Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-06-28T13:43:43Z No. of bitstreams: 1 Andresa Priscila de Souza Ramos.pdf: 2860451 bytes, checksum: 3fdb7bbde9ea81f39f6781ff6c556c67 (MD5) / Made available in DSpace on 2016-06-28T13:43:43Z (GMT). No. of bitstreams: 1 Andresa Priscila de Souza Ramos.pdf: 2860451 bytes, checksum: 3fdb7bbde9ea81f39f6781ff6c556c67 (MD5) Previous issue date: 2016-02-29 / Plant growth promoting bacteria shows beneficial mechanisms that can promote better development of the host plant. The interaction between host and bacteria can be affected by several factors, such as plant genotype, plant specie, and the niche with microorganims lived. The citros culture have a great socieoeconomic importance for the country, which is one of the largest producers and exporters of citrus fruits. The aim of this study were to evaluate the bacterial communities associated with diferente hybrid citrus genotype, and observed the bacterial density, associated with niches diferents and evaluate the potencial ability to become culture inoculantes. through the study of production growth promoters production substances. Bacterial strains were isolated from the rhizosphere niches, roots, stems and leaves of 13 different genotypes rootstock hybrids of citrus and evaluated its production capacity of amylase, cellulase, pectinase (polygalacturonase and pectate lyase), inorganic phosphate solubilization, IAA producing dependent pathway of tryptophan, the potential biological nitrogen fixation and production of siderophores. Data were analyzed using statistical nonparametric test of Kruskal-Wallis and Wilcoxon using the R software. The results showed that bacteria associated with citrus plants have great potential, and the population density varies from the niche and host plant. Of the 183 isolates from 80% of the isolates were positive for the production of IAA, producing up 85μg.mL-1, 55.7% produced one or more types of enzymes, solubilized 48% phosphate, 37% were positive for nitrogen fixation and 60% for the production of siderophores. Confirming the potential to be potential inoculants culture. / Bactérias promotoras de crescimento de plantas mostram mecanismos benéficos que podem promover o melhor desenvolvimento da planta hospedeira. A interação entre hospedeiro e bactéria pode ser afetada por diversos fatores, como o genótipo da planta, a espécie, e o nicho do qual o micro-organismo foi originado. A cultura dos citros tem grande importância socioeconômica para o país, que é um dos maiores produtores e exportadores dos frutos. Diante disso, os objetivos deste trabalho foram estudar a comunidade bacteriana associada a diferentes genótipos híbridos de citros, observar a densidade bacteriana associada a diferentes nichos e avaliar a potencial capacidade de se tornarem futuros inoculantes para a cultura, através do estudo da produção de sustâncias promotoras de crescimento, e avaliar a diversidade genética através do BOXPCR. Foram estudados isolados bacterianos dos nichos rizosfera, raízes, caules e folhas de 13 diferentes genótipos de híbridos de porta-enxertos de citros e avaliada sua capacidade de produção das enzimas amilase, celulase, pectinases (poligalacturonase e pectato liase), solubilização de fosfato inorgânico, produzir AIA via dependente de triptofano, a potencial fixação biológica de nitrogênio e produção de sideróforos. Os dados foram analisados através do teste estatistico não-paramétrico de Kruskal-Wallis e Wilcoxon utilizando o software R. Os resultados demostraram que bactérias associadas a plantas de citros apresentam grande potencial e que a densidade populacional varia em relação ao nicho e ao hospederio. Dos 183 isolados obtidos, 80% do isolados foram positivos para a produção de AIA, produzindo até 85μg.mL-1, 55,7% produziram um ou mais tipos de enzimas, 48% solubilizaram fosfato, 37% foram positivos para fixação de nitrogênio e 60% para a produção de sideróforos. Confirmando o potencial para serem potenciais inoculantes da cultura.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-04-10T17:01:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Iasmim Lucas da Silva.pdf: 2916095 bytes, checksum: c23cab722c74173251220dd40ba0f0cc (MD5) / Made available in DSpace on 2017-04-10T17:01:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Iasmim Lucas da Silva.pdf: 2916095 bytes, checksum: c23cab722c74173251220dd40ba0f0cc (MD5) Previous issue date: 2016-07-28 / Facepe / A L-asparaginase (E.C. 3.5.1.1) é essencial para um curso normal do ciclo celular, estando presente em bactérias, fungos, animais e plantas e não é encontrada em seres humanos. Importante para o tratamento da leucemia linfoblástica aguda dentre outras doenças leucêmicas e a doença de Hodgkin. As Pseudomonas são ocorrentes no solo e água, apresentam a capacidade de produzir enzimas, como é o caso da L-asparaginase do Tipo I e II. Com isso, o objetivo deste trabalho foi obter melhores parâmetros para otimização, purificação parcialmente da L-asparaginase excretada por Pseudomonas sp. isolada da rizosfera da Caesalpinia. pyramidalis. No presente estudo visando otimizar a produção do complexo de L-asparaginase foi realizado um planejamento experimental central composto (23) com 4 pontos centrais, sendo as variáveis pH (4, 7 e 10), temperatura (30°C, 40°C e 50°C) e concentração da L-asparagina (0,2%, 0,5% e 0,8%) estudadas. Posteriormente realizou-se a análise estatística das variáveis e a análise da variância (ANOVA) dos resultados obtidos que foram avaliados através do programa Statistica, versão 7.0 (StatSoft Co., USA). Em seguida, realizou-se a purificação da enzima L-asparaginase excretada por Pseudomonas sp. através da técnica de cromatografia de troca iônica e uma eletroforese SDS-PAGE. Posteriormente, foi realizada a caracterização da enzima purificada, avaliando os efeito e estabilidades em diferentes valores de pH e temperaturas, por fim a enzima em diferentes substratos e íons. Finalizando foram efetuadas as identificações morfológica e molecular da bactéria, utilizando o Microscópio Eletrônico de Varredura (MEV) e a nível de espécie usou-se o primer 16 S respectivamente. Os resultados obtidos no procedimento de otimização da atividade de L-asparaginase foram obtidos nos ensaios com concentração de L-asparagina a 0,8%, pH 4 e temperatura de 30°C, com valores de atividades 0,5604 U/mL e 0,0632 mg de proteína/mL, respectivamente. Os melhores dados da cromatografia de troca iônica mostraram que das 57 frações coletadas, as de melhor atividade para L-asparaginase, foram as frações de valores 1,1412 U/mL e 1,1417 U/mL, com um rendimento de 0,64% e 18,6 U/mg de atividade específica. Realizou-se uma eletroforese PAGE-SDS para obter o peso molecular da proteína, apresentando como peso molecular de 32 kDa. Na caracterização enzimática, a enzima se mantem estável em pH 7, em relação a temperatura, tivemos a melhor estabilidade 30 °C, a L-asparaginase apresentou melhor efeito nos substratos L-asparagina seguido da L-glutamina e por fim o nitrato de sódio (NaNO3), cloreto de Mercúrio (HgCl2), cloreto de magnésio (MgCl2) e ureia apresentaram os melhores efeitos de íons. Em relação à identificação bacteriana encontrou-se colônias em forma de bastonetes curtos, com longos flagelos e a nível de espécie foi denominada com sendo Pseudomonas aeruginosa. Portanto, os resultados obtidos da L-asparaginase caracterizada, isolada da P. aeruginosa, mostraram resultados significativos e extremamente importantes para a indústria biotecnológica, apresentando assim a viabilidade e confiabilidade do estudo realizado. / L-asparaginase (E.C. 3.5.1.1) is essential for the normal course of cell cycle being present in bacteria, fungi, animals and plants and is not found in humans. Important for the treatment of acute lymphoblastic leukemia among other leukemic disease and Hodgkin's disease. The Pseudomonas are occurring in soil and water, have the capacity to produce enzymes, such as L-asparaginase Type I and II. Thus, the aim of this study was to obtain the best parameters for optimization, purification part of L-asparaginase secreted by Pseudomonas sp. isolated from the rhizosphere of Caesalpinia pyramidalis. In the present study to optimize the production of L-asparaginase complex was performed a central experimental design compound (23) with four central points and the variables pH (4, 7 and 10), temperature (30°C, 40°C and 50°C) and concentration of L-asparagine (0,2%, 0,5% and 0,8%) studied. Later there was the statistical analysis of the variables and analysis of variance (ANOVA) of the results that were evaluated through Statistica, version 7.0 (StatSoft Co., USA). Then there was the purification of L-asparaginase enzyme secreted by Pseudomonas sp. by ion exchange chromatography technique and SDS-PAGE electrophoresis. Afterwards, the characterization of the purified enzyme was carried out to evaluate the effect and stability at different pH values and temperatures, and finally the enzyme on different substrates and ions. Finalizing were made the morphological and molecular identification of bacteria using the Scanning Electron Microscope (SEM) and the kind of level we used the primer 16S respectively. The results of the optimization procedure for L-asparaginase activity in the assays were obtained with a concentration of L-asparagine to 0,8%, pH 4 and 30°C, with activity values 0,5604 U/mL and 0,0632 mg of protein/mL, respectively. The best data ionic exchange chromatography showed that the 57 collected fractions, the best activity to L-asparaginase were fractions values 1,1412 U/mL and 1,1417 U/mL, with a yield of 0,64 % and 18,6 U/mg specific activity. We performed a SDS-PAGE electrophoresis for the molecular weight of the protein, presenting as molecular weight of 32 kDa. In the enzyme characterization, the enzyme is stable at pH 7 with respect to temperature had better 30 °C stability, L-asparaginase had a better effect on the L-asparagine substrate followed by L-glutamine and finally sodium nitrate (NaNO3), mercury chloride (HgCl2), magnesium chloride (MgCl2) and urea showed the best effects ions. Regarding the bacterial identification met colonies in the form of short rods with flagella long and species level was named as being Pseudomonas aeruginosa. Therefore, the results of characterized L-asparaginase, isolated from P. aeruginosa, showed significant results and extremely important for the biotechnology industry, thus presenting the viability and reliability of the study performed.

Made available in DSpace on 2015-04-20T12:31:19Z (GMT). No. of bitstreams: 1 Rogerio Sebastiao Correa da Costa.pdf: 1343873 bytes, checksum: 23be9ef21982a872728d073af4a7e99c (MD5) Previous issue date: 2010-12-10 / The deforested areas in the Amazonia occupy more than 70 million hectares, the majority in advanced stage of degradation. Allied to this, the majority of the soils present high acidity and high aluminum saturation, which limits its uses in the regional agriculture. An efficient alternative for soil utilization is the use of soil microorganisms aiming at a better exploitation of the nutrients for the plants. The arbuscular mycorrhizal fungi (AMF) increase the capacity of the plant to absorb water and nutrients of the soil favoring its nutrition. The Agroforestry Systems have for base, to approach the structure and dynamics of a natural forest. The objective of this study was to evaluate the mycorrhizal interactions in the plant rhizosphere of two Agroforestry Systems. The interactions among chemical and physical characteristics of the soil and leaves nutrients with the mycorrhizal variable in component species of two Agroforestry Systems (AFs) were analyzed at distinct times and localities. Roots colonization by arbuscular mycorrhizae fungi ranged from 6.8 to 99.6% on plants of AFS in the Brasileirinho community and 0.4 to 52.4% in the AFS in the São Miguel community. Hyphae predominated in the roots of all the species, with 80 to 100% of the observed fungal structures, in both the AFs. The arbuscules, in the Agroforestry System of the Brasileirinho, were the mycorrhizal structure that more was correlated with rhizospheric and leaves nutrients cntents. The rhizospheric pH, in both the AFs, was the component of the soil that more influenced the mycorrhiza variables, correlating in such a way positively as negative with the mycorrhizal structures. The Fe in the rhizospheric soil, in both the AFSs, was the element that more influenced the nutrients-mycorrhiza relations in all the species. The leaves nutrients that were more related with the AMF were the Mg and K in the AF of the Brasileirinho and P and Ca in the AFS of São Miguel / As áreas desmatadas na Amazônia ocupam mais de 70 milhões de hectares, a maioria em estádios avançados de degradação. Aliado a isso, maioria dos solos apresenta acidez elevada e alta saturação com alumínio, o que limita os seus usos na agricultura regional. Uma alternativa eficiente para o uso dos solos é a utilização de microrganismos do solo visando um melhor aproveitamento dos nutrientes pelas plantas. Os fungos micorrízicos arbusculares aumentam a capacidade da planta de absorver nutrientes do solo e água, favorecendo sua nutrição. Os Sistemas Agroflorestais tem por base se aproximar da estrutura e dinâmica de uma floresta natural. O objetivo deste trabalho foi avaliar as interações micorrízicas na rizosfera das plantas de dois Sistemas Agroflorestais. Foram analisadas as interações entre as características químicas e físicas do solo e foliares com as variáveis micorrízicas em espécies componentes de dois Sistemas Agroflorestais, em épocas e localidades distintas. A colonização radicular por fungos micorrízicos arbusculares variou de 6,8 a 99,6 % nas plantas do SAF da comunidade do Brasileirinho e de 0,4 a 52,4% no SAF da comunidade de São Miguel. As hifas predominaram nas raízes de todas as espécies, respondendo entre 80 a 100 % das estruturas fúngicas observadas, em ambos os SAFs. Os arbúsculos, no SAF do Brasileirinho, foi a estrutura micorrízica que mais se correlacionou com os teores de nutrientes rizosféricos e foliares. O pH rizosférico, em ambos os SAFs, foi o componente do solo que mais influenciou as variáveis micorrízicas, correlacionando tanto positivamente como negativamente com a estruturas micorrízicas. O Fe presente no solo rizosférico, em ambos os SAF, foi o elemento que mais influenciou as relações nutrientes-micorrizas em todas as espécies. Os nutrientes foliares que mais se relacionaram com os FMA foram os teores de magnésio e potássio no SAF do Brasileirinho e fósforo e cálcio no SAF de São Miguel

A Araucaria angustifolia é uma espécie vegetal de vital importância para a manutenção da Floresta Ombrófila Mista (Floresta de Araucária), ecossistema no qual ocorre naturalmente. A araucária apresenta elevado valor sócio-econômico e ambiental e, devido à intensa exploração predatória a que foi submetida, hoje se encontra criticamente ameaçada de extinção. A destruição dessa preciosa espécie vegetal ocasionaria graves consequências ao ecossistema, que poderiam envolver a redução da diversidade de inúmeros animais, plantas e micro-organismos, os quais estão intimamente associados e apresentam grande dependência de A. angustifolia. Pesquisas envolvendo particularmente a caracterização de rizobactérias promotoras do crescimento de plantas (RPCP), nunca foram realizadas em A. angustifolia. Assim, o objetivo do presente estudo foi realizar o isolamento, a seleção e a caracterização de rizobactérias com potencial de promoção do crescimento e biocontrole de fungos fitopatogênicos em A. angustifolia. Os isolados obtidos foram selecionados quanto à capacidade para a promoção do crescimento por meio da produção de ácido indolacético (AIA), solubilização de fosfato inorgânico, produção de fosfatases, fixação assimbiótica de N2, este último através da análise de acúmulo total de nitrogênio em meio de cultura e análise de redução do acetileno (ARA). Também foram selecionadas rizobactérias através da avaliação de mecanismos indiretos de ação, como produção de sideróforos e antagonismo a Fusarium oxysporum, Cylindrocladium candelabrum e C. pteridis, fitopatógenos de espécies arbóreas. Os isolados mais promissores foram caracterizados através da análise do perfil de ácidos graxos da membrana celular (FAME), BOX-PCR e sequenciamento parcial do gene 16S rRNA. Foram isoladas 97 rizobactérias, sendo submetidas aos testes fenotípicos. Todos os isolados apresentaram ao menos uma característica positiva para os mecanismos de promoção do crescimento avaliados, excetuando-se a produção de quitinases. Dentre os isolados obtidos, 18 produziram AIA, 27 foram capazes de solubilizar fosfato inorgânico, 37 foram positivos para sideróforos e 83 foram hábeis produtores de fosfatases. Com relação à fixação assimbiótica de N2, 20 isolados foram positivos para a formação de película em meio de cultura livre de nitrogênio, no entanto, nenhum diferiu significativamente do controle, sem nitrogênio, quando analisado o acúmulo deste elemento em meio de cultura. Por outro lado, 3 isolados foram capazes de reduzir acetileno a etileno. Quarenta e cinco isolados formadores de endósporos apresentaram antagonismo a F. oxysporum. Os isolados B4, B15, B27, B35, B36, B42 e RISP2, caracterizados como Bacillus spp., destacaram-se como bons antagonistas aos fitopatógenos avaliados. Também foi verificada a presença de estirpes com múltiplos mecanismos de ação. A análise genotípica dos isolados mais promissores através da técnica de BOX-PCR apresentou a formação de dois grandes grupos, expressando nitidamente o agrupamento dos isolados formadores de endósporo. A técnica FAME e o sequenciamento do gene 16s rRNA concordaram em caracterizar os melhores isolados como pertencentes às famílias Bacillaceae (9 isolados), Enterobacteriaceae (11) e Pseudomonadaceae (1). Até onde sabemos, este é o primeiro estudo a relacionar a espécie Ewingella americana a diversos mecanismos de promoção do crescimento. Osresultados obtidos demonstram grande potencial de aplicabilidade dessas rizobactérias como biofertilizantes e no controle biológico de fitopatógenos em A. angustifolia. / Araucaria angustifolia is a plant species of vital importance for the maintenance of the Mixed Ombrophylous Forest (Araucaria Forest) ecosystem in which it occurs naturally. Araucaria has a high socio-economic and environmental value, and due to its intense predatory exploitation, it is critically endangered. The destruction of this precious plant species would cause serious consequences to the ecosystem, which could involve a reduction of the diversity of many animals, plants and microorganisms, which are closely associated and reveal a great dependence on A. angustifolia. Studies involving plant growth promoting rhizobacteria (PGPR) and their characterization have never before been performed in A. angustifolia. The objective of this study was to isolate, select and characterize rhizobacteria with potential for growth promotion and biocontrol of plant pathogenic fungi in A. angustifolia. The isolates were selected by means of their potential to produce indole acetic acid (IAA), to solubilize inorganic phosphate, to produce phosphatase, and for asymbiotic N2 fixation. The latter was estimated by analyzing the accumulation of nitrogen in the culture medium and by the acetylene reduction assay (ARA). Rhizobacteria were also selected through the evaluation of indirect action mechanisms such as siderophore production and antagonism to Fusarium oxysporum, Cylindrocladium candelabrum and C. pteridis, all pathogens of trees. The most promising isolates were characterized by analyzing the fatty acid methyl ester (FAME), by BOX-PCR and partial sequencing of the 16S rRNA gene. Ninety seven rhizobacteria were isolated and subjected to the phenotypic tests. All isolates presented at least one positive feature, characterizing them as potential PGPR, except for the production of chitinases. Among these isolates, 18 produced IAA, 27 were able to solubilize inorganic phosphate, 37 were positive for siderophores and 83 were phosphatases producers. Regarding the asymbiotic N2 fixation, 20 isolates were effective in the pellicle formation in nitrogen free culture medium, however, none differed significantly from the control, when analyzed for the accumulation of N in the culture medium. Furthermore, three isolates were able to reduce acetylene to ethylene. Forty five isolates were endospore formers and antagonistic to F. oxysporum. The isolates B4, B15, B27, B35, B36, B42 and RISP2, characterized as Bacillus spp. stood out as good antagonists of the evaluated plant pathogens. We also observed the presence of strains with multiple mechanisms of action. Genotypic analysis of the most promising isolates by the BOX-PCR technique showed the formation of two major groups, expressing clearly the grouping of isolates forming endospores. Both techniques, FAME and PCR, resulted in the same grouping of the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the bacterium Ewingella americana among the PGPR. The results demonstrate a potential application of these rhizobacteria as biofertilizers and for biological control of plant pathogens in A. angustifolia.

Os fungos representam cerca de 75% da biomassa microbiana em áreas florestais, desempenhando funções importantes, desde a mineralização dos resíduos orgânicos até a disponibilização de nutrientes para plantas por meio das associações micorrízicas, o que influencia a ciclagem de nutrientes e, consequentemente, o crescimento das árvores. O objetivo desse trabalho foi avaliar a comunidade de fungos do solo, da rizosfera e do sistema radicular de Eucalyptus grandis e Acacia mangium plantados em monocultivos e em consórcio, e encontrar respostas para os padrões observados por meio da correlação com os atributos físicos, químicos, biológicos e a profundidade do solo. A coleta das amostras foi realizada na Estação Experimental de Ciências Florestais de Itatinga, em 2016, quando as plantas estavam com 2 anos de idade. Foram coletadas amostras em quatro tratamentos: monoculturas de E. grandis e de A. mangium e consórcios de E. grandis e de A. mangium, nos quais foram construídas trincheiras para coleta das amostras nas camadas de 0-10, 10-20, 20-50 e 50-100 cm de profundidade. Foram caracterizados os atributos físicos e biológicos do solo e os atributos químicos do solo, da rizosfera e das raízes. Para a avaliação micorrízica, foi quantificado o número de esporos de fungos micorrízicos arbusculares (FMA) e as taxas de colonização radicular por FMA e por fungos ectomicorrízicos. Foi avaliada a morfologia das estruturas das micorrizas arbusculares e ectomicorriza (ECM). A estrutura da comunidade de fungos do solo e da rizosfera foi avaliada por meio da técnica de Terminal restriction fragment length polymorphism (T-RFLP). Para isso, o DNA foi amplificado utilizando os primers ITS1f-FAM e ITS4 e a restrição dos fragmentos foi realizada com a enzima HaeIII. A abundância de cópias do gene ITS do solo e da rizosfera foi quantificada por PCR quantitativo (qPCR), utilizando os primers ITS1f e 5.8s. Os atributos físicos, químicos e biológicos tiveram poucas variações entre os tratamentos avaliados, sendo as maiores diferenças encontradas entre as profundidades. O número de esporos (<29) e as taxas de colonização micorrízica (<48%) foram baixos em todos os tratamentos, e se reduziram com o aumento da profundidade. As plantas de A. mangium não formaram micorrizas arbusculares. Nas raízes de E. grandis, não houve a formação de arbúsculos, mas foi verificada a presença de hifas enroladas (hyphal coils), estrutura de micorriza do tipo Paris. A anatomia das ECM confirmou a colonização destes fungos nas raízes das plantas estudadas. O qPCR mostrou maior abundância de genes ITS na rizosfera em relação ao solo, assim como nas camadas superficiais (0-10 cm) em relação às mais profundas (10 cm abaixo). A Análise de Coordenadas Principais revelou diferenças na estrutura das comunidades de fungos nos tratamentos estudados, principalmente para a região da rizosfera, diferenciando o perfil de fungos do monocultivo de E. grandis dos demais tratamentos, assim como a influência da A. mangium na estruturação da comunidade. A análise de redundância mostrou a influência de alguns atributos químicos nas taxas de colonização e estruturação da comunidade. Dessa forma, conclui-se que em sistema de consórcio, uma espécie de planta parece ser mais influente do que a outra na estruturação da comunidade de fungos e essa influência é mais evidente na rizosfera. Além disso, os atributos químicos são fatores importantes na organização da comunidade fúngica. / The fungi represent about 75% of the microbial biomass in forest areas, performing important functions, from the mineralization of the organic residues to the availability of nutrients to plants through mycorrhizal associations, which influences the nutrient cycling and, consequently, the growth of trees. The objective of this work was to evaluate the community of fungi of the soil, rhizosphere and root system of Eucalyptus grandis and Acacia mangium planted in monocultures and consortium, and to find explanations for the observed patterns through the correlation with physical and chemical soil attributes and soil depth. The samples were collected at the Experimental Station of Forest Sciences of Itatinga in 2016, when the plants were 2 years old. Samples were collected in four treatments: monocultures of E. grandis and A. mangium and consortia of E. grandis and A. mangium, in which trenches were constructed to collect samples in the 0-10, 10-20, 20 -50 and 50-100 cm deep. The physical and biological attributes of the soil and the chemical attributes of soil, rhizosphere and roots were characterized. For the mycorrhizal evaluation, the number of spores of arbuscular mycorrhizal fungi (AMF) and the rates of root colonization by AMF and ectomycorrhizal fungi were quantified. The morphology of arbuscular mycorrhizal and ectomycorrhizal (ECM) structures was evaluated. The structure of the soil and rhizosphere fungi community by was evaluated by the technique of Terminal restriction fragment length polymorphism (T-RFLP). For this, the DNA was amplified using primers ITS1f-FAM and ITS4 and restriction of the fragments was performed with the enzyme HaeIII. The abundance of ITS gene copies of soil and rhizosphere was quantified by quantitative PCR (qPCR), using primers ITS1f and 5.8s. The physical, chemical and biological attributes had few variations among the evaluated treatments, being the greatest differences found between the depths. The number of spores (<29) and mycorrhizal colonization rates (<48%) were low in all treatments, and reduced with increasing depth. A. mangium plants did not form FMA. In the roots of E. grandis, there was no formation of arbuscules, but we found the presence of hyphal coils, mycorrhizal structures of the Paris type. The anatomy of the ECM confirmed the colonization of these fungi in the roots of the studied plants. The qPCR showed higher abundance of ITS genes in the rhizosphere in relation to the soil, as well as in the superficial layers (0-10 cm) in relation to the deeper ones (10 cm below). The Principal Coordinates Analysis revealed differences in the structure of the fungal communities in the treatments studied, especially for the rhizosphere region, differentiating the fungal profile of the E. grandis monoculture from the other treatments, as well as the influence of A. mangium on the structure of the community. The redundancy analysis showed the influence of some chemical soil attributes on the rates of colonization and community structuring. Thus, it is concluded that in a consortium system, one plant species seems to be more influential than the other in structuring the fungal community, and this influence is more evident in the rhizosphere. In addition, chemical attributes are important factors in the organization of the fungal community.

A cultura de cana-de-açúcar ocupa posição de destaque nos cenários nacional e internacional devido principalmente a produção de etanol como fonte de energia renovável e menos nociva ao ambiente. Entretanto um dos obstáculos à produtividade é a ocorrência de várias doenças dentre elas a escaldadura das folhas causada por Xanthomonas albilineans. Bactérias endofíticas e rizosféricas pertencentes ao gênero Burkholderia tem sido isoladas com alta frequência em diferentes culturas como, por exemplo, a cana-de-açúcar. Nas últimas décadas, estas bactérias têm recebido especial atenção devido ao seu potencial como promotoras de crescimento vegetal, como agentes de biorremediação, mas muito pouco é explorado quanto ao potencial biotecnológico dessas bactérias como agentes de biocontrole de doenças. Visto que essas bactérias vivem em um ambiente altamente competitivo e sujeito a flutuações ambientais, representam uma fonte altamente significativa de metabólitos secundários bioativos, como as bacteriocinas sintetizadas ribossomicamente e outros peptídeos não ribossomais. Portanto, os objetivos principais deste trabalho foram determinar a frequência de linhagens de Burkholderia spp. endofíticas e da rizosfera de cana-deaçúcar capazes de produzir bacteriocinas e outros metabólitos secundários e por meio de mutagênese aleatória por transposon, identificar genes associados a produção desses metabólitos. Os resultados de caracterização permitiram concluir que as bactérias endofíticas e rizosféricas pertencentes ao gênero Burkholderia avaliadas apresentaram grande potencial em produzir metabólitos com atividade antibacteriana in vitro; sendo capazes de controlar X. albilineans importante patógeno da cultura de cana-de-açúcar. Para uma das linhagens foi obtida uma biblioteca de mutantes, a qual foi parcialmente caracterizada quanto à alteração da atividade antibacteriana. Foram identificados doze mutantes que apresentaram perda da atividade antibacteriana. A análise das sequências flanqueadoras do transposon para os doze mutantes permitiu a identificação de genes associados a produção de bacteriocinas, a regulação da expressão gênica, a enzimas possivelmente associadas ao metabolismo secundário, ao metabolismo geral da célula e a proteínas hipotéticas. A identificação e clonagem de tais genes permitirão uma maior compreensão da produção desses compostos e futuras aplicações biotecnológicas. / The sugarcane crop has an important role in international and national scenery mainly because the ethanol production as a sustainable energy source and less harmful to the environment. However one of the obstacles to the productivity is the occurrence of several diseases among them leaf scald caused by Xanthomonas albilineans. Endophytic and rhizospheric bacteria that belong to the Burkholderia genus have been isolated in high frequency in different cultures, such as sugarcane. In the last decades, these bacteria have been receiving attention due their potential as plant growth promoters, bioremediation agents. However, the biotechnological potential of these bacteria as agents of diseases biocontrol is very poorly evaluated. Since these bacteria live in a highly competitive environment and subject to environmental fluctuations, they may represent a highly significant source of bioactive secondary metabolites, as ribosomal synthesized bacteriocins and other nonribosomal peptides. Therefore, the main aim of this work was to determine the frequency of bacteriocin and secondary metabolites production by endophytic and rhizospheric isolates of Burkholderia spp. from sugarcane. Also, the genes associated to synthesis of these metabolites were identified by random mutagenesis based on Tn5 transposon. The results showed that endophytic and rhizospheric Burkholderia spp. present in vitro potential to production of metabolites with antibacterial activity; being inhibited X. albilineans, an important pathogen of sugarcane crops. For one of the Burkholderia strain it was obtained a mutant library, which was partially characterized according to antibacterial activity. Twelve mutants that showed the loss of antibacterial activity were identified and further evaluated. Also, the analysis of the transposon flanking sequences for these mutants indicated that genes associated to the bacteriocin production, regulation of gene expression, enzymes possibly associated to the secondary metabolism, general metabolism of the cell and hypothetical proteins are related to loss of inhibition ability. The identification and cloning of such genes will allow a better understanding of the production of theses compounds and further biotechnological applications.

La pollution des sols par l’Arsenic (As) est une préoccupation environnementale grandissante dans plusieurs pays du monde : États-Unis, Canada, Mexique, Chine, Vietnam et France. L’émergence des techniques écologiques dites « phytoremédiation » constitue à l’heure actuelle une des solutions aux problématiques de décontamination des sols pollués par l’As. Aussi, elle permet de rallier le respect de l’état écologique des sites d’une part et le coût financier de traitement d’autre part. Pour cela, l’appréhension des interactions entre le végétal et l’As à l’interface racine-sol est nécessaire afin d’accélerer la mise en place de la phytoremédiation et de tester son efficacité. D’où l’objectif général de ce travail est d’étudier l’effet de quatres espèces végétales Holcus lanatus, Dittrichia viscosa, Lotus corniculatus et Plantago lanceolata sur la mobilité de l’As dans un sol à contamination géochimique en As (2000 mg kg-1). Notre étude a permis au départ de comparer et de caractériser la tolérance et la capacité accumulatrice des espèces séléctionnées à l’égard de l’As en milieu de culture et en sol contaminé chimiquement par l’As. Ensuite, l’étude de la mobilisation de l’As dans la rhizosphère a été réalisée par deux approches d’étude complémentaires : l’approche « rhizopot-rhizons » et l’approche « tapis racinaire ». L’approche « rhizopot-rhizons » nous a permis d’identifier d’intéressantes corrélations entre l’As et les paramètres suivis (NO3-, SO42- , Ca, Mg, P, pH, COD) tout au long de la période de croissance. Nous avons identifié et caractérisé plusieurs processus ifluençant la mobilisation de l’As dans la rhizosphère (alcalinisation/acidification, compétition anionique…). L’avantage de l’approche « rhizopot-rhizons » réside dans le fait que les plantes se sont développées sans prétraitement du substrat de croissance (contamination artificielle, ajout de solutions nutritives…). Par ailleurs, l’approche « tapis racinaire » a permis d’identifier quelques anions organiques dans les racines de P. lanceolata et de mettre en évidence le rôle du Phosphore dans la mobilisation de l’As dans la rhizosphère. Elle permet d’amplifier l’effet du système racinaire sur la mobilité de l’As dans le sol d’une part, et de récupérer entièrement le système racinaire tout en limitant les artefacts liés à la contamination des racines par le sol d’autre part. / The pollution of soils with Arsenic (As) is an increasing environmental concerns in several countries in the world: USA, Canada, Mexico, China, Vietnam and France. Recently, the emergence of green technologies sush as “phytoremediation” is considered an attractive soil remediation strategy due to its low cost and soil preservation. For this reason, the understanding of interactions between plants and As at the soil-root interface is necessary in order to accelerate phytoremediation implementation and to test its effectiveness. Hence the aim of this work was to study the effect of four plant species Holcus lanatus, Dittrichia viscosa, Plantago lanceolata and Lotus corniculatus on As mobility in a natural As contaminated soil (up to 2000 mg kg-1). First, we compared and characterized the tolerance behaviour of the four selected species to As and their accumulation capacity in hydroponic and in natural soil conditions. Then, the study of As mobilization in the rhizosphere was conducted by using two approaches "rhizopot-rhizons” and "root mat”. We identified by the “rhizopot-rhizons” approach, some interesting correlations between As and the monitored parameters (NO3-, SO42-, Ca, Mg, P, pH, COD) over the vegetative growing period. We observed several processes affecting As mobilization in the rhizosphere (alkalization / acidification, anionic competition ...). The advantage of this approach is that the plants have grown normally without growth substrate pretreatment (artificial contamination, addition of nutrient solutions ...).Whereas, the "root mat" approach allowed the identification of some organic anions in the root system of P. lanceolata and highlighted the role of phosphorus on As mobilization due mainly toto the easy roots collection at the end of experiment.

MSCAGR / Department of Plant Production / Chickpea (Cicer aritienum L.) is an ancient crop that originated in South-Eastern Turkey and belongs to the genus Cicer, tribe Cicereae, and family Fabaceae. Chickpea has the ability to fix atmospheric nitrogen (N) for its growth. However, chickpea productivity not only depends on N2 fixation or dry matter accumulation, but also the effectiveness of nutrient partitioning to seed, a key component to overall yield. There is a dearth of information on the effect of P with rhizobial inoculation in response of nutrients accumulation in the rhizosphere, shoots and grain of chickpea, especially when determined at different growth stages in the African continent. This study contributes knowledge on this crucial aspect which will likely lead to more other similar research reports in other settings. Therefore, the objectives of this study was to evaluate the effect of P fertilizer rates and rhizobial inoculation on yield and nutrients accumulation in two chickpea genotypes. Field experiments were conducted in winter 2017 and 2018 at University of Venda, Thohoyandou and University of Limpopo’s experiment farm, Syferkuil. Treatments consisted of a factorial combination of two rates of P fertilizer (0 and 90 kg P ha-1), two desi chickpea genotypes (ACC1 and ACC5) and two rhizobial inoculation levels (with and without rhizobiam strain). The treatments were laid out in a randomized complete block design (RCBD) and replicated three times on 22 April 2017 and 11 April 2018 (Syferkuil), 13 April 2017 and 29 April 2018 (Thohoyandou). Macronutrients including P, K, Ca, Mg were determined using the citric acid method. The total N concentration were determined by the micro-Kjeldahl method in both soil, shoots and grain. Zn was extracted using a di-ammonium ethylenediaminetetraacetic acid (EDTA) solution. The content of macronutrients (P, K, Ca, Mg, Ca, and Zn) in soil, shoots and grain was determined by first subjected to wet digestion (Mehlich, 1984). From the digest, various elements were read using relevant procedures. P contents was determined colorimetrically using a spectrophotometer. Yield and yield components were assessed at harvest maturity. Genotypes affected the accumulation of mineral elements in rhizosphere soil, shoots, grain and yield. Accession 5 performed better in most of nutrients elements compared to accession 1 in both seasons and sites. Application of phosphorus alone, and in combination with rhizobium inoculation increased the concentration of majority of nutrients in the rhizosphere. When the test accessions were grown at the Syferkuil and Thohoyandou study location in 2017, they showed significant differences in the concentration of N, P and K while Ca, Mg and Zn were similar in the rhizosphere. The concentrations of N, P and K were markedly higher in the rhizosphere of ACC5 compared to ACC1. In fact, the concentration of P was two-fold greater in the rhizosphere of ACC5 than ACC1. Accession 5 exhibited a markedly higher shoot dry weight, number and dry weight of pods, 100-seed weight, grain yield and harvest index compared to ACC1. P plus rhizobium inoculation, P, rhizobium inoculation affected grain yield and yield components of chickpea genotypes. This preliminary finding show that the combination of P and rhizobium inoculation affected the nutrients accumulation in the rhizosphere, shoots, grain, yield and yield components in both locations. Moreover, Thohoyandou had the highest nutrients accumulation on the rhizosphere, shoots, grain, yield and yield components compared to Syferkuil. / NRF

In situ assays of N2 fixation activity, using the acetylene reduction technique, were performed in four successional stages of a Northern Wasatch Mountain subalpine forest ecosystem, elevation 2,800 m. Emphasis was made on rhizosphere fixation in association with Antennaria microphylla and Achillea millefolium. The vegetation period was approximately 100 days. Assays were performed in Saran bags. A defined amount of propane was injected at initiation of the assay and acetylene was generated from CaC2. Samples were analyzed for ethylene and propane. Data were evaluated assuming that the ethylene production was directly proportional to the increase in the ratio of ethylene to propane in the samples. Input of N by soil free-living N2 fixers in the meadow, the aspen, the fir and the spruce was 0.5, 0.3, 0.2 and 0.3 kg N ha-1 y-1, respectively. A higher activity in the presence of plant, as compared to the soil activity, was measured in 10 of the 16 assays, however, the increase was significant at three testings only. This might indicate a contribution by rhizosphere N2 fixation, but to an extent lower than was expected and therefore not detectable with the method used Leakage of gases from the test device was not corrected for in the method used for evaluation of data. This introduces and overestimation of the obtained activities that increases exponentially with a more rapid effusion rate. Correction for effusion from a closed device can be made provided quantitative analyses of the tracer gas. To determine a small difference between enclosures with and without plants the accuracies in the effusion rates must be high. Quantitative analyses were not required for evaluation according to the method used and therefore, the obtained effusion rates have too wide standard deviations for correction of the effusion rate. It was shown that the determined effusion rates with corresponding standard deviations might obscure a low rhizosphere N2 fixation activity. Acetylene reduction assays performed in open devices can not easily be corrected for diffusion of gases. The initial very rapid diffusion from an open device leads to a vastly overestimated acetylene reduction activity, when the diffusion is not corrected for.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21150号 / 農博第2276号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5124(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 北山 兼弘, 教授 小杉 緑子, 教授 本田 与一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM