Submitted by Bruna Rodrigues (bruna92rodrigues@yahoo.com.br) on 2017-10-04T13:31:39Z No. of bitstreams: 1 DissRKR.pdf: 1036463 bytes, checksum: 2c845de1e471e5e42f4a7e8591961fc9 (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-10T20:14:17Z (GMT) No. of bitstreams: 1 DissRKR.pdf: 1036463 bytes, checksum: 2c845de1e471e5e42f4a7e8591961fc9 (MD5) / Approved for entry into archive by Ronildo Prado (producaointelectual.bco@ufscar.br) on 2017-10-10T20:14:25Z (GMT) No. of bitstreams: 1 DissRKR.pdf: 1036463 bytes, checksum: 2c845de1e471e5e42f4a7e8591961fc9 (MD5) / Made available in DSpace on 2017-10-10T20:21:54Z (GMT). No. of bitstreams: 1 DissRKR.pdf: 1036463 bytes, checksum: 2c845de1e471e5e42f4a7e8591961fc9 (MD5) Previous issue date: 2017-05-02 / Não recebi financiamento / Soil is an environment with great microbial diversity. Filamentous fungi and bacteria have been known for decades for the ability to stimulate plant development. Recently, studies have shown that yeasts are also capable of benefiting plants, for example, through the solubilization of inorganic phosphates. This work aims at a better understanding of the ability of rhizospheric yeasts in the solubilization of tricalcium phosphate. It also aims to study the influence of factors (pH, temperature and agitation) in the process. Initially, isolates of corn and sugarcane rhizosphere yeasts were made. The isolates were evaluated for the ability to solubilize tricalcium phosphate in solid medium BDYA. The isolate with the highest solubilization capacity was identified and evaluated for the solubilization capacity of tricalcium phosphate in NBRIP liquid medium at different temperature levels (20, 25 and 30 ° C), initial pH (3, 5 and 7) and agitation (0, 100, 150 and 200 rpm). Solubilization evaluations were also performed at 24-hour intervals, ranging from 1 to 7 days. Six isolates were obtained. All presented a positive result for solubilization. The yeast 6S01 (Torulaspora globosa) had the highest solubilization capacity. After five days, T. globosa solubilized 26 to 35% of the total phosphorus in the NBRIP culture medium. There was no significant difference for any of the environmental parameters tested. Assays performed in periods of 1 to 7 days showed that the yeast solubilized a maximum amount of phosphorus in 48 hours (42%). This makes it interesting for industrial and agricultural applications, since it has a fast solubilization process and can be used in a wide range of conditions. It was possible to understand that a higher cell growth is not related to the efficiency in the solubilization, and that acidification of the medium may be one of the main mechanisms used by yeast in the in vitro solubilization of tricalcium phosphate. / O solo é um ambiente com grande diversidade microbiana. Fungos filamentosos e bactérias são conhecidos há décadas pela capacidade de estimular o desenvolvimento vegetal. Recentemente, trabalhos mostram que leveduras também são capazes de beneficiar plantas, por exemplo, através da solubilização de fosfatos inorgânicos. Esse trabalho visa um maior entendimento da capacidade de leveduras rizosféricas na solubilização de fosfato tricálcico. Também visa o estudo da influência de fatores (pH, temperatura e agitação) no processo. Inicialmente, foi feito um isolamento de leveduras de rizosfera de milho e cana-de-açúcar. Os isolados foram avaliados quanto à capacidade de solubilizar fosfato tricálcico em meio sólido BDYA. O isolado com maior capacidade de solubilização foi identificado e avaliado quanto à capacidade de solubilização de fosfato tricálcico em meio líquido NBRIP, em diferentes níveis de temperatura (20, 25 e 30°C), pH inicial (3, 5 e 7) e agitação (0, 100, 150 e 200 rpm). Também foram feitas avaliações da solubilização em intervalos de 24 horas, de 1 a 7 dias. Foram obtidos seis isolados. Todos apresentaram resultado positivo para solubilização. A levedura 6S01 (Torulaspora globosa) apresentou a maior capacidade de solubilização. Após cinco dias, T. globosa solubilizou 26 a 35% do fósforo total no meio de cultura NBRIP. Não houve diferença significativa para nenhum dos parâmetros ambientais testados. Ensaios realizados em períodos de 1 a 7 dias mostraram que a levedura solubilizou uma quantidade máxima de fósforo em 48 horas (42%). Isso a torna interessante para aplicações industriais e agrícolas, visto que possui rapidez no processo de solubilização e pode ser empregada numa ampla faixa de condições. Foi possível compreender que um maior crescimento celular não está relacionado à eficiência na solubilização, e que a acidificação do meio pode ser um dos principais mecanismos utilizados pela levedura na solubilização in vitro do fosfato tricálcico.

Bacterial volatile organic compounds (VOCs) are signal molecules that may have beneficial roles in the soil-plant-microbiome ecosystem. In this Ph.D. thesis, I aimed to assess and characterize the role of bacterial VOCs in plant tolerance to drought and in the biocontrol of fungal pathogens. I started by studying two root endophytic bacteria isolated from pepper plants cultivated under desert farming conditions. They showed an enhancement of pepper tolerance to drought stress and an amelioration of its physiological status. Moreover, they induced the expression of a vacuolar pyrophosphatase proton pump (V-PPase), implicated in the regulation of the vacuolar osmotic pressure, facilitating water uptake. Besides, the exposure of Arabidopsis thaliana plants, grown under salinity stress, to the volatile 2,3-butanediol, described for its plant growth promotion (PGP) potential, enhanced the plants tolerance to salinity, proving the potential involvement of this volatile in the osmotic stress resistance mechanism. Then, I studied VOCs released by three bacteria associated to healthy rice plants. Their released VOCs mixtures modified the color pattern of Magnaporthe oryzae, the agent of the rice blast disease, and protected rice from the pathogen infection. A significant reduction of melanin production, sporulation and appressoria formation was measured in presence of the bacterial VOCs, without major effects on mycelial proliferation. 1-butanol-3-methyl, one of the nine VOCs co-produced by the studied bacteria, proved its potential of reducing M. oryzae melanin in vitro. In vivo tests confirmed the infection inhibition effects mediated by the rice-bacterial VOCs, with a reduction of 94% of the disease incidence. Lastly, I compared the genomes of the five bacteria considered in the previous experimental studies. The PGP traits and the VOCs pathways identified from the genome analyses confirmed the effects observed with the in vitro and in vivo assays, revealing a complex mode of promotion and protection offered by the studied plant-associated bacteria. In conclusion, plant-associated bacterial VOCs can play potentially important roles in modulating plant drought tolerance and reducing fungal virulence. Such biological resources represent novel tools to counteract the deleterious effects of abiotic and biotic stresses and have the potential to be exploited for sustainable approaches in agriculture.

Mangrove forests are highly productive ecosystems widespread in tropical and subtropical coastlines, with a coverage of 75% of the world’s tropical shorelines. Mangrove plants developed specific physiological and morphological adaptation to thrive in such unique environments. Together with plant adaptations, mangroves develop a tight partnership with microorganisms, mainly bacteria and fungi, that form the so-called mangrove-microbiome. Plant-associated microorganisms are generally recruited by the root system (root tissues and rhizosphere) and the colonization process starts with the release of root-related exudates detected by the surrounding edaphic microorganisms that are attracted in the rhizosphere zone. Then, root surface selects those microorganisms that can enter the tissues as endophytes. The microorganisms recruited belowground can migrate through the plant tissues by using the plant vessels and may colonize the aboveground compartments of the plant. Here, I aimed to evaluate the environmental origin and compartmentalization of the mangrove microbiome. To do this, I sampled bulk sediments, sea water, and mangrove plant compartments (root rhizosphere and endosphere, pneumatophores, shoot, leaves, flowers and propagules) of 20 gray mangrove trees (Avicennia marina L.) across two sites on the Red Sea coast of the King Abdullah University of Science and Technology (KAUST), Saudi Arabia. By high-throughput sequencing of the bacterial 16S rRNA gene, I showed that the bacterial assembly in A. marina plant compartments follows a clear niche partition process in which bacterial communities are actively recruited from the surrounding ecosystem (sediment and sea water) by the root system, and further distributed across the different plant organ and compartments. Moreover, the composition of microbiome detected had many similitudes with others previously described around the world, suggesting that certain bacteria represent a mangrove “core microbiome”. The conservation of microbiome composition, mainly driven by environmental and host selection, that beneficial bacteria provide to the plant and contribute to its growth and fitness by several mechanisms. Thus, the characterization and identification of mangrove microbiome can meliorate our knowledge regarding plant–microbe interactions, as well as put the bases for the development of Nature-based Solution (NBS) to enhance reforestation and rehabilitation of mangrove ecosystems

No description available.

Climate change is expected to increase the frequency of mild winters and warm springs, which can induce premature plant development. This premature development results in a high risk of exposure of young plant tissues to cold stress leading to severe reductions in plant growth and agricultural production. Plants are associated with complex bacterial communities that can activate acclimation processes and positively affect plant performance at low temperatures. Beneficial effects of plant colonization by cold-tolerant bacteria include the modulation of cold-related genes and the reduction in cellular damage under cold stress, but scarce information is available on mechanisms stimulated by bacterial endophytes in tomato plants against cold stress. The aims of this work were i) to analyze the taxonomy and potential functions of plant-associated microbial communities in cold regions, ii) to understand metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress, and iii) to identify possible genomic traits of cold-tolerant endophytic bacteria responsible for plant growth promotion and cold stress mitigation. The first chapter includes an introduction on cold stress and acclimation processes in plants, and the second chapter defines the aims of the project. In the third chapter, the taxonomic and functional characterization of plant-associated microbial communities of alpine, Arctic, and Antarctic regions was reviewed, highlighting the main environmental factors affecting their taxonomic structure. e. The key findings of this chapter are the functional roles of microbial communities in plant growth and survival in cold environments, and the suggestion of potential biotechnological applications of ubiquitous and endemic cold-tolerant microorganisms. In the fourth chapter, metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress were studied by metabolomic analyses, and compounds possibly associated with cold stress mitigation were found. 14 Tomato seeds were inoculated with two bacterial endophytes isolated from Antarctic Colobanthus quitensis plants (Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10) or with Paraburkholderia phytofirmans PsJN, while mock-inoculated seeds were used as control. The metabolic composition of tomato plants was analyzed immediately after cold stress exposure (4°C for seven days) or after two and four days of recovery at 25°C. Under cold stress, the content of malondialdehyde, phenylalanine, ferulic acid, and p-coumaric acid was lower in bacterium-inoculated compared to mock-inoculated plants, indicating a reduction of lipid peroxidation and the stimulation of phenolic compound metabolism. The content of two phenolic compounds, five putative phenylalanine-derived dipeptides, and three further phenylalanine-derived compounds was higher in bacterium-inoculated compared to mock-inoculated samples under cold stress. Thus, the presented work suggests that psychrotolerant endophytic bacteria can reprogram polyphenol metabolism and stimulate the accumulation of secondary metabolites, like 4-hydroxybenzoic and salicylic acid, which are involved in cold stress mitigation, and phenylalanine-derived dipeptides possibly involved in plant stress responses. In the fifth chapter, functional and genomic traits of Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10 were studied. In the framework of the present study, Ewingella sp., Pseudomonas sp., and the bacterial consortium showed plant growth-promoting activity on tomato seedlings at low temperatures. Ammonia was produced by both bacterial isolates and their consortium, while indole-3-acetic acid and proteases were produced by Ewingella sp. and Pseudomonas sp., respectively. Ewingella sp. and Pseudomonas sp. genomes (51.57% and 60.63% guanine-cytosine, 4,148 and 5,983 predicted genes, respectively) encompassed genes related to amino acid metabolism, plant hormone metabolism (auxin, cytokinins, ethylene, and salicylic acid), nitrogen metabolism, lytic activities (amylases, cellulases, and proteases). Traits related to plant growth promotion included genes for iron transport, phosphate metabolism, potassium transport, siderophore metabolism and 15 transport, and zinc transport. Moreover, Ewingella sp. and Pseudomonas sp. encompassed genes related to cold tolerance, such as cold shock and heat shock-related proteins, lipid desaturases, and genes related to polyamine metabolism, proline metabolism, proline and glycine betaine transport, reactive oxygen species detoxification, and trehalose metabolism. Thus, in this chapter, it was discovered that Antarctic cold-tolerant endophytes include multiple genomic and functional traits to survive under cold conditions and some of them can contribute to promote the host plant growth at low temperatures. These findings indicate that plant-associated bacteria of cold regions have a great biotechnological potential to mitigate cold stress in crop plants. In particular, Antarctic bacterial endophytes encompass genomic traits responsible for plant growth promotion and protection against cold stress, and they can mitigate cold stress in tomato plants by a complex reprogramming of plant metabolism. Although further metabolomic and functional studies are required to verify compound annotations and to better clarify the role of phenylalanine-derived compounds and phenylalanine-derived dipeptides in cold stress mitigation, these results provided a better understanding of metabolic changes stimulated by psychrotolerant endophytic bacteria in cold-stressed tomato plants. Thus, the validation of cold stress mitigation activated by psychrotolerant endophytic bacteria under field conditions will pave the way for the further development of endophytic bacterial inoculants as sustainable products to protect crops against cold stress.

Os manguezais são ecossistemas encontrados na transição entre os ambientes terrestre e marinho apresentando uma biodiversidade funcional única e conseqüentemente flora e fauna específica. Por se tratar de um ambiente inóspito e pouco explorado é de grande importância o estudo desta comunidade a fim de se obter isolado com potencial biotecnológico. Sabe-se que tecidos vegetais são habitados por microrganismos denominados endofíticos, cuja interação com a planta hospedeira pode conferir características vantajosas ao mesmo. Este trabalho teve como objetivo estudar a comunidade bacteriana endofítica cultivável de manguezais, além de determinar a produção enzimática e a utilização de isolados na promoção de crescimento vegetal. Para tanto, foram coletadas amostras de ramo das espécies vegetais Rhizophora mangle, Avicenia nitida, Laguncularia racemosa para o isolamento de bactérias endofíticas. Os locais e épocas amostrados foram manguezais do litoral paulista em Bertioga (local com e sem impacto ambiental) e em Cananéia (local considerado preservado) durante o verão e inverno de 2007 e 2008. Foi obtido grande número de isolados em todos os locais e épocas avaliados, porém a análise estatística não apresentou diferença significativa entre as variáveis avaliadas, com exceção da planta L. rancemosa no período do verão em Bertioga impactado a qual apresentou baixa freqüência. Dentre os isolados obtidos selecionou-se para estocagem aproximadamente 1000 isolados os quais foram submetidos a testes enzimáticos in vitro a fim de realizar uma triagem inicial. Destes, 75% apresentaram atividade para pelo menos uma das enzimas avaliadas. Selecionou-se então isolados com atividade enzimática para no mínimo três ou mais enzimas para testes mais específicos chegando a um número de 115 isolados. A identificação dos isolados foi realizada por seqüênciamento parcial do gene 16S rDNA. Dentre os isolados identificados, os gêneros mais freqüentes foram: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas entre outros. Os resultados demonstraram que dos isolados avaliados: 69% são produtores de fosfatase, 69% de protease, 60% de endoglicanase, 58% de lípase, 43% de amilase e 21% de esterase. Além destes resultados, dos 115 isolados avaliados, 35% apresentou a capacidade de sintetizar AIA (ácido indol acético) e 45% a capacidade de fixar nitrogênio atmosférico. Os isolados com os melhores resultados quanto a fixação de nitrogênio e síntese de AIA foram inoculados em plântulas de cana-de-açúcar e monjoleiro. Os experimentos foram desenvolvidos em casa de vegetação e em viveiro respectivamente. As bactérias selecionadas não promoveram o crescimento de cana-de-açúcar, sendo inclusive observado um crescimento superior da testemunha em relação aos tratamentos com inoculação bacteriana. Já com monjoleiro foi observado que o isolado de Enterobacter sp. apresentou aumento na massa da matéria fresca e seca da parte aérea e da raiz. Assim, os dados apresentados demonstraram potencial de aplicação das bactérias endofíticas isoladas dos manguezais brasileiro, tanto na busca de novos compostos, como enzimas, para aplicação industrial, quanto no desenvolvimento de inoculantes visando a promoção de crescimento de espécies vegetais utilizadas na agricultura e reflorestamento / The mangrove is an ecosystem of transition between the terrestrial and marine environments, featuring a unique functional biodiversity, providing consequently a specific flora and fauna. Due to its inhospible characteristics, few study explore its very importance the study of this community to get isolated with biotechnological potencial. It is known that the plant tissues are inhabited by microorganisms known as endophytes and this interaction may confer advantageous for both. The aiming of this work was the evaluation of the potencial of the mangrove endophytic bacteria to enzymatic production as well their potencial to plant growth promotion. The bacterial endophytic isolades were obtained from branches from Rhizophora mangle, Avicenia nitida and Laguncularia rancemosa mangrove species. The vegetal samples were colleted in the mangroves at Bertioga (with and without environmental impact) and Cananeia (a preserved area) during two different time, summer a winter on 2007 and 2008. We obtained large numbers of isolates in all areas and times evaluated, but the statistical analysis showed no significant difference between the variables, except the plant L. rancemosa in the summer, Bertioga impacted area, that presented low frequency of isolation. Arbitrarily, 1000 were stored and all those evaluated to enzymatic production. Of these, 75% presented activity for at least one of the evaluated enzymes. It was selected 115 isolates that showed activity for at least three or more enzymes. The molecular identification of these isolates were performed by partial sequencing of the 16S rDNA. The predominant identified genera were: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erwinia, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas and others. Among the evaluated isolates: 69% produce phosphatase, 69% produce protease, 60% produce endoglucanase, 58% produce lipase, 43% produce amylase and 21% produce esterase. Even, 35% and 45% are able to produce IAA and nitrogen fixation respectively. Isolates that showed good characteristics to IAA and nitrogen fixation were inoculated with sugar cane and monjoleiro (Acacia polyphylla) seedlings. All experiments were conducted in greenhouse. None selected bacteria promoted the sugar cane growther, being the development of control treatment better than bacterial inoculation. It was observed an increase in their fresh and dried biomass. Thus, the results show the huge potential of endophytic bacteria from mangrove to the discovery news compounds, as enzymes, to industrial application and to the development of new inoculants aiming the plant growth promotion of agriculture and reforestation

O uso de fertilizantes minerais é de grande importância para que a cultura atinja o seu potencial produtivo e torne a atividade de produção viável economicamente, no entanto o uso excessivo é danoso ao meio ambiente, trazendo riscos à saúde humana e à biodiversidade local. A utilização de bactérias promotoras de crescimento de plantas (BPCP) tem se mostrado uma alternativa promissora e sustentável, visando melhorar a produtividade e reduzir o uso de fertilizantes. Essas bactérias colonizam a rizosfera e tecidos internos da planta e são capazes de estimular o desenvolvimento e sanidade de sua hospedeira através de mecanismos como disponibilização de nutrientes, produção de fitohormônios e controle de patógenos. Este estudo teve por objetivo avaliar o efeito da inoculação de três linhagens de BPCP em milho e o impacto causado na comunidade bacteriana associada à cultura. As linhagens utilizadas foram Burkholderia ambifaria RZ2MS16, Bacillus sp. RZ2MS9, ambas isoladas da rizosfera de guaranazeiro, e Azospirillum brasilense Ab-v5, um inoculante comercial. Primeiramente, foi realizado um ensaio de antibiose entre RZ2MS9 e Ab-v5, constatando não haver inibição. Após, um experimento de promoção de crescimento em condições de campo, foi realizado, com plantas de milho inoculadas com: (i) RZ2MS16; (ii) RZ2MS16 e Ab-v5; (iii) Ab-v5; (iv) RZ2MS9; (v) RZ2MS9 e Ab-v5; e (vi) tratamento controle. As sementes foram inoculadas e, 60 dias após o plantio, a altura da planta, altura até a inserção da espiga e o diâmetro do colmo foram medidos. A inoculação com Ab-v5 e a coinoculação de RZ2MS9 com Ab-v5 promoveram o incremento de 3% em altura da planta, além disso, esse consórcio promoveu incremento de 9% no diâmetro do colmo, todos comparados ao tratamento controle. Usando o DNA total da folha e raíz do milho, o fragmento 16S rRNA bacteriano foi sequenciado, através da plataforma Ion Torrent, para avaliar o efeito da inoculação na comunidade bacteriana associada à ambos os tecidos. A inoculação foi capaz de modular a comunidade bacteriana associada à folha, com a análise de coordenadas principais (PCoA) explicando 39,51% da variação. Não foi observada modulação na comunidade bacteriana associada à raiz. Foi observada diferença na estrutura da comunidade bacteriana quando ambos os nichos foram comparados, independente de inoculação, com a PCoA explicando 80,97% dessa variação. Assim observa-se que estudos dessa natureza são de grande importância para o melhor entendimento da interação entre as BPCP e a comunidade bacteirana associada à planta hospedeira, e dos mecanismos que levam ao desenvolvimento da cultura. / The use of mineral fertilizers is of great importance to the crop reaches its potential yield and become the production activity economically feasible, however, its excessive use is harmful to the environment, and brings risk to human health and local biodiversity. The use of plant growth-promoting bacteria (PGPB) has been shown as a promising and sustainable alternative, aiming to improve productivity and reduce fertilizer use. These bacteria colonize the rhizosphere and plant internal tissues and are able to stimulate their host development and health through mechanisms such as nutrient availability, phytohormone production and pathogen control. This study aimed to evaluate the inoculation effect of three PGPB strains in maize and the impact on associated bacterial community. The strains inoculated were Burkholderia ambifaria RZ2MS16, Bacillus sp. RZ2MS9, both isolated from guarana rhizosphere, and Azospirillum brasilense Ab-v5, a commercial inoculant. First, an anthibiosis assay was conducted between RZ2MS9 and Ab-v5 strains, showing no inhibition. Then, a growth-promotion assay was performed under field conditions, with maize plants inoculated with: (i) RZ2MS16; (ii) RZ2MS16 and Ab-v5; (iii) Ab-v5; (iv) RZ2MS9; (v) RZ2MS9 and Ab-v5; and (vi) control. The seeds were inoculated and, 60 days after sowing, the plant height, height to the cob insertion and stem diameter were measured. The inoculation with Ab-v5 and the co-inoculation with RZ2MS9 plus Ab-v5 increased the plant height in 3%, furthermore, the co-inoculation increased stem diameter in 9%, all compared to control. Using total DNA of maize\'s leaf and root, bacterial 16S rRNA fragment was sequenced by Ion Torrent platform, to evaluate the effect of inoculation in associated bacterial community of both tissues. The inoculation was able to modulate the leaf bacterial community, with principal coordinates analysis (PCoA) explaining 39.51% of variation. It was not found modulation on root\'s bacterial community. Difference in the bacterial community structure was observed when both niches were compared, regardless inoculation, with PCoA explaining 80.97% of this variation. Therefore, it is noted that studies of this nature are of great importance for a better understanding of the interaction between PGPB and bacterial community associated to the host plant and mechanisms leading to crop development.

Os manguezais são ecossistemas encontrados na transição entre os ambientes terrestre e marinho apresentando uma biodiversidade funcional única e conseqüentemente flora e fauna específica. Por se tratar de um ambiente inóspito e pouco explorado é de grande importância o estudo desta comunidade a fim de se obter isolado com potencial biotecnológico. Sabe-se que tecidos vegetais são habitados por microrganismos denominados endofíticos, cuja interação com a planta hospedeira pode conferir características vantajosas ao mesmo. Este trabalho teve como objetivo estudar a comunidade bacteriana endofítica cultivável de manguezais, além de determinar a produção enzimática e a utilização de isolados na promoção de crescimento vegetal. Para tanto, foram coletadas amostras de ramo das espécies vegetais Rhizophora mangle, Avicenia nitida, Laguncularia racemosa para o isolamento de bactérias endofíticas. Os locais e épocas amostrados foram manguezais do litoral paulista em Bertioga (local com e sem impacto ambiental) e em Cananéia (local considerado preservado) durante o verão e inverno de 2007 e 2008. Foi obtido grande número de isolados em todos os locais e épocas avaliados, porém a análise estatística não apresentou diferença significativa entre as variáveis avaliadas, com exceção da planta L. rancemosa no período do verão em Bertioga impactado a qual apresentou baixa freqüência. Dentre os isolados obtidos selecionou-se para estocagem aproximadamente 1000 isolados os quais foram submetidos a testes enzimáticos in vitro a fim de realizar uma triagem inicial. Destes, 75% apresentaram atividade para pelo menos uma das enzimas avaliadas. Selecionou-se então isolados com atividade enzimática para no mínimo três ou mais enzimas para testes mais específicos chegando a um número de 115 isolados. A identificação dos isolados foi realizada por seqüênciamento parcial do gene 16S rDNA. Dentre os isolados identificados, os gêneros mais freqüentes foram: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas entre outros. Os resultados demonstraram que dos isolados avaliados: 69% são produtores de fosfatase, 69% de protease, 60% de endoglicanase, 58% de lípase, 43% de amilase e 21% de esterase. Além destes resultados, dos 115 isolados avaliados, 35% apresentou a capacidade de sintetizar AIA (ácido indol acético) e 45% a capacidade de fixar nitrogênio atmosférico. Os isolados com os melhores resultados quanto a fixação de nitrogênio e síntese de AIA foram inoculados em plântulas de cana-de-açúcar e monjoleiro. Os experimentos foram desenvolvidos em casa de vegetação e em viveiro respectivamente. As bactérias selecionadas não promoveram o crescimento de cana-de-açúcar, sendo inclusive observado um crescimento superior da testemunha em relação aos tratamentos com inoculação bacteriana. Já com monjoleiro foi observado que o isolado de Enterobacter sp. apresentou aumento na massa da matéria fresca e seca da parte aérea e da raiz. Assim, os dados apresentados demonstraram potencial de aplicação das bactérias endofíticas isoladas dos manguezais brasileiro, tanto na busca de novos compostos, como enzimas, para aplicação industrial, quanto no desenvolvimento de inoculantes visando a promoção de crescimento de espécies vegetais utilizadas na agricultura e reflorestamento / The mangrove is an ecosystem of transition between the terrestrial and marine environments, featuring a unique functional biodiversity, providing consequently a specific flora and fauna. Due to its inhospible characteristics, few study explore its very importance the study of this community to get isolated with biotechnological potencial. It is known that the plant tissues are inhabited by microorganisms known as endophytes and this interaction may confer advantageous for both. The aiming of this work was the evaluation of the potencial of the mangrove endophytic bacteria to enzymatic production as well their potencial to plant growth promotion. The bacterial endophytic isolades were obtained from branches from Rhizophora mangle, Avicenia nitida and Laguncularia rancemosa mangrove species. The vegetal samples were colleted in the mangroves at Bertioga (with and without environmental impact) and Cananeia (a preserved area) during two different time, summer a winter on 2007 and 2008. We obtained large numbers of isolates in all areas and times evaluated, but the statistical analysis showed no significant difference between the variables, except the plant L. rancemosa in the summer, Bertioga impacted area, that presented low frequency of isolation. Arbitrarily, 1000 were stored and all those evaluated to enzymatic production. Of these, 75% presented activity for at least one of the evaluated enzymes. It was selected 115 isolates that showed activity for at least three or more enzymes. The molecular identification of these isolates were performed by partial sequencing of the 16S rDNA. The predominant identified genera were: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erwinia, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas and others. Among the evaluated isolates: 69% produce phosphatase, 69% produce protease, 60% produce endoglucanase, 58% produce lipase, 43% produce amylase and 21% produce esterase. Even, 35% and 45% are able to produce IAA and nitrogen fixation respectively. Isolates that showed good characteristics to IAA and nitrogen fixation were inoculated with sugar cane and monjoleiro (Acacia polyphylla) seedlings. All experiments were conducted in greenhouse. None selected bacteria promoted the sugar cane growther, being the development of control treatment better than bacterial inoculation. It was observed an increase in their fresh and dried biomass. Thus, the results show the huge potential of endophytic bacteria from mangrove to the discovery news compounds, as enzymes, to industrial application and to the development of new inoculants aiming the plant growth promotion of agriculture and reforestation

O uso de fertilizantes minerais é de grande importância para que a cultura atinja o seu potencial produtivo e torne a atividade de produção viável economicamente, no entanto o uso excessivo é danoso ao meio ambiente, trazendo riscos à saúde humana e à biodiversidade local. A utilização de bactérias promotoras de crescimento de plantas (BPCP) tem se mostrado uma alternativa promissora e sustentável, visando melhorar a produtividade e reduzir o uso de fertilizantes. Essas bactérias colonizam a rizosfera e tecidos internos da planta e são capazes de estimular o desenvolvimento e sanidade de sua hospedeira através de mecanismos como disponibilização de nutrientes, produção de fitohormônios e controle de patógenos. Este estudo teve por objetivo avaliar o efeito da inoculação de três linhagens de BPCP em milho e o impacto causado na comunidade bacteriana associada à cultura. As linhagens utilizadas foram Burkholderia ambifaria RZ2MS16, Bacillus sp. RZ2MS9, ambas isoladas da rizosfera de guaranazeiro, e Azospirillum brasilense Ab-v5, um inoculante comercial. Primeiramente, foi realizado um ensaio de antibiose entre RZ2MS9 e Ab-v5, constatando não haver inibição. Após, um experimento de promoção de crescimento em condições de campo, foi realizado, com plantas de milho inoculadas com: (i) RZ2MS16; (ii) RZ2MS16 e Ab-v5; (iii) Ab-v5; (iv) RZ2MS9; (v) RZ2MS9 e Ab-v5; e (vi) tratamento controle. As sementes foram inoculadas e, 60 dias após o plantio, a altura da planta, altura até a inserção da espiga e o diâmetro do colmo foram medidos. A inoculação com Ab-v5 e a coinoculação de RZ2MS9 com Ab-v5 promoveram o incremento de 3% em altura da planta, além disso, esse consórcio promoveu incremento de 9% no diâmetro do colmo, todos comparados ao tratamento controle. Usando o DNA total da folha e raíz do milho, o fragmento 16S rRNA bacteriano foi sequenciado, através da plataforma Ion Torrent, para avaliar o efeito da inoculação na comunidade bacteriana associada à ambos os tecidos. A inoculação foi capaz de modular a comunidade bacteriana associada à folha, com a análise de coordenadas principais (PCoA) explicando 39,51% da variação. Não foi observada modulação na comunidade bacteriana associada à raiz. Foi observada diferença na estrutura da comunidade bacteriana quando ambos os nichos foram comparados, independente de inoculação, com a PCoA explicando 80,97% dessa variação. Assim observa-se que estudos dessa natureza são de grande importância para o melhor entendimento da interação entre as BPCP e a comunidade bacteirana associada à planta hospedeira, e dos mecanismos que levam ao desenvolvimento da cultura. / The use of mineral fertilizers is of great importance to the crop reaches its potential yield and become the production activity economically feasible, however, its excessive use is harmful to the environment, and brings risk to human health and local biodiversity. The use of plant growth-promoting bacteria (PGPB) has been shown as a promising and sustainable alternative, aiming to improve productivity and reduce fertilizer use. These bacteria colonize the rhizosphere and plant internal tissues and are able to stimulate their host development and health through mechanisms such as nutrient availability, phytohormone production and pathogen control. This study aimed to evaluate the inoculation effect of three PGPB strains in maize and the impact on associated bacterial community. The strains inoculated were Burkholderia ambifaria RZ2MS16, Bacillus sp. RZ2MS9, both isolated from guarana rhizosphere, and Azospirillum brasilense Ab-v5, a commercial inoculant. First, an anthibiosis assay was conducted between RZ2MS9 and Ab-v5 strains, showing no inhibition. Then, a growth-promotion assay was performed under field conditions, with maize plants inoculated with: (i) RZ2MS16; (ii) RZ2MS16 and Ab-v5; (iii) Ab-v5; (iv) RZ2MS9; (v) RZ2MS9 and Ab-v5; and (vi) control. The seeds were inoculated and, 60 days after sowing, the plant height, height to the cob insertion and stem diameter were measured. The inoculation with Ab-v5 and the co-inoculation with RZ2MS9 plus Ab-v5 increased the plant height in 3%, furthermore, the co-inoculation increased stem diameter in 9%, all compared to control. Using total DNA of maize\'s leaf and root, bacterial 16S rRNA fragment was sequenced by Ion Torrent platform, to evaluate the effect of inoculation in associated bacterial community of both tissues. The inoculation was able to modulate the leaf bacterial community, with principal coordinates analysis (PCoA) explaining 39.51% of variation. It was not found modulation on root\'s bacterial community. Difference in the bacterial community structure was observed when both niches were compared, regardless inoculation, with PCoA explaining 80.97% of this variation. Therefore, it is noted that studies of this nature are of great importance for a better understanding of the interaction between PGPB and bacterial community associated to the host plant and mechanisms leading to crop development.

Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. / Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.