Les systèmes à deux composants jouent un rôle prépondérant dans l'adaptation des bactéries à leur environnement. L'objectif de ce travail de thèse était d'identifier et de caractériser des systèmes à deux composants chez la bactérie phytostimulatrice Azospirillum nécessaires à l'adaptation à la rhizosphère de sa plante-hôte. L'analyse de la distribution génomique des gènes appartenant à la famille des systèmes à deux composants dans les génomes d'Azospirillum disponibles a révélé l'existence d'un grand nombre de gènes codant des hisitidine kinases hybrides, et une analyse plus approfondie a montré une organisation multidomaines complexe de cette famille de protéines. Afin de comprendre leur rôle chez Azospirillum, nous avons, dans un premier temps, sélectionné et inactivé quatre gènes codant des histidine kinases hybrides présentant une architecture multidomaines complexe. A l'aide d'une approche multidisciplinaire combinant génétique, biochimie et phylogénie, nous avons mis en évidence pour la première fois chez Azospirillum, un système atypique à trois-composants nommé PreSKR contrôlant un grand nombre de processus impliqués dans la survie et la colonisation de la rhizosphère, qui agirait en modulant le taux intracellulaire de c-di-GMP. Dans un second temps, nous nous sommes focalisés sur une histidine kinase hybride exprimée au contact de la plante hôte ; cette protéine, appelée RsiK, s'avère être impliquée dans la perception de surfaces et la régulation de la formation de biofilms. L'analyse du régulon par RNA-seq a révèlé que 78 gènes étaient contrôlés par ce système. La prévalence de la famille des histidine kinases hybrides chez Azospirillum couplée à l'approche fonctionnelle réalisée sur deux d'entre elle souligne l'importance des phosphorelais encore largement méconnus chez les bactéries rhizosphériques / Bacterial two-component systems play an important role in the ability of bacteria to adapt to various environments. The aim of this thesis was to identify and characterize two-component systems involved in the adaptation of the phytostimulatory bacteria Azospirillum to its host plant. Analysis of the genomic distribution of genes encoding two-component systems across Azospirillum available genomes revealed the existence of a high number of genes encoding hybrid histidine kinases, and further analyses highlighted a complex multi-domain organization of this family of proteins. In order to understand their role in Azospirillum, as a first step we selected and inactivated four genes encoding complex hybrid histidines kinases. Using a multidisciplinary approach which combines genetics, biochemistry and phylogeny, we brought to light for the first time in Azospirillum, an atypical three-component system named PreSKR which controls a wide variety of processes involved in survival and rhizosphere colonization likely by modulating c-di-GMP levels. As a second step, we focused on a gene encoding a hybrid histidine kinase named RsiK which is induced in contact with its host plant. RsiK is involved in surface sensing and biofilm formation regulation. Transcriptomic analysis of rsiK regulon by RNA-seq showed that 78 genes were under the control of this system. The prevalence of genes encoding hybrid histidine kinase family in Azospirillum, coupled with the functional characterization of two of them, highlight the importance of phosphorelays, still largely unrecognized in rhizospheric bacteria

Orientador: Sandra Cristina de Oliveira / Resumo: Está dissertação teve como objetivo principal, averiguar os efeitos da inoculação de sementes de amendoim, com as bactérias Bradyrhizobium sp. e Azospirillum brasilense de forma isolada e em conjunto (coinoculação). Para tanto foram realizadas analises bioquímicas das folhas, afim de analisar os teores de compostos fotossintetizantes, açucares solúveis totais, ureídeos, feofitinas totais e compostos nitrogenados e também foi foram feitas avaliações referentes a parâmetros fitotécnicos. Primeiramente foi realizada uma revisão bibliográfica por meio de levantamentos de dados sobre o panorama da produção de amendoim a nível mundial, nacional e algumas cidades localizadas no Oeste paulista, região a qual é destaque na produção de amendoim. Além do levantamento bibliográfico, foi realizado um experimento à campo e em laboratório, a fim de investigar os efeitos da inoculação e coinoculação em sementes de amendoim com bactérias promotoras do crescimento de plantas. Na parte experimental utilizou-se o delineamento experimental de blocos ao acaso com quatro repetições e sete tratamentos, sendo: Controle (sem inoculação); Az 2 mL kg-¹ (Azospirillum brasilense na dose de 2 mL para 1 kg de sementes); Az 4 mL kg-¹ (Azospirillum brasilense na dose de 4 mL para 1 kg de sementes); Bd 2 mL kg-¹ (Bradyrhizobium sp. na dose de 2 mL para 1 kg de sementes); Bd 4 mL kg-¹ (Bradyrhizobium sp. na dose de 4 mL kg-¹ para 1 kg de sementes); Az + Bd 2 mL kg-¹ (Azospirillum brasilense + Bradyrhizobium sp.... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by Tatiana da Silva Santos (ta.tianasantos@hotmail.com.br) on 2019-01-29T23:13:44Z No. of bitstreams: 1 Dissertaçao SANTOS Tatiana.pdf: 1460829 bytes, checksum: 860ca0a833bcae646664af005f5e7c5b (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2019-01-30T10:20:56Z (GMT) No. of bitstreams: 1 santos_ts_me_ilha.pdf: 1460829 bytes, checksum: 860ca0a833bcae646664af005f5e7c5b (MD5) / Made available in DSpace on 2019-01-30T10:20:56Z (GMT). No. of bitstreams: 1 santos_ts_me_ilha.pdf: 1460829 bytes, checksum: 860ca0a833bcae646664af005f5e7c5b (MD5) Previous issue date: 2018-10-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O manejo eficiente do solo pode provocar mudanças benéficas na qualidade física, química e microbiológica do solo refletindo em alta produtividade de grãos de milho. O nitrogênio (N) é o nutriente mais exigido pela planta de milho. A aquisição de fertilizantes nitrogenados pode ser responsável por 50% dos custos variáveis da lavoura. Dessa forma, a utilização de bactérias diazotróficas é considerado uma fonte alternativa economicamente viável e sustentável para aquisição desse nutriente por meio da fixação biológica de N por esses microrganismos. Em vista do exposto, o objetivo deste trabalho foi verificar o desenvolvimento, componente produtivo e produtividade de grãos de milho sob diferentes manejos de solo e inoculado via foliar por Azospirillum brasilense e/ou Bacillus amyloliquefaciens. O experimento foi conduzido durante o ano agrícola de 2016/17 em um Latossolo Vermelho distroférrico localizado na Fazenda de Ensino, Pesquisa e Extensão (FEPE) – Setor de Produção Vegetal pertencente à Faculdade de Engenharia de Ilha Solteira, município de Selvíria - MS. O delineamento experimental utilizado foi o de blocos casualizados, esquema de faixas. Foram estabelecidos 12 tratamentos com 4 repetições. Os tratamentos foram constituídos por 3 tipos de sistemas de manejo do solo: Sistema convencional (SC), cultivo mínimo (CM) e sistema plantio direto (SPD). Os tratamentos de inoculação via foliar de bactérias diazotróficas consistiram em: Azospirillum brasilense (AB), Bacillus amyloliquefaciens (BA), Azospirillum brasilense + Bacillus amyloliquefaciens (AB + BA) e sem inoculação (SI). Não houve efeito significativo da aplicação de bactérias sobre a altura de planta, diâmetro do colmo, diâmetro de espiga e comprimento de espiga. Houve interação para a altura de inserção da primeira espiga e produtividade de grãos. A inoculação com Bacillus amyloliquefaciens promoveu acréscimos à altura de inserção da primeira espiga quando utilizado o cultivo mínimo (1,31 m). A inoculação com Azospirillum brasilense e Bacillus amyloliquefaciens proporcionou aumento da produtividade de grãos no preparo convencional. / Efficient soil management can lead to beneficial changes in the physical, chemical and microbiological quality of the soil, reflecting high yields of corn grains. Nitrogen (N) is the nutrient most required by the corn plant. The acquisition of nitrogen fertilizers can account for 50% of the variable costs of the crop. Thus, the use of diazotrophic bacteria is considered an economically viable and sustainable alternative source for the acquisition of this nutrient through the biological fixation of N by these microorganisms. In view of the above, the objective of this research was to verify the development, productive component and productivity of corn grains under different soil management and foliar inoculation by Azospirillum brasilense and / or Bacillus amyloliquefaciens. The experiment was conducted during the agricultural year of 2016/17 in a Distroferric Red Oxisol located at the Teaching, Research and Extension Farm (FEPE) - Vegetable Production Sector belonging to the Faculty of Engineering of Ilha Solteira, Selvíria - MS. The experimental design was a randomized complete block design. Twelve treatments with four replicates were established. The treatments were constituted by 3 types of soil management systems: Tillage (SC), minimum tillage (CM) and notillage (SPD). The treatments of foliar inoculation of diazotrophic bacteria consisted of: Azospirillum brasilense (AB), Bacillus amyloliquefaciens (BA), Azospirillum brasilense + Bacillus amyloliquefaciens (AB + BA) and No Inoculation (SI). There was no significant effect of the application of bacteria on plant height, stalk diameter, ear diameter and ear length. There was interaction for the first ear insertion height and grain yield. The inoculation with Bacillus amyloliquefaciens promoted increases at the insertion height of the first ear when using the minimum culture (1,31 m). Inoculation with Azospirillum brasilense and Bacillus amyloliquefaciens provided an increase in grain yield in conventional tillage. / CAPES: Código de Financiamento 001

Orientador: Ricardo Antonio Ferreira Rodrigues / Banca: Orivaldo Arf / Banca: Gisele Herbst Vazquez / Resumo: A produtividade média de feijão no Brasil é baixa. Alternativas que possam aumentar a produtividade de forma sustentável devem ser preconizadas, tais como a redução do uso de fertilizantes nitrogenados pela adoção da fixação biológica de nitrogênio. Neste cenário, uma técnica alternativa de co-inoculação ou também denominada de inoculação mista com bactérias simbióticas e assimbióticas tem sido estudada em leguminosas, no entanto existem poucos estudos em campo com feijoeiro e sobre a influência da quantidade de água irrigada. Dessa forma, o objetivo deste trabalho foi avaliar a eficiência da inoculação e co-inoculação de feijão com Rhizobium tropici e Azospirillum brasilense sob duas lâminas de irrigação na produção e qualidade fisiológica de sementes. O experimento foi desenvolvido no período de outono/inverno dos anos de 2012 e 2013, no município de Selvíria-MS. O delineamento experimental foi de blocos casualizados em esquema fatorial 5x2, com quatro repetições. Os tratamentos foram constituídos pela combinação de cinco formas de fornecimento de nitrogênio (testemunha sem inoculação com 40 kg ha-1 de nitrogênio em cobertura; testemunha sem inoculação com 80 kg ha-1 de N em cobertura; inoculação de A. brasilense com 40 kg ha-1 de N em cobertura; inoculação de R. tropici com 40 kg ha-1 de N em cobertura; e co-inoculação de A. brasilense e R. tropici com 40 kg ha-1 de N em cobertura) e duas lâminas irrigação (recomendada e 75% da recomendada). A aplicação de nitrogênio em cobertura foi realizada utilizando a ureia. Concluiu-se que a co-inoculação, a inoculação de R. tropici, a inoculação de A. brasilense associadas a 40 kg ha-1 de nitrogênio em cobertura e a testemunha sem inoculação com aplicação de 80 kg ha-1 de N em cobertura não proporcionam acréscimos de produtividade em relação a testemunha não inoculada com 40 kg ha-1 ... / Abstract: The average yield of beans in Brazil is low. Alternatives that can increase yield in a sustainable manner should be preconized, such as reducing the use of nitrogen fertilizers through the adoption of biological nitrogen fixation. In this scenario, an alternative technique of co-inoculation or also called the mixed inoculation with symbiotic and non-symbiotic bacteria has been studied in leguminous, however there are few studies in field with common bean under the influence of the amount of water irrigated. Thus the objetive of this study was to evaluate the efficiency of inoculation and co-inoculation of common bean with Rhizobium tropici and Azospirillum brasilense under two irrigation depths on production and physiological seed quality. The experiment was performed in the fall/winter of the years 2012 and 2013, in Selviria-MS. A randomized block design in a 5x2 factorial scheme with four repetitions was used. The treatments were constituted by the combination of five forms of nitrogen supply (control non-inoculated with 40 kg ha- 1 of nitrogen in topdressing; control non-inoculated with 80 kg ha- 1 of N in topdressing; A. brasilense inoculation with 40 kg ha-1 of N in topdressing; R. tropici inoculation with 40 kg ha-1 of N in topdressing; and co-inoculation of A. brasilense and R. tropici with 40 kg ha- 1 of N in topdressing) and two irrigation depths (recommended and 75% of the recommended). Was concluded that the co-inoculation, the inoculation of R. tropici, the inoculation of A. brasilense associated with 40 kg ha-1 of nitrogen in topdressing and the control non-inoculation with application of 80 kg ha-1 of N topdressing not increase of grain yield compared to control non-inoculation with 40 kg ha-1 of nitrogen in topdressing. Considering the development conditions of the experiment, the use of 75% of the recommended irrigation depth provides similar or greater grain yield than the ... / Mestre

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-03-23T14:47:27Z No. of bitstreams: 1 2016 - Jo?o Paulo de Lima Aguilar.pdf: 2057822 bytes, checksum: e5eac0160e0ba66e10d0af7b9cf5437f (MD5) / Made available in DSpace on 2017-03-23T14:47:27Z (GMT). No. of bitstreams: 1 2016 - Jo?o Paulo de Lima Aguilar.pdf: 2057822 bytes, checksum: e5eac0160e0ba66e10d0af7b9cf5437f (MD5) Previous issue date: 2016-02-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / This work was carried out in Area Horticulture, Crop Science Department of Agronomy Institute of the Universidade Federal Rural do Rio de Janeiro. It was divided in to two stages, on initially in germination greenhouses and the second stage was performed in a protected environment. Statice seeds were inoculated with freeze-dried peat inoculant Azospirillum Sp245 (2,8x109 ufc g-1) as treatment concentration (Az), and compared to the control seeds, with out Azospirillum. In the first step, were seeded 18 polypropylene trays with 50 cells with a volume of 17ml each, with the substrate BIOMIX? seedlings. The following parameters were analyzed: percentage of germination (%Germ), root volume (RV), total length (CTT.), length of aerial parts (CPA), total of fresh material (MFT), total of dry material (MST), and length of roots (CR.).This step was planned in a completely randomized design. In the second stage, seeds were inoculated and sown in a protected environment, at the same time of the beginning of the first stage. They were divided into two plots of 20 m long with six blocks in each bed. Each treatment contained 21 plants. In the interval of 21 days, the plants were fertilized with ?torta de mamona? and ?Bokashi?, alternately with an average of 15 days between the applications. The parameters selected for evaluation were as follows: number of leaves(NF), root length (CR), shoot length (CPA), fresh matter of roots(MFR.), dry matter of root(MSR), root length(CR), root volume (RV), fresh matter of the aerial parts (MFPA), dry matter or aerial parts (MSPA), and rate between aerial part and roots(MSPA/MSR). The experiment began on August 15th2015andfinishedon November 28th2015. The first step lasted 42 days and the second step 63days, in a total of 105days. In the first step, the use of Azospirillum has influenced on all parameters. In the second step, the parameters: number of leaves, shoot/root ratio, fresh weigh to roots, dry wheight and root, and root volume were positively influenced by the treatment. We can cite three possible causes to explain why the other analyzes were not significant. The lack of water in certain periods; high temperature or heat stroke in plant closure period may have aborted the flower stalks; and competition with existing weeds in place, especially Cyperaceaae. The use Azospirillum Sp245 as inoculants proved to be avaliable alternative in the cultivation of statice (Limonium sinuatum) as a cut flower / Este trabalho foi realizado na ?rea de Horticultura, do departamento de Fitotecnia do Instituto de Agronomia da Universidade Federal Rural do Rio de Janeiro. Foi dividido em duas etapas, uma inicial em estufas de germina??o e a segunda em ambiente protegido. Sementes de statice foram inoculadas com o inoculante turfoso contendo Azospirillum Sp245 (2,8x109 ufc g-1) como tratamento (Az) e comparadas com sementes n?o tratadas (inoculadas). Na primeira etapa de avali??o, foram semeadas 18 bandejas de polipropileno, com 50 c?lulas com volume de 17 ml, preenchidas com o substrato BIOMIX? mudas, sendo analisados os seguintes par?metros: percentual de germina??o (%Germ), volume de raiz (VR), comprimento total (CTT), comprimento da parte a?rea (CPA), mat?ria fresca total (MFT), mat?ria seca total (MST) e comprimento da raiz (CR). O delineamento experimental foi inteiramente casualizado. Na segunda etapa, sementes que foram inoculadas e semeadas na mesma ?poca do in?cio da primeira etapa, foram plantadas numa ?rea em ambiente protegido, divididos em dois canteiros com 20 m de comprimento e com seis blocos em cada canteiro. Cada tratamento continha 21 plantas. No intervalo de 21 dias, as plantas foram fertilizadas com torta de mamona e bokashi, alternadamente com uma m?dia de 15 dias entre as aplica??es. Os par?metros que foram selecionados para serem avaliados foram os seguintes:n?mero de folhas (NF), rela??o entre a parte a?rea e as ra?zes (RPA/RZ), comprimento da raiz (CR), comprimento parte a?rea (CPA), massa fresca da raiz (MFR), massa seca da raiz (MSR), comprimento da raiz (CR), volume da raiz (VR), massa fresca da parte a?rea (MFPA), massa seca da parte a?rea (MSPA), in?cio da produ??o das flores (IF), peso das hastes florais (PHF), n?mero de espiguetas (NE) e n?mero de flores por espigueta (NFE). O experimento teve in?cioem 15/08/2015 e foi finalizado em 28/11/2015. A primeira etapa foi avaliada durante 42 dias e a segunda etapa durante 63 dias, totalizando 105 dias. Na primeira etapa, o inoculante ? base de Azospirillum teve influ?ncia em todos os par?metros analisados. Na segunda etapa, os par?metros n?mero de folhas, rela??o parte a?rea/peso de raiz, massa fresca da raiz, massa seca da raiz e volume de raiz foram influenciados positivamente pelo tratamento.Podemos citar tr?s poss?veis causas para n?o haver signific?ncia nas outras an?lises: falta de ?gua em determinados per?odos; alta temperatura/insola??o no per?odo de fechamento das plantas, o que pode ter abortado as hastes florais; e a competi??o com as plantas espont?neas existentes no local, principalmente ciper?ceas. O uso do inoculante com a estirpe Sp245 de Azospirillum demostrou ser uma alternativa vi?vel no cultivo de statice (Limonium sinuatum) como flor de corte.

Orientador: Antonio Carlos Monteiro / Banca: Ernani Pinto Júnior / Banca: Eliana Gertrudes Macedo Lemos / Banca: Ana Teresa Lombardi / Banca: Jackson Antonio Marcondes de Souza / Resumo: A microalga Haematococcus pluvialis é conhecida como o microrganismo que apresenta maior rendimento final de astaxantina em sua biomassa, quando submetida a fatores de estresse celular eficientes. Contudo ainda apresenta uma característica de crescimento desfavorável quando comparada a outras espécies de microalgas cultivadas em escala comercial, visto que possui uma taxa de crescimento intrínseca, resultando em baixa densidade celular ao final dos cultivos. Assim, o rendimento final do pigmento é relativamente baixo. Neste sentido, este estudo teve por objetivo avaliar a resposta de Haematococcus pluvialis ao processo de indução à carotenogênese por meio de estresse luminoso e nutricional, e promover o aumento de biomassa vegetativa da microalga através da adição da rizobactéria promotora de crescimento em plantas, Azospirillum brasilense V6, estirpe produtora do fito hormônio AIA (ácido indol - acético), em cultivos estanques, visando obter maior rendimento final de astaxantina. O trabalho foi dividido em duas fases. Primeiramente, no cultivo referente ao controle (contendo apenas H. pluvialis) foram aplicadas duas combinações de fatores de estresse celular para indução a síntese de astaxantina pela microalga, visando a escolha do método de estresse a ser aplicado na fase seguinte. As células de H. pluvialis foram submetidas ao estresse por alta intensidade luminosa (350 μmol photons m-2 s-1) ou ao mesmo estresse luminoso aliado a suplementação com CO2 (4%) na aeração fornecida na fase exponencial de crescimento. Avaliou-se, entre outros fatores de resposta celular, a concentração final de astaxantina na biomassa após 10 dias de estresse. As concentrações do pigmento foram 4,15 e 17,66 mg g-1, respectivamente, para as culturas submetidas ao estresse luminoso e para... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The microalgae Haematococcus pluvialis is known as the microorganism that has a highest final yield of astaxanthin in its biomass, when induced by efficient stress factors. However, still presents a characteristic of unfavorable growth when compared to other species of microalgae grown on a commercial scale, once it has an intrinsic growth rate that provides low cell density in the culture. Thus, the final yield of the pigment is relatively low. Thus, this study aimed to evaluate the response of Haematococcus pluvialis submitted to carotenogenesis induction process through light and nutrient stress, and promotion of vegetative microalgae biomass increase through the addition of the growth promoter bacteria in plants, Azospirillum brasilense V6, a strain that produce the phyto hormone IAA (indole - acetic acid). The experiments were performed in "batch" systems, aiming to obtain higher final yield of astaxanthin. The work was divided into two phases. First, to the control (containing only H. pluvialis) it was applied two combinations of stress factors to induce cellular synthesis of astaxanthin by the microalgae, to define the method of stress to be applied in the next phase. The cells of H. pluvialis were submitted to stress by high light intensity (seven times the original) or the same light stress combined with CO2 supplementation (4%) in the aeration provided in the exponential growth phase. We evaluated, among other factors of cellular response, the final concentration of astaxanthin in the biomass after 10 days of stress. The pigment concentrations were 4.15 and 17.66 mg g-1, respectively, for cultures submitted to light stress and to those who received the same light stress add with the increase of C / N ratio in the culture medium. So, for the next experimental phase, the combination... (Complete abstract click electronic access below) / Doutor

Le but de ce travail était d'identifier les fonctions régulées par quorum-sensing (QS) chez la bactérie phytostimulatrice Azospirillum. Les effets phytobénéfiques in vitro des souches B518 et TVV3 (isolées du riz) ne sont pas altérées par l'inactivation des molécules signal impliquées dans le QS. La combinaison d'une approche ciblée et d'une approche globale par protéomique montre que le QS régule des fonctions liées à l'adaptation à la plante, notamment à la colonisation racinaire chez B518. Chez TVV3, aucune fonction régulée par QS n'a pu être identifiée mais les gènes impliqués dans le QS sont localisés dans un environnement atypique, constitué de gènes prophagiques. La mise en évidence d'un prophage chez TVV3 a conduit à la caractérisation de phages tempérés chez dix autres souches et au séquençage du premier génome d'un bactériophage isolé d'Azospirillum. Ce travail montre que la régulation de type QS est souche spécifique et révèle la prévalence des phages chez Azospirillum.

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

Previous studies have shown that chemotaxis plays an important role in the colonization of the wheat roots surfaces by Azospirillum brasilense and a chemotaxis operon shown to control motility and chemotaxis in A. brasilense has been isolated. This study looked at the effects of mutations in individual genes coding for chemotaxis proteins from this operon on the ability of the cells to colonize the surface of sterile wheat roots. Using both quantitative and qualitative assays, the study shows differences in the colonization ability of the mutants relative to the wild type: the cheB, cheR, cheBR, and cheOp mutants were significantly impaired in wheat root colonization. Interestingly, the cheA mutant was not affected in its ability to colonize the wheat root surface relative to the wild type. Future studies will look for the factors that compensate for cheA impairment in the rhizosphere.

Nitrogen (N) is an essential plant nutrient, widely applied as N-fertilizer to improve yields of agriculturally important crops. An alternative to fertilizer use could be the exploitation of plant growth-promoting bacteria, capable of enhancing growth and yield of many plant species. Azospirillum and Gluconacetobacter are root colonizing, free-living, N2-fixing bacteria (diazotrophs) with the potential to transfer fixed N to associated plants. The purpose of this study was to evaluate the agronomic efficiency of two diazotrophs, Azospirillum lipoferum and Gluconacetobacter azotocaptans, inoculated onto wheat. Physiological parameters and yield components were evaluated. The objectives of this study were to: 1) determine the survival of each diazotroph species on wheat seeds over time; 2) determine the survival of A. lipoferum and G. azotocaptans inoculated on wheat seed treated with a fungicide seed treatment, Dividend® XL RTA®; 3) determine if inoculation of wheat with the diazotrophs under controlled conditions causes an increase in dry matter, N2-fixation and N uptake; 4) determine if fertilizer N applied at three levels influences atmospheric N2-fixation by A. lipoferum or G. azotocaptans; 5) determine if inoculation of wheat with A. lipoferum or G. azotocaptans under field conditions causes any increase in dry matter, N2-fixation and N uptake; 6) determine if N-fertilization levels under field conditions influenced N2-fixation by A. lipoferum or G. azotocaptans. In order to meet these objectives lab, growth chamber, and field studies were completed. Laboratory investigations revealed that the decline in recovery of colony forming units (CFU) of G. azotocaptans was not significantly different (P<0.05) for any seed treatment. There was a general decrease in CFU over time regardless of seed treatment. Analysis of the recovered CFU of A. lipoferum over time showed that there was a significant difference (P<0.05) between both the non-sterilized seed and the Dividend® XL RTA® treated seed when compared sterilized seed. Recovery of CFU on sterilized seed declined at a more rapid rate compared to the other two seed treatments. Gluconacetobacter azotocaptans and A. lipoferum were not negatively influenced by the Dividend® XL RTA® seed treatment. Also, both diazotrophs were able to compete with other microorganisms that may have been on the seed coat of unsterilized seeds. Azospirillum lipoferum and G. azotocaptans were able to fix atmospheric N, but, there were no significant (P<0.05) differences between the diazotroph species. Additions of fertilizer N enhanced N2-fixation, in both the growth chamber and field studies. As the amount of fertilizer N increased, so did the %Ndfa and N uptake. In the growth chamber study, inoculated wheat, and fertilized with 12.2 and 24.5 µg N g-1 had the highest %Ndfa of 25.5%, and wheat fertilized with 24 µg N g-1 had the highest N uptake (1.3 g pot-1) at maturity. In the field study, inoculated wheat fertilized with of 80 kg N ha-1 had significantly higher (P<0.05) %Ndfa (10.5%) compared to wheat grown with the other fertilizer levels, which also corresponded to the highest N uptake in wheat plants (47 kg ha-1). The diazotrophs also affected the partitioning of N in the wheat plants differently. Wheat inoculated with A. lipoferum had significantly higher (P<0.05) amounts of N accumulated in heads of plants, and wheat inoculated with G. azotocaptans had significantly higher (P<0.05) amounts of N accumulated in stems of plants. However, this trend was not evident in the field study.