O processo de fixação biológica de nitrogênio (FBN) é a principal forma de entrada de nitrogênio (N) em ecossistemas naturais e é intermediado por microorganismos diazotróficos simbióticos ou de vida livre. A produção de biofertilizantes com bactérias diazotróficas é a principal alternativa ao uso de fertilizantes nitrogenados solúveis. Apesar da simbiose Rhizobium-leguminosas ser eficiente em promover o crescimento das plantas, a inoculação de bactérias diazotróficas em gramíneas vem apresentando resultados questionáveis, principalmente devido à baixa eficiência e incompatibilidade entre os atuais inoculantes bacterianos e as plantas. Os biofertilizantes para gramíneas utilizam bactérias endofíticas as quais desenvolvem interações pouco conhecidas com as plantas. Uma possibilidade para melhorar a eficiência da FBN em gramíneas é a aplicação de bactérias epifíticas de menor seletividade em relação às bactérias endofíticas. Os objetivos deste trabalho foram isolar novos genótipos de bactérias diazotróficas da filosfera de bambu da Mata Atlântica, verificar seu potencial biotecnológico in vitro e avaliar sua eficiência agronômica como biofertilizante em cana-de-açúcar em casa-de-vegetação. Foram obtidos 120 isolados bacterianos os quais foram caracterizados morfológica e filogenéticamente. Com relação ao potencial biotecnológico, 48 isolados bacterianos apresentaram resposta positiva nos testes in vitro de redução de acetileno (ARA) e para produção in vitro de ácido indol-3-acético (AIA) na presença de L-triptofano. Desses 48 isolados, 50% apresentaram capacidade de solubilizar fosfato de cálcio, 8% de produzir quitinases, 16% de produzir ACC desaminase e 17% de produzir sideróforos. O sequenciamento do gene rRNA 16S indicou que 75% dos isolados da filosfera de bambu foram similares à classe Gammaproteobacteria (Proteobacteria) , e a família Enterobacteriaceae. Dentre eles, 32% dos isolados apresentaram sequências similares a Klebsiella sp. e 2% foram similares a Serratia e Enterobacter. Das 48 sequências analisadas, 37% delas não foram classificadas quanto ao gênero, podendo então representar novos gêneros ou espécies. Dez isolados contendo o gene nifH foram selecionados para experimentos de eficiência agronômica em casa-de-vegetação usando bactérias inoculadas via pulverização foliar ou encapsuladas em micro-esferas de alginato e associados a diferentes níveis de adubação nitrogenada. A inoculação por meio de pulverização nas folhas de cana-de-açúcar resultou em aumento significativo de massa seca de parte aérea e concentração de N na parte aérea das plantas na sua fase inicial de desenvolvimento. A maior taxa de fixação nas folhas inoculadas foi observada sete dias após a inoculação no tratamento sem adição de N mineral. A inoculação no solo com os mesmos 10 isolados encapsulados em matriz polimérica em conjunto com diferentes níveis de adubação nitrogenada mostrou aumentos significativos de massa seca da parte aérea, raízes e concentração de N na parte aérea da cana-de-açúcar em comparação aos controles, principalmente nas fases tardias do desenvolvimento das plantas. O solo que recebeu as bactérias encapsuladas apresentaram elevadas taxas de FBN, oscilando entre 0 e 4 g de N g-1 h-1, 7 dias após a inoculação. Os resultados sugerem que as bactérias selecionadas possuem alto potencial biotecnológico para promover o crescimento das plantas em momentos diferentes do seu ciclo de desenvolvimento, dependendo do tipo de abordagem para inoculação. / The process of biological nitrogen fixation (BNF) is the most important form of nitrogen (N) input in natural ecosystems and is mediated by symbiotic or free-living diazotrophic microorganisms. The production of biofertilizers containing diazotrophic bacteria is the main alternative to the use of soluble nitrogen fertilizers, improving plant growth by nitrogen fixation or other plant growth promoting mechanisms. Despite the efficiency of the symbioses Rhizobium-legumes in promoting plant growth, the inoculation of diazotrophic bacteria in grasses, such as sugarcane, has shown variable results, mainly due to the low efficiency and incompatibility between the current bacterial strains used in inoculants and plant genotypes. Most of the biofertilizers for grasses uses endophytic bacteria that develop complexes interactions with the host plant, which are not totally understood. A possibility to improve the efficiency of BNF in grasses is the application of epiphytic diazotrophic bacteria that are less selective as compared to endophytes. The aims of this work were to isolate new genotypes of diazotrophic bacteria from the phyllosphere of bamboo from Atlantic Forest, determine their biotechnological potential based on in vitro assays, and evaluate their agronomic efficiency as biofertilizer for sugarcane under greenhouse conditions. A total of 120 bacterial isolates were obtained and characterized morphologically and phylogenetically. Regarding the biotechnological potential, 48 isolates showed positive responses under in vitro the acetylene reduction assay (ARA) and indole-acetic-acid (IAA) production in vitro assay in the presence of L-tryptophan. Among the 48 isolates evaluated, 50% were able to solubilize calcium phosphate, 8% produced chitinases, 16% were able to produce ACC deaminase, and 17% produced siderophores. The sequencing of the rRNA 16S gene revealed that 75% of the isolates were phylogenetically related to the family Enterobacteriaceae (Gammaproteobacteria) . The genus Klebsiella accounted for 32% of the isolates, whereas Serratia and Enterobacter accounted for 2%. Aproximatelly 37% of the isolates were assembled unclassified Bacteria. Ten isolates containing the nifH gene were selected for agronomic efficiency test under greenhouse conditions, using bacteria inoculated via foliar spraying, or encapsulation in alginate beads and inoculation in the soil, associated with different doses of nitrogen fertilizer. The inoculation on the sugarcane leaf surfaces resulted in significant increases in root biomass and N concentration in the shoots at the early stage of plant development. The highest N fixation rates in inoculated leaves were observed 7 days after inoculation in the absence of mineral N. The soil inoculation with the same 10 isolates immobilized in polymeric matrix in addiction to different rates of nitrogen fertilization showed significant increases in shoot and root biomass and N concentration in the shoots of sugarcane, when compared to the controls, mostly at later stages of plant development. The soil inoculated with encapsulated bacteria showed high rates of BNF even when nitrogen fertilizer was applied, ranging between 0 and 4 g of N g-1 h-1 seven days after the inoculation. The results suggest that the selected bacteria have high biotechnological potential to promote sugarcane growth at different stages of development, depending on the inoculation approach.

ABSTRACT The success of land plants can be attributed to the evolution of beneficial associations between plant roots and soil microbes. Root-microbe mutualisms extend the range of plant nutrient acquisition delivered through the hyphal network of mycorrhiza, an ancient and widespread plant symbiosis, or by the more recent adaptive innovation of nitrogen-fixing nodule symbioses. A plant’s genetic toolkit governs its selection of beneficial symbionts and the developmental extent of these intimate interactions. However, the evolutionary origins and function for only a few symbiotic signaling components have been explored. The central aim of this dissertation is to resolve the evolutionary events that contributed two, novel genetic components for establishing root symbioses, NPF1B and NPF1C. The Medicago truncatula (Mt) LATD/NIP/NPF1.7C transporter functions in root and nodule meristems and is a member of the large NPF1 gene subfamily. Here, I propose that LATD/NIP’s role in establishing nitrogen-fixing symbioses is derived from the ancient mycorrhizal signaling pathway. I used a comparative phylogenomic approach to investigate the evolutionary origins of the NPF1 gene across flowering plants and then asked whether diversifying or purifying selection forces influenced NPF1 gene retention. I postulated that such gene retention correlates with the adaptive traits of mycorrhizal or nitrogen-fixing root nodule symbiosis; to test this I measured trait correlation within my dataset. I found that the NPF1 phylogeny is comprised of five well-supported angiosperm clades, A, B, C, D1 and D2, that arose by successive duplications and have unequal gene retention. NPF1B is present as a single copy gene or lost entirely, while the other major NPF1 clades expanded to multiple genes within angiosperms. The NPF1A, B and C genes are under strong purifying selection while the NPF1D genes display positive, diversifying selection. My data revealed a statistically significant correlation of NPF1A, B, C, and D2, but not NPF1D1, gene retention with the ability of a species to form mycorrhizal associations. Additionally, the retention of the NPF1B, C, D1, D2, but not NPF1A, genes within a species is statistically correlated with its ability to form nitrogen-fixing symbiosis. Supporting this correlation, NPF1B genes are expressed in plant root tissues with and without mycorrhizal fungi yet available datasets failed to detect NPF1B expression in nodule tissues whereas the NPF1C genes are expressed in both symbiotic and non-symbiotic plant root tissues. In support of functional conservation, expression of legume LATD/NIP cDNAs from Cicer arietinum (Ca) and Lotus japonicus (Lj) restored, in part, the root and nodule defects of the Mtlatd mutant and resulted in the formation of peculiar hybrid lateral root-nodule structures while, in wild-type M. truncatula, significantly augmented root development. In L. japonicus, the disruption of LATD/NIP alters the number of lateral roots and nodules My thesis data support the hypothesis for an ancestral NPF1 gene function in establishing mycorrhizal associations in angiosperms and, consequent to the monocot-eudicot divergence, co-opted this function for accommodating nitrogen-fixing symbioses in eudicots. Successive duplications then yielded the NPF1B and NPF1C genes that, by neofunctionalization and natural selection, further refined their roles in root organogenesis and symbiosis; a prerequisite for the evolution of nodule organs.

Australia is amongst the more efficient milk producers in the world.Milk production in the region of south-western Victoria relies mainly on rainfed white clover/perennial ryegrass pastures.As the demand for efficient and competitive milk production increases, the value of N2 fixation must be maximised. The objective of this thesis was to assess N2 fixation in grazed dairy pastures in south-western Victoria. Several tests and experiments were conducted and results noted. Studies revealed low white clover yields to be the major factor limiting N2 fixation in the region. For N2 fixation to have a significant impact on pasture quality and production, problems associated with legume persistence need to be addressed. Strategies may include the breeding of white clover cultivars with greater tolerance to water stress, improved winter production and increased competitiveness with companion species. Alternatively, the introduction of different legume species, better suited to the environment, may be appropriate. Where N2 fixation is unlikely to satisfy N demands, it may be necessary to introduce the strategic use of supplementary feeds or nitrogenous fertilisers. However, this would need to be carefully considered to ensure high input costs did not jeopardise the competitive advantage of low input pasture-based systems / Masters Thesis

To understand the soil microbial community structures of different locations and altitudes, as well as the fluctuation of microbial populations foward seasonal change, this study investigated the biodiversity of soil microbiota of eight sampling sites along the Lao-Nong River basin during 4/11/2003~3/21/2004. The results showed that the soil properties were different among these sampling sites. The soil temperatures were ranging in between 24~32¢J, except Tianchr, the highest altitude sampling site (7~19¢J) ; pH values were 4.00 (Tianchr) ~8.20 (Tauyuan) ; moisture contents were 2.31% (Liouguei) ~70.23% (Tianchr) ; total organic carbon contents were 0.38% (Linkou) ~36.21% (Tianchr) ; total organic nitrogen contents were 0.067% (Tauyuan) ~0.672% (Tianchr); the C/N ratio were 7.0~25.0, except Tianchr (42 in average) . In microbial community structures analysis, bacteria were the most abundant organisms among all soil samples (106~108 CFU per gram of soil) . The amount of other microbes were as follow: nitrogen-fixing microbes 105~107 CFU, actinomycetes 104~107 CFU, fungi 104~106 CFU, cellulolytic microbes 104~106 CFU, and the least one was phosphate- solubilizing microbes 103~105 CFU. For molecular biological microbiota analysis, the bead-beating DNA extraction method resulted the richest bacterial 16S rDNA DGGE profile. The16S rDNA DGGE profiles of various samples taken from different seasons showed somewhat seasonal variations that might relate to the specific climates of Taiwan, such as the plum rains and typhoons. Moreover, we also successfully cultured and preserved many microbes with specific biological functions. These microorganisms will provide a useful resource for further research and development in the biotechnology industries.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 48-53). / viii, 53 leaves, bound ill. (some col.) 29 cm

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Australian National University, 2004. / Title from PDF title page (viewed on July 2, 2005). Includes bibliographical references (p. 172-196).

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-19Bitstream added on 2014-06-13T18:44:27Z : No. of bitstreams: 1 cicero_eas_dr_jabo.pdf: 499254 bytes, checksum: 2953fb8dcadff8a6c63acdb3bda489bc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis por meio da caracterização morfológica e molecular identificando as diferentes subclasses dos genes cry3, cry7, cry8, cry9 e cry35 e determinar a patogenicidade contra Sphenophorus levis, que é uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1128 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis e três linhagens padrões. O material genético foi extraído pela matriz de troca iônica “Instagene Matrix” e submetido a PCR com iniciadores gerais cry3, cry7, cry8, cry9 e cry35 identificando-se 45 isolados com produto de amplificação para coleópteros, os quais juntamente com as linhagens padrões de B. thuringiensis var. tenebrionis, var. morrissone e var. tolworthi e testemunhas foram utilizados para a realização do bioensaio. A análise discriminante alocou os isolados em três grupos quanto à toxicidade de B. thuringiensis e os grupos ficaram assim definidos: um grupo de BAIXA efetividade, com 19,60% do total dos isolados, nesse grupo ficaram as testemunhas e uma linhagem padrão B. thuringiensis var. morrissone e isolados do LGBBA; um grupo de MÉDIA efetividade, com 66,67% do total dos isolados, nesse grupo ficaram duas linhagens padrões B. thuringiensis var. tolworthi e var. morrissone e isolados do LGBBA e um grupo com ALTA efetividade com 13,72% do total dos isolados do LGBBA, os quais podem ser considerados promissores no controle biológico de S. levis. / Molecular characterization and selection of B. thuringiensis isolates exhibiting potential control on Sphenophorus levis larvae. Bacillus thuringiensis is characterized by the production of toxic proteins to insects from a number of different orders which are coded by the cry genes. This work was developed aiming to select B. thuringiensis isolates using morphological and molecular analysis so as to identify different cry genes subclass for cry3, cry7, cry8, cry9 and cry35 and also to determine the pathogenicity levels against Sphenophorus levis larvae, that is one of the most important sugar-cane pests. As much as 1128 bacterial isolates together with three control strains were used and for the phase contrast microscopy and molecular analysis. The genetic materials were obtained using the Instagene Matrix that is an ionic matrix and the PCR primers used were developed for cry3, cry7, cry8, cry9 and cry35 genes. A total of 45 isolates were selected showing possible amplicons for coleopterans. These isolates together with the standard strains B. thuringiensis var. tenebrionis, var. morrissone a var. tolworthi were used for bioassays. A discriminating analysis has allocated the bacterial isolates within three groups of B. thuringiensis toxicity and these groups were defined as one with LOW effectivity, comprising 19,6% of the isolates and the control strains together with one of the control strains B. thuringiensis var. morrissone. Another group has revealed a MEDIAN effectivity with a total of 66.7% of the isolates including two other control strains B. thuringiensis var. tolworthi e var. morrissone and a third group with HIGH effectivity with 13.7% of the isolates which were considered as promising B. thuringiensis isolates for the control of S. levis larvae.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-13Bitstream added on 2014-06-13T19:22:43Z : No. of bitstreams: 1 kishi_lt_dr_jabo_prot.pdf: 679937 bytes, checksum: fee4252e785e3aad08913f7c07f58933 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bactérias simbiontes conhecidos como rizóbios interagem com raízes de leguminosas e induzem a formação de nódulos fixadores de nitrogênio. Em rizóbios, genes essenciais para simbiose são compartimentalizados em ilhas simbióticas ou em ilhas no cromossoma. Para o entendimento da estrutura e evolução dessas ilhas simbióticas, é necessário analisar seu contexto genético e sua organização. O genoma parcial de B. elkanii SEMIA 587 apresenta hoje um total de 5.821.111 pb seqüenciados contendo 62,6 % de GC em 3941 Contigs, onde foram identificados 5381 ORFs. Uma suposta ilha simbiótica foi localizada através da identificação de genes relacionados à fixação e nodulação em comparação à ilha simbiótica de B. japonicum, encontrando 267 ORFs em B. elkanii, onde 17 ORFs foram identificadas como transposases. Assim, através destes resultados parciais pode ser possível averiguar que B. elkanii provavelmente sofreu um rearranjo genético no cromossoma, já que ele apresenta um tamanho menor que B. japonicum em relação ao tamanho do genoma. / Symbiont bacteria, commonly known as rhizobia interact with root legume and induce the formation of Nitrogen-fixing nodules. Among rhizobia, essential genes for symbioses are compartmentalized either in symbiotic islands or in chromosomal islands. For better understanding of the structure and evolution of these symbiotic islands, it is necessary to analyze their genetic context and organization. The work had as objective to analyze genes related to the symbiotic island in the partial genoma of the B. elkanii strain 587 has 5.821.111 bp sequenced, containing 62,6 % of GC in 3941 Contigs, been identified 5381 ORFs. A purported symbiotic island was localized through of the identification of related genes of fixing nitrogen and nodulation in comparison with the symbiotic island of B. japonicum, founding 267 ORFs in B. elkanii, where 17 ORFs were identified as transposases. So through these partial results could be possible to infer that B. elkanii probably shows a symbiotic island with genetic rearrange into chromosome, because it showed lower genome size than B. japonicum.