent less than 3 % of arable crops in France and Europe. Nevertheless, legumes have several major assets (i) as plant proteins for animal feed and human food and (ii) as plants fixing atmospheric N2 and thereby saving nitrogen inputs in cropping systems, and (iii) as diversification crops. The analysis of forage and grain legume producing systems in France showed that the dominant system results in a lock-in of the different factors limiting legume insertion into the current systems. But legumes could have a major role to play in the agroecological transition towards more sustainable agricultural, food and energy systems. The multi-level perspective in the theory of transitions helped us to identify ways of unlocking legume production. Four groups of research questions were thus identified: (i) analysing the lock-ins in the current systems and identifying ways of moving towards systems that include more legumes (ii) identifying new and more diversified outlets for legumes in a sustainable food chain, (iii) assessing and improving the ecosystem services provided by legumes at cropping system and territory scales and (iv) optimising legume crop management in a global agroecological approach.

Burkholderia legume symbionts (also called a-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group''. We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, a-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from beta to alpha-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.

Les plantes dites "fixatrices symbiotiques d'azote", comme toutes les plantes, ont besoin d'azote, mais ont la propriété remarquable de pouvoir puiser cet élément à partir de l'atmosphère, tandis que les autres plantes cultivées doivent recevoir des engrais azotés. En fait, ce n’est pas la plante elle-même qui est capable de fixer l'azote de l'air, mais des bactéries spécifiques avec lesquelles elles vivent en association au niveau d'organes particuliers : les nodosités. C'est le cas de deux principaux groupes de plantes : les légumineuses (ou fabacées) qui s'associent avec les Rhizobia et des plantes appartenant à huit familles d'angiospermes (dites actinorhiziennes) qui s'associent avec l'actinomycète Frankia. La plante forme la nodosité qui abrite la bactérie et lui fournit l'énergie nécessaire ; ces bactéries réduisent l'azote gazeux de l'air en ammoniac et cèdent l'azote fixé à la plante. Cette association à bénéfices réciproques est appelée symbiose. Les partenaires de ces symbioses ainsi que leur fonctionnement et leur importance écologique seront décrits, puis seront présentées et discutées leurs utilisations commerciales actuelles, les applications futures (fertilité des sols, revégétalisation de sols dégradés, et foresterie) ou les perspectives d'utilisation pour d'autres services écosystémiques (réduction du gaz à effet de serre N2O, solubilisation des phosphates, dégradation des xénobiotiques...)

We compiled quantitative estimates on symbiotic N2 fixation by trees in agroforestry systems (AFS) in order to evaluate the critical environmental and management factors that affect the benefit from N2 fixation to system N economy. The so-called ''N2-fixing tree'' is a tripartite symbiotic system composed of the plant, N2-fixing bacteria, and mycorrhizae-forming fungi. Almost 100 recognised rhizobial species associated with legumes do not form an evolutionary homologous clade and are functionally diverse. The global bacterial diversity is still unknown. Actinorrhizal symbioses in AFS remain almost unstudied.Dinitrogen fixation in AFS should be quantified using N isotopic methods or long-term system N balances. The general average ± standard deviation of tree dependency on N2 fixation (%Ndfa) in 38 cases using N isotopic analyses was 59 ± 16.6 %. Under humid and sub-humid conditions, the percentage was higher in young (69 ± 10.7 %) and periodically pruned trees (63 ± 11.8 %) than in freegrowing trees (54 ± 11.7 %). High variability was observed in drylands (range 10-84 %) indicating need for careful species and provenance selection in these areas. Annual N2 fixation was the highest in improved fallow and protein bank systems, 300-650 kg [N] ha-1. General average for 16 very variable AFS was 246 kg [N] ha-1, which is enough for fulfilling crop N needs for sustained or increasing yield in low-input agriculture and reducing N-fertiliser use in large-scale agribusiness. Leaf litter and green mulch applications release N slowly to the soil and mostly benefit the crop through long-term soil improvement. Root and nodule turnover and N rhizodeposition from N2-fixing trees are sources of easily available N for the crop yet they have been largely ignored in agroforestry research. There is also increasing evidence on direct N transfer from N2- fixing trees to crops, e.g. via common mycelial networks of mycorrhizal fungi or absorption of tree root exudates by the crop. Research on the below-ground tree-cropmicrobia interactions is needed for fully understanding and managing N2 fixation in AFS.

Les symbioses entre les légumineuses et les bactéries fixatrices d’azote (rhizobium) représentent un enjeu majeur pour la réduction des intrants azotés en agriculture. La réduction de l'azote moléculaire (N2) en ammonium (NH4+) par la nitrogénase bactérienne, puis son assimilation par la cellule végétale constituent la fonction centrale des nodosités symbiotiques. Ce processus, très coûteux en énergie, présente le paradoxe de se produire dans un organe très appauvri en oxygène, ce qui conduit à fortement réduire la respiration mitochondriale, principale source d'ATP. La question se posait donc de savoir par quels mécanismes l'ATP nécessaire au fonctionnement cellulaire est régénéré au sein de la nodosité. Sur la base d'observations récentes faites dans les racines en hypoxie, nous avons posé l'hypothèse selon laquelle, dans la nodosité fixatrice d'azote, une partie de l'ATP est régénérée par le biais d'une respiration cyclique (respiration nitrate-NO) en quatre étapes: 1) le nitrate (NO3-) est réduit en nitrite (NO2-) dans le cytosol par la nitrate réductase (NR); 2) le NO2- est transporté du cytosol dans la mitochondrie par l'intermédiaire d'un (ou plusieurs) transporteur(s); 3) le NO2- est réduit en oxyde nitrique (NO) par les électrons de la chaîne respiratoire (permettant ainsi le fonctionnement de la respiration et la régénération de l'ATP); et 4) le NO diffuse librement vers le cytosol où il est oxydé en NO3- par la leghémoglobine (Lb). Au cours de ce travail de thèse, mené sur le modèle Medicago truncatula-Sinorhizobium meliloti, nous avons d'abord montré que les nitrate réductases et les chaînes de transfert d'électron des deux partenaires, végétal et bactérien, sont impliquées dans la production de NO dans les nodosités fonctionnelles. Nous avons également montré que l'activité nitrate réductase est nécessaire au maintien d'un état énergétique élevé permettant la fixation de l'azote. Dans un second temps, nous avons entrepris de caractériser le transport du nitrite chez des mitochondries purifiées de racines. L’objectif était ensuite d’identifier un ou plusieurs candidats pour le transport du nitrite dans les mitochondries. Pour cela, sur la base d'homologies de séquence avec des transporteurs déjà identifiés et de séquences d'adressage putatives, une recherche in silico des protéines prédites de M. truncatula a été conduite. Cette analyse a permit de sélectionner 6 candidats avec une localisation possible aux mitochondries. L’analyse par spectrométrie de masse des protéines présentes sur les membranes des mitochondries a révélé 3 autres candidats. L'expression des 9 candidats retenus a été caractérisée en situation de normoxie et d’hypoxie dans les racines et les nodosités. Cela a permis de mettre en évidence deux profils intéressants. Enfin, l'étude de l’adressage subcellulaire et l’expression tissulaire au cours du développement de la nodosité ont été débutés pour certains des candidats retenus sur leur profil d’expression et ceux obtenus par spectrométrie de masse., Symbiosis between legume plants and nitrogen-fixing bacteria (rhizobium) constitute a major issue to reduce nitrogen fertilization in crops. Central function of nodules is the enzymatic reduction of atmospheric nitrogen (N2) into ammoniac (NH4+) by the bacterial nitrogenise, and the subsequent assimilation in the plant cell. This highly energy-consuming process is a paradox as occurring in hypoxied tissue where mitochondrial respiration -the main ATP source- is strongly reduced. The question rose how the ATP required for cellular functioning is generated into nodules. Based on recent observations on hypoxied roots, we rose the hypothesis that, in nodules, ATP is formed trough a cyclic respiration -called nitrate-NO respiration- that consists in four steps: 1) nitrate reductase (NR) reduces nitrate (NO3-) into nitrite (NO2-) in the cytosol ; 2) NO2- is transported from cytosol into mitochondria using transporter(s) protein(s) ; 3) electrons of mitochondrial transfer chain reduces NO2- into nitric oxide (NO), thus allowing respiration functioning and ATP formation ; and 4) NO freely diffuses from mitochondria to cytosol to be oxidized back into NO3- through leghemoglobine (Lb) action. During this thesis, we showed, using Medicago truncatula-Sinorhizobium meliloti system as a model, that NRs and electron transfer chains from both plant and bacterial partners were involved in NO production of functioning nodules. NR activity was also showed to be crucial to maintain high energetic status required for nitrogen fixation. The second aim was to characterized nitrite transport across purified root mitochondria. Then, mitochondrial nitrite transporter candidate(s) were investigated. An in silico strategy searching for predicted proteins in M. truncatula genome, potentially localized to mitochondria, that shared homologous sequences with known transporters was conducted. 6 candidates were chosen at the end of this analysis. Membrane mitochondria proteins were identified using mass spectrometry (MS) analysis, revealing 3 new candidates. The expression profile in normoxied and hypoxied roots and nodules was characterized for the 9 candidates retained. Two of them showed particularly promising patterns. Finally, sub-cellular localization and tissular expression during nodule development were analyzed for some candidates that shared interesting expression patterns.

International audience; The diversity of rhizobia associated with introduced and native Acacia species in Algeria was investigated from soil samples collected across seven districts distributed in arid and semi-arid zones. The in vitro tolerances of rhizobial strains to NaCl and high temperature in pure culture varied greatly regardless of their geographical and host plant origins but were not correlated with the corresponding edaphoclimatic characteristics of the sampling sites, as clearly demonstrated by principal component analysis. Based on 16S rRNA gene sequence comparisons, the 48 new strains isolated were ranked into 10 phylogenetic groups representing five bacterial genera, namely, Ensifer, Mesorhizobium, Rhizobium, Bradyrhizobium, and Ochrobactrum. Acacia saligna, an introduced species, appeared as the most promiscuous host because it was efficiently nodulated with the widest diversity of rhizobia taxa including both fast-growing ones, Rhizobium, Ensifer, and Mesorhizobium, and slow-growing Bradyrhizobium. The five other Acacia species studied were associated with fast-growing bacterial taxa exclusively. No difference in efficiency was found between bacterial taxa isolated from a given Acacia species. The tolerances of strains to salinity and temperature remains to be tested in symbiosis with their host plants to select the most adapted Acacia sp.-LNB taxa associations for further revegetation programs.

International audience; Responses of the Medicago truncatula-Sinorhizobium interaction to variation in N₂-fixation of the bacterial partner were investigated. * Split-root systems were used to discriminate between local responses, at the site of interaction with bacteria, and systemic responses related to the whole plant N status. * The lack of N acquisition by a half-root system nodulated with a nonfixing rhizobium triggers a compensatory response enabling the other half-root system nodulated with N₂-fixing partners to compensate the local N limitation. This response is mediated by a stimulation of nodule development (number and size) and involves a systemic signaling mechanism related to the plant N demand. In roots co-infected with poorly and highly efficient strains, partner choice for nodule formation was not modulated by the plant N status. However, the plant N demand induced preferential expansion of nodules formed with the most efficient partners when the symbiotic organs were functional. The response of nodule expansion was associated with the stimulation of symbiotic plant cell multiplication and of bacteroid differentiation. * A general model where local and systemic N signaling mechanisms modulate interactions between Medicago truncatula and its Sinorhizobium partners is proposed.

The excessive concentrations of toxic heavy metals in mine tailings and their very low N content make soil reclamation strategies by phytostabilization difficult. Our objective was to test if the symbiotic association between the legume Anthyllis vulneraria subsp. carpatica and the bacteria Mesorhizobium metallidurans originating from highly polluted mine tailings is able to increase N concentration in soils with contrasting Zn, Pb and Cd contents. Plants of A. vulneraria subsp. carpatica from a mine site and of a non-metallicolous subsp. praeopera from non-polluted soil were inoculated with a metallicolous or a non-metallicolous compatible Mesorhizobium spp. and grown on low and high heavy metal-contaminated soils. In contaminated soil, many nodules were observed when the metallicolous A. vulneraria was inoculated with its rhizobium species M. metallidurans, whereas the non-metallicolous A. vulneraria died after a few weeks regardless of the rhizobium inoculant. Eighty percent of the total nitrogen was derived from biological nitrogen fixation through the association between metallicolous A. vulneraria and the rhizobium grown on metal-enriched soil. The ability of the metallicolous A. vulneraria to develop a high nitrogen fixing potential opens new possibilities for promoting a low-maintenance plant cover and for stabilizing the vegetation in high heavy metal-contaminated soils.

Background Arbuscular mycorrhizal (AM) fungi, which engage a mutualistic symbiosis with the roots of most plant species, have received much attention for their ability to alleviate heavy metal stress in plants, including cadmium (Cd). While the molecular bases of Cd tolerance displayed by mycorrhizal plants have been extensively analysed in roots, very little is known regarding the mechanisms by which legume aboveground organs can escape metal toxicity upon AM symbiosis. As a model system to address this question, we used Glomus irregulare-colonised Medicago truncatula plants, which were previously shown to accumulate and tolerate heavy metal in their shoots when grown in a substrate spiked with 2 mg Cd kg-1. Results The measurement of three indicators for metal phytoextraction showed that shoots of mycorrhizal M. truncatula plants have a capacity for extracting Cd that is not related to an increase in root-to-shoot translocation rate, but to a high level of allocation plasticity. When analysing the photosynthetic performance in metal-treated mycorrhizal plants relative to those only Cd-supplied, it turned out that the presence of G. irregulare partially alleviated the negative effects of Cd on photosynthesis. To test the mechanisms by which shoots of Cd-treated mycorrhizal plants avoid metal toxicity, we performed a 2-DE/MALDI/TOF-based comparative proteomic analysis of the M. truncatula shoot responses upon mycorrhization and Cd exposure. Whereas the metal-responsive shoot proteins currently identified in non-mycorrhizal M. truncatula indicated that Cd impaired CO2 assimilation, the mycorrhiza-responsive shoot proteome was characterised by an increase in photosynthesis-related proteins coupled to a reduction in glugoneogenesis/glycolysis and antioxidant processes. By contrast, Cd was found to trigger the opposite response coupled the up-accumulation of molecular chaperones in shoot of mycorrhizal plants relative to those metal-free. Conclusion Besides drawing a first picture of shoot proteome modifications upon AM symbiosis and/or heavy metal stress in legume plants, the current work argues for allocation plasticity as the main driving force for Cd extraction in aboveground tissues of M. truncatula upon mycorrhization. Additionally, according to the retrieved proteomic data, we propose that shoots of mycorrhizal legume plants escape Cd toxicity through a metabolic shift implying the glycolysis-mediated mobilization of defence mechanisms at the expense of the photosynthesis-dependent symbiotic sucrose sink.

Following acquisition of a rhizobial symbiotic plasmid, adaptive mutations in the virulence pathway allowed pathogenic Ralstonia solanacearum to evolve into a legume symbiont under plant selection., Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis., Author Summary Most leguminous plants can form a symbiosis with members of a group of soil bacteria known as rhizobia. On the roots of their hosts, some rhizobia elicit the formation of specialized organs, called nodules, that they colonize intracellularly and within which they fix nitrogen to the benefit of the plant. Rhizobia do not form a homogenous taxon but are phylogenetically dispersed bacteria. How such diversity has emerged is a fascinating, but only partly documented, question. Although horizontal transfer of symbiotic plasmids or groups of genes has played a major role in the spreading of symbiosis, such gene transfer alone is usually unproductive because genetic or ecological barriers restrict evolution of symbiosis. Here, we experimentally evolved the usually phytopathogenic bacterium Ralstonia solanacearum, which was carrying a rhizobial symbiotic plasmid into legume-nodulating and -infecting symbionts. From resequencing the bacterial genomes, we showed that inactivation of a single regulatory gene allowed the transition from pathogenesis to legume symbiosis. Our findings indicate that following the initial transfer of symbiotic genes, subsequent genome adaptation under selection in the plant has been crucial for the evolution and diversification of rhizobia.

Les légumineuses fourragères et à graines ont joué un rôle historique important dans la naissance de l'agriculture. Depuis les années 1950, l'effet conjugué de l'intensification des élevages (appuyée par des importations massives de soja par l'Europe), et du soutien politique européen aux productions de céréales et oléagineux (appuyées par une production massive d'engrais azotés élaborés à partir d'énergies fossiles importées) ont réduit la part des légumineuses dans nos agricultures française et européenne. Aujourd'hui, des attentes socio-économiques sont croissantes pour (i) des systèmes de production végétale et animale plus durables prenant en compte les coûts des intrants et les impacts environnementaux, et (ii) pour une traçabilité et sécurité alimentaire des produits consommés. Ce contexte nouveau est favorable au développement des légumineuses qui présentent, (i) pour les producteurs l'atout d'une capacité à fixer l'azote atmosphérique par une symbiose avec des bactéries rhizobiacées et celui d'une bonne insertion technique des différents systèmes de culture, et (ii) pour les utilisateurs humains ou animaux, l'atout d'une production de protéines de bonne valeur nutritionnelle. Ce développement qui contribuera à davantage d'autonomie de l'UE pour les ressources en protéines, nécessitera une forte coordination des décideurs politiques avec les acteurs de la filière et ceux de la société civile., Forage and grain legumes played a key role in history of agriculture. Since the 1950s, the joint effect of animal production intensification (helped by massive import of soybean by Europe), and of the political support of Europe to cereals and oil seed crops (helped by a massive industry of nitrogen fertilizers which uses fossil energy) have reduced the contribution of legumes in European and French agricultures. New socio-economic demands now increase for (i) sustainable crop and animal production systems, taking into account inputs costs and environmental impacts, and (ii) for a traceability and food and feed security. The new context is favourable to an increase of forage and grain legumes acreage because of their ability to symbiotically fix dinitrogen with Rhizobium bacteria, of their favourable insertion in sustainable cropping systems and of their high nutritional value for animals and humans. This take-off which contribute to a higher autonomy of EU for protein resources, requires a strong coordination of different stakeholders, including research, seed and processing industries, policy-makers and society.

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti–Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new webbased portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources. © 2008 Elsevier B.V. All rights reserved.

The diversity among 191 bacterial strains isolated from stem and root nodules (151 and 40 strains, respectively) of Sesbania rostrata grown in different geographical areas in Senegal and in The Philippines was studied by using DNA-DNA hybridization techniques (S1 nuclease method), by determining DNA base compositions, by performing legume nodulation tests, and by determining nitrogenase activity. The following conclusions were drawn. (i) All of the strains produced stem and root nodules on S. rostrata. (ii) Most of the organisms (184 strains) belonged to the genus Azorhizobium; their guanine-plus-cytosine contents ranged from 66 to 68 mol%, they fixed N2 under free-living conditions, and they produced effective nodules on the stems and roots of S. rostrata. (iii) The seven other strains probably belonged to the genus Rhizobium, since guanine-plus-cytosine contents ranged from 59 to 63 mol% and they did not fix N2 under free-living conditions; three strains produced effective root nodules, but their stem nodules exhibited very low activity or were ineffective, and the four remaining strains produced ineffective nodules on both stems and roots. (iv) The genetic diversity among the 184 Azorhizobium strains allowed us to divide them into two genomic species; genomic species 1 constituted the major group (175 strains) and corresponded to Azorhizobium caulinodans since all of the strains were more than 79% related to type strain ORS 571, and genomic species 2 contained nine strains that were only 44 to 53% related to type strain ORS 571 (difference between the denaturation temperatures of homologous and heterologous hybrids, more than 6°C) and more than 76% related to reference strains SD02 and SG28 (difference between the denaturation temperatures of homologous and heterologous hybrids, less than 3°C). The species that were distinct from A. caulinodans cannot be named until they can be differentiated by phenotypic tests.

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.

Two indigenous bradyrhizobia strains displaying different natural behaviours towards water regime (strain ORS 3257, nodulating more frequently in favourable-water conditions and strain ORS 3260, in limited-water conditions) were studied for their competitivity for nodulation of cowpea (Mouride cultivar) under favourable and limited water conditions in non-sterile soil. The nodule occupancy was studied by PCR-RFLP analysis. Both strains showed good competition with other indigenous rhizobia populations under favourable- and limited-water conditions. Competition between the inoculated strains in the mixture varied between water regimes. In non-limited-water conditions, strain ORS 3257 was the best competitor, whereas in limited-water conditions, strain ORS 3260 was the best competitor. Results indicated that screening of strains according to their environmental origin could ensure successful rhizobia Inoculation.

Chapitre 9 : Plant microbe interactions and soil quality Partie : 9-2; International audience

International audience; We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega of- ficinalis and G. orientalis in northern regions so far

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C1 compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060T=CNCM I 2342T=LMG 21967T) is proposed for this group of root-nodule-forming bacteria.

Nous avons effectué des inoculations ponctuelles sur le système racinaire de Medicago laciniata, cultivée in vitro La bactérie symbiotique Sinorhizobium meliloti et le champignon mycorhizien arbusculaire Glomus sp. S043 ont été utilisés. Nous avons étudié la réponse chimique du tissu racinaire en présence et en l'absence de la bactérie ou du champignon par observation en microscopie visible et à épi-fluorescence. L'inoculation ponctuelle de Sinorhizobium meliloti ou Glomus sp. S043 induit une accumulation de flavonoïdes localisée autour du point d'inoculation. Les autres zones de la racine ne présentent aucune accumulation de flavonoïdes. Il n'a été observé d'accumulation de tannins ni chez le contrôle ni dans les racines inoculées. Le rôle de ces flavonoïdes est discuté.

Summary • The influence of two fluorescent pseudomonads strains (HR13 and HR26) on the ectomycorrhizal symbiosis between Pisolithus alba and Acacia holosericea is reported here. • We measured ectomycorrhizal establishment, fungal growth in the soil (by HPLC) and soil microbial biomass (using the fumigation–extraction method) in treatments with or without pseudomonads. • Bacteria inoculated with the fungal symbiont stimulated ectomycorrhizal formation and shoot or root biomass. Only HR13 significantly increased fungal biomass in the soil. The bacteria stimulated fungal growth and production of phenolic compounds. Sequence analysis of the two fluorescent Pseudomonas revealed 99% homologuey between HR13 and P. monteilii, and 98% between HR26 and P. resinovorans. • It is clear that some bacteria (Mycorrhiza Helper Bacteria) can stimulate the establishment of the ectomycorrhizal symbiosis in tropical conditions.