Abstract Data collated from around the world indicate that, for every tonne of shoot dry matter produced by crop legumes, the symbiotic relationship with rhizobia is responsible for fixing, on average on a whole plant basis (shoots and nodulated roots), the equivalent of 30-40 kg of nitrogen (N). Consequently, factors that directly influence legume growth (e.g. water and nutrient availability, disease incidence and pests) tend to be the main determinants of the amounts of N2 fixed. However, practices that either limit the presence of effective rhizobia in the soil (no inoculation, poor inoculant quality), increase soil concentrations of nitrate (excessive tillage, extended fallows, fertilizer N), or enhance competition for soil mineralN (intercropping legumes with cereals) can also be critical. Much of the N2 fixed by the legume is usually removed at harvest in high-protein seed so that the net residual contributions of fixed N to agricultural soils after the harvest of legume grain may be relatively small. Nonetheless, the inclusion of legumes in a cropping sequence generally improves the productivity of following crops. Whilesome of these rotational effects may be associated with improvements in availability ofN in soils, factors unrelated to N also play an important role. Recent results suggest that one such non-N benefit may be due to the impact on soil biology of hydrogen emitted from nodules as a by-product of'N, fixation.

Abstract Data collated from around the world indicate that, for every tonne of shoot dry matter produced by crop legumes, the symbiotic relationship with rhizobia is responsible for fixing, on average on a whole plant basis (shoots and nodulated roots), the equivalent of 30-40 kg of nitrogen (N). Consequently, factors that directly influence legume growth (e.g. water and nutrient availability, disease incidence and pests) tend to be the main determinants of the amounts of N2 fixed. However, practices that either limit the presence of effective rhizobia in the soil (no inoculation, poor inoculant quality), increase soil concentrations of nitrate (excessive tillage, extended fallows, fertilizer N), or enhance competition for soil mineralN (intercropping legumes with cereals) can also be critical. Much of the N2 fixed by the legume is usually removed at harvest in high-protein seed so that the net residual contributions of fixed N to agricultural soils after the harvest of legume grain may be relatively small. Nonetheless, the inclusion of legumes in a cropping sequence generally improves the productivity of following crops. Whilesome of these rotational effects may be associated with improvements in availability ofN in soils, factors unrelated to N also play an important role. Recent results suggest that one such non-N benefit may be due to the impact on soil biology of hydrogen emitted from nodules as a by-product of'N, fixation.

Sanders, Donald W. The University of Manitoba, March, 2017. The effect of preceding crop on soybean (Glycine max) grain yield, mycorrhizal colonization, and biological nitrogen fixation. Major Professor:Yvonne Lawley. Manitoba has seen a twenty-fold increase in soybean acres seeded since 2000, with over 1.6 million acres seeded in 2016. This change presents unique opportunities and challenges to improve crop rotations in Manitoba. This experiment studied the effect of four crop sequences on soybean yield, mycorrhizal colonization, and biological nitrogen fixation. In the first year of this experiment, spring wheat, canola, corn and soybeans were grown at three sites in Manitoba (Carman, Portage la Prairie, and Kelburn). In the second year, soybeans were grown on these same plots as a test crop. This two-year sequence of crops was done twice at each site, in 2012-13 and 2013-14. To determine mycorrhizal colonization, root samples were collected at the V3 stage and then analyzed microscopically for mycorrhizal infection. Nitrogen fixation was estimated using the natural abundance method using soybeans collected at the R5 and R6 stage and canola as a reference crop. Soybean following soybean had significantly higher grain yield than all other crop sequences at one site year, and significantly lower grain yield than all other crop sequences at another site year. There were no other differences in soybean test crop yield between crop sequences. Crop sequence significantly affected mycorrhizal colonization. Soybean following canola had significantly lower mycorrhizal colonization than soybean following soybean or corn. Soybean following spring wheat also had significantly lower mycorrhizal colonization than soybean following soybean or corn. Soil test phosphorus levels also significantly affected mycorrhizal colonization, with increasing soil phosphorus resulting in decreased mycorrhizal colonization. Crop sequence significantly affected biological nitrogen fixation. Soybean following soybean or corn often had significantly greater biological nitrogen fixation than soybean following spring wheat or canola. Soil test nitrate levels affected biological nitrogen fixation, with increasing soil nitrate resulting in decreased biological nitrogen fixation. Soil test nitrate levels were affected by the carbon to nitrogen ratio of the preceding crop, with a higher carbon to nitrogen ratio associated with decreased soil nitrate. These results indicate that although there is often not a yield penalty associated with specific rotations, crop sequence has a strong impact on mycorrhizal colonization and biological nitrogen fixation. The soil organisms associated with those processes affect soil phosphorus uptake and nitrogen fixation. Producers should consider the importance of crop rotation when seeking to maximize productivity through symbiotic processes with mycorrhizae and nodule forming bacteria. / May 2017

This investigation has determined the presence of biological nitrogen fixation in two reservoirs in the southwestern United States: Lake Arlington and Lake Ray Hubbard. Subsequent tests have gathered baseline data on the effects of various biological, chemical, and physical parameters on in situ nitrogen fixation in these reservoirs. Of specific importance is the relationship between nitrogen fixation arid occasional blooms of blue-green algae which produce such problems as testes and odors in these water-supply impoundments.

Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

Nitrogen is a critical nutrient for growth and reproduction in living organisms. Although the Earth’s atmosphere is composed of ~80% nitrogen gas (N2), it is inaccessible to most living organisms in that form. Biological nitrogen fixation, however, can be performed by microbes that harbor the enzyme nitrogenase. This enzyme converts N2 into bioavailable ammonia (NH3) and accounts for at least half of the “fixed”nitrogen on the planet. The other major contributor to ammonia production is the industrial Haber-Bosch process. While the Haber-Bosch process has made significant advances in sustaining the global food supply through the generation of fertilizer, it requires high temperature and pressure and fossil fuels. This makes nitrogenase an ideal system for study, as it is capable of performing this challenging chemistry under ambient conditions and without fossil fuels. Nitrogenase requires energy and electrons to convert N2into NH3. The work presented here examined how the enzyme receives electrons to perform the reaction. It was discovered that some microbes employ a novel mechanism that adjusts the energy state of the electrons so that nitrogenase can accept them. Further, the slowest step that takes place in nitrogenase once the electrons are taken up was identified. Finally, by capitalizing on fundamental knowledge, a biohybrid system was designed to grow nitrogen-fixing bacteria in association with electrodes for light-driven production of fixed nitrogen that has potential to be used as a fertilizer for plant growth. Gaining an in-depth understanding of nitrogenase provides insight into one of the most challenging biological reactions, and the newfound knowledge may be a catalyst in developing more efficient systems for sustainable ammonia production.

Submitted by RODRIGO ALBERTO REPKE null (rodrigorepke@hotmail.com) on 2016-10-27T12:42:23Z No. of bitstreams: 1 Tese FINAL (Corrigida).pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-11-01T16:01:05Z (GMT) No. of bitstreams: 1 repke_ra_dr_bot.pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) / Made available in DSpace on 2016-11-01T16:01:05Z (GMT). No. of bitstreams: 1 repke_ra_dr_bot.pdf: 1048387 bytes, checksum: 7e4212c9a987cc6df565f0d8dc5423a4 (MD5) Previous issue date: 2016-09-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O triticum aestivum é uma espécie de ciclo anual, pertencente à família Poaceae. Cultivado em regiões de climas subtropical e temperado, ocupa a segunda maior área plantada no mundo. Para obtenção de altas produtividade dentre outros cuidados, é essencial o fornecimento de nutrientes na quantidade demandada pelas cultivares de alto potencial produtivo, com destaque para o nitrogênio (N). A fixação biológica de nitrogênio é realizada por microrganismos simbióticos associados com raízes das plantas. Entre os microrganismos simbióticos que fixam nitrogênio associados com raízes de plantas, destacam-se os do gênero Azospirillum brasilense. O objetivo no presente trabalho foi avaliar a eficiência do uso da inoculação Azospirillum brasilense no desenvolvimento e componentes produtivos da cultura do trigo. O estudo foi dividido em dois experimentos sendo o primeiro em ambiente protegido e o segundo em ambiente não protegido, ambos na Faculdade de Ciências Agronômicas - Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Botucatu, SP, no ano agrícola 2013. O delineamento utilizado foi o de blocos inteiramente casualizados para ambiente protegido e blocos casualizados para ambiente não protegido, com 10 tratamentos em esquema fatorial 3x3+1. Sendo os tratamentos compostos pelo uso de duas estirpes (Ab-V5 e Ab-V6) isoladamente e em combinação das mesmas (Ab-V5+Ab-V6), todos aplicados em três concentrações 65, 130 e 195 milhões de unidades formadoras de colônia (UFC) de bactérias. Mais a testemunha, sem inoculação de bactérias. Para o experimento em ambiente protegido, cada parcela experimental foi constituída por plantas cultivadas em um recipiente de amianto com capacidade para 0,462 m3 de solo. Foram 7 linhas de 0,88 m cada, espaçadas em 0,15 m, com 40 sementes por metro. Já no experimento em ambiente não protegido, a parcela experimental foi composta por 10 linhas de 2,0 m cada, espaçadas em 0,17 m, e 45 sementes por metro, onde ambos os experimentos teve uma densidade de semeadura em 266 sementes m2 almejando uma densidade de 250 plantas m2. Para atender a necessidade de água da cultura durante todo o ciclo, adotou-se o sistema de irrigação localizada por gotejamento, monitorada por tensiômetros de mercúrio. Após a emergência das plântulas até a colheita, foram realizadas avaliações de crescimento em diversos componentes morfológicos e de produtividade das plantas de trigo. Os dados obtidos foram submetidas à análise de variância pelo teste F a 5% de probabilidade, sendo os dados quantitativos submetidos a análise de regressão em função das doses de A.brasiliense os dados qualitativos em função das estirpes pelo teste de Tukey. Em ambiente protegido, a inoculação das sementes com Azospirillum brasilense promove maior crescimento das plantas de trigo, sem influencia na produção de grãos. Em condições de ambiente não protegido o crescimento das plantas é influenciado pela inoculação de Azospirillum brasilense. O uso de bactérias promotoras de crescimento em plantas aumentam o desenvolvimento das plantas de trigo, o que representa uma estratégia viável, além dos benefícios ambientais associados à redução no uso de fertilizantes nitrogenados. / Triticum aestivum is a yearly crop species belonging to the Poaceae family. It is cultivated in regions with subtropical and temperate climate, and takes up the second largest planted area in the world. Nutrient provision in the demanded amount by high productive potential cultivars, mainly nitrogen (N), is essential to obtain high productivity. The biological fixation of nitrogen is done by symbiotic microorganisms associated with plan roots. Azospirillum brasilense is one of the genera in which the symbiotic microorganisms fixate nitrogen associated with plant roots. This study aimed to evaluate the agronomical efficiency of Azospirillum brasilense inoculation on the growth and productive components of wheat cultivation. The study was divided into two experiments: the fist protected environment, and the second non-protected environment conditions, both in the School of Agriculture - Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus of Botucatu, SP, Brazil, during 2013 crop year. The experiment had completely randomized block design for the fist experiment in protected environment, and random blocks for the second experiment in non-protected environment, with 10 treatments in 3x3+1 factorial scheme. The treatments consisted of two separate (V5 and V6) and combined (V5+V6) stocks and three concentrations (65, 130 and 195 million UFC of bacteria) were applied. The control treatment had no bacterial inoculation. For the protected environment, each experimental plot consisted of plants cultivated in an asbestos recipient with the following dimensions: 0.50 m of height, 0.88 m of width and 1.05 m of length, and 0.462 m3 of soil. Seven 0.88-m rows with 0.15 m spacing and 40 seeds per meter were utilized. In the field experiment, the experimental plot consisted of ten 2.0-m rows with 0.17 m spacing and 45 seeds per meter. For both experiments, the seeding density was 266 seeds m2, aiming a density of 250 plants m2. To meet water cultivation requirements throughout the cycle, dripping irrigation was used and monitored by mercury tensiometers. After seedling emergence until harvest, growth evaluations of several morphological and productivity components of wheat plants were carried out. The obtained data were submitted to analysis of variance by F test at 5% probability and quantitative data were submitted to regression analysis in function of A.brasiliense doses whereas qualitative data in function of stocks were submitted to Tukey’s test. In protected environment, the inoculation of Azospirillum brasilense seeds provides greater growth of wheat plants, without influencing grain production. Under non-protected environment conditions, the plant growth is influenced by the inoculation of Azospirillum brasilense. The use of growth-promoting bacteria in plants increase the development of wheat plants, which represents a viable strategy, besides the environmental benefits related to the reduced utilization of nitrogen fertilizers.

Enterobactérias são comumente encontradas em simbiose com plantas, promovendo o crescimento vegetal, através da redução do nitrogênio atmosférico a amônia. Entretanto, são os principais patógenos na medicina humana e veterinária. O objetivo deste estudo foi buscar marcadores genéticos e fisiológicos que caracterizem espécies de enterobactérias isoladas de cana-de-açúcar (n=24) e de origem clínica (n=15). Foram submetidas a testes moleculares e fisiológicos como: capacidade de reduzir N2; perfil de susceptibilidade a antibióticos; atividade hemolítica; produção de substâncias promotoras de crescimento e liberação de enzimas extracelulares. Atividades metabólicas e fisiológicas altamente conservadas, sustentadas pelo ponto vista genético, permitem a distribuição destas espécies em diferentes ecossistemas, e, em condições favoráveis, esta adaptação pode contribuir para o estabelecimento de processos patogênicos ou simbióticos em plantas. Então, enterobacterias ambientais podem se tornar patogênicas, enquanto isolados clínicos podem se adaptar a condições ambientais. / Enterobacteria are commonly found in symbiosis with plants, promoting their growth by reducing atmospheric nitrogen to ammonia. However, they are also major pathogens in human and veterinary medicine. The aim of this study was to characterize physiological and genetic markers that distinguish enterobacteria species isolated from sugarcane (n= 24) and clinical sources (n= 15). The samples were characterized regarding their molecular and physiological assays as: ability to reduce N2, antibiotic susceptibility profile; screen for hemolytic activity; production of plant-growth promoting compounds and release of extracellular enzymes. Finally, highly conserved physiological and metabolic activities are supported on genetic backgrounds that allow a wide distribution along different ecosystems and, in favorable conditions, this adaptation could contribute to the establishment of a pathogenic process or symbiosis in plants. So, environmental enterobacteria isolates can become pathogenic, whereas clinical strains can adapt to environmental conditions.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-03-05T18:23:35Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Leandro Tullio.pdf: 1410480 bytes, checksum: b92ca948d0810d08208334b6703b86c9 (MD5) / Made available in DSpace on 2018-03-05T18:23:35Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Leandro Tullio.pdf: 1410480 bytes, checksum: b92ca948d0810d08208334b6703b86c9 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O pH é o principal determinante da diversidade microbiana do solo. Rhizobium freirei é um notável endossimbionte de feijão e reconhecido por sua elevada capacidade de tolerância a estresses ambientais, como o pH ácido. O objetivo deste trabalho foi analisar alterações na expressão gênica, em nível transcricional e traducional, de R. freirei cultivada em pH ácido. A bactéria foi cultivada em caldo TY até alcançar a fase exponencial em pH 6,8 e pH 4,8 e, submetida à extração de proteínas totais. As proteínas foram separadas por eletroforese bidimensional e os perfis proteicos obtidos foram analisados em imagens de alta resolução. Os spots diferencialmente expressos (p ≤ 0,05) foram selecionados para análise em espectrometria de massa e identificação in silico. A expressão diferencial dos genes gshB, gstA e ropB foi analisada após 0, 30 e 60 min de exposição de R. freirei ao pH ácido. De acordo com os resultados, o principal mecanismo de tolerância de R. freirei ao pH ácido parece estar associado à alteração de vias metabólicas centrais, devido ao aumento na expressão de proteínas envolvidas com vias oxidativas (ZwF, KDPG e PckA), relacionadas ao Ciclo do Ácido Tricarboxílico. A maior expressão destas enzimas sugere um aumento no consumo de carboidratos e consequente formação de NADH, que pode ser corroborado pelo aumento na expressão da subunidade NuoC do Complexo 1 da cadeia respiratória, que cataliza a conversão de NADH em NAD+ acoplada ao transporte de prótons para o periplasma, reduzindo a acidificação citosólica. O Complexo 1 é considerado o principal formador de radical superóxido, além disso a maior expressão das enzimas antioxidativas AhpC e GstA em nível traducional, bem como a maior expressão de gstA em nível transcricional, sugere fortemente que o pH ácido induz ao estresse oxidativo em R. freirei. Por outro lado, a menor expressão da proteína AccC pode estar relacionada a um direcionamento do metabolismo central ao consumo de ácidos em detrimento à biossíntese de ácidos graxos, processo em que AccC é fundamental. A menor expressão da subunidade β do Complexo ATPase (AtpD) pode estar direcionada à redução na acidificação do pH interno, por meio da diminuição na entrada de prótons. Tal abrangência de vias metabólicas envolvidas sugere que a resposta adaptativa ao pH ácido possui caráter multigênico, cuja predominância de proteínas citoplasmáticas reforça a importância do metabolismo central para sua capacidade de tolerar pH acidificado. Ao contrário de trabalhos que enfocam genes ou mecanismos específicos de tolerância, nossos resultados mostraram, pela primeira vez, que o metabolismo central parece ser o principal mecanismo de tolerância ao pH ácido em R. freirei e que há fortes indícios da correlação entre estresse ácido e oxidativo. / The pH is the main controller of microbial diversity of soil. Rhizobium freirei is a remarkable bean endosymbiont and recognized by its ability to tolerate environmental stresses, such as acidic pH. The aim of this study was to analyze changes in gene expression at transcriptional and translational level of R. freirei grown in acidic pH. Bacteria was grown in TY medium until exponential phase in two treatments: pH 6,8 and pH 4,8. The protein extraction was performed and the extract was separated by two-dimensional electrophoresis. High-resolution images of the profiles were analyzed and the spots that showed statistical difference in the relative volume (%vol) between treatments were selected for identification by mass spectrometry (MS) and in silico identification. Differential expression of R. freirei was assessed by analysis of gshB, gstA and ropB genes after 0, 30 and 60 min of exposure in acidic pH. According to the results, the changing of central metabolic pathways seems to be the main mechanism of tolerance to acid stress in R. freirei, due to the increased expression of proteins involved in oxidative pathways (Zwf, aldolase KDPG and PckA) related to the Tricarboxilic Acid cicle (TCA). The increased expression of these proteins suggests higher carbohydrate consumption and NADH formation, reinforced by elevated expression of NuoC subunit of respiratory chain Complex 1, that converts NADH into NAD+ coupled to proton transport to periplasm and consequent decreases the cytosolic acidification. The Complex 1 of the respiratory chain is the main superoxide former; moreover the higher expression of the antioxidative enzymes AhpC and GstA at translational level, and the higher expression of gstA at transcriptional level, strongly suggests an oxidative stress induced by acid pH condition. On the other hand, the decreased AccC protein expression supports the trend of the central metabolism to acid-consuming reactions, in contrast to fatty acids biosynthesis pathways, where AccC is essential. Down-regulation of the β subunit of ATPase Complex can be related to regulation of cytosolic acidity by reduction of proton entry through ATPase Complex. Such broad range of pathways involved in the response to acid conditions suggests an adaptive response of multigenic character mainly involving cytoplasmic proteins, and reinforces the importance of the central metabolism for R. freirei ability to tolerate acidic pH conditions. In contrast to studies focused in few genes or specific mechanisms of tolerance, our results shows by the first time that the central metabolism appears to be the main mechanism of acid tolerance in R. freirei and suggests a correlation between acid and oxidative stress responses.

Grandes esforços têm sido empreendidos para preservar recursos naturais e desenvolver sistemas de produção agrícola mais sustentáveis, reduzindo o uso de fertilizantes. Uma das alternativas ao uso de fertilizantes nitrogenados na agricultura são os inoculantes com bactérias diazotróficas. Os micro-organismos diazotróficos mais estudados para produção de inoculantes são os simbiontes noduladores de leguminosas. Para outros grupos de plantas, os diazotróficos mais utilizados são assimbióticos. Estudos conduzidos na Mata Atlântica e na Amazônia tem mostrado que a fixação biológica de nitrogêncio (FBN) assimbiótica associada a filosfera pode contribuir com aportes de N significativos, e que esses biomas podem abrigar uma grande diversidade de bactérias diazotróficas, sendo ambientes apropriados para prospectar diazotróficos eficientes para o uso na agricultura. O objetivo deste trabalho foi isolar novos genótipos de bactérias diazotróficas assimbióticas da filosfera e serapilheira de espécies arbóreas da floresta Amazônica, e estimar suas taxas de FBN e de síntese de ácido indolacético in vitro, bem como identificar taxonomicamente os isolados considerados mais promissores para uso biotecnológico. Bactérias diazotróficas de vida-livre foram isoladas da filosfera e serapilheira de três espécies arbóreas (Rinorea pubiflora, Amphirrhox longifolia e Chamaecrista xinguensis), e cultivadas em meio isento de N. Um total de 86 isolados foram obtidos. A taxa de fixação de nitrogênio foi estimada para todos os isolados, através do ensaio de redução de acetileno (ARA), pelo complexo nitrogenase. Para todos os isolados, também foi determinada a taxa de produção de ácido indolacético (AIA), na presença de L-triptofano. Os dados obtidos foram submetidos à análise de variância e as médias comparadas pelo teste de Scott-Knott e pelo teste de Duncan (p<0,05), através do software estatístico R. Os isolados que apresentaram as maiores taxas de redução de acetileno e de síntese de AIA foram identificados através do sequenciamento parcial do gene rRNA 16S. Cerca de 84,8% dos isolados apresentaram atividade de nitrogenase in vitro, e o teste estatístico desmonstrou não haver diferenças significativas entre as taxas de redução de acetileno entre os isolados, exceto o isolado 10RF, o qual apresentou uma taxa de redução de acetileno maior do que a dos demais isolados (p<0,05). Todos os isolados produziram AIA in vitro, e houve diferenças significativas na síntese de AIA entre os isolados, e quanto origem dos mesmos. Isolados bacterianos de Amphirrox longifolia sintetizaram mais AIA in vitro, em comparação aos isolados das outras duas espécies arbóreas. Cerca de 22% dos isolados apresentaram taxas de fixação de nitrogêncio maiores que 0.025ng N &mu;g-1 h-1, e 20% dos isolados produziram mais de 21,87 &mu;g/mL-1 de AIA. 21 isolados, considerados promissores para uso agrícola (fixadores de N e produtores de AIA), foram identificados através do sequenciamento do gene rRNA 16S, e afiliados aos gêneros Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium e Stenotrophomonas. Aproximadamente 71 % dos isolados considerados promissores para promoverem o crescimento de plantas foram isolados da filosfera, indicando ser este um ambiente propício à prospecção de bactérias diazotróficas promotoras de crescimento de plantas. / Great efforts have been made to preserve natural resources and develop more sustainable agricultural production systems, reducing the use of fertilizers. One of the alternatives to the use of nitrogen fertilizers in agriculture is the use of inoculants with diazotrophic bacteria. The most studied diazotrophic microorganisms for the production of inoculants are nodule-forming symbionts of legumes. For other groups of plants, the most used diazotrophs are asymbiotic. Several studies in the Atlantic and Amazon forests have shown that the biological nitrogen fixation (BNF) associated with the phyllosphere may contribute with significant N inputs, and that these biomes can harbor a great diversity of diazotrophic bacteria, making them important environments for the prospection of efficient diazotrophs for agricultural use. The objective of this work was to isolate new genotypes of asymbiotic diazotrophic bacteria from the phyllosphere and litter of tree species of the Amazon forest, and to estimate their rates of BNF and indoleacetic acid biosynthesis in vitro, as well as to identify taxonomically the most promising isolates for biotechnological use. Asymbiotic diazotrophic bacteria were isolated from the phyllosphere and litter of three tree species (Rinorea pubiflora, Amphirrhox longifolia and Chamaecrista xinguensis), and cultivated in N-free medium. A total of 86 isolates were obtained. The nitrogen fixation rate was estimated for all isolates measuring the nitrogenase activity by the acetylene reduction assay (ARA). The rates of indoleacetic acid (IAA) production in the presence of L-tryptophan was also determined for all isolates. The data obtained were submitted to analysis of variance and the means were compared by the Scott-Knott test and the Duncan test (p<0.05) using the statistical software R. The isolates showing the highest rates of acetylene reduction and IAA biosynthesis were identified through the partial sequencing of the 16S rRNA gene. Approximately 84.8% of the isolates were positive for nitrogenase activity in vitro, and the statistical analyses showed that there were no significant differences between the acetylene reduction rates among the isolates, except the isolate 10RF, which showed a higher acetylene reduction rate than the other isolates (p<0.05). All isolates produced IAA in vitro, and there were significant differences in the biosynthesis rates among the isolates, as well as regarding the origin of the isolate. Bacterial isolates from Amphirrox longifolia synthesized higher amounts of IAA in vitro as compared to the isolates from the other two tree species. Approximately 22% of the isolates showed nitrogen fixation rates greater than 0.025 ng N &mu;g-1 h-1, and 20% of the isolates produced more than 21.87 &mu;g mL-1 of IAA. 21 isolates, considered promising for agricultural use (N-fixers and IAA producers), were identified by sequencing the 16S rRNA gene, and affiliated to the genera Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium and Stenotrophomonas. Approximately 71% of the isolates considered promising to promote plant growth were isolated from the phyllosphere, suggesting that this environment is propitious to the prospection of diazotrophic plant growth promoting bacteria.