Blooms of nitrogen fixing cyanobacteria (NFC) occur in many freshwater and marine systems, including the Baltic Sea. By fixing dissolved nitrogen, they circumvent general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for non-diazotrophic primary producers and ultimately supporting secondary production. Elucidating trophic links between primary consumers and NFC is essential for understanding role of these blooms for secondary production. However, until recently, there was no reliable method to quantify individual prey species for zooplankter feeding in situ. The development of PCR-based methods to detect prey-specific DNA in the diet of consumers, including microscopic animals, allows identification and quantification of trophic linkages in the field. Using molecular diet analysis in combination with egg production measurements, biochemical markers of growth and condition; and stable isotope approach, we explored a possibility to determine (1) whether cyanobacteria are grazed and assimilated by mesozooplankters (Papers I and II), (2) which species/groups are particularly efficient consumers of cyanobacteria (Papers II and III), and (3) how feeding on cyanobacteria affects zooplankton growth and development (Paper I and III). Taken together, these laboratory and field observations, provided evidence that NFC contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii (Paper I). The favorable growth conditions for juvenile copepods observed during NFC blooms were hypothesized to be mediated by picoplankton that take up bioavailable nitrogen exuded from cyanobacterial cells. This hypothesis found support in Paper II that provided quantitative estimates for the direct picocyanobacteria → mesozooplankton pathway, with highest weight-specific consumption observed in nauplii. Further, using field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic proper, we found that NFC nitrogen is assimilated and transferred to zooplankton via both direct grazing and indirectly through grazing on small-sized phyto- and bacterioplankton (Paper III). Finally, these and other findings emphasizing the importance of NFC for Baltic Sea secondary production during growth season were synthesized to show that diazotrophic nitrogen enters food webs already at bloom initiation (Paper III) and is transferred via multiple pathways to pelagic and benthic food webs and, ultimately, to fish (Paper IV). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Accepted.</p>

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Ce travail de thèse a pour but d'étudier la fixation d'azote marine dans les environnements riches mais déficitaires en azote (N), comparé au phosphore (P) dans un rapport N:P<16, grâce à l'utilisation d'approches complémentaire en culture in vitro et sur le terrain in situ. La première partie de ce travail a consisté à évaluer la réponse de la fixation d'azote de la cyanobactérie unicellulaire diazotrophe Crocosphaera watsonii face à des concentrations micromolaires en azote inorganique dissous (DIN) supposées inhiber l'activité de fixation d'azote : - suite à un apport sporadique, ou, - après une longue période d'acclimatation. Les résultats de ces études n'ont pas permis d'observer une inhibition des activités de fixation d'azote de cet organisme, laissant supposer que ce processus pourrait être actif dans une zone de l'Océan ayant ces même caractéristiques biogéochimiques : le Sud Est Tropical de l'Océan Pacifique (ETSP). En effet, cette zone est une des trois plus grandes zones de minimum d'oxygène (OMZ) de l'Océan et d'intenses processus de pertes de N (dénitrification et anammox) y ont lieu, résultant en un déficit de N par rapport au P. Des études présumaient que des processus inverses, de gains de N par la fixation d'azote, pourraient y être actifs mais aucune mesure à l'échelle du bassin n'y avait été faite car la fixation d'azote n'était supposée se produire que dans les environnements oligotrophes, comme les gyres subtropicaux. Dans le cadre d'un projet international, des missions océanographiques ont pu avoir lieu dans cette zone en Février 2010 pendant un évènement El Niño et en Mars-Avril 2011 pendant un évènement La Niña. / The objectif of these thesis was to study dinitrogen fixation in marine environments rich but deficient of nitrogen (N) compared to phosphorus (P) in a ratio N:P<16, by using complementary approaches in culture in vitro and in the field in situ. The first part of this work was to evaluate the response of nitrogen-fixing unicellular Cyanobacteria Crocosphaera watsonii faced with micromolar concentrations of dissolved inorganic nitrogen (DIN) supposed to inhibit nitrogen fixation activity : - after sporadic input, or, - after a long period of acclimatization. The results of these studies have failed to observe the inhibition of nitrogen fixation activities of this organism, suggesting that this process could be active in an area of the Ocean with these same biogeochemical characteristics : the Eastern Tropical South Pacific (ETSP). Indeed, this area is one of the three largest oxygen minimum zones (OMZ) of the Ocean, where intense processes of N losses (denitrification and anammox) took place, resulting in a deficit of N compared to P. Studies assumed that the inverse process, gain of N by nitrogen fixation, could be active in the ETSP but no measurements across the basin have been performed because nitrogen fixation was assumed to occur only in oligotrophic environments, such as the subtropical gyres. In the framework of an international project, cruises took place in this area in February 2010 during a El Niño event and in March-April 2011 during a La Niña event. Results of these two cruises have confirmed that nitrogen fixation was unexpectedly active with an intensity comparable to those reported in oligotrophic areas.

Made available in DSpace on 2017-07-10T17:36:59Z (GMT). No. of bitstreams: 1 Flavia_Bordignon_Hendges.pdf: 1440040 bytes, checksum: 03c9ee2c7068a9ec8b86a685f1cf1d86 (MD5) Previous issue date: 2015-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Plants are constantly exposed to a variety of environmental stresses which cause reductions in productivity. Plants subjected to water stress undergo changes in the biochemical structure of your cells, including the production of reactive oxygen species (ROS) or free radicals and the plant in response to these free radicals, synthesize antioxidants. The objective of this study was to evaluate the antioxidant effect of inoculation with Herbaspirillum seropedicae bacteria associated with the wheat genotypes CD 120 submitted to drought and nitrogen fertilization. They were assessed physiological parameters such as membrane stability index (IEM), relative water content (RWA), fresh and dry biomass and enzymatic activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and Glutathione S-transferase (GST), and proline contents and lipid peroxidation (MDA). The design was completely randomized in 4x2 factorial design with three replications, the first factor relates to the conditions of inoculation / fertilization (C0: control, C1: fertilization with ammonium sulfate, C2: inoculation with bacteria and C3: fertilization combined with inoculation) and the second factor relates to the water conditions with or without stress (at booting stage of the plants were submitted to water deficit for 8 days while the control plants were still being irrigated usually). Plants inoculated with H. seropedicae subjected to drought stress, show lower increases in IEM (19%) in the MDA (41%) and proline (6.7 times) compared with the control (30%, 70% and 25 times, respectively). Regarding the antioxidant effect, the drought caused greater reduction in specific activity of CAT regardless of fertilization conditions / applied inoculation. For SOD stressed plants and in the presence of bacteria, the levels of enzyme increased, and GST, the effect was reversed. However, the activity of APX remained unchanged, both the application of stress as the presence of inoculation. Bacterial presence increased the fresh and dry biomass and weight of 100 grains. Although little antioxidant effect observed there was no damage to the tissue and had no effect on yield under water stress conditions, yet the antioxidant effect, this was not capable of restoring / As plantas estão constantemente expostas a uma grande variedade de estresses ambientais que ocasionam reduções na produtividade. Plantas submetidas a déficit hídrico sofrem alterações na estrutura bioquímica de suas células, entre elas a produção de espécies reativas de oxigênio (EROs) ou radicais livres e a planta, em resposta a esses radicais livres, sintetizam agentes antioxidantes. O objetivo deste trabalho foi avaliar o efeito antioxidante da inoculação com a bactéria Herbaspirillum seropedicae associada ao genótipo de trigo CD 120 submetidas ao déficit hídrico e a adubação nitrogenada em cobertura. Foram avaliados parâmetros fisiológicos como índice de estabilidade de membrana (IEM), teor relativo de água (TRA), biomassa fresca e seca e atividade enzimática das enzimas antioxidantes: Catalase (CAT), Superóxido Dismutase (SOD), Ascorbato Peroxidase (APX) e Glutationa S-Transferase (GST), além de teores de prolina e peroxidação lipídica (MDA). O delineamento utilizado foi inteiramente ao acaso, em esquema fatorial 4x2 com 3 repetições, sendo que o primeiro fator refere-se as condições de inoculação/fertilização (C0: controle, C1: fertilização com sulfato de amônio, C2: inoculação com a bactéria e C3: fertilização combinada com inoculação) e o segundo fator refere-se as condições hídricas com ou sem estresse (no estádio do emborrachamento as plantas foram submetidas a défict hídrico por 8 dias enquanto que as plantas controle permaneciam sendo irrigadas normalmente). Plantas inoculadas com H. seropedicae submetidas ao estresse hídrico, apresentaram menores acréscimos no IEM (19%), no MDA (41%) e prolina (6,7 vezes) em comparação com o controle (30%, 70% e 25 vezes, respectivamente). Em relação ao efeito antioxidante, a seca promoveu redução acentuada da atividade especifica da CAT independentemente das condições de fertilização/inoculação aplicadas. Para a SOD, plantas estressadas e na presença da bactéria, aumentaram os níveis desta enzima, e para a GST, o efeito foi inverso. Porém, a atividade da APX permaneceu inalterada, tanto pela aplicação do estresse quanto pela presença da inoculação. A presença bacteriana aumentou a biomassa seca e fresca e a massa de 100 grãos. Apesar do pouco efeito antioxidante observado não houve dano ao tecido e não houve efeito na produtividade sob condição de déficit hídrico, contudo o efeito antioxidante, este não foi capaz de restabelecer

Made available in DSpace on 2017-07-10T17:37:49Z (GMT). No. of bitstreams: 1 Deniele_Marini.pdf: 772563 bytes, checksum: 60b795ca9bfd2ea49314eba25691e403 (MD5) Previous issue date: 2012-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Studies on the inoculation of Azospirillum on grass species has shown benefits to growth and development of plants, through the production of plant hormones and biological nitrogen fixation that lead to other positive effects, and could thus reduce the need for nitrogen fertilization. However, strains efficient and promising genotypes should be associated for obtain benefits with this association. Therefore, the present study aimed to evaluate the response of corn hybrids to its association with A. brasilense and nitrogen fertilization.The experimental design was randomized blocks in factorial 2 x 2 x 5 with four replications. The treatments consisted of two corn hybrids (H1-30F53 and H2-CD386); inoculation of a commercial product based on A. brasilense, and five nitrogen rates (0, 40, 80, 120 and 160 kg ha-1). Were evaluated the parameters: plant height, ear height, diameter stem, dry mass of leaves, stem, ear and total shoot, leaf area, leaf N content and yield components (ear length, ear diameter, number of rows per ear, number of grains per row, weight of ear, weight of 100 grains and productivity). There was interaction between inoculation and nitrogen in the vegetative stage to the stem diameter and between hybrids and nitrogen for productivity. Hybrids responded differently to the variables and the application of increasing doses of N provided increments for biometric variables, leaf N content and yield components. Effect of inoculation with A. brasilense was found on leaf area and total dry mass of shoots in the reproductive stage, which had increased in the presence of inoculation / Estudos com a inoculação de Azospirillum em espécies de gramíneas têm demonstrado benefícios ao crescimento e desenvolvimento das plantas, por meio da produção de hormônios vegetais e da fixação biológica de nitrogênio que acarretam em outros efeitos positivos, podendo assim reduzir a necessidade de adubação nitrogenada para as culturas. Contudo, estirpes eficientes e genótipos promissores, devem ser associados para que se obtenham benefícios com essa associação. Nesse sentido, o presente trabalho teve por objetivo avaliar a resposta de híbridos de milho à associação com A. brasilense e adubação nitrogenada. O delineamento experimental empregado foi de blocos ao acaso em esquema fatorial 2 x 2 x 5 com quatro repetições. Os tratamentos foram constituídos por dois híbridos de milho (H1-30F53 e H2-CD386); inoculação ou não de produto comercial a base de A. brasilense, e cinco doses de N (0, 40, 80, 120 e 160 kg ha-1). Foram avaliados os parâmetros: altura de planta, altura de inserção de espiga, diâmetro do colmo, massa seca de folhas, colmo + bainha, espiga e total de parte aérea, área foliar, teor de N foliar e os componentes de produção (comprimento de espiga, diâmetro de espiga, número de fileiras por espiga, número de grãos por fileira, massa da espiga, massa de 100 grãos e produtividade). Houve interação entre inoculação e adubação nitrogenada no estádio vegetativo para o diâmetro do colmo e entre híbridos e adubação nitrogenada para a produtividade. Os híbridos responderam de forma diferenciada para as variáveis analisadas e a aplicação de doses crescentes de N proporcionou incrementos para variáveis biométricas, teor de N foliar e componentes de produção. Houve influência da inoculação com A. brasilense apenas sobre a área foliar e massa seca total de parte aérea no estádio reprodutivo, que apresentaram incremento em presença da inoculação

Made available in DSpace on 2017-07-10T17:37:49Z (GMT). No. of bitstreams: 1 Janaina_Dartora.pdf: 971140 bytes, checksum: 59416a74903d62a087d8ae6beec5412e (MD5) Previous issue date: 2012-02-29 / The aim of this work was to evaluate the development and productivity of maize in response to combined of bacteria Azospirillum brasilense and Herbaspirillum seropedicae in association with nitrogen fertilization. The experimental design was a randomized complete block with four replications and treatments arranged in a 4x5 factorial. The first factor is from the inoculation of seeds: non-inoculated, strain of Azospirillum brasilense (Ab-V5), strain of Herbaspirillum seropedicae (SmR1) and the combination of strains (Ab-V5+SmR1); and the second factor of nitrogen (N) rates: 0, 40, 80, 120 e 160 kg ha-1 of N. Were evaluated biometric variables: basal diameter stem, plant height, shoot dry weight and leaf area; NPK content in leaves and grains; and production components: height of ear height, ear length, ear diameter, number of rows per ear, number of grains per row, weight of ear and productivity. There was no interaction between the factors under study, and was observed a positive effect of inoculation on the development and production of maize, especially with combined inoculation of strains, wich resulted in increased crop yield of 922 kg ha-1 compared to control. The application of increasing doses of N provided increased growth and productivity of maize until the dose of 160 kg ha-1 de N. Inoculation effect was observed only on the levels of N in leaf and P in grain, while the nitrogen fertilization only influenced the P content of leaf / O presente trabalho foi desenvolvido com o objetivo de avaliar o desenvolvimento e a produtividade do milho em resposta à inoculação combinada das bactérias A. brasilense e H. seropedicae em associação à adubação nitrogenada. O experimento foi conduzido em delineamento experimental de blocos ao acaso em esquema fatorial 4 x 5, com quatro repetições. O primeiro fator constitui-se da inoculação das sementes: testemunha sem inoculação, estirpe de A. brasilense (Ab-V5), estirpe de H. seropedicae (SmR1) e a combinação das estirpes (Ab-V5 + SmR1); e o segundo fator das doses de nitrogênio (N): 0, 40, 80, 120 e 160 kg ha-1 de N. Foram avaliadas as variáveis biométricas diâmetro basal do colmo, altura de planta, massa seca de parte aérea e área foliar; teor de NPK nas folhas e grãos; e os componentes da produção altura de inserção, comprimento de espiga, diâmetro de espiga, número de fileiras por espiga, número de grãos por fileira, massa de espiga e produtividade. Verificou-se que não houve interação entre os fatores em estudo, tendo sido observado efeito positivo da inoculação quanto ao desenvolvimento e produção da cultura do milho, especialmente com a inoculação combinada das estirpes, que proporcionou incremento na produtividade da cultura de 922 kg ha-1 em relação à testemunha. A aplicação de doses crescentes de N em cobertura proporcionou incremento no crescimento e produtividade do milho até a dose de 160 kg ha-1 de N de forma independente. Quanto à análise de nutrientes, foi verificado efeito da inoculação apenas sobre os teores de N nos grãos e de P foliar, enquanto a adubação nitrogenada influenciou apenas o teor de P foliar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In view of the use of corn for various purposes, principally for animal feed in the form of silage or grain to make animal feed, this work aimed to evaluate the effects of nitrogen fertilization in different doses (0, 60, 120, 240 and 480 kg h N-¹) in the form of urea, in association of different purposes hybrids and inoculated with Azospirillum brasilense strains Ab-V5 and Ab-V6. Were evaluated the development, plant silage productivity, nutritional value, morphological structure and grain yield. The experimental area was carried out in two agricultural crops, 2013/2014 and 2014/2015, sowed in October, harvest for silage at the end of January and early February and grain harvest in March, in both years. Inoculation with A. brasilense increased silage productivity (dry matter per area), improved the dry matter content at ensiling, increased the corn cob contents in the silage, decreased the hemicelulose content and ash and increased the buffering capacity of forages. The inoculation had interaction with hybrids used increasing their productivity and changing the stem percentage ensiled. Fertilizations from 120 kg ha-¹ N are enough to potentially raise the productivity of the crop for silage and combined with hybrids of silage purposes are more appropriate, as the largest biomass productivity without significant changes in the silage quality. For grain yield, significant interaction between inoculation with A. brasilense and AS 1572 hybrid was founded, increasing grain production (1 ton ha-¹) and it was associated with the increment in number of grains per cob. The hybrid with grain production purpose, the AG 9030, showed higher grain mass, compared to hybrids with mixed purposes, AS 1572. Nitrogen fertilization increased the crop yield by raising the grain weight. Inoculation caused no effects on the external morphological structure of plants. / Tendo em vista a utilização da cultura do milho para diversos fins, principalmente para alimentação animal na forma de silagem ou grãos para compor rações, este trabalho avaliou os efeitos da adubação nitrogenada em diferentes doses (0, 60, 120, 240 e 480 kg ha-¹ de N) na forma de ureia, associada à utilização de híbridos de diferentes propósitos inoculados com Azospirillum brasilense estirpes Ab-V5 e Ab-V6. Avaliou o desenvolvimento, produtividade das plantas para silagem, bem como o valor nutritivo da mesma, além da estrutura morfológica e a produtividade de grãos. O cultivo da área experimental foi realizado em duas safras agrícolas, safra 2013/2014 e safra 2014/2015, tendo a semeadura realizada no mês de outubro, a colheita para silagem no final do mês de janeiro e início de fevereiro e a colheita de grãos no mês de março, em ambos os anos. A inoculação com A. brasilense incrementou a produtividade de matéria seca de silagem por hectare, elevou o teor de MS no momento da ensilagem, aumentou a participação de espigas na massa ensilada, diminuiu o teor de hemicelulose e cinzas na silagem e elevou a capacidade tamponante da forragem. Ainda, a inoculação apresentou interação com os híbridos utilizados aumentando a sua produtividade e alterando o percentual de colmo ensilado. Adubações a partir de 120 kg ha-¹ de N são suficientes para elevar potencialmente a produtividade da cultura para silagem. A utilização de híbridos de fins silageiros é a mais indicada, visto a maior produtividade de biomassa sem alterações significativas na qualidade da silagem. Para produção de grãos houve interação significativa entre a inoculação com A. brasilense com o híbrido AS 1572 onde foi encontrada maior produção de grãos (uma tonelada ha-¹), sendo o aumento associado ao acréscimo do número de grãos por espiga. O híbrido com finalidade de produção de grãos, o AG 9030, apresentou maior massa de grãos, quando comparado ao híbrido com fins mistos, o AS 1572. A adubação nitrogenada incrementou a produtividade da cultura por elevar a massa dos grãos. A inoculação não provocou efeitos na estrutura morfológica externa das plantas.

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.

A Brachiaria brizantha é considerada uma das forrageiras preferidas entre os agropecuaristas por possuir elevada produção de forragem, tolerância ao calor e ao déficit hídrico, alta resposta à aplicação de fertilizantes, produção em grande massa de raízes e sementes, resistência à cigarrinha das pastagens (exceto as pertencentes ao gênero Mahanarva) e boa competição com plantas invasoras. É considerada a principal fonte de alimento para bovinos, sendo utilizada tanto na cria, recria, como na engorda dos animais. As bactérias fixadoras de nitrogênio ou diazotróficas são procariotos capazes de reduzir o N2 a NH3, forma assimilável pelos organismos, e também podem produzir hormônios vegetais, como ácido-indol-acético, que estimulam o crescimento radicular da planta. Estes micro-organismos apresentam grande importância para a manutenção dos ecossistemas. Sua associação com as raízes de plantas e seu efeito promotor quando associados à Brachiaria brizantha possibilitaria a recuperação de áreas de pastagens que apresentam deficiência de nitrogênio, o que é um mecanismo ainda pouco explorado. Com o objetivo de estudar esta possibilidade, foram escolhidas três áreas (Nova Odessa-SP, São Carlos- SP e Campo Verde-MT), preferencialmente onde o nitrogênio era limitante, constituídas por pastagem de Brachiaria brizantha para a amostragem de solo e raiz. Os três locais demonstraram a ocorrência de diazotróficos, após o isolamento e cultivo das bactérias em meio de cultivo semisólido sem adição de nitrogênio na forma combinada (JNFb). Foram obtidas 110 estirpes bacterianas e, após sorteio aleatório, 72 isolados foram mantidos para realização de testes a fim de se avaliar o potencial biotecnológico das bactérias. Destes, 10 demonstraram atividade da nitrogenase quando submetidos ao método de aumento na concentração de nitrogênio total (Ntotal) em meio de cultura. 57 isolados foram capazes de reduzir o gás acetileno a etileno quando submetidos à técnica de redução de acetileno. As estirpes bacterianas C4 (Pseudomonas sp.) e C7 (Azospirillum sp.), isoladas da rizosfera de Brachiaria brizantha da área de Campo Verde-MT, se destacaram das demais por apresentar atividade da nitrogenase muito superior até a de bactérias diazotróficas que foram incluídas na avaliação como testemunhas positivas. Outros 68 isolados produziram o hormônio vegetal ácido-indol-acético quando cultivados em meio de cultivo LB, na presença de triptofano. A produção variou de 0,39µg/mL a 195 µg/mL de AIA. Todos os 72 isolados foram utilizados em experimento em casa de vegetação para avaliar o efeito de inoculação em B. brizantha quando com eles inoculada. Avaliaram-se a matéria seca da parte aérea e raiz e o teor de nitrogênio total da parte aérea através do método micro-Kjeldhal. Nenhum isolado diferiu significativamente do controle sem inoculação bacteriana que continha a mesma dose de nitrogênio fornecido às plantas. O seqüenciamento parcial do gene 16S rRNA dos 72 isolados permitiu a caracterização de sete grupos genotípicos: Stenotrophomonas sp, Pseudomonas sp., Xanthomonas sp., Bacillus sp., Rhizobium sp, Sphingomonas sp. e Azospirillum sp. O gênero Stenotrophomonas sp. predominou (69%) nas três áreas de estudo. / Brachiaria brizantha is considered one of preferred fodders among farmers for having high forage yield, tolerance to heat and drought, high response to fertilizer application, large production of root mass and seeds, resistance to grassland leafhopper (except those belonging to the genus Mahanarva) and good competition with weeds. It is considered the main source of food for cattle, being used in the raising, breeding, and fattening of animals. The nitrogen fixing bacteria or diazotrophs are prokaryotes able to reduce N2 to NH3, which is assimilated by organisms, and may also produce plant hormones such as indole-acetic acid, which stimulates root growth. These micro-organisms have great importance for the maintenance of ecosystems. Their association with plant roots and their promoting effect when combined with Brachiaria brizantha enable recovery of nitrogen-deficient grazing areas, which is a mechanism still little explored. Therefore, three areas were chosen (Nova Odessa-SP, Sao Carlos-SP and Campo Verde-MT), preferably where nitrogen was limiting, consisting of Brachiaria brizantha from which samples of soil and roots were collected. The three sites showed the occurrence of diazotrophs after the isolation and cultivation of bacteria in semi-solid culture medium with no nitrogen added in the combined form (JNFb). It was obtained 110 bacterial strains and, after the raffle random, 72 were kept isolated for testing in order to assess the biotechnological potential of bacteria. From which, 10 showed nitrogenase activity when subjected to the method of total nitrogen concentration increase (N-total) in the culture medium. 57 isolates were able to reduce acetylene to ethylene when subjected to the acetylene reduction technique. The strains C4 (Pseudomonas sp.) and C7 (Azospirillum sp.), isolated from the rhizosphere of Brachiaria brizantha in the area of Campo Verde-MT, stood out from the others by presenting nitrogenase activity far superior to that of diazotrophs recommended as positive controls. Other 68 isolates produced the plant hormone indole-acetic acid when grown in LB culture medium in the presence of tryptophan. Production ranged from 0.39 g/mL to 195 g/mL of IAA. All 72 isolates were used in an experiment in a greenhouse to evaluate the effect of inoculation on B. brizantha. Evaluations were carried out on dry matter of shoot and root and total nitrogen content of the shoot through the micro-Kjeldahl method. None of the isolates differed significantly from the control without bacterial inoculation which contained the same amount of nitrogen supplied to plants. Partial sequencing of the 16S rRNA of the 72 isolates allowed the characterization of seven genotype groups: Stenotrophomonas sp., Pseudomonas sp., Xanthomonas sp., Bacillus sp., Rhizobium, Sphingomonas sp. and Azospirillum sp. The genus Stenotrophomonas sp. predominated (69%) in the three study areas.

A Mata Atlântica é um importante bioma da costa brasileira, apresenta grande diversidade de plantas e animais, porém, pouco se sabe sobre a diversidade microbiana. Da mesma forma, pouco se sabe sobre o papel funcional desses microrganismos. Vários microrganismos estão envolvidos na ciclagem do nitrogênio na Mata Atlântica, e dentre eles os diazotróficos são de particular interesse, pois contribuem para o aporte direto de nitrogênio nos ecossistemas. O objetivo deste trabalho foi avaliar a diversidade de bactérias diazotróficas que nodulam leguminosas em duas parcelas permanentes do Parque Estadual da Serra do Mar em diferentes altitudes. Nódulos de raízes foram coletados nas quatro estações do ano. As bactérias foram isoladas do interior dos nódulos, resultando em 105 isolados. A análise de diversidade genética destas bactérias foi feita utilizando-se BOX-PCR e sequenciamento parcial do gene 16S rRNA. A capacidade de nodulação dos isolados foi determinada através da formação de nódulos em caupi (Vignia unguiculata). Os resultados indicaram que há uma diferença na distribuição espacial e temporal dos nódulos nas áreas estudadas. A maior quantidade de nódulos foi encontrada na parcela de Picinguaba, em estações com menores índices pluviométricos. Os isolados apresentaram uma grande diversidade fenotípica, sendo separados em 6 grupos com características culturais semelhantes. Os perfis de BOX-PCR formaram 8 grupos genotípicos com mais de 80% de similaridade, agrupando tanto isolados de Picinguaba quanto de Santa Virgínia. Os perfis dos géis do BOX-PCR apresentaram variação no número e mobilidade das bandas. Os dados foram transformados em uma matriz binária de presença e ausência que possibilitou a análise de agrupamento hierárquicos com 8 grupos genotípicos com mais de 80% de similaridade, agrupando tanto isolados de Picinguaba quanto de Santa Virgínia. Através do sequenciamento parcial de fragmentos de gene rRNA 16S verificou-se que a estrutura das comunidades diazotróficas de Picinguaba e Santa Virgínia não apresentaram diferença estatisticamente significativa, indicando que não há seleção de populações bacterianas especificas nas áreas.Dos isolados testados, 88% apresentaram capacidade de nodular caupi, porém alguns não foram eficientes em promover o crescimento das plantas. Nas duas áreas predominou UTOs filogeneticamente associados ao gênero Paenibacillus nos nódulos, sugerindo que essas bactérias são importantes para nodulação de leguminosas na Mata Atlântica. / The Atlantic Rainforest is a major biome of the Brazilian coast, which harbors great diversity of flora and fauna, but little is known about its microbial diversity. Furthermore, little is known about the functional role of these abundant microorganisms. Several microorganisms are involved in cycling of the Atlantic Rainforests nitrogen, among them the diazotrophs which are of particular interest because they contribute to the direct input of nitrogen to ecosystems. The aim of this study was to evaluate the diversity of legumes nodulating diazotrophs in two permanent plots of the Serra do Mar State Park at different altitudes. Root nodules were collected throughout the four seasons. The bacteria were isolated from inside of the nodules, resulting in 105 isolates. The analysis of genetic diversity was performed using BOX-PCR and rRNA 16S sequencing. The nodulation capacity of the isolates was determined by the nodule formation in cowpea (Vigna unguiculata). The results indicated that there are spatial and temporal differences distribution in the areas studied. The largest number of nodules was found in the Picinguaba plot during seasons of low rainfall. The isolates showed a wide phenotypic diversity, hence divided into six groups with similar cultural characteristics. The BOX-PCR profiles formed eight genotypic groups with more than 80% similarity, when comparing the isolates from Picinguaba those from Santa Virginia. The BOX-PCR gel profiles showed variation in number and mobility of bands. The data was transformed into a binary matrix of presence and absence that allowed the hierarchical cluster analysis of eight genotypic groups with more than 80% similarity, again comparing both isolates Picinguaba and Santa Virginia. Through partial sequencing of 16S rRNA gene fragments no statistically significant difference was found between the diazotrophs community structures of Picinguaba the and that of Santa Virginia , indicating no selection for specific bacterial populations in these areas. In both isolates tested, 88% showed cowpea nodulating capacity, but some were not effective in promoting plant growth. In both areas OTUs phylogenetically associated with nodule of genus Paenibacillus predominated, suggesting that these bacteria are important for legume nodulation in the Atlantic Rainforest.