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.

Philosophiae Doctor - PhD / Frankia is one of two partners in the globally distributed N2-fixing actinorhizal symbiosis between this filamentous soil-dwelling actinomycete and almost 300 species of host plants from eight diverse angiosperm families. The actinorhizal symbiosis is a major contributor to nitrogen reservoirs in terrestrial ecosystems, and allows actinorhizal plants to perform the role of pioneers in newly formed and nitrogen-poor soils. Frankia are differentiated into four main host-infection groups (1: Alnus/Comptonia/Myrica-infective, 2: Rosaceae/Datisca/Coriaria-infective, 3: Elaeagnaceae/Gymnostoma-infective and 4: Casuarina-infective), and there is a large degree of phylogenetic clustering within these HIGs. Of these host lineages, species from the genus Morella, from the family Myricaceae, are notable as they have the ability to establish effective partnerships with Frankia from more than one host-infection group. Africa houses 16 of the world’s 33 currently accepted Morella species, and Morella is the continents only genus containing endemic actinorhizal species. Despite this, the diversity of Frankia in symbiosis with African Morella has never been explored. To address this lack of knowledge I investigated Frankia in root nodules of six Morella species from the Cape flora of Southern Africa, as well as in rhizosphere soils from selected hosts. Partial nif H gene fragments recovered from 202 root nodules yielded 26 unique sequences, which phylogenetic analysis assigned to Frankia Cluster I (the Alnus host infection group) and Frankia Cluster III (the Elaeagnus host infection group)1. Nineteen nif H sequences were assigned to three sub-clusters within Frankia Cluster III (CC-3, CC-4 and CC-5), and the remaining seven sequences to two sub-clusters within Cluster I (CC-1 and CC-2), one of which (CC-1) is novel to the current study. Identical sequences were recovered from nodules collected at geographically distant locations, suggesting a cosmopolitan distribution within the region for some subgroups from both clusters, but more localized distribution (or tighter host-specificity) for others. Soil pH correlated with strain presence in nodules, with Cluster I sequences being associated with hosts growing in acidic soils exclusively. Furthermore, three Morella species from the Cape flora of southern Africa are promiscuous in their natural habitats, with host infection group influenced by habitat edaphic conditions. In order to explore the correlation between soil characteristics and Frankia presence in nodules, nif H soil libraries were created from selected host rhizospheres. While Cluster III sequences from these libraries corresponded closely to sequences found in nodules from the same sites, the dominant Cape Cluster I group (CC-1) was absent from all six libraries, even when present in nodules recovered from the same soils. Whether this was due to low abundance of -but strong selection for- these strains by hosts under particular conditions, or due to the absence in soil of hyphal forms of these strains could not be determined. Cluster III strains are known to be better able to persist saprophytically than their relatives from other host-infection groups. A second group of Cluster I strains, detected at only one sampling site, was present in that site's corresponding soil library. An Alnus-infective subgroup, cluster AI, which has been detected in soils collected on five continents, was also detected in the of the Cape soil libraries but never in nodules, raising questions as to this group’s ability to persist in soil in the absence of known suitable hosts. Ten Frankia strains representing all three of the numerically dominant subgroups (CC-1, CC-3 and CC-4, found in 186 of 202 root nodules) were isolated from four Morella species. These isolates represent six of the most abundant unique nodular nif H sequences found in the field survey, and display morphological and cultural characteristics typical of Frankia. Phylogenetic analysis confirmed their identity as Frankia, and multilocus analysis revealed that the isolates belong to three genospecies. Two of these genospecies fall into existing groups within the Elaeagnus-infective Cluster III, while the remaining genospecies is a novel addition to the otherwise well-described Alnus-infective Cluster I. Whole genome sequencing of a representative from each of the Cape genospecies allowed for basic annotation and genome descriptions, which agreed in each case with what has been previously found for strains from the Elaeagnus and Alnus host-infection groups, respectively. Similarly, the organization of nitrogenase gene clusters in each of the sequenced strains mirrors that found in other strains from their respective host-infection groups, indicating that this gene cluster is highly conserved in different Frankia lineages. For the first time the diversity of Frankia nodulating endemic African Morella, and present in root-associated soils of these species, has been explored. This is also the first study to report isolation and description of Frankia strains from actinorhizal plants endemic to Africa.

Causes and consequences of woody encroachment into grass dominated systems have been widely studied, however functional mechanisms which promote encroachment are largely unknown. Many expansive woody species are shrubs with rhizobial or actinorhizal N-fixing symbiotic associations. Morella cerifera L. (Myricaceae) is an actinorhizal N-fixing shrub which rapidly expands into grasslands on the barrier islands off the coast of Virginia, USA. The objective of this research was to determine physiological drivers of woody encroachment resulting in increased woody cover of M. cerifera on Southeastern, US barrier islands. Variations in physiology and resource use efficiencies (RUE) of M. cerifera and co-occurring shrubs were determined, and edaphic characteristics beneath shrub thicket canopies and in open areas were quantified as indications of resource availability. Analysis of dune vegetation and soils showed severe freshwater limitation and reduced plant height of dune forbs suggesting dunes represent an upper elevational boundary for M. cerifera distribution. Soil N availability was higher beneath shrubs compared to open areas, and both physiology and isotope effects showed facilitation of the non-fixing shrub, Baccharis halimifolia, by M. cerifera which may lead to increased rates of woody encroachment as B. halimifolia colonizes expanding thicket edges. Morella cerifera and other N-fixers had higher %refixation within stems which resulted in higher carbon use efficiency (CUE) and water use efficiency of N-fixing shrubs compared to non-fixers. Results of an N-fertilization experiment suggest B. halimifolia has higher dependence on and demand for soil nutrients compared to M. cerifera. Morella cerifera showed no signs of resource deficiency or reduced physiological capacity even at 0 ppm total Nsoil. Morella cerifera transitioned from utilizing solely fixation derived N to soil N as N concentrations increased providing another mechanism leading to increased CUE and, indirectly, overall RUE. In summary greater RUE, lower resource demand, and greater resource availability for M. cerifera compared to co-occurring shrubs may result from symbiotic root associations with bacteria and fungi. While expansion of M. cerifera thickets is limited to lower elevational interdunal depressions, expansion may continue and result in increased rates of woody encroachment through facilitation of co-occurring shrubs.

In the nutrient-poor soils characteristic of coastal environments, symbiotic association with the nitrogen-fixing root endosymbiont Frankia is essential to establishment and survival of the woody shrub Morella cerifera. Nutrient deficiency quickly becomes severe unless seedlings are infected by Frankia soon after germination. However, the means of arrival of Frankia prior to shrub establishment has not been determined. Using sterilized lab-grown M. cerifera seedlings and fecal samples collected from passerine birds on the Eastern Shore of Virginia, viability of avian dispersal of the bacteria was tested. Although passerine fecal samples did produce nodules on some sterilized M. cerifera seedlings, these experimental inoculations did not lead to significantly higher likelihood of nodulation, relative to sterilized reference seedlings. Non-sterilized seedlings displayed greatest percent nodulation; results suggest that passerines contribute to Frankia dispersal, but also that the actinomycete is contained on or within viable seeds or fruits of M. cerifera, and therefore may be co-dispersed directly from the parent plant.

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.

I quantified seasonal variations in corticular photosynthesis in 1st through 5th order branches of Myrica cerifera L. (Myricaceae) in order to determine whether corticular photosynthesis contributes to whole plant carbon gain by reducing respirational CO2 loss. Maximum % refixation was 110 ± 39 % of CO2 efflux in the dark (Rd) in 1st order branches during winter, minimum was 18 ± 3 % in 5th order branches during summer. Variations in % refixation paralleled changes in photosynthetically active radiation (PAR). As light attenuated with increasing branch order % refixation decreased. Increased PAR in the winter due to a more sparse canopy lead to increases in % refixation. Total chlorophyll content and chlorophyll a:b ratios were consistent with shade acclimation as branch order increased. Corticular photosynthesis may be a mechanism to enhance shrub expansion due to increased whole plant carbon use efficiency (CUE) and water use efficiency (WUE) attributed to refixation.

Sustainable landscaping includes utilization of plants requiring few inputs. We chose four species showing potential for use in arid landscapes: Purshia mexicana, Shepherdia argentea, Shepherdia rotundifolia, and Alnus maritima. We sowed seeds of S. rotundifolia, S. argentea and P. mexicana in three substrates with various water-holding properties due to differing amounts of organic matter (OM). S. rotundifolia germination was maximized in a calcined clay (66.2%) containing no OM and had low germination (12.7 - 21.8%) in the other substrates. S. argentea germination (42.3 to 53.7%) was similar in all substrates. Poor seed quality of P. mexicana resulted in inconclusive results. Our results suggest that germination of some species is enhanced by substrates with excellent drainage properties. We also investigated effects of different rates of controlled-release fertilizer (CRF) on symbiotic nodule formation in seaside alder. We found that lower than prescribed rates of CRF enhanced nodulation without compromising nitrogen status.

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.

Les deux systèmes nodulaires symbiotiques les plus importants au niveau agronomique et environnemental sont, d'une part, les symbioses Rhizobium-légumineuses qui concernent environ 14 000 espèces, et d'autre part, les symbioses entre les plantes actinorhiziennes (environ 200 espèces) et l'actinomycète du sol Frankia. La plupart des plantes actinorhiziennes sont capables de fixer des quantités d'azote comparable à celles des Légumineuses ; ce sont généralement des plantes pionnières capables de coloniser des environnements pauvres en éléments minéraux. Elles représentent donc un atout écologique important. Si la symbiose Rhizobium-légumineuse est très étudiée, les mécanismes moléculaires à l'origine de la formation des nodules actinorhiziens restent actuellement peu connus. Ainsi, chez les Légumineuses, les flavonoïdes sont des molécules-clefs du processus de nodulation, alors que chez les plantes actinorhiziennes, l'implication des flavonoïdes dans la nodulation reste imprécise. L'objectif de cette thèse était de comprendre l'implication des flavonoïdes au cours de l'interaction symbiotique entre l'arbre actinorhizien tropical Casuarina glauca et son symbiote Frankia. L'analyse d'une base de données d'unigènes couplée à celle de données d'expression de puces à ADN a permis l'identification de huit genes de C. glauca impliqués dans la voie de biosynthèse des flavonoïdes. L'étude de leur expression dans les racines par PCR quantitative au cours d'une cinétique d'infection de C. glauca par Frankia a montré que les transcrits de la chalcone isomerase et de l'isoflavone reductase s'accumulaient très tôt après l'inoculation, suggérant ainsi une implication des isoflavonoïdes dans la symbiose actinorhizienne. Nous avons alors utilisé une stratégie d'ARN interférent pour réduire l'expression du gène de la chalcone synthase, la première enzyme de la voie de biosynthèse des flavonoïdes. La réduction de l'expression du gène de la chalcone synthase a provoqué une réduction significative du taux de flavonoïdes dans les racines ainsi qu'une très forte diminution du taux de nodulation chez les plantes transformées. Une restauration du taux de nodulation a pu être obtenu en présence de naringenin, une molécule centrale de la voie de biosynthèse des flavonoïdes.Nos résultats apportent donc, pour la première fois, une évidence directe de l'implication forte des flavonoïdes au cours de la nodulation des plantes actinorhiziennes. / Nitrogen-fixing root nodulation, confined to four plant orders, encompasses more than 14,000 Leguminosae species, and approximately 200 actinorhizal species forming symbioses with rhizobia and Frankia bacterial species, respectively. Most actinorhizal plants are capable of high rates of nitrogen fixation comparable to the nitrogen fixing symbiosis between legumes and Rhizobium. As a consequence, these plants are able to grow in poor and disturbed soils and are important elements in plant community worldwide. The basic knowledge of the symbiotic interaction between Frankia and actinorhizal plants is still poorly understood, although it offers striking differences with the Rhizobium-legume symbiosis. In the symbiosis between legumes and Rhizobium, flavonoids are key molecules for nodulation. In actinorhizal plants, the involvement of flavonoids in symbiosis is poorly understood, but because of the similarities of the infection process between some actinorhizal plants and legumes, flavonoids were proposed to act as plant signals for the bacteria Frankia. The objective of this thesis was to investigate the involvement of flavonoids during the actinorhizal nodulation process resulting from the interaction between the tropical tree Casuarina glauca and the actinomycete Frankia.Eight C. glauca genes involved in flavonoid biosynthesis were identified from a unigene database and their expression patterns were monitored by quantitative real-time PCR during the nodulation time course. Our results showed that chalcone isomerase and isoflavone reductase transcripts accumulated preferentially early after inoculation with Frankia, suggesting thus for the first time that isoflavonoids are implicated in actinorhizal nodulation. To go deeper in the understanding of the role of these molecules in actinorhizal symbiosis, we used RNA interference strategy to silence chalcone synthase, the enzyme that catalyzes the first committed step of the flavonoid pathway. Knockdown of chalcone synthase expression led to a strong reduction of specific flavonoids levels and resulted in a severely impaired nodulation. Nodule formation could be rescued by supplementation of plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of a strong implication of flavonoids during actinorhizal nodulation.

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.