La déficience des sols en éléments minéraux, particulièrement le phosphore (P) est une limitation majeure pour la croissance et le développement des légumineuses fixatrices d’azote. L’application des fertilisants phosphatés est une pratique traditionnelles pour satisfaire les besoins des plantes en P. Ainsi, pour tester l’efficacité d'utilisation du P pour la fixation symbiotique de l’azote (FSN) sous déficience en P, plusieurs lignées recombinantes (RILs) de haricot contrastantes dans leurs tolérance au déficit en P ont été utilisées. L’objectif principal de cette étude est d’évaluer l’aptitude de ces RILs pour l’amélioration de la fertilité phosphatée des sols déficients en P et sa relation avec la croissance et la nodulation de la légumineuse. Pour atteindre ces objectifs, des expériences ont été réalisées sous serre et en champs d'agriculteurs. Sous conditions contrôlées (serre) et non contrôlées (champs), les résultats ont montré une diminution du pH des sols associée à une augmentation du P assimilable. Une telle augmentation de la disponibilité du P a eu un effet positif sur la nodulation et la croissance de certaines des RILs testées. Aussi, l’amélioration de l’aptitude de ces RILs à fixer l’azote atmosphérique et l’élévation de la libération des protons H+ par les racines nodulées ont été quantifiés en milieu contrôlé. Ainsi la diminution du pH du sol a permis de réduire l’indisponibilité du P dans la solution du sol en le transformant en une forme directement biodisponible pour ces plantes. En outre, les résultats ont montré des différences significatives entre les différentes RILs en termes de la biomasse aérienne et nodulaire selon les sites d'observation. Nous concluons que, outre leur aptitude de fixation d’azote, l’utilisation efficace du phosphore pour sauver des engrais minéraux et de réduire les risques de pollution et pourrait améliorer la disponibilité des sols P. / The deficiency of soil minerals, particularly phosphorus (P) is a major limitation for growth and development of nitrogen-fixing by legumes. The application of phosphate fertilizers is a traditional practice to meet the needs of plant P. Thus, to test the effectiveness of use of P for symbiotic nitrogen fixation (NSF) under P deficiency, several recombinant inbred lines (RILs) of beans in their contrasting tolerance to P deficiency have been used. The main objective of this study is to assess the ability of these RILs to improve the fertility of soils deficient in phosphorus (P) and its relationship with growth and nodulation of the legume. To achieve these objectives, some experiments were conducted in greenhouses and fields. Under controlled conditions (greenhouse) and uncontrolled conditions (field), the results showed decreasing in soil pH associated with Proton release H+ and P acquisition. The increasing in available P had a positive effect on nodulation and growth of some RILs tested. Also, improving the ability of these RILs to fix atmospheric nitrogen and release H+ by nodulated roots were quantified in a controlled environment. Thus the decreasing in soil pH has reduced the un-availability of P in soil solution by transforming it into a bio-available form to the plants. In addition, the results showed significant differences between RILs in biomass and nodulation as observation in sites. We conclude that, in addition to their ability to fix nitrogen, effective use of phosphorus to save mineral fertilizer and reduce the pollution risks and could improve the availability of soil P.

Beijerinckia sp., bactéria de vida livre fixadora de nitrogênio, comumente encontrada em solos tropicais lateríticos. A cianoficina produzida por cianobactérias é a única reserva nitrogenada intracelular descrita até hoje. O presente trabalho teve como principal objetivo verificar o acúmulo de material nitrogenado intracelular associado à Fixação Biológica de Nitrogênio em cinco isolados de Beijerinckia da rizosfera de cana-de-açúcar (Saccharum sp.). Os resultados mostraram um aumento na concentração de proteína celular total concomitantemente a atividade da nitrogenase durante a fase estacionária de todos os isolados. A fixação de nitrogênio durante esta fase sugere que o destino do nitrogênio fixado seriam os grânulos de armazenamento. A análise química desta reserva confirmou a presença de arginina em teor muito elevado em relação aos demais aminoácidos sugerindo uma reserva nitrogenada diferente da cianoficina. Em recombinantes de Escherichia coli confirmou-se um possível gene envolvido no armazenamento de material nitrogenado em Beijerinckia sp. / Beijerinckia sp. bacteria free-living nitrogen-fixing, commonly found in tropical lateritic soils. The cyanophycin produced by cyanobacteria is the only intracellular nitrogen reserve described to date. This study aimed to verify the intracellular buildup of nitrogen associated with Biological Nitrogen Fixation in five Beijerinckia isolated from the rhizosphere of sugarcane (Saccharum sp.). The results showed an increase in total cellular protein concentration concomitantly nitrogenase activity during the stationary phase of all isolates. The nitrogen fixation during this phase suggests that the fate of fixed nitrogen would be the storage granules. Chemical analysis of the reserve confirmed the presence of very high content of arginine in relation to other amino acids suggesting a different reserve of cyanophycin. In recombinant Escherichia coli confirmed a possible gene involved in nitrogen storage material in Beijerinckia sp.

La respiration anaérobie peut être un trait essentiel dans le mode de vie, la colonisation de l'environnement et la survie. Jusqu'à présent, la seule respiration anaérobie confirmée chez Agrobacterium spp. est la dénitrification. De façon intéressante, cette voie est inégalement répandue chez les agrobactéries. Ces observations m'ont amené à mon hypothèse, à savoir la respiration anaérobie et notamment la dénitrification pourraient expliquer la coexistence d'agrobactéries et leur distribution dans des niches spécifiques de la rhizosphère. Ma thèse visait à explorer les stratégies de respiration anaérobie d’Agrobacterium spp. et de les relier à l'adaptation de niche écologiques différentes. Les objectifs de ma thèse étaient (1) de caractériser tous les gènes impliqués dans la dénitrification chez A. fabrum C58 in vitro, (2) d'explorer les gènes de la dénitrification nécessaires à la colonisation des racines du maïs et (3) de découvrir de nouvelles respirations anaérobies pendant la colonisation racinaire du maïs (Figure 16). Réaliser des mutants et les tester dans des conditions particulières est le moyen classique de déterminer l'implication d'un gène dans une voie spécifique. Cependant, cette méthode implique une vision à priori et des connaissances solides sur les gènes cibles et ne peut pas être appliquée à toutes les situations. Nous avons alors dû développer une méthode plus adaptée pour identifier les gènes essentiels impliqués dans la croissance dans des conditions anaérobies spécifiques. - Gènes de dénitrification chez A. fabrum C58 in vitro. Pour compléter la voie de dénitrification chez A. fabrum C58 et identifier tous les gènes et régulateurs impliqués dans la dénitrification, nous avons adopté deux stratégies : Premièrement, une vision à priori pour (1) identifier la nitrate réductase impliquée dans la première étape de la dénitrification et (2) valider le rôle d'un ARN non codant dans le contrôle de la dénitrification. Pour ce faire, nous avons construit un mutant napA de A. fabrum C58 et un mutant de l'ARN non codant NopR et nous avons évalué leur croissance et leur capacité à produire du N2O dans des conditions anoxiques. Deuxièmement, pour identifier tous les gènes impliqués dans la dénitrification, nous avons construit une banque de transposons mutants de C58 et testé sa croissance dans des conditions de dénitrification in vitro en présence de nitrate ou de nitrite. - Rôle des gènes de la denitrification de A. fabrum C58 dans la colonisation racinaire du maïs. Il est bien connu que le séquençage de transposons (Tn-Seq) est une méthode très puissante pour déterminer les gènes nécessaires à la croissance bactérienne en présence de leur hôte. Pour déterminer les gènes de dénitrification impliqués dans la colonisation des racines en anoxie, nous avons utilisé la banque construite chez C58 et l’avons inoculée sur les plants de maï cultivées sur un sol fertile et cultivées dans des conditions inondées mimant des conditions anaérobies. Le séquençage des cellules d ‘A. fabrum C58 récupérées mettra en évidence les gènes impliqués dans la colonisation anaérobie de cette niche spécifique. - Découverte de nouvelles voies de respiration anaérobie chez A. fabrum C58. Pour découvrir de nouvelles voies de respiration anaérobie, nous avons mis en place des tests de croissance de C58 dans des conditions anoxiques en présence de sources de C et de N en tant qu'accepteurs terminaux d'électrons. De façon interéssante, en cultivant des souches WT et mutée dans le gène napA au contact de la racine de maïs dans des conditions anoxiques (chapitre 1), nous avons montré une croissance des deux souches. Ce résultats suggère que les exsudats de racine servent d'accepteurs d'électrons terminaux pour la croissance anaérobie de C58. Pour déterminer quels composés exsudés du maïs peuvent servir de TEA, les principaux métabolites ont été identifiés par HPLC et certains ont été testés en tant que TEA dans des conditions anoxiques / Anaerobic respiration may be an essential trait in lifestyle, environment colonization and survival. Until now, the only confirmed anaerobic respiration in Agrobacterium spp. is denitrification. Interestingly, this pathway is unequally widespread among Agrobacteria. These observations led me to my hypothesis which is anaerobic respiration and notably denitrification could explain the coexistence of Agrobacteria and their distribution in specific niches in the rhizosphere. My thesis was undertaken to explore the anaerobic respiration strategies of Agrobacterium spp. and to relate them to niche adaptation. The objectives of my thesis were to (1) characterize all the genes involved in denitrification in A. fabrum C58 in vitro, (2) explore the genes of denitrification that are needed during maize root colonization and (3) discover new anaerobic respirations that occur during maize root colonization (Figure 16). Mutational analysis is the classic way to determine the involvement of a gene in specific pathway. However, this method implies an a priori view and solid knowledge on target genes and cannot be applied for every situation. We have to develop a more adapted method to identify essential genes involved in growth in specific anaerobic conditions. - Denitrification genes in A. fabrum C58 in vitro. To complete denitrification pathway in A. fabrum C58 and identify all the genes and regulators involved in the denitrification function, we adopted two strategies: Firstly, an a priori view to (1) identify the nitrate reductase involved in the first step of denitrification and (2) validate the role of a non-coding RNA in denitrification control. To do so, we constructed a mutant of napA of A. fabrum C58 and a mutant of the non-coding RNA NopR and we evaluated their growth and capacity to produce N2O under anoxic conditions. Secondly, to identify all the genes involved in denitrification, we constructed a mutant transposon library of C58 and tested its growth under denitrification conditions in vitro in the presence of either nitrate or nitrite. - Role of A. fabrum C58 denitrifying genes in the root colonization of maize. It is well known that Transposon-sequencing (Tn-Seq) is a very powerful method to determine genes required for bacterial growth in the presence of their host. To determine denitrifying genes involved in root colonization under anaerobic conditions, we used the library constructed in C58 and performed in planta assays. The mutant library was inoculated on maize plants grown on fertile-ground and cultured under flooded conditions miming anaerobic conditions. Sequencing the recovered A. fabrum C58 cells will evidence the genes involved in this anaerobically specific niche colonization. - Discovery of new anaerobic respiration pathways in A. fabrum C58. To discover new anaerobic respiration pathways, we set-up growth assays of C58 under anoxic conditions in the presence C and N sources as terminal electrons acceptors. Interestingly, by culturing WT and NapA-deficient strains in contact with maize root under anoxic conditions (Chapter 1), we showed growth of both strains, suggesting that root exudates serve as terminal electrons acceptors for anaerobic growth of C58. To determine which maize exuded compounds can serve as TEAs, primary metabolites were identified by HPLC and some were tested as TEAs under the set-up conditions

Orientador: Ian Castro-Gamboa / Resumo: A utilização de produtos naturais pela humanidade, como agentes terapêuticos, é descrita desde primórdios da antiguidade e representam um importante aliado no tratamento de doenças. Com isso a busca por novas ferramentas analíticas mais robustas, com abordagem metabolômica se faz necessária, de forma a otimizar e acelerar a busca por novas moléculas e substâncias com atividade biológica promissora. Nesse contexto, esse trabalho tem como alvo de estudo, os fungos Fusarium solani e Purpureocillium lilacinum isolados da rizosfera de Senna spectabilis. Essas espécies de microrganismos apresentam muitos estudos químicos relatados na literatura, o que influenciou o interesse em realizar os estudos de OSMAC (One Strain Many Compounds). Para, assim, verificar a variação metabólica que acontece com esses fungos em diferentes tipos de cultivo (variando meio de cultura e agitação) incluindo o co-cultivo. Para os extratos obtidos também foram realizados os ensaios biológicos citotóxico e antibacteriano. Foram utilizadas ferramentas analíticas do estado da arte como GC-MS (Cromatografia Gasosa acoplada a Espectrometria de Massas) e LC-MS (Cromatografia Líquida acoplada a Espectrometria de Massas) fazendo uso das ferramentas de bioinformática Molecular Networking (ou Rede Molecular) com a plataforma GNPS (Global Natural Product Social Molecular Networking) e o UNIFI (Sistema de informação científica – Waters Corporation). A espectrometria de massas (MS) foi uma grande aliada que ajudou a d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of natural products by mankind, as therapeutic agents, has been described since ancient times and represents an important ally in the treatment of diseases. Thus, the search for new more robust analytical tools, with a metabolomic approach is necessary, in order to optimize and accelerate the search for new molecules and substances with promising biological activity. In this context, this study is aimed at studying Fusarium solani and Purpureocillium lilacinum fungi isolated from the rhizosphere of Senna spectabilis. These species of microorganisms have many chemical studies reported in the literature, which influenced the interest in carrying out the studies of OSMAC (One Strain Many Compounds). Thus, to verify the metabolic variation that happens with these fungi in different types of cultivation (varying culture medium and agitation) including co-culture. For the extracts obtained, cytotoxic and antibacterial biological tests were also performed. State-of-the-art analytical tools such as GCMS (Gas Chromatography coupled to Mass Spectrometry) and LC-MS (Liquid Chromatography coupled to Mass Spectrometry) were used alongside the bioinformatics tools Molecular Networking with the platform GNPS (Global Natural Product Social Molecular Networking) and UNIFI (Scientific Information System - Waters Corporation). Mass spectrometry (MS) was a great ally that helped to demonstrate the variation of the metabolites profile of these microorganisms in different cultures, in whic... (Complete abstract click electronic access below) / Doutor

No description available.

Plants in constructed wetlands serve as carriers for attached microbial growth. They mainly transfer oxygen and release exudates into the root zone. As a result, an area exists around the roots (rhizosphere) in which bacteria are stimulated by root growth. Our goals were to ascertain whether stimulating the microbial community only has a local effect on the rhizoplane, and to establish the importance of this stimulation for wastewater purification in the root zone. Observations were carried out in a planted and an unplanted laboratory batch reactor incubated with an artificial wastewater with a high concentration of ammonia. Samples were taken at intervals of 10 mm away from the roots. The chemical and physical conditions and enzyme activities in soil sections at various distances from the roots affecting the efficiency of microbial nitrogen removal were characterized. An influence on the nitrification and denitrification process was detected via several parameters up to a range of different root distances: microbial parameters such as the total bacterial number, respiratory activity, protein and DNA amount seem to be affected by roots up to a distance of 50 mm from the roots, whereas the oxygen concentration, DOC and redox potential are unaffected at a distance exceeding 20-30 mm. This indicates that improved nitrogen removal is also possible in the wider root surroundings. Given the average root-to-root distance of 35 mm, the root-influenced area could therefore be expanded to the whole rooted zone in a constructed wetland.

<p dir="ltr">Plant secondary metabolites (PSMs) are specialized compounds produced in response to a range of insect herbivores and microbes, making them important in shaping tri-trophic interactions. However, despite being well-studied in the context of plant-insect coevolution, it is unclear how PSMs impact microbial communities associated with plants and the insect herbivores that feed on them. The overarching goal of this dissertation was to better understand how variation in plant defensive responses, particularly expression of PSMs, influences the composition and functional potential of microbial communities associated with plant tissues (roots and leaves) and insect herbivores. Monarchs (<i>Danaus plexippus</i>) and their milkweed hosts (<i>Asclepias spp.)</i> are well-studied for mechanisms of plant defense and insect counter defense, but little is known about the role of associated microbial communities in this iconic system. To address this knowledge gap, a combination of metabarcoding and metagenomics was used to characterize the taxonomic composition and functional gene profiles of bacterial communities associated with plant tissues (i.e., phyllosphere and rhizosphere) and monarch caterpillars fed on multiple milkweed species (<i>A. curassavica</i>, <i>A. syriaca</i>, and <i>A. tuberosa</i>). Findings show the composition of phyllosphere, rhizosphere, and monarch microbiomes vary across milkweed species in terms of diversity and relative abundance of bacterial taxa. Furthermore, phyllosphere and rhizosphere microbiomes were shown to have distinct functional gene profiles and presence of potential PSM metabolism genes that also varied across milkweed species. Rhizosphere microbiomes had a greater overall capacity for PSM metabolism compared to the phyllosphere, having more genes, and associated metabolic pathways involved in degradation or detoxification of known classes of PSMs. However, plant associated microbiomes were not generally affected by monarch feeding, evidenced by few changes in taxonomic composition or abundance of genes predicted to be involved in PSM metabolism. Interestingly, monarch microbiomes shared >90% of their taxa with their host plants, but there was little evidence of PSM metabolism genes present in functional gene profiles. Overall, this dissertation lays the foundation for understanding how PSMs shape all the microbial communities associated with monarchs and their milkweed hosts. Findings suggest plant defensive responses affect the assembly, functional potential and ultimately the evolution of plant and insect microbiomes.</p>

No description available.

Les champignons de la rhizosphère ont une grande influence sur le développement et la croissance des plantes. Certains de ces micro-organismes protègent les plantes contre les pathogènes, atténuent l'impact des stress abiotiques ou facilitent la nutrition des plantes. Ces organismes s'influencent mutuellement et forment des réseaux complexes d'interactions. Déterminer le fonctionnement du microbiome fongique de la rhizosphère des plantes cultivées est une étape nécessaire pour optimiser l'efficacité de la production végétale. Nous avons testé les hypothèses suivantes : (1) la diversification des systèmes de culture influe sur le microbiome fongique de la rhizosphère du canola; (2) le canola a un core microbiome, soit un ensemble de champignons toujours associés au canola quelles que soit les conditions du milieu; et (3) que certains de ces taxons ont une influence déterminante sur la structure des communautés (taxons nodaux) dans le core microbiome. Pour ce faire, en 2013 et 2016, nous avons échantillonné à la floraison, la phase de canola (Brassica napus) du système de culture, qui est l'un des deux types de canola (Roundup Ready® et Liberty Link®), utilisés dans le cadre d'une expérience de terrain à long terme (6 ans). Lacombe (Alberta), Lethbridge (Alberta) et Scott (Saskatchewan). En utilisant le séquençage d’amplicon par illumina, nous avons obtenus des résultats qui montrent que la diversification des cultures a un impact significatif sur la structure des communautés fongiques de la rhizosphère. Nous avons également découvert et décrit un core microbiome constitué de 47 OTU (Unité Taxonomique Opérationnelle) en 2013 et identifié Preussia funiculata, Schizothecium sp., Mortierella sp., Nectria sp. ainsi que deux taxons inconnus (OTU12 et OTU298) comme taxons nodaux parmi ce core microbiome. Cependant ce core microbiome s’est montré variable, et nous n’avons pu identifier qu’un OTU y appartenant en 2016 : Olpidium Brassicae. Nos résultats permettent de confirmer l’impact de la diversité culturale sur le microbiome fongique du canola et sont présentés comme une base pour le développement de stratégies d'ingénierie écologique pour l'amélioration de la production de canola. / The fungi in the rhizosphere have a large influence on plant development and growth. Some of these micro-organisms protect plants against pathogens, mitigate the impact of abiotic stress, or facilitate plant nutrition. These organisms influence each other and form complex webs of interactions. Deciphering the structure and function of the fungal microbiome of crop plant rhizosphere is a necessary step toward optimizing the efficiency of plant production. We tested the hypotheses that (1) the diversification of cropping systems influences the fungal microbiome of canola rhizosphere, (2) canola has a fungal core microbiome, i.e. a set of fungi that are always associated with canola, and (3) that some taxa have a determining influence on the structure of the communities (hub-taxa) within the core microbiome. In 2013 and 2016 we used the canola (Brassica napus) phase of five cropping system at blooming stage, from the less to the most diversified, that included one of two types of canola (Roundup Ready® and Liberty Link®), in an existing long-term (6 years) field experiment. The experiment has a randomized complete block design with four blocks, and is replicated at three locations: Lacombe (Alberta), Lethbridge (Alberta) and Scott (Saskatchewan). Our results show that crop diversification has significant impact on the structure of rhizosphere fungal communities. We also discover and described a canola core microbiome made of 47 OTUs in 2013 and identified Preussia funiculata, Schizothecium sp., Mortierella sp., Nectria sp. and two other unidentified taxa (OTU12 and OTU298) as the hub-taxa among this core. However this core microbiome was variable and could identify only one member in 2016 : Olpidium brassicae. Our results confirmed the effect of crop diversification upon the fungal microbiome of canola and are presented as a basis for the development of ecological engineering strategies for the improvement of canola production.

Soil degradation is a common occurrence in Haiti that is mainly caused by the cultivation of marginal lands and deforestation, which both contribute to the excessive erosion rate seen in the country today. The Central Plateau of Haiti is a mountainous region in which a majority of the population is rural and practices subsistence agriculture on hillsides and steeply-sloping land. Essential plant nutrients, such as nitrogen (N) and phosphorus (P), are commonly a limiting factor in crop production, yet fertilizer is unavailable or is too expensive for smallholder farmers to purchase. This study was conducted to a) evaluate organic matter and nutrient stocks of various soils in the Central Plateau region, along with other chemical and physical characteristics and b) to evaluate the phosphorus-scavenging ability of commonly-grown crops to isolate those that may benefit subsequent smallholder yields. Soils from four locations in the Central Plateau were assessed for organic matter in labile and non-labile fractions as well as for cation exchange capacity (CEC), total organic carbon (C) and N, pH, texture, and other characteristics. Results indicated that most of the soil (92%) was contained within aggregates, and organic matter was mainly present in stable, slowly-decomposing fractions. Seven species were evaluated in a controlled-environment pot experiment for bulk and rhizosphere soil P and pH, plant dry weight, and above- and below-ground P tissue content as indicators of the species' ability to solubilize P from the soil. Velvet bean (Mucuna pruriens (L.) DC) produced the most biomass and was able to take up the most P, though lablab (Lablab purpureous (L.) Sweet), took up comparable amounts of P. / Master of Science / LTRA-6 (A CAPS program for the Central Plateau of Haiti)