Auswahl der wissenschaftlichen Literatur zum Thema „Alnus-Frankia symbiosis“

A unique case of microbial symbiont capable of dormancy within its living host cells has been reported in actinorhizal symbioses. Some Frankia strains, named Sp+, are able to sporulate inside plant cells, contrarily to Sp− strains. The presence of metabolically slowed-down bacterial structures in host cells alters our understanding of symbiosis based on reciprocal benefits between both partners, and its impact on the symbiotic processes remains unknown. The present work reports a metabolomic study of Sp+ and Sp− nodules (from Alnus glutinosa), in order to highlight variabilities associated with in-planta sporulation. A total of 21 amino acids, 44 sugars and organic acids, and 213 secondary metabolites were detected using UV and mass spectrometric–based profiling. Little change was observed in primary metabolites, suggesting that in-planta sporulation would not strongly affect the primary functionalities of the symbiosis. One secondary metabolite (M27) was detected only in Sp+ nodules. It was identified as gentisic acid 5-O-β-d-xylopyranoside, previously reported as involved in plant defenses against microbial pathogens. This metabolite significantly increased Frankia in-vitro sporulation, unlike another metabolite significantly more abundant in Sp− nodules [M168 = (5R)-1,7-bis-(3,4-dihydroxyphenyl)-heptane-5-O-β-d-glucopyranoside]. All these results suggest that the plant could play an important role in the Frankia ability to sporulate in planta and allow us to discuss a possible sanction emitted by the host against less cooperative Sp+ symbionts.

Although most vascular plants have symbiotic relationships with soil microbes, and there is an extensive theoretical literature on the evolution of mutualism, there has been little experimental examination of the evolution of mutualism between plants and their microbial symbionts. We inoculated red alder (Alnus rubra Bong.) seedlings from three high- and three low-elevation populations with crushed nodule suspensions containing the nitrogen fixing bacterium Frankia from either the parent trees (familiar strains) or the other plant population sampled within the parent watershed (unfamiliar strains). The inoculated seedlings were planted on three high- and three low-elevation sites. Growth was monitored over the second and third year following planting, after which the whole plants were harvested. The proportion of nitrogen derived from fixation was estimated from the ratio of stable nitrogen isotopes in the harvested leaves. On low-elevation sites, which had high soil nitrogen, plants with familiar Frankia strains were half the size and derived less fixed nitrogen from their symbionts compared with plants inoculated with unfamiliar Frankia strains. On high-elevation sites, which had low soil nitrogen, the type of inoculum had little effect on plant performance, although plants with familiar inoculum were consistently larger than plants with unfamiliar inoculum. These results suggest that the degree of mutualism in this symbiosis depends on environmental conditions and may decrease with time.Key words: coevolution, Frankia, Alnus rubra, mutualism, nitrogen fixation, symbiosis.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

ABSTRACT Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N2 fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

Nodulation kinetics were analysed in two nitrogen-fixing actinorhizal symbioses that show different pathways for infection: Alnus acuminata H. B. K., which is infected by Frankia ArI3, and Discaria trinervis (Hooker et Arnot) Reiche, which is infected by Frankia BCU110501. Both pairs are incompatible in cross-inoculation experiments. The dose–response effects in nodulation were studied in A. acuminata seedlings using different concentrations of compatible and incompatible bacteria in co-inoculation experiments. Restriction fragment length polymorphism PCR analysis and plant-trapping analysis showed no co-occupation in A. acuminata nodules when plants were co-inoculated with Frankia BCU110501 and Frankia ArI3. Despite the lack of co-occupation, the noninfective BCU110501 could modify the nodulation parameters of the non-host A. acuminata when infective ArI3 was present in the inoculum. The results suggest that although BCU110501 was not able to induce nodulation in A. acuminata, its interaction with the plant could induce autoregulation as if some level of infection or partial recognition could be achieved. We explored the possibility that physiological complementation of the heterologous Frankia BCU110501 for nodulation of A. acuminata originated in the homologous Frankia ArI3 in the presence of compatible root exudates. Despite the possibility of full activation between bacteria and the host, there was no co-infection of Frankia BCU110501 in Alnus or of Frankia ArI3 in Discaria either. These negative results suggest a physical recognition barrier in actinorhizal symbiosis that operates after early interactions, involving something other than root exudates and diffusible factors of bacterial or plant origin, regardless of the infection pathway.

To address issues of dominance and diversity of Frankia spp. strains, we sequenced 16S rRNA genes from root nodules and strains collected from Alnus incana subsp. rugosa (Du Roi) R.T. Clausen and Myrica pensylvanica Loisel. stands. Of 22 strains isolated previously from A. incana, 16 had the same partial rDNA sequence; the remaining 6 strains composed five additional groups. The groups identified by 16S rDNA analysis corresponded to phenotypic groups established previously by one- and two-dimensional polyacrylamide gel analysis, colony and hyphal morphology, and carbon source utilization patterns. Dominance of one strain was also evident in nodules collected from a single M. pensylvanica stand. The dominant strain had a partial 16S rDNA sequence identical to that of Frankia alni strain CpI1.Key words: Frankia, Myrica, Alnus, actinorhizal, root nodules, nitrogen fixation, symbiosis, 16S rRNA.

The actinobacteria Frankia spp. are able to induce the formation of nodules on the roots of a large spectrum of actinorhizal plants, where they convert dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living Frankia alni cells and on Alnus glutinosa nodule bacteria, using whole-genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia spp. genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to nitrogen fixation were highly induced, nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster), and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified, suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We also showed that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae. Finally, comparison with rhizobia transcriptome suggested that F. alni is metabolically more active in symbiosis than rhizobia.

Frankia BCU110501 induces nitrogen-fixing root nodules in Discaria trinervis (Gillies ex Hook. & Arn.) Reiche (Rhamnaceae) via intercellular colonisation, without root hair deformation. It produces diffusible factors (DFs) that might be involved in early interactions with the D. trinervis roots, playing a role in the nodulation process. The induction of root nodule development in actinorhizal symbiosis would depend on the concentration of factors produced by the bacteria and the plant. A detailed analysis of nodulation kinetics revealed that these DFs produce changes at the level of initial rate of nodulation and also in nodulation profile. Diluted Frankia BCU110501 inoculum could be activated in less than 96 h by DFs produced by Frankia BCU110501 cells that had been previously washed. Biochemical characterisation showed that Frankia BCU110501 DFs have a molecular weight of <12 kDa, are negatively charged at pH 7.0 and seem to contain a peptide bond necessary for their activity. Frankia BCU110501, belonging to Frankia Clade 3, does not induce nodules in Alnus acuminata H.B.K. ssp. acuminata but is able to deform root hairs, as do Frankia strains from Clade 1. The root hair deforming activity of Frankia BCU110501 DFs show the same biochemical characteristics of the DFs involved in nodulation of D. trinervis. These results suggest that Frankia symbiotic factors have a basic structure regardless of the infection pathway of the host plant.

Les patrons de distribution chez les micro-organismes reposeraient sur leurs capacités à disperser dans le temps et dans l'espace, en lien avec des facteurs abiotiques comme les propriétés du sol, le climat, et des interactions biotiques, notamment avec l'hôte dans le cas des symbiontes, mais aussi sur les traits d'histoire de vie propres aux micro-organismes, telle que la capacité à sporuler. Frankia sp. est une actinobactérie sporulante et fixatrice d'azote à la biogéographie complexe, car vivant à la fois de façon saprophytique dans le sol, en symbiose racinaire (nodosité) avec les plantes actinorhiziennes dont les aulnes (Alnus, Betulaceae). Deux types de souches de Frankia génétiquement différentes ont été décrites dont la distinction phénotypique majeure réside dans la capacité à maintenir (Sp+) ou non (Sp-) leur sporulation in planta. Cette sporulation endophytique est à notre connaissance unique dans un contexte symbiotique et son implication dans la biogéographie de Frankia, reste peu connue. Ces travaux de thèse intègrent à la fois des approches descriptives et expérimentales, sur le terrain et au laboratoire, afin d'accroître la compréhension du rôle écologique de la sporulation in planta de Frankia. Dans un premier temps, nous avons étendu la description de la phylobiogéographie des souches de Frankia Sp+ afin de tester la validité du patron de distribution centré sur les milieux froids des zones de haute altitude et de haute latitude de l'hémisphère nord. Un intérêt tout particulier a été porté sur les aires géographiques où une plus forte diversité de Frankia était attendue, dans la zone d'origine de l'aulne et ses refuges glaciaires. Dans un second temps, nous avons étudié l'influence du partenaire végétal dans la distribution observée des Frankia Sp+ et l'implication du trait Sp+ dans la capacité d'association à l'hôte. Des croisements expérimentaux ont été réalisés au laboratoire afin de découpler les effets de l'espèce-hôte et du climat, et tester les implications du trait Sp+ en termes d'infectivité, compétitivité et spectre d'hôte. Enfin, nous avons étudié les conséquences écosystémiques de l'expansion subalpine du complexe symbiotique Alnus/Frankia, au niveau de la diversité microbienne et du fonctionnement du cycle de l'azote, en fonction du phénotype de sporulation des souches associées. Des analyses pédologiques, en association avec des mesures de nitrification, dénitrification et fixation d'azote, ainsi que des analyses de diversité microbienne (globale et fonctionnelle), ont été réalisées dans différentes aulnaies Sp+, Sp- ou mixte, à différents stades de colonisation de l'aulne. Les résultats obtenus démontrent une prédominance des souches Sp+ associées aux espèces d'aulne des milieux froids sur les 3 continents de la zone Holarctique, avec une diversité nouvelle dans l'aire d'origine et les zones refuges de l'aulne. Les croisements effectués révèlent une infectivité et compétitivité plus forte des Sp+ par rapport aux Sp-. De plus, contrairement aux Sp- à spectre d'hôte très large, les Sp+ présentent un spectre limité entraînant des incompatibilités d'association suggérant une dépendance forte à une espèce-hôte donnée. Les modifications des communautés microbiennes du sol en réponse à l'expansion du complexe symbiotique Alnus/Frankia ont été démontrées, en lien avec la stimulation du cycle de l'azote dans les milieux sub-/alpins. Les premiers résultats sur l'efficience comparée de la fixation d'azote in natura des souches Sp- par rapport aux Sp+ suggèrent que 100% de l'azote de l'aulne est obtenu par le biais de la fixation. Aucun patron n'est mis en évidence entre souches Sp+ et Sp-, suggérant un effet plus complexe de la saisonnalité, de l'âge de l'arbre et de celui de la nodosité. Les résultats obtenus nous permettent de mieux appréhender les facteurs guidant la biogéographie de Frankia et de discuter de l'évolution de ces patrons de distribution en réponse au réchauffement climatique
Microbial biogeography would be based on the ability of microorganisms to disperse across time and space, as a function of abiotic factors such as soil properties, climate, and of biotic interactions, in particular with the host in the case of symbionts, but also on life history traits such as the ability to sporulate. Frankia sp. is a spore-forming and nitrogen-fixing actinobacterium that has a complex biogeography given its abilities for both saprophytic life and root symbiotic interaction with actinorhizal plants such as alders (Alnus, Betulaceae). Two distinct groups of Frankia lineages have been described according to a major phenotypic divergence, based on the presence (Sp+) or the absence (Sp-) of spores in planta.. To the best of our knowledge, this endophytic sporulation is an original trait in a symbiotic context and very little is known about its incidence in Frankia biogeography. This work integrates descriptive and experimental approaches on both field and laboratory areas, in order to improve the understanding of the ecological role of Frankia in planta sporulation. First, we have extended the description of the phylobiogeography of Sp+ Frankia strains to validate the previously proposed distribution pattern focused on cold environements at high altitude or high latitude. A phylogeny has been computed using a large number of nodular strains coming from the 3 continents of the Northern Hemisphere and 10 different Alnus species. Special attention was paid to geographic areas where a higher diversity was expected, in Asia, and in its glacial refuges. Second, we studied the influence of the host-plant on the distribution of Fankia Sp+ and the incidence of Sp+ in the symbiotic interaction. Experimental crosses have been performed to disentangle host and climate effects and to test the incidence of the Sp+ trait in terms of infectivity, competitiveness and host-range. Finally, we studied the ecological consequences of the Alnus/Frankia symbiotic complex, on the microbial diversity and on the nitrogen cycle functionning, with respect to the sporulation of Frankia and to the Alnus expansion on sub-/alpine grasslands. Soils analyses were performed in association with measures of nitrification and denitrification, as well as global and functional microbial diversity analyses, in Sp+, Sp- or mixed alder stands and at different colonization stages. In each part of this work, alder ectomycorhizae were analyzed to compare the distribution pattern between the two symbionts and to highlight potential interactions with the Sp+ trait of Frankia. Our results show the dominance of Sp+ strains in nodules of alder species from cold environments over the 3 continents of the Holarctic zone, with original diversity patterns in alder area of origin and in glacial refuges. Even if these strains are genetically homogenous, host-specific clusters were observed in the phylogeny. Crosses revealed that Sp+ strains were more infective and competitive than Sp- strains. Moreover, unlike Sp- strains that harbor a wide host-range, Sp+ strains have a narrower specificity leading to association’s incompatibilities and suggesting strong host dependence. For the first time, modifications of microbial communities were revealed in response to the Alnus-Frankia symbiotic complex colonization and were linked to a stimulation of the nitrogen cycle in the sub-/alpine grasslands. The first comparative results of nitrogen fixation between Sp+ and Sp- strains in natura suggest a maximal efficiency of fixation, representing almost 100% of the alder nitrogen. However, unlike previous reports in literature, no pattern was observed between Sp+ and Sp- strains, suggesting a complex effect of seasonality, alder age as well as that of nodules. Altogether, the previous results contribute to a better understanding of the Frankia biogeography drivers and allow us to discuss the expected evolution of distribution pattern in response to the global warming

Frankia est une actinobactérie capable d'établir une symbiose racinaire avec les plantes actinorhiziennes dont le genre Alnus. Seulement certaines souches de Frankia sont capables de sporuler in planta, ce qui est illustré par la présence (Sp+) ou l'absence (Sp–) de sporanges dans les cellules végétales de la nodosité. C’est à notre connaissance un cas unique de sporulation endophyte. Cependant la description et l’interprétation écologique de ce trait d’histoire de vie (THV) original étaient incomplètes. Notre contribution à l’étude de la sporulation in planta des Frankia infectives de l’aulne intègre des approches théorique, descriptive et expérimentale, pour préciser (i) l’influence relative de la souche bactérienne, de l’espèce de la plante-hôte et des conditions pédoclimatiques sur ce THV, (ii) le rôle de la variabilité environnementale sur la distribution, la diversité et la sélection du trait, ainsi que (iii) les coûts et bénéfices associés pour les deux partenaires. Nous avons démontré pour la première fois que la sporulation in planta est un THV (i) spécifique de certaines lignées de Frankia, (ii) majeur pour en comprendre l'histoire évolutive et (iii) significativement corrélé à des caractéristiques génétiques des souches. Nous avons également confirmé que l’occurrence du trait varie selon l’environnement. Nous avons enfin établi un modèle de l'évolution du trait abordant sa valeur adaptative. L’ensemble des réflexions menées et des résultats obtenus nous permet de discuter de la sporulation in planta dans le cadre d’un continuum de stratégies symbiotiques, et plus généralement de discuter de l’écologie évolutive des symbioses entre microorganismes et plantes
Frankia sp. is a telluric actinobacteria able to establish a root symbiosis with actinorhizal plant such as Alnus sp. Only some Frankia strains are able to sporulate in-planta, as spores can be present in (Sp+) or absent from (Sp–) the vegetal cells of the root nodule. It is to our knowledge a unique case of endophytic sporulation. However, the description and the ecological interpretation of this original life-history trait (LHT) were scarce. Our contribution to the study of the in-planta sporulation of Alnus-infective Frankia sp. combines theoretical, descriptive and experimental approaches to precise (i) the relative effect of the bacterial strain, the host-plant species and the pedoclimatic conditions on this LHT, (ii) the effect of the of the environmental variability on the distribution, diversity and selection of the trait, and (iii) the associated costs and benefits for the two symbiotic partners. We demonstrated for the first time that the in-planta sporulation is a LHT (i) specific to some Frankia lineages, (ii) major to understand their evolutionary history and (iii) significantly correlated to particular genetic features. We also shown that the occurrence of the trait varies according to the environment We also proposed a model of the evolution of the trait taking its fitness into account. We bring all the previous considerations and results to discuss the inplanta sporulation trait within a continuum of symbiotic strategies and more generally to discuss the evolutionary ecology of plant-microbe symbioses

The occurrence in Scotland of arbuscular mycorrhizal fungi (AMF) infective on Alnus glutinosa and the effects on early growth of inoculation of seedlings with AMF and Frankia were investigated. AMF characteristic of G/omiw-like fungi were detected microscopically in A. glutinosa roots from mineral and loam but not from highly organic soils. Both GJomus- and Gigaspora-like fungi were detected only in roots from sandy soils. Glasshouse experiments showed that Glomus and Gigaspora species gave higher colonisation of roots, and were of higher compatibility with the host plant, than Acutelospora or Scutettospora species. Colonisation by AMF increased nodulation by Frankia and vice versa. Inoculation with AMF alone and dual inoculation with Gigaspora rosea and Frankia inhibited growth of young A. glutinosa seedlings. Initially after inoculation, AMF colonisation was poor allowing the host to meet the requirements of both developing symbioses. Mycorrhizal plants inoculated with Frankia 15 days after AMF inoculation, were under significant stress and this delayed the beneficial effects of AMF and Frankia symbiosis. Seedlings compensated for the cost of symbiosis by developing a larger, leafy shoot in a period of 25-30 days after AMF inoculation, with or with out, Frankia nodulation. A significant increase in root length and branching detected after inoculation with AMF and Frankia must impose additional energy requirements. However, the stimulation of lateral root branching that follows AMF colonisation may facilitate nutrient uptake, thus helping the plant to overcome the disadvantages of early retardation of shoot growth. Competition with adjacent plant species for below ground space may also be enhanced thus facilitating competition for growing space with other plant species. These factors may be part of a strategy that assists the survival of very young seedlings in the field. The data obtained suggest that delaying Frankia inoculation for 3-4 weeks after inoculation with selected AMF should improve the production in Scottish nurseries of robust, well nodulated and mycorrhizal seedlings.

Les principaux responsables de la contamination des sols par les métaux sont les exploitations minières et les activités industrielles. Évidemment, de tels ajouts de contaminants au niveau du sol affecteront directement sa microflore qui comprend plusieurs microorganismes d'importance environnementale comme les bactéries fixatrices d'azote. Les méthodes actuelles de décontamination des sols s'avèrent coûteuses et fréquemment dommageables pour l'environnement en plus d'être limitées au niveau du volume de sol pouvant être traité. C'est pourquoi dans les dernières années les efforts de recherche se concentraient à développer de nouvelles biotechnologies de décontamination utilisant les capacités naturelles de certains microorganismes (bioremédiation) et de certaines plantes (phytoremédiation). C'est dans cette optique que pourraient être utilisés les bactéries du genre Frankia spp. et les aulnes. Frankia spp. se retrouve dans le sol partout à l'échelle mondiale. Elle fait partie de la classe des actinomycètes et possède la capacité de fixer l'azote en mode saprophyte et symbiotique (symbiose actinorhizienne). La symbiose actinorhizienne a été observée chez plus de deux cent espèces végétales sur tous les continents sauf en Antarctique et s'avère aussi efficace dans la fixation d'azote atmosphérique que la symbiose entre les Rhizobium et les légumineuses. Les aulnes possèdent également une distribution géographique très vaste. Ce sont des plantes pionnières (arbres ou arbustes) qui enrichissent graduellement le sol par l'apport de matière organique et de matière azotée. Leurs utilisations actuelles sont très variées (foresterie, agroforesterie, revégétalisation de sites contaminés, etc.). Parmi les espèces d'aulnes, l'aulne noir (Alnus glutinosa), est utilisé sur plusieurs sites d'Europe contaminés en métaux. À la vue des informations disponibles sur Frankia spp. et A. glutinosa, l'hypothèse que Frankia spp. et sa symbiose avec A. glutinosa tolèreraient les métaux dans leur environnement et pourraient servir en revégétalisation et/ou phytoremédiation a été posée. De cette hypothèse découlent deux objectifs spécifiques et originaux : 1- Évaluer la résistance de Frankia spp. face aux métaux et 2- Évaluer l'impact des métaux sur la symbiose et sur la plante hôte. Dans le cadre de ce projet, une méthode de criblage en milieu de culture liquide a été développée afin de déterminer la tolérance de cinq souches de Frankia sp. envers douze métaux. La méthode de révélation choisie impliquait la réduction d'un sel de tétrazolium par le métabolisme respiratoire des souches. Ces travaux ont permis de mettre en évidence une diversité au niveau de la tolérance des souches de Frankia sp. face aux métaux. La tolérance de la symbiose actinorhizienne face aux métaux a été étudiée grâce à la souche Frankia ACN14a et A. glutinosa. La culture hydroponique de ces organismes dans des conditions gnotobiotiques et dans un environnement contrôlé (cycle de luminosité, température et humidité) a permis d'observer plusieurs phénomènes intéressants. Les résultats globaux démontrent que la symbiose serait davantage affectée par les métaux lors de son établissement (dénombrement des nodules) que lors de son développement (poids frais des nodules). De plus, la symbiose avec Frankia ACN14a favoriserait un développement supérieur des parties aériennes de l'aulne. Aussi, aucun métal testé (aux concentrations permettant la survie de la plante) n'a aboli complètement la symbiose et la capacité fixatrice d'azote de Frankia ACN14a dans les nodules. Le dosage de l'activité nitrogénase a été mesuré via la réduction de l'acétylène en éthylène par chromatographie en phase gazeuse. Finalement, la quantité de métal se retrouvant dans les parties aériennes a été quantifiée par ICP-MS et les résultats ont démontré la possibilité que l'aulne noir accumule des métaux au niveau des tiges et feuilles. À long terme, ces résultats pourraient être transposés à grande échelle et servir à l'élaboration de stratégies de décontamination employant les aulnes noirs et Frankia spp..

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

Les mesures quantitatives de l'azote fixe sont utilisees pour mettre en evidence les interactions entre les deux partenaires de la symbiose aulne/frankia d'une part et celles-existant entre le milieu et ce systeme fixateur d'autre part a l'aide des methodes 15n. Dans leur milieu naturel, les aulnes peuvent avoir une grande capacite fixatrice et contribuer ainsi a la formation des sols par un apport d'n organique facilement mineralisable. Les capacites fixatrices sont renforcees lorsque cet arbre est utilise en culture mixte avec d'autres arbres non-fixateurs comme le peuplier. La production et la capacite fixatrice des aulnes dependent de l'espece et a l'interieur meme de l'espece, de l'origine de la plante hote. Cette capacite est differente suivant le site etudie. Aussi, la selection de plantes hautement performantes au niveau de la fixation ne peut se faire que pour un sol donne. La selection du partenaire bacterien pose le probleme de l'introduction d'une nouvelle souche dans un sol, de ses chances de survie et du maintien de ses capacites fixatrices. Deux souches de frankia peuvent coexister sur le meme systeme racinaire ce qui peut laisser esperer qu'une souche introduite pourrait subsister malgre la competition au niveau de la nodulation avec les souches natives du sol. Mais, la survie de cette souche est largement dependant des conditions de milieu. La souche ai15(sp#) introduite dans un sol donne contenant des souches indigenes de type sp#+ perdait ses capacite de nodulation, contrairement aux resultats obtenus a partir d'un milieu inerte inocule avec 2 souches. Par son influence sur sa capacite fixatrice d'azote, le sol est bien un facteur determinant dans le choix final du meilleur couple symbiotique alnus/frankia

I examined the effect of Frankia spp. and ectomycorrhizal fungi on green alder (Alnus viridis ssp. crispa), growing in nutrient-poor soil and saline conditions. The first experiment involved inoculating green alder growing in low fertility soil with three species of ectomycorrhizal fungi (Lactarius torminosus, Lactarius theiogalus, Hebeloma crustuliniforme) alone or in combination, with and without Frankia spp. on. Frankia spp. inoculation significantly increased plant performance compared to non-Frankia treatments. However, nodulated plant total biomass decreased with an increasing number of fungi. The second experiment examined the effect of Hebeloma crustuliniforme and Frankia spp. on green alder exposed to 0, 50 and 100 mM NaCl. Frankia spp. inoculation showed significant increase on plant performance but Hebeloma crustuliniforme did not. Plant mass, root:shoot ratio, nodule allocation and total nitrogen fixation decreased with NaCl exposure. A decrease in root:shoot ratio caused by salt was more moderate in nodulated plants compared to non-nodulated plants.
October 2016