Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-10-29Bitstream added on 2014-06-13T18:54:07Z : No. of bitstreams: 1 toledo_bfb_me_jabo.pdf: 494396 bytes, checksum: 73007bbe5afed3d92412924b6ebe8b81 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A crescente demanda de alimentos causada pelo aumento populacional, aliado a alto custo de fertilizantes industrializados e impacto ambiental causado por eles leva, mundialmente, à utilização em grande escala de inoculantes de bactérias fixadoras de nitrogênio. Os inoculantes utilizados no Brasil (coleção SEMIA- IPAGRO) ainda não estão suficientemente caracterizados geneticamente. O objetivo deste trabalho foi avaliar e confrontar as seqüências parciais dos genes 16S rDNA e nifH de 26 estirpes padrões já classificadas, com 70 estirpes de rizóbios recomendadas e autorizadas para a produção de inoculantes no Brasil. Para esta finalidade, a partir das amostras de DNA extraídos destas bactérias foram realizadas reações de PCR com oligonucleotídeos iniciadores relativos à região codificadora do gene 16S rDNA e do nifH, sendo então seqüenciadas com o objetivo de detectar diferenças nucleotídicas entre as diferentes bactérias estudadas. Para a comparação dos resultados do seqüenciamento com a consulta de similaridade de nucleotídeos no BLASTn, foram geradas árvores filogenéticas através de ferramentas de bioinformática. Foi observado que a classificação taxonômica das estirpes SEMIA recomendadas para inoculação de leguminosas previamente disponível na FEPAGRO, com base em propriedades morfológicas e especificidade hospedeira, não foi confirmada em todas as estirpes pelos sequenciamentos parciais dos genes estudados. Sugerimos revisão da classificação destas estirpes. Concluímos também que a consulta de similaridade ao BLASTn pelo seqüenciamento parcial dos genes 16S rDNA e nifH é, na maioria dos casos, consistente com a classificação proposta pela construção de árvores filogenéticas destas sequências. Estas ferramentas apresentaram-se muito confiáveis para obtenção de classificação em nível de gênero das estirpes estudadas. / The growing demand for food caused by population growth, combined with high cost of fertilizers and industrial environmental impacts caused by them leading, worldwide, the large scale use of inoculants different nitrogen-fixing bacteria. The inoculants used in Brazil (SEMIA-IPAGRO collection) are not yet sufficiently characterized genetically. The purpose of this study was to evaluate and compare the sequences of partial 16S rDNA and nifH of 26 strains already classified, with 70 strains of rhizobia recommended and authorized for the production of inoculants in Brazil. For this purpose, from DNA samples taken from these bacteria were performed with PCR reactions with primers on the coding region of the gene 16S rDNA and nifH, and sequencing with the aim of detecting nucleotide differences between different bacteria studied. To compare the results of the consultation of similarity of sequences of nucleotides in BLASTn, phylogenetic trees were generated through bioinformatics tools. It was observed that the taxonomic classification of strains SEMIA recommended for inoculation of legumes previously available in FEPAGRO, based on morphological properties and host specificity, it wasn’t confirmed in all strains by partial sequencing of the genes studied. We suggest reviewing the classification of these strains. We concluded that the similarity of the consultation BLASTn by partial sequencing of 16S rDNA and nifH is, in most cases, consistent with the classification proposed by the construction of phylogenetic trees of these sequences. These tools were very reliable for obtaining classified in the genus level of strains studied.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / New technologies are intended to assist the farmer in the inoculation process, as is the case of antecipated inoculation (pre-inoculation) provided by using osmoprotectors. In search of higher yields, the kind of inoculation and management with nitrogen fertilizer also need to be clarified. In this study, two experiments were conducted to evaluate the agronomic performance of two inoculation strategies with Bradyrhizobium alone (conventional inoculation) or combined with Azsopirillum brasilense (co-inoculation). The first experiment, we evaluated the pre-inoculation (0, 7 and 10 days before seeding). The second experiment evaluated the response to supply urea (0, 75 and 150 kg N ha-1) split into two periods of three soybeans cultivars development stage (BMX Ativa RR, TEC 6029 IPRO and BMX Potência RR). We evaluated the interaction of nodulation, of morphophysiological components and grain yield at conditions of Santa Maria. Co-inoculation and conventional inoculation, both plus an osmoprotectors, showed increases in grain yield relative to treatment not inoculated 31 and 25%, respectively. With seven days before to sowing, use of osmoprotectors for both types of inoculation, did not differ statistically from the other treatments inoculated the same day of sowing. Co-inoculation provided increment in grain yield of 240 kg ha-1 compared to conventional inoculation. BMX Ativa, TEC 6029 IPRO and BMX Potência when co-inoculated showed increases in grain yield 6, 4 e 12%, respectively. The addition of 150 kg N ha-1 for the two indeterminate cultivars had an increases, but without economic return. The cultivars tested when inoculated conventionally not respond positively to the application of urea. Therefore, these results confirm the effectiveness of the use osmoprotectores and co-inoculation ond the seeds and low economic returns provided by the use of nitrogen fertilizer. It emphasizes the use of activation and protection substances on rhizobial inoculants for the formulation, as well as more studies concerning the efficiency of co-inoculation with rhizobacteria promote plant growth to obtain higher yields. / Novas tecnologias têm surgido para auxiliar o agricultor no processo de inoculação, como é o caso da pré-incoulação (inoculação antecipada) possibilitada pelo uso de osmoprotetores. Na busca por produtividades superiores, o tipo de inoculação e o manejo com fertilizante nitrogenado também precisam ser melhor esclarecidos. No presente trabalho, foram conduzidos dois experimentos para avaliar o desempenho agronômico de duas estratégias de inoculação com bactérias do gênero Bradyrhizobium isoladamente (inoculação convencional) ou combinadas com Azospirillum brasilense (co-inoculação). O primeiro experimento, testou a pré-inoculação (0, 7 e 10 dias anteriores à semeadura). O segundo experimento, testou à resposta ao fornecimento de ureia (0, 75 e 150 kg de N ha-1) fracionado em dois momentos do estádio de desenvolvimento de três cultivares de soja (BMX Ativa RR, TEC 6029 IPRO e BMX Potência RR). Avaliou-se os componentes da nodulação, morfofisiológicos e da produtividade de grãos nas condições edafoclimáticas de Santa Maria. A co-inoculação e a inoculação convencional, ambas acrescidas do osmoprotetor, apresentaram acréscimos na produção de grãos em relação ao tratamento não inoculado de 31 e 25%, respectivamente. Com sete dias anteriores à semeadura, o uso do osmoprotetor para os dois tipos de inoculação, não diferiu estatisticamente dos demais tratamentos inoculados no mesmo dia da semeadura. A co-inoculação proporcionou incremento na produtividade de grãos de 240 kg ha-1 quando comparado à inoculação convencional. Os cultivares BMX Ativa, TEC 6029 e BMX Potência quando co-inoculadas apresentaram incrementos na produtividade de 6, 4 e 12% respectivamente. Com a adição de 150 kg de N ha-1, os dois cultivares indeterminados apresentaram incremento na produtividade de 300 kg ha-1, porém, sem retorno econômico. Os cultivares testados quando inoculados convencionalmente não respondem positivamente à aplicação de ureia. Portanto, esses resultados confirmam a eficácia do uso de osmoprotetores e co-inoculação de sementes e o baixo retorno econômico proporcionado pelo uso de fertilizante nitrogenado. Enfatiza-se o uso de substâncias de ativação e proteção para os rizóbios na formulação de inoculantes, assim como, mais estudos quanto a eficiência da co-inoculação com rizobactérias promotoras de crescimento de plantas para obtenção de maiores produtividades.

Nitrogen-fixing bacteria in soybean nodules convert atmospheric nitrogen to plant-available forms in exchange for carbon from the plant, but other non-nitrogen-fixing bacteria also reside in nodules, and their role in the nodule is not well understood. This study was conducted to determine the effect of three non-nitrogen-fixing Pseudomonas spp. strains isolated from nodules on soybean, and we hypothesized these strains benefit soybean. A greenhouse study in which two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were treated with three fluorescent Pseudomonas spp. strains (referred to in this study as Bullseye, Pancake, and Starfish) and an uninoculated control. Soybeans were harvested at two time points: the R2/R3 growth stage and the R6 growth stage. Following each harvest, measures of growth, yield, and nitrogen fixation were taken, and data were analyzed using two non-parametric, multivariate analyses: multiple response permutation procedure (MRPP) and permutational multivariate analysis of variance (PERMANOVA). Both analyses showed soybeans of both cultivars treated with Pancake differed from controls following the first harvest but not the second. When analyzed individually, most metrics for growth, yield, and nitrogen fixation following the first harvest were not significantly different between Pancake and control treatments, but Pancake treatment means were still generally higher than controls. If metrics are considered collectively in conjunction with the results of the multivariate analyses, the results show Pancake generally increased soybean growth and nitrogen fixation. These findings support the hypothesis that non-nitrogen-fixing bacteria from nodules benefit plants, and such bacteria have the potential to serve as biofertilizers. / Master of Science in Life Sciences / Soybeans are one of the most commonly grown crops in the world, and nitrogen-fixing bacteria colonize the roots of soybeans and initiate the formation of spherical nodules attached to the roots. Inside the nodules, these bacteria convert atmospheric nitrogen to plant-available forms in exchange for sugar from the plant, and such bacteria reduce the need to add nitrogen fertilizer to agricultural fields. Other non-nitrogen-fixing bacteria also reside in nodules, but their role in the nodule is not well understood. If these bacteria benefit soybeans, they have the potential to serve as biofertilizers (microbial inoculants that promote plant growth). This study was conducted to determine whether non-nitrogen-fixing bacteria isolated from nodules benefit soybean. A greenhouse study in which two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were grown in soil and were either left uninoculated or were inoculated with one of three strains of bacteria from the genus, Pseudomonas (referred to in this study as Bullseye, Pancake, and Starfish). Following harvest, measures of growth, yield, and nitrogen fixation were taken, and data showed the bacteria generally benefited the soybean plants. Although, these results showed the bacteria benefitted the plants, field trials and further testing in the greenhouse should be conducted before using these bacteria as commercial biofertilizers. Additionally, the effects of other non-nitrogen-fixing nodule bacteria on soybeans should also be tested to identify other beneficial strains, and the cost of production should be compared to the potential gains of using such bacteria before they are developed into biofertilizers.

Diazotrophic microbes reside in soybean nodules; however, other non-nitrogen fixing bacteria are a part of the interior nodule microbiome. Results from a previous greenhouse study show that a novel species of Pseudomonas associates with soybean nodules as a plant-growth promoting rhizobacteria (PGPR). This study observes the soybean growth promoting potential of Pseudomonas spp. in a field setting. Additionally, this study observed differences in soybean growth promotion based on amending the plant with isolated strains or a mixed culture of the species' strains. Two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were either amended with isolated strains of the novel Pseudomonas spp. (referred to as PAMW1 and BUMW2 in this study), a mix of the two strains, or an uninoculated control. The study recorded measurements to observe growth, yield, and nitrogen fixation differences. The study uses two-way factorial ANOVAs and non-parametric, multivariate analyses to determine differences in growth promotion among samples. Soybean amended with PAMW1 has greater shoot mass, biomass, and height than other treatments. Through nonmetric multidimensional scaling (NMS), samples amended with a mixed culture or PAMW1 may be different regarding growth promotion relative to the non-amended samples. Univariate results support the hypothesis that the novel Pseudomonas spp. benefit soybean in a field setting. However, it is inconclusive whether a mixed culture amendment of multiple strains alters the overall growth promotion of soybean compared to samples amended with isolated strains. / Master of Science / Soil hosts a relatively abundant and diverse community of microorganisms. Moreover, the area of soil that interacts closely with plant roots and their associated exudates, called the rhizosphere, has a significantly greater microbial abundance than surrounding bulk soil. Interactions between microbes and the plant often promote plant growth because of secondary metabolites produced by these beneficial microbes. One particular bacterial species, belonging to the Pseudomonas genus, was discovered and extracted from the soybean nodule interior. Nitrogen-fixing bacteria predominantly reside in the soybean nodule, yet this microorganism cannot fix nitrogen. Although trace amounts of non-nitrogen-fixing bacteria reside in the soybean nodule, this novel species has a relatively high abundance. This study determines the benefits of this species in the soybean nodule. Following positive results in a greenhouse study, this field experiment observes variance in soybean growth and productivity based on their received bacterial amendment. For this study, two soybean cultivars were either amended with an isolated strain of this species, a mix of the two strains, or left uninoculated to serve as a control. Numerous recorded measurements serve as indices of soybean growth and productivity. The results suggest that this novel Pseudomonas species benefits the plant by significantly improving biomass. With further research, this species can potentially serve as an environmentally sensitive and sustainable alternative to fertilizers through its ability to promote soybean growth.

Pine rocklands are endangered ecosystems unique to south Florida, the Bahamas and Cuba. As a result of their karstic calcium carbonate­rich soil, these systems are limited in phosphorus and nitrogen, making symbiotic associations critical to plant growth. Four leguminous species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima, and Abrus precatorious) were used to determine the relationship between rhizobial partners and plant performance, and the symbiosis related gene nifH was amplified to characterize the diversity of rhizobial symbionts. Plants were grown in soils from four different south Florida pine rocklands, and a salinity treatment was added to determine how storm surge and sea level rise could affect this symbiotic relationship. While plant performance and nodulation were highly impacted by soil type, salinity did not represent a significant effect. Phylogenetic analysis determined that all four plant species were found to associate with Bradyrhizobium spp. and no rhizobial shift between salinity treatment and soil type was found.