Cercocarpus betuloides is an actinorhizal plant commonly found in the chaparral vegetation association of Arizona. Information needed to assess the contribution of actinorhizal plants to the nitrogen economy of chaparral ecosystems is lacking. Several physical and chemical factors are known to influence symbiotic N-fixation. To improve understanding of the influence some of these factors have on C. betuloides a series of greenhouse and laboratory studies were conducted to assess the response of C. betuloides to phosphorus supplementation, water availability and temperature. Seedlings of C. betuloides grown in P-supplemented soil produced nearly twice as much dry matter (1.30 vs. 0.77 g pot⁻¹) as did plants grown without added P. Phosphorus supplementation affected nodulation more than dry matter production. Only one of three control seedlings was nodulated, while all nine seedlings grown in P-supplemented soil were nodulated. A higher incidence of nodulation greatly improved the N status of the seedlings. The amount (18.9 mg pot⁻¹) and concentration (1.55%) of N in plant tissue of nodulated seedlings in the P-supplementation treatment were higher than the amount (7.9 mg pot⁻¹) and concentration (1.20%) of N in tissue of control plants. Dry matter production, nodulation, whole plant nitrogenase activity and N-accretion were greatest in seedlings grown at a soil water potential of -0.19 MPa and declined at higher and lower soil water potentials. Dependent variables exhibited a quadratic response to soil water content and a cubic response to soil water potential. Optimum temperature for nitrogenase activity in nodules of C. betuloides appears to be between 30 and 35°C. Nitrogenase activity declined at higher and lower temperatures. Nitrogenase activity responded positively when nodules exposed to lower than optimum temperatures were then exposed to optimum temperatures, but did not recover after being exposed to higher than optimum temperatures. Temperature and soil water potential conditions in the rooting zone of C. betuloides are not known but it seems likely that conditions favorable for N-fixation occur during a portion of the growing season. When temperature and soil moisture conditions are favorable for nitrogenase activity, N-fixation may be limited by P availability.

The depiction of the phylogenetic structure of the genus Frankia is still troublesome and the evolutionary forces guiding the speciation, dispersion and diversity are not well documented. The current phylogeny has been defined on the basis of the comparative analysis of the 16S rRNA gene sequence while de genomospecies definition is still subjected to DNA-DNA hybridization trials. Aiming to bring to light the genomic variability of the genus and its translation into the ecological and specific diversity, our studies consisted in, firstly, evaluating the specific diversity within the genus and the ability of the Amplified Fragment Length Polymorphism technique (AFLP) to describe Frankia genomospecies and their phylogenetic liaisons. Moreover this technique was also tested for the study of the non isolated Frankia directly in the actinorhizal nodules. Secondly, we defined a MLSA (Multilocus Sequence analysis) scheme which allowed us to establish a phylogeny of the genus by using a hundred of strains and for the first time to describe the phylogenetic divergence of a group of non culturable strains exhibiting the particular ability (phenotype) of sporulating in planta (Sp+). The Sp+ strains are distributed into two divergent clades whose structure is highly correlated to the host genotype. The importance of genetic markers having impact over ecology of the strains has been revised. In this regard we have studied the phylogenetic analysis and the occurrence of the genetic components for the siderophore production and of the sodF gene in Frankia.

Actinorhizal plants are in symbiosis with the nitrogen-fixating soil bacterium Frankia, which forms nodules in the plant root. However, several studies also report other endophytic bacteria appearing in the nodules, but their function and interaction with the host plant or Frankia is not yet understood. This thesis used a bioinformatic approach to investigate the metagenomes of eighteen actinorhizal nodule samples to find out which bacteria are present, how the microbiomes differed from each other, and if the genomes of non-Frankia inhabitants could give indications of any functions. The results showed that the bacterial composition, richness, and diversity differed among the samples, especially between the samples sequenced from the field versus those primarily cultivated in a greenhouse. All samples had a substantial number of sequencing reads belonging to potential endophytes, such as strains of Enterobacteria, Pseudomonas, Streptomyces, Micromonospora, Mycobacteria and Pseudonocardia. There seemed to be a common microbial community shared among the plants on a family level, since no significant difference was found in the core microbiomes between the field and greenhouse groups. Some sequences found in the metagenomes were annotated as potential functions of the fellow travellers, such as antibiotic synthesis, proteins involved in regulating abiotic stresses, but also probable plant damaging compounds rather associated with pathogens than symbionts.

La définition de la structure phylogénétique du genre Frankia est encore problématique, les forces évolutives guidant son spéciation, dispersion et donc la génération de sa diversité ne sont pas complètement documentées. La phylogénie actuelle du genre a été définie par l’analyse comparative de la séquence du 16S rRNA. Par ailleurs, la définition des espèces génomiques a été gênée par la faible applicabilité de la technique d’hybridation ADN-ADN. Dans le cadre de cette thèse nos travaux ont consisté à étudier la variabilité génomique dans le genre et sa conséquente traduction en variabilité spécifique et écologique. Dans un premier temps, nous avons évalué la diversité spécifique du genre ainsi que l’utilité de la technique AFLP (Amplified Fragment Length Polymorphism) pour la définition des espèces génomiques. De plus, notre protocole fut aussi utilisé pour analyser souches non isolées en appliquant le protocole directement sur des nodosités actinorhiziennes. Dans un deuxième temps, un schéma MLSA (Multilocus Sequence Analysis) nous a permis de redéfinir la phylogénie du genre sur une centaine de souches, et pour la première fois de décrire la divergence phylogénétique d’un groupe de souches non-isolées présentant un phénotype unique de sporulation in planta (Sp+). Les souches Sp+ sont distribuées dans deux clades très divergents dont la structuration est fortement corrélée au génotype de la plante hôte et au phénotype Sp+/Sp- de la souche. L’intérêt de marqueurs génétiques présentant un intérêt pour l’écologie des souches a été révisé. Dans ce but nous avons étudié la présence, distribution et phylogénie de sodF et des différents composants génétiques impliquées dans la production des siderophores chez Frankia. / The depiction of the phylogenetic structure of the genus Frankia is still troublesome and the evolutionary forces guiding the speciation, dispersion and diversity are not well documented. The current phylogeny has been defined on the basis of the comparative analysis of the 16S rRNA gene sequence while de genomospecies definition is still subjected to DNA-DNA hybridization trials. Aiming to bring to light the genomic variability of the genus and its translation into the ecological and specific diversity, our studies consisted in, firstly, evaluating the specific diversity within the genus and the ability of the Amplified Fragment Length Polymorphism technique (AFLP) to describe Frankia genomospecies and their phylogenetic liaisons. Moreover this technique was also tested for the study of the non isolated Frankia directly in the actinorhizal nodules. Secondly, we defined a MLSA (Multilocus Sequence analysis) scheme which allowed us to establish a phylogeny of the genus by using a hundred of strains and for the first time to describe the phylogenetic divergence of a group of non culturable strains exhibiting the particular ability (phenotype) of sporulating in planta (Sp+). The Sp+ strains are distributed into two divergent clades whose structure is highly correlated to the host genotype. The importance of genetic markers having impact over ecology of the strains has been revised. In this regard we have studied the phylogenetic analysis and the occurrence of the genetic components for the siderophore production and of the sodF gene in Frankia.

La sporulation est un phénomène présent chez de nombreux microorganismes, généralement impliqué dans les mécanismes de dispersion et/ou résistance en conditions environnementales défavorables. La sporulation observée chez certaines souches de Frankia (genre actinobactérien fixateur d'azote) lors de leur interaction symbiotique avec les plantes actinorhiziennes est donc paradoxale dans un contexte où la bactérie bénéficie d'une niche écologique favorable à son développement. Ces souches particulières de Frankia, dites Sp+, représentent un modèle unique de symbiote capable de sporulation au sein même des cellules de son hôte. Le rôle écologique et le sens évolutif de cette sporulation in-planta reste à ce jour peu élucidé. Les deux principaux objectifs de ce travail de thèse visent donc à (i) comprendre l'influence de la sporulation in-planta sur les capacités symbiotiques des souches Sp+, en termes d'infectivité et de compétitivité et (ii) appréhender l'impact de cette sporulation sur le fonctionnement du complexe symbiotique par une méthode de profilage métabolique. Ces travaux ont permis de confirmer les particularités symbiotiques des souches Sp+ (infectivité et compétitivité accrues) et de montrer des différences significatives dans le métabolisme primaire et secondaire du complexe symbiotique associées à la présence de spores de Frankia / Sporulation is a phenomenon present in many microorganisms, usually involved in the mechanisms of dispersion and/or resistance to unfavorable environmental conditions. Sporulation occurs in some Frankia strains (a diazotrophic actinobacteria) during their symbiotic interaction with actinorhizal plants, which is paradoxical in a context where the bacterium has a favorable ecological niche for its development. These particular Frankia strains, called Sp+, represent a unique model of symbiont capable of sporulation within the host cells. The ecological role and the evolutionary meanings of this in-planta sporulation still remain understood. The two main objectives of this thesis aimed to (i) understand the influence of in-planta sporulation on the symbiotic capacity of Sp+ strains in terms of infectivity and competitiveness and (ii) understand the impact of this sporulation on the functioning of the symbiotic complex by a metabolic profiling approach. These studies have confirmed the symbiotic characteristics of Sp+ strains (greater infectivity and competitiveness) and have shown significant differences in the primary and secondary metabolism of the symbiotic complex associated with the presence of Frankia spores