Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Entre as leguminosas forrageiras potencialmente importantes para as condições edafoclimaticas do Brasil, o gênero Stylosanthes destaca-se como alternativa para a melhoria da fertilidade do solo em pastagens consorciadas com gramíneas, tanto pela sua adaptação ao sistema quanto pela capacidade de estabelecer simbiose com bactérias fixadoras de nitrogênio. O objetivo deste trabalho foi isolar, caracterizar e avaliar a eficiência na fixação biológica de nitrogênio por bactérias isoladas de nódulos de estilosantes (Stylosanthes ssp.). Foram realizadas coletas de plantas de Stylosanthes ssp. em dois municípios de Roraima (Boa Vista e Bonfim), sendo exsicatas das plantas enviadas para identificação botânica no Museu Integrado de Roraima. As raízes das plantas foram lavadas com água e ao todo 564 nódulos foram coletados e isolados em meio de cultura YMA. Adicionalmente, também foram utilizados no estudo 34 isolados que haviam sido obtidos previamente de Stylosanthes sp. Após a purificação, os isolados foram morfologicamente caracterizados em meio YMA, considerando-se o tempo de crescimento, alteração do pH no meio de cultura, forma e aspecto das colônias e muco. Realizou-se então um agrupamento dos isolados utilizando-se o coeficiente de Jaccard. Em seguida, conduziu-se um experimento pelo período de 20 dias para a autenticação de 89 isolados de rizóbio em experimento de casa de vegetação inoculando-se plantas de feijão-caupi. Os isolados foram selecionados por representarem os grupos morfológicos das bactérias e, também, as espécies de Stylosanthes de onde haviam sido obtidos. Quarenta e quatro isolados que nodularam o feijão-caupi foram posteriormente utilizados para o estudo da eficiência simbiótica em Stylosanthes capitata cv. lavradeiro. Este experimento foi conduzido em casa de vegetação utilizando-se a estirpe BR BR 446 (recomendada para o Stylosanthes) como controle positivo, além de um tratamento nitrogenado (180,75mg / N por semana) e um controle sem inoculação e sem nitrogênio mineral. O plantio foi realizado em potes contendo uma mistura de areia e solo (1:1) autoclavados e semanalmente aplicou-se solução nutritiva e água quando necessário. A coleta foi realizada aos 65 dias após a emergência das plantas e foram avaliados número e massa de nódulos secos, matéria seca da parte aérea e nitrogênio acumulado na parte aérea. Dos 564 nódulos foram obtidos 258 isolados que somados aos demais 34 isolados (anteriormente obtidos) totalizaram 292. Os isolados apresentaram grande diversidade morfológica e foram agrupados em 87 grupos com 70% de similaridade. A maioria dos isolados 46% apresentou característica de crescimento rápido em meio de cultura YMA e apenas 0,7% com crescimento lento. A autenticação indicou que apenas 44 das 89 bactérias testadas formaram nódulos em feijão-caupi, sendo na sua maioria bactérias de crescimento rápido. Isto indica que possa haver especificidade de nodulação entre os isolados de rizóbio e as espécies de estilosantes e diverge da literatura que indica que estilosantes preferencialmente nodula com bactérias de crescimento lento. O experimento de eficiência simbiótica indicou que cerca de 50% dos isolados proporcionaram número de nódulos em Stylosanthes capitata igual à estirpe recomendada e proporcionaram produção de matéria seca da parte aérea com tendência superior ao tratamento nitrogenado e a estirpe recomendada. As estirpes St4A29, StG57, St1A8, StF29 e St6E2-3 apresentaram melhor desempenho e possuem potencial para futuras pesquisas de recomendação de estirpes. / Among potentially important forage legumes for edaphoclimatic conditions in Brazil, the genus Stylosanthes appears as alternative for improving the soil fertility in consortium with grasses, both by the adaptation to the system as well as the ability to establish symbiosis with nitrogen-fixing bacteria. The aim of this study was isolate, characterize and assess the biological nitrogen fixation efficiency of the bacterial isolated from nodules of Stylosanthes ssp. Samplings of Stylosanthes ssp. plants were taken from two municipalities of Roraima State (Boa Vista and Bonfim), and plants exsiccates were sent for botanical identification at Integrated Museum of Roraima. The plants roots were washed with water and a total of 564 nodules were collected and isolated on YMA medium. Additionally, 34 isolates previously obtained from Stylosanthes sp. were also included in the study. After purification, the isolates were morphologically characterized, considering the time to growth, culture medium pH reaction, shape and aspect of the colonies and mucus. A clustering analysis was further performed with the Jaccard index. An authentication experiment was carried on during 20 days in glasshouse with 89 isolates inoculated in cowpea. The isolates were selected based on morphological groups and also Stylosanthes species that they had been isolated. Forty-four isolates that nodulated cowpea were after used for symbiotic efficiency evaluation in Stylosanthes capitata (cv. Lavradeiro). This experiment was performed in glasshouse where it was used the strain BR 446 (indicated for Stylosanthes inoculation) as positive control, besides a treatment with nitrogen (180,75mg / N per week) and a control without inoculation or nitrogen. The sowing was done in pots containing a mixture of sand and soil (1:1) autoclaved. Harvest was done 65 days after plant germination and were evaluated the nodules number and dry mass, plant dry matter and nitrogen accumulation in the dry matter. From 564 nodules, 258 isolated were purified and, with the additional 34 isolates (previously obtained), 292 isolates were consider for the study. The isolates showed high morphological diversity and were clustered in 87 groups, with threshold 70%. Most of the isolates 46% presented fast growing on YMA medium and only 0,7% were slow-growing. The authentication revealed that 44 of the 89 bacteria tested were able to form nodules in cowpea, and most of this isolates were fast-growing. This result indicated that could exist nodulation specificity between rhizobium isolates and Stylosanthes species, and diverge of the previous observations that had indicated Stylosanthes as mainly nodulated by slow-growing bacteria. Symbiotic efficiency experiment indicated that about 50% of the isolates were able to form nodules in Stylosanthes capitata similar to recommend strain and trend to higher contribution for biomass production was observed in comparison with nitrogen treatment and recommended strain as well. The strains St4A29, StG57, St1A8, StF29 and St6E2-3 have shown best performance and have potential for future researches to strains recommendation.

V. faba fixes nitrogen effectively (Richards & Soper, 1979), however nitrogen fixation is reportedly energetically expensive and water deficit sensitive. Research was designed to determine whether medium nitrogen applications would result in increased productivities in V. faba, particularly during water deficits. Non-nodulated and nodulated V. faba were subjected to gradual water deficit imposition, and were supplied with a variety of medium nitrogen nutrition. Nitrogen fixing V. faba exhibited greater productivities than V. faba which were supplied with low medium nitrate concentrations (0.8 roM N), even during water deficits. Plant performance parameters (growth; net photosynthesis; nitrogen assimilatory enzyme activities; osmotic adjustment) were greater in nodulated than in non-nodulated 'no nitrate' supplied V. faba throughout water deficits, inferring water deficit tolerance for nitrogen fixation. However significantly greater plant performance paramaters were exhibited in V. faba when supplied with increasingly concentrated medium nitrogen nutrition (> 0.8 roM N) than when reliant on nitrogen fixation. In contrast to the bulk of previous literature, NR activities were maintained in V. faba until water deficits became severe, inferring a role for nitrate assimilation in nitrogenous osmotica production. Medium ammonia additions resulted in the exhibition of significantly increased root biomasses; cumulative leaf areas (important for a green manure crop); heights; and nitrogen assimilation in V. faba throughout water deficits, and accordingly in increased osmotic adjustment (including compatible solute accumulation), protein concentrations and vegetative yields. Greater plant productivities in v. faba when supplied with medium ammonia additions were attributed in part to lower associated assimilatory costs for ammonia than nitrate nutrition (Raven, 1992). Results indicated increased metabolism as opposed to storage of medium ammonia, and therefore potentially alleviated 'sink size' feedback inhibition of photosynthesis and nitrogen metabolism in V. faba when supplied with medium ammonia additions. Furthermore ammonia supplied V. faba may have been predisposed towards water deficit tolerance. In summary V. faba exhibited significantly greater nitrogen assimilation; osmotic adjustment; net photosynthesis; and growth when supplied with increasingly concentrated medium nitrogen nutrition (and particularly with medium ammonia additions) than when reliant on nitrogen fixation, both during periods of adequate irrigation and during water deficits.

The association between root-nodulating bacteria and leguminous plants is advantageous due to their ability to alter atmospheric nitrogen into a useful form in a process known as biological nitrogen fixation (BNF). Research has shown that BNF is the most efficient way to supply the large amounts of nitrogen needed by plants to produce high-yielding crops. As a result, there have been numerous studies into the diversity and identity of the associated nitrogen-fixing bacterial symbionts. Recent advances in molecular microbiology together with the isolation of rhizobia from previously uninvestigated legumes have led to major revisions of rhizobial taxonomy, most notably the inclusion of bacteria from the ß-Proteobacteria in the genera Burkholderia and Ralstonia. In this study, the diversity of root nodule bacteria associated with Phaseolus coccineus and Phaseolus vulgaris species in South Africa was investigated. A selection of rhizobial isolates were characterised by SDS-PAGE of whole cell proteins and rep-PCR DNA fingerprint analyses. These results were supplemented by partial 16S rDNA sequencing of a select number of isolates to confirm their identity. Where isolates displayed unexpected genus associations, partial nodA sequencing was performed to determine whether these were incidental contaminants or true nodulators. Based on 16S rDNA sequence analysis, the majority of isolates investigated were fast-growers belonging to the genus Rhizobium. A few isolates showed close relationship to species of the ß-Proteobacteria genus, Burkholderia. Both the SDS-PAGE analyses and the combined rep-PCR analyses were able to resolve isolates down to strain level, but the comparison of the SDS-PAGE and 16S rDNA sequencing data confirmed that bacterial discrimination using SDS-PAGE is not useful at the genus level and higher, as isolates showing affinity to Burkholderia were mingled with isolates showing similarity to Rhizobium. These isolates were separate from the Rhizobium isolates in the combined rep-PCR dendrogram. While there were discrepancies between results obtained from SDS-PAGE and rep-PCR analyses, results from the combined rep-PCR analysis correlated with many of the results obtained in the SDS-PAGE analysis. Both geographic location and host plant species appear to have affected the grouping of isolates. Many clusters consisted of isolates from the same location or the same host plant species in both the SDS-PAGE dendrogram as well as the combined rep-PCR dendrogram. The nodA sequencing demonstrated that the majority of isolates tested contain the nodA gene indicating that they are capable of nodulation. There was a large strain diversity observed for the isolates of this study and a number of the root-nodulating bacteria of the Phaseolus spp. appear to constitute several novel nodulating genotypes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Long term management plans for restoration of natural flow conditions through the Everglades increase the importance of understanding potential nutrient impacts of increased freshwater delivery on coastal biogeochemistry. The present study sought to increase understanding of the coastal marine system of South Florida under modern conditions and through the anthropogenic changes in the last century, on scales ranging from individual nutrient cycle processes to seasonal patterns in organic material (OM) under varying hydrodynamic regime, to century scale analysis of sedimentary records. In all applications, carbon and nitrogen stable isotopic compositions of OM were examined as natural recorders of change and nutrient cycling in the coastal system. High spatial and temporal variability in stable isotopic compositions were observed on all time scales. During a transient phytoplankton bloom, ä15N values suggested nitrogen fixation as a nutrient source supporting enhanced productivity. Seasonally, particulate organic material (POM) from ten sites along the Florida Reef Tract and in Florida Bay demonstrated variable fluctuations dependent on hydrodynamic setting. Three separate intra-annual patterns were observed, yet statistical differences were observed between groupings of Florida Bay and Atlantic Ocean sites. The POM ä15N values ranged on a quarterly basis by 7‰, while ä13C varied by 22‰. From a sediment history perspective, four cores collected from Florida Bay further demonstrated the spatial and temporal variability of the system in isotopic composition of bulk OM over time. Source inputs of OM varied with location, with terrestrial inputs dominating proximal to Everglades freshwater discharge, seagrasses dominating in open estuary cores, and a marine mixture of phytoplankton and seagrass in a core from the boundary zone between Florida Bay and the Gulf of Mexico. Significant shifts in OM geochemistry were observed coincident with anthropogenic events of the 20th century, including railroad and road construction in the Florida Keys and Everglades, and also the extensive drainage changes in Everglades hydrology. The sediment record also preserved evidence of the major hurricanes of the last century, with excursions in geochemical composition coincident with Category 4-5 storms.

O presente estudo teve por objetivo analisar os efeitos da inoculação de Acinetobacter sp. ICB117 no desenvolvimento da cana-de-açúcar variedade SP791011. As plantas foram cultivadas a partir de toletes com uma gema e fornecidas pelo Centro de Tecnologia Canavieira. Aos 30 dias de idade, foram submetidas aos seguintes tratamentos: controle (C) sem inoculação e solução de Hoagland sem nitrogênio; nitrato (N) sem inoculação e solução de Hoagland com nitrato (15 mM); bactéria (B) inoculação bacteriana e solução de Hoagland sem nitrogênio; bactéria + nitrato (BN) inoculação bacteriana e solução de Hoagland com nitrato (15 mM). Antes da realização da inoculação, foi inserido em ICB117 um plasmídio que contém um gene de expressão para o GFP. O material vegetal foi coletado mensalmente (2 coletas), sendo os seguintes parâmetros analisados: altura, nº de folhas, massa seca, composição de aminoácidos na seiva xilemática, razão C/N das raízes e folhas, atividade da nitrato redutase foliar, assimilação máxima de CO2 e estimativa da população endofítica de ICB117. / Acinetobacter sp. ICB117 is a nitrogen-fixing bacterium isolated from Brazilian sugarcane (Saccharum sp. variety SP791011) which releases amino acids, polyamines and indole-3-acetic acid (IAA) when grown in vitro. The present study aimed to evaluate the effects of Acinetobacter sp. ICB117 (a diazotrophic endophytic bacterium) inoculation on the growth of Saccharum sp. variety SP791011 (sugarcane). Sugarcane plants were obtained from stools with one bud, provided by CTC (Centro de Tecnologia Canavieira). Plants with 30 days old were submitted to the following treatments: control (C) - no bacterium inoculation and Hoagland solution without nitrogen; nitrate (N) - no bacterium inoculation and Hoagland solution with nitrate (15 mM); bacteria (B) - bacterium inoculation and Hoagland solution without nitrogen; Bacteria + nitrate (BN) - bacterial inoculation and Hoagland solution with nitrate (15 mM). Previously to the inoculation procedure, Acinetobacter sp. ICB117 was transformed with plasmid containing a gene that expresses the Green Fluorescent Protein (GFP). Plant material were sampled monthly (2 sampling times) in order to evaluate the following parameters: hight, number of leaves, dry mass, amino acids composition on xylem sap, leaf and root nitrogen and carbon content, leaf nitrate reductase activity, photosynthetic CO2 assimilation and ICB117 endophytic population estimative. The results showed a very similar pattern along experimental period: inoculation with bacterium increased plant total dry mass (evidenced by roots), number of leaves and the assimilation of CO2, but it did not influece significantly the shoot size of the plants and the leaf nitrate reductase activity. The endophytic ICB117 population was larger in plants treated without nitrate than in plants with nitrate inputs. Among the different treatments, alterations were not verified in the amino acids composition on xylem sap prevailing arginine in the sap of all the plants. In the roots, the carbon nitrogen (C/N) ratio was smaller in the plants treated with nitrate (N and BN); in the leaves, the C/N ratio was smaller only in the plants treated with nitrate without inoculation (N).

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.

Made available in DSpace on 2016-01-26T18:56:25Z (GMT). No. of bitstreams: 1 REBESCHINI DISSERTACAO revisao final apos dissertacao (26-06-12).pdf: 764338 bytes, checksum: 7548acbd68bd69e21dcebc69ed592c15 (MD5) Previous issue date: 2012-06-19 / Bean represents an important protein source in the human diet in developing countries of tropical and subtropical regions. It is one of the major components of the Brazilian diet and an important protein source. Regarding the nitrogen supply for the bean, we can mention the use of mineral fertilizers and biological fixation of N2. The fixation efficiency is variable, ranging from 2 to 100 kg N ha-1. Thus it is justified the researches that involve the use of mineral nitrogen fixation with N. The objective of this study was to evaluate doses of nitrogen in the bean cultivation in field conditions, associated with seed inoculation. The study was conducted under field conditions in two traditional bean cultivation area in Paraná northern. The experiment was consisted by plots with or without inoculation of Rhizobium tropici in the seeds and use five N rates (0, 20, 40, 60 and 80 kg ha-1) in cover. The experimental design employed was randomized blocks, being the factorially treatments disposed in five levels of nitrogen with and without inoculation. Evaluations were made about the nodulation, N content in leaves, shoot dry matter, N uptake, number of pods per plant and grain yield. The results showed that application of nitrogen coverage or use of the inoculant did not provide income gains in the bean. Nodulation was affected by the application of mineral nitrogen coverage. The performance of bean differed in the two cultivated areas, which may have been influenced by climatic conditions and management at the sites studied. / O feijão representa uma importante fonte protéica na dieta humana dos países em desenvolvimento das regiões tropicais e subtropicais. No Brasil, o feijão é um dos componentes básicos da dieta alimentar da população e importante fonte de proteína. Com relação ao fornecimento de nitrogênio (N) para o feijoeiro pode ser destacado o uso de fertilizantes minerais e a fixação biológica de N2. A eficiência da fixação biológica é muito variável, oscilando de 2 a 100 kg de N ha-1. Desta forma justificam-se trabalhos que busquem associar o uso do nitrogênio mineral com a fixação biológica de N. O objetivo deste trabalho foi o de avaliar doses de nitrogênio em cobertura no cultivo do feijão em condições de campo, associado a inoculação de sementes. O estudo foi conduzido em condições de campo em duas áreas de cultivo tradicional de feijoeiro, na região norte do Paraná. O experimento foi constituído por parcelas com ou sem inoculação de Rhizobium tropici nas sementes e utilização de cinco doses de N (0, 20, 40, 60 e 80 kg ha-1) em cobertura. O delineamento experimental empregado foi de blocos ao acaso sendo os tratamentos arranjados fatorialmente em 5 doses de nitrogênio com e sem inoculante. Foram feitas avaliações quanto a nodulação, teor de N nas folhas, matéria seca da parte aérea, N acumulado, número de vagens por planta e rendimento de grãos. Os resultados encontrados revelaram que a aplicação do nitrogênio em cobertura ou utilização do inoculante não proporcionou ganhos de rendimento no feijoeiro. A nodulação foi afetada pela aplicação de nitrogênio mineral em cobertura. O desempenho do feijoeiro diferenciou-se nas duas áreas cultivadas, o que pode ter sido influenciado pelas condições climáticas e de manejo nos locais estudados.

Made available in DSpace on 2016-07-18T17:51:09Z (GMT). No. of bitstreams: 1 REBESCHINI DISSERTACAO revisao final apos dissertacao (26-06-12).pdf: 764338 bytes, checksum: 7548acbd68bd69e21dcebc69ed592c15 (MD5) Previous issue date: 2012-06-19 / Bean represents an important protein source in the human diet in developing countries of tropical and subtropical regions. It is one of the major components of the Brazilian diet and an important protein source. Regarding the nitrogen supply for the bean, we can mention the use of mineral fertilizers and biological fixation of N2. The fixation efficiency is variable, ranging from 2 to 100 kg N ha-1. Thus it is justified the researches that involve the use of mineral nitrogen fixation with N. The objective of this study was to evaluate doses of nitrogen in the bean cultivation in field conditions, associated with seed inoculation. The study was conducted under field conditions in two traditional bean cultivation area in Paraná northern. The experiment was consisted by plots with or without inoculation of Rhizobium tropici in the seeds and use five N rates (0, 20, 40, 60 and 80 kg ha-1) in cover. The experimental design employed was randomized blocks, being the factorially treatments disposed in five levels of nitrogen with and without inoculation. Evaluations were made about the nodulation, N content in leaves, shoot dry matter, N uptake, number of pods per plant and grain yield. The results showed that application of nitrogen coverage or use of the inoculant did not provide income gains in the bean. Nodulation was affected by the application of mineral nitrogen coverage. The performance of bean differed in the two cultivated areas, which may have been influenced by climatic conditions and management at the sites studied. / O feijão representa uma importante fonte protéica na dieta humana dos países em desenvolvimento das regiões tropicais e subtropicais. No Brasil, o feijão é um dos componentes básicos da dieta alimentar da população e importante fonte de proteína. Com relação ao fornecimento de nitrogênio (N) para o feijoeiro pode ser destacado o uso de fertilizantes minerais e a fixação biológica de N2. A eficiência da fixação biológica é muito variável, oscilando de 2 a 100 kg de N ha-1. Desta forma justificam-se trabalhos que busquem associar o uso do nitrogênio mineral com a fixação biológica de N. O objetivo deste trabalho foi o de avaliar doses de nitrogênio em cobertura no cultivo do feijão em condições de campo, associado a inoculação de sementes. O estudo foi conduzido em condições de campo em duas áreas de cultivo tradicional de feijoeiro, na região norte do Paraná. O experimento foi constituído por parcelas com ou sem inoculação de Rhizobium tropici nas sementes e utilização de cinco doses de N (0, 20, 40, 60 e 80 kg ha-1) em cobertura. O delineamento experimental empregado foi de blocos ao acaso sendo os tratamentos arranjados fatorialmente em 5 doses de nitrogênio com e sem inoculante. Foram feitas avaliações quanto a nodulação, teor de N nas folhas, matéria seca da parte aérea, N acumulado, número de vagens por planta e rendimento de grãos. Os resultados encontrados revelaram que a aplicação do nitrogênio em cobertura ou utilização do inoculante não proporcionou ganhos de rendimento no feijoeiro. A nodulação foi afetada pela aplicação de nitrogênio mineral em cobertura. O desempenho do feijoeiro diferenciou-se nas duas áreas cultivadas, o que pode ter sido influenciado pelas condições climáticas e de manejo nos locais estudados.

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.

Leguminous plants are able to fix nitrogen by establishing a symbiotic relationship with soil dwelling bacteria, called rhizobia. The model plant Medicago truncatula forms a partnership with Sinorhizobium meliloti whereby the plant gains bioavailable nitrogen and in exchange the bacteria gains carbohydrates. This process occurs within nodules, which are structures produced on the roots of the plants within which nitrogen is fixed. M. truncatula incomplete root elongation (MtIRE) was localized to the infection zone, which is zone II of indeterminate nodules. It was shown to encode a signaling kinase so it was anticipated to play a role in nodulation. Mutants of MtIRE in the R108 background, mutagenized with the Tnt1 retrotransposon, were obtained from reverse screen, and were assessed to determine if a disrupted MtIRE gene was the cause of nitrogen fixation defective nodules. Mutant line NF1320, having a mutant phenotype, showed typical Mendelian segregation of 3:1 when backcrossed to R108. Experimental results show that MtIRE gene is not the cause of the mutant phenotype, but was linked to the causative locus. MtIRE co-segregated with the mutant phenotype 83%. Southern blot and the first version of the M. truncatula genome (version 3.5) reported a single MtIRE gene and this was shown to be on chromosome 5 but the latest version of the M. truncatula genome (version 4.0) showed a second copy of the gene on chromosome 4. The genome sequence is based on the A17 reference genome. Both genes are 99% identical. Genetic markers that originate from flanking sequence tags (FSTs) on both chromosome 4 and 5 were tested in an attempt to find an FST that co-segregated with the mutant phenotype 100%. An FST derived from a Tnt1 insertion in Medtr4g060930 (24F) co-segregated with the mutant phenotype closely, with 76% co-segregation. Medtr4g060930 (24F) is on chromosome 4, making it likely that the Tnt1 inserted in the MtIRE gene is also on chromosome 4, and thus the defective gene is on chromosome 4.