Campylobacter jejuni is a commensal microorganism of the poultry gastrointestinal tract. Broilers, layers, ducks, turkeys, and quails can be colonized by Campylobacter without illness occurring. The vast majority of human Campylobacter infections are recognized as being foodborne. For 2008, preliminary FoodNet data showed that the Campylobacter incidence of infection, 12.68 per 100,000 of the U.S. population, is the second highest, only behind Salmonella at 16.20 per 100,000. To further understand Campylobacter’s role as a foodborne pathogen, analysis at the molecular level is needed. Microbial molecular typing allows for identification and differentiation of bacterial strains beneath the species level. In this study, the “gold standard” method for molecular subtyping, Pulsed Field Gel Electrophoresis (PFGE), along with Diversilab® repetitive element Polymerase Chain Reaction (rep-PCR) and 16S-23S Internal Spacer Region Denaturing Gradient Gel Electrophoresis (ISR DGGE) were used for the molecular typing of Campylobacter jejuni isolates obtained during different stages of commercial broiler production and processing. In addition, the C. jejuni isolates were tested for resistance to antimicrobials commonly used in both veterinary and human medicine. Antimicrobial resistance testing was carried out using a broth dilution system. The majority of recovered isolates came from post-harvest carcass rinsates. Carcass rinses were obtained at post-evisceration, post-chill stages. All isolates (n = 46) were identified by the Polymerase Chain Reaction as Campylobacter jejuni. Three genotypes (n = 44, n = 1, n = 1) were identified by PFGE. The 46 rep-PCR products grouped into seven clusters and two outliers. Clustering of rep-PCR products by sample source was not observed. No relatedness trends were observed for isolates recovered from the same source. The combination of PFGE and Diversilab rep-PCR methods provides highly discriminatory molecular typing results. These results provide practical epidemiological information that shows postevisceration and post-chill stages are still important targets for intervention studies. The very high occurrence of C. jejuni isolates exhibiting genotype A suggests it may differentially express certain gene(s) that enable this strain to more favorably survive under the different harsh environmental conditions encountered during production and processing. In addition, phenotypic testing revealed all of the isolates were not resistant to the antimicrobials azithromycin, ciprofloxacin, erythromycin, gentamycin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin at any of the concentrations tested. All the C. jejuni isolates exhibited an indistinguishable two-band 16S-23S ISR DGGE profile. Overall, these C. jejuni commercial broiler pre- and post-harvest isolates exhibited an extremely low degree of molecular and phenotypic variability.

Orientador: Laura Maria Mariscal Ottoboni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:29:04Z (GMT). No. of bitstreams: 1 Carlos_Camila_M.pdf: 4715140 bytes, checksum: f65583df741bb75c1b397fb4581da9ef (MD5) Previous issue date: 2010 / Resumo: Coliformes e enterococos fecais de origem humana ou animal na água podem indicar a presença de patógenos de veiculação hídrica como, por exemplo, Salmonella e Giardia. A identificação da fonte de contaminação fecal (lançamento de esgoto doméstico, escoamento de fezes animais de criação no solo, de animais silvestres, aves e outros) é importante para a implantação de medidas efetivas de gerenciamento e remediação de águas superficiais. Dessa forma, o desenvolvimento de métodos para identificação da fonte de contaminação fecal é de fundamental importância para as ações de controle, para preservar a integridade dos corpos d'água e para proteger a saúde da população. Até o momento, não existe um método único e universal para este tipo de análise e, no Brasil, as pesquisas nessa área são praticamente nulas. Assim sendo, este trabalho teve como objetivo a obtenção de marcadores moleculares hospedeiro-específicos em Escherichia coli, que permitam a identificação da fonte animal de contaminação fecal em águas superficiais. Neste trabalho foram utilizadas 174 linhagens de origem humana, 50 de origem bovina, 39 de origem suína, 16 de origem aviária, 29 de origem ovina, 16 de origem caprina, 44 de esgoto, 36 de reservatórios com contaminação esperada de origem humana e 30 de rios e reservatórios com contaminação esperada de origem animal. A determinação do grupo filogenético de todas as linhagens foi realizada pela detecção dos genes chuA e yjaA e do fragmento Tspe4.C2 por PCR. Os resultados mostraram que a distribuição dos grupos filogenéticos principais A, B1, B2 e D foi diferente entre os hospedeiros analisados, o que permitiu a predição da fonte de contaminação fecal da maioria dos pontos de amostragem. Cem linhagens de humanos e todas as linhagens de origem animal foram analisadas por BOX- e (GTG)5-PCR. O BOX-PCR apresentou uma taxa global de classificação correta de 63,70%, o (GTG)5-PCR de 49,10% e quando os dois métodos foram utilizados a taxa foi de 57,61%. Outras 32 linhagens de humanos e 28 de esgoto também foram analisadas por BOX-PCR, sendo que, 59,4% das linhagens de humanos foram corretamente classificadas e 85,2% das linhagens de esgoto foram classificadas como de humanos. Vinte linhagens de humanos, 15 de bois e 16 de galinhas foram analisadas por espectroscopia no infravermelho com transformada de Fourier (FTIR). Utilizando-se um modelo PLS-DA com a segunda derivada do espectro na região 2816 e 3026 cm-1 foi possível separar completamente as linhagens segundo sua origem animal. Assim sendo, a espectroscopia FT-IR foi considerada a técnica mais promissora para futuros estudos de rastreamento de fonte de contaminação fecal. / Abstract: The detection of fecal coliforms and enterecocci in water indicates the presence of waterborne pathogens such as Salmonella and Giardia. The identification of the source of fecal contamination is important for the effective management of superficial water pollution. The development of methods for the identification of the source of fecal contamination is essential to preserve the quality of the water systems and to protect the public health. Until now, there is no universal method for this analysis and, in Brazil, there is no research in this area. In this way, the aim of this work was to obtain host-specific molecular markers in Escherichia coli for the identification of the source of fecal contamination in superficial water. For this work it was used, 174 strains from humans, 50 from cows, 39 from pigs, 29 from sheep, 16 from goat, 16 from chickens, 44 from sewage, 36 from water reservoirs whose the expected contamination source is human and 30 from water reservoirs and rivers whose the expected contamination source is animal. The determination of the phylogenetic groups of all strains was performed by the detection of the genes chuA and yjaA and the fragment Tspe4.C2 by PCR. The results showed that the distribution of the phylogenetic groups A, B1, B2 and D differs among the hosts analyzed, which allowed the prediction of the contamination source of most of the environmental samples. One hundred strains from humans, 50 from cows, 39 from pigs, 29 from sheep, 16 from goat and 16 from chickens were analyzed by BOX- and (GTG)5-PCR. The BOX-PCR presented an overall rate of correct assignment of 63.70%, the (GTG)5-PCR of 49.10% and, when the two methods were used, the rate was 57.61%. Thirty two other strains from humans and 28 strains from sewage were analyzed by BOX-PCR and compared with the profiles previously obtained. 59.4% of the human strains were correctly assigned as human and 85.2% of the sewage strains were assigned as human. Twenty strains from humans, 15 from cows and 16 from chickens were analyzed by Fourier transform infrared spectroscopy (FT-IR). The use of a PLS-DA model with the second derivative of spectra at the region 2816 and 3026 cm-1 made it possible to completely discriminate the strains according to the animal source. Therefore, the FT-IR spectroscopy was considered the most promising method for the identification of fecal contamination. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

This thesis deals with automatic bacteria genotyping by rep-PCR method. Its theoretical part presents various methods of DNA typing, basic information on electrophoresis and modern electrophoretic approaches, including their problems, misleading data distortion. In order to automate typing, there has been introduced a program for phylogenetic sample classification from rep-PCR, also applicable for data from chip capillary electrophoresis. The program consists of three main parts: digitization, bandmatching and clustering apparatus to bacterial type classification. The result of the algorithm is a phylogenetic tree, which indicates the cluster of sapmles according to bacterial type. The program has a graphical user interface for possible use in the Children's Hospital. Finally, the program is tested with data from the Children´s Hospital.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-18T13:33:35Z No. of bitstreams: 2 Dissertação - Bruna Alicia Rafael de Paiva - 2014.pdf: 5696996 bytes, checksum: 7e1928a13da15787585830bd9a4f4ae1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-18T13:34:11Z (GMT) No. of bitstreams: 2 Dissertação - Bruna Alicia Rafael de Paiva - 2014.pdf: 5696996 bytes, checksum: 7e1928a13da15787585830bd9a4f4ae1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-18T13:34:11Z (GMT). No. of bitstreams: 2 Dissertação - Bruna Alicia Rafael de Paiva - 2014.pdf: 5696996 bytes, checksum: 7e1928a13da15787585830bd9a4f4ae1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli (Xap) and Xanthomonas fuscans subsp. fuscans (Xff), is one of most important bacterial etiology diseases from the common bean plant. This pathogen causes a great loss in the production, mainly in the “water” harvest, when the environmental conditions are helpful to the development of this disease and to its dissemination. Among the strategies about integrated handling of diseases, the genetic resistance is the main measure of control and, for obtaining some cultivate with lasting resistance and of notorious specter, it is important to the know the genetic pathogen diversity. The present work has the goal of studying the diversity and the genetic structure around and inside the population of Xap and Xff using the markers rep-PCR; to connect the genetic pathogen diversity with its geographical distribution inside Brazilian producing common bean plant regions. There were obtained 42 Xap and Xff isolated and four Xanthomonas isolated not pathogens of to the bean plant, originated in the States of São Paulo, Goiás, Paraná and Rio Grande do Sul. The genetic profiles obtained by primers BOX, ERIC and REP produced 12 haplotipes matched (HC), where the HC 3, specific xff, was more frequent in PR and GO, and was not found in RS. The dendrogram produced through the analysis showed that Xap, Xff and isolated not pathogens are genetically different. In the analysis of genetic structure, the total genetic diversity (Ht), among three populations was 0,2385 revealing that there was variability from a population to the other one. Higher values of the Shannon rate (0,3648) show that Xap population is more different genetically. From a total of 51 locos, 94,12 % are polymorphic, inside Xap population they are all polymorphicand inside Xff, only 18,18 % are polymorphic. The genetic differentiation coefficient (Gst = 0,5194) revealed diversity inside and among the patogenic populations, confirming AMOVA’s result, where 51,98 % of the diversity was among the populations and 48,06 % inside them. Then, the technique helped how to find differences between Xap and Xff and measure the values of genetic diversity among and inside populations. The results of this study give us much information about the pathogens diversity, which is considered useful to identify and to characterize the resistant germoplasm in a more efficient way. / O crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli (Xap) e Xanthomonas fuscans subsp. fuscans (Xff), é uma das doenças de etiologia bacteriana mais importantes do feijoeiro-comum. Este patógeno provoca grandes perdas na produção, principalmente na safra das “águas”, quando as condições ambientais são favoráveis ao desenvolvimento da doença e à disseminação do patógeno. Dentro das estratégias do manejo integrado de doenças, a resistência genética é a principal medida de controle, e para a obtenção de cultivares com resistência duradoura e de amplo espectro é necessário o conhecimento da diversidade genética do patógeno. O presente trabalho teve como objetivos, estudar a diversidade e a estrutura genética entre e dentro das populações de Xap e Xff usando marcadores rep-PCR; relacionar a diversidade genética do patógeno com sua distribuição geográfica dentro das regiões produtoras de feijoeiro-comum no Brasil. Foram obtidos 42 isolados de Xap e Xff, e quatro isolados de Xanthomonas não patogênicas ao feijoeiro, oriundos dos estados de São Paulo, Goiás, Paraná e Rio Grande do Sul. Os perfis genéticos obtidos pelos primers BOX, ERIC e REP geraram 12 haplótipos combinados (HC), em que o HC 3, específico de Xff, foi mais frequente nos estados do PR e GO , porém não foi identificado no RS. O dendrograma gerado através da análise conjunta mostrou que Xap, Xff e isolados não patogênicos foram geneticamente distintos. Na análise de estruturação genética, a diversidade genética total (Ht) entre as populações foi de 0,2385, mostrando que houve variabilidade entre as populações. Valores mais altos do índice de Shannon (0,3648) revelam que a população de Xap é mais diversa geneticamente. De um total de 51 locos em todas populações, 94,12% foram polimórficos, sendo que, dentro de Xap todos foram polimórficos, e dentro de Xff apenas 18,18% foram polimórficos. O coeficiente de diferenciação genética (GST = 0,5194) revelou diversidade tanto dentro como entre as populações patogênicas, confirmando o resultado obtido pela AMOVA, em que 51,98% da diversidade está distribuída entre as populações e 48,06% dentro destas. Portanto, o uso da técnica rep-PCR permitiu diferenciar Xap e Xff e mensurar os valores de diversidade genética entre e dentro de populações. Os resultados deste estudo fornecem informações sobre a diversidade dos patógenos, o que auxiliará para melhor identificar e caracterizar o germoplasma resistente.

Chronic infection with P. aeruginosa is expected in patients with cystic fibrosis (CF), but the ability to delay, prevent, or better manage infection with multiply-resistant P. aeruginosa (MRPA) can potentially increase quality of life and extend survival. The Texas Children’s Hospital CF Care Center has identified an endemic MRPA strain (dominant clone), and this study aimed to identify risk factors for acquisition of the clone as well as determine differences in patient outcome associated with subsequent infection with the clone. The study included 71 patients with CF with documented MRPA infection. Designation of patients as members of the dominant clone or a non-dominant clone group was based on molecular typing by rep-PCR of MRPA isolates from respiratory cultures. Patient data was collected from Port CF, the national patient registry of the CF Foundation. Patient demographic information and clinical parameters prior to MRPA infection were analyzed by logistic regression as potential risk factors. Differences in patient outcome including change in BMI, change in FEV1, and hospitalization rate were evaluated by MANOVA. Recent hospitalization (< 90 days) was a statistically significant (p = 0.035) risk factor for acquisition of the dominant clone. Patients hospitalized < 90 days prior to MRPA diagnosis were four times more likely to be infected with the dominant clone, and patients hospitalized 91-180 days prior were almost three times more likely. Increased hospitalization rates were seen in the dominant clone group both pre- (11 more days/year) and post-infection (14 more days/year) as compared to the non-dominant clone group. Patients infected with the endemic strain exhibited poorer outcomes in terms of nutritional status (3.73% decrease/year in BMI %ile) and lung function (3.7% decrease/year in FEV1 %ile). Significant overlap in hospitalization episodes of patients known to be infected with the dominant clone and patients subsequently infected with the dominant clone was observed. Recent hospitalization was a significant risk factor for infection with the dominant MRPA clone, and following infection, patients infected with the endemic strain exhibited declines in nutritional status and lung function and increased hospitalization rates. The results suggest potentially increased virulence and transmissibility of the endemic MRPA strain.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-28Bitstream added on 2014-06-13T20:37:45Z : No. of bitstreams: 1 rodrigues_lmr_me_botfca.pdf: 618458 bytes, checksum: cb654c56905a6abeae0bdd7484f37739 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve como objetivo avaliar a agressividade de linhagens de Ralstonia solanacearum provenientes de solanáceas, plantas ornamentais e eucalipto, em plantas de batata, tomate e fumo, bem como caracterizar as linhagens por meio de técnicas moleculares. Vinte e duas linhagens foram utilizadas nos ensaios de avaliação da agressividade, em experimentos conduzidos em casa-de-vegetação evidenciaram alta severidade da doença pelas linhagens de R. solanacearum quando inoculadas em plantas de tomate e batata, sendo a batata mais afetada nas inoculações. Todas as linhagens mostraram-se agressivas, sendo que o fumo mostrou baixa suscetibilidade ao ataque das bactérias. As linhagens mais agressivas em plantas de tomate foram IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 e IBSBF 2000, pertencentes às biovares I, II e III. As linhagens mais agressivas às plantas de fumo foram IBSBF 309, IBSBF 2131 e IBSBF 292T, pertencentes à biovar I. Foi efetuado também ensaio de microbiolização in vitro em sementes de eucalipto, a fim de se identificar possíveis linhagens patogênicas a esta espécie vegetal e concluiu-se que todas as linhagens utilizadas infectaram plantas de eucalipto ou afetaram seu crescimento. A caracterização molecular de 41 linhagens de Ralstonia solanacearum, provenientes de diversas plantas hospedeiras, incluindo solanáceas, bananeira, helicônia, plantas ornamentais e eucalipto, foi efetuada empregando-se ERIC e BOX-PCR e os resultados mostraram grande diversidade genética entre as linhagens. A análise de PCR-RFLP da região espaçadora 16S-23S DNAr permitiu distinguir os isolados pertencentes à biovar III das demais biovares (I, II, IIA e IIT), quando digeridos com as enzimas Taq I e Hin6 I. A análise de sequenciamento de parte dos genes Endoglucanase (Egl) e MutS possibilitou a classificação em filotipos e os resultados... / This study aimed to evaluate the aggressiveness of strains of Ralstonia solanacearum from solanaceus, ornamental and eucalyptus plants, on potato, tomato and tobacco, and to characterize the strains through molecular techniques. Twenty-two strains were used in this study to evaluate the aggressiveness and, the experiments conducted in a greenhouse revealed the high susceptibility of tomato and potato plants, with the potato being the most affected on through the inoculations. All isolates proved to be aggressive and higher tolerance to the attack of bacteria was verified on tobacco plants. Strains more aggressive on tomato were IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 and IBSBF 2000, belonging to biovars I, II and III. The more aggressive strains on the tobacco plants were IBSBF 309, IBSBF 292T and IBSBF 2131 belonging to biovar I. Tests in vitro of microbiolization of eucalyptus seeds were also performed in order to identify possible pathogenic strains to this species and the results showed that all strains used cause infection on emerging plants or affected their growth. To molecular characterization of 41 strains of Ralstonia solanacearum from several host plants including solanaceous, banana, heliconia, ornamentals and eucalyptus were employed to ERIC and BOX-PCR, and the results showed high genetic diversity among strains. The analysis of PCR-RFLP of 16S-23S spacer region rDNA allowed us to distinguish the isolates belonging to biovar III from the others (biovars I, II, IIA and IIT) when digested with enzymes Taq I and Hin6 I. The sequence analysis of the partial of Endoglucanase (Egl) and MutS genes allowed the classification in phylotypes and the results revealed a predominance of the phylotype II in Brazil, and four isolates were classified in the phylotype I, all belonging to biovar III

Made available in DSpace on 2016-06-02T20:21:33Z (GMT). No. of bitstreams: 1 DissLS.pdf: 2088581 bytes, checksum: 7c2ce32f768263b79d4fae9da49f7321 (MD5) Previous issue date: 2004-06-30 / Universidade Federal de Sao Carlos / Não consta.

Resumo: O presente trabalho teve como objetivo avaliar a agressividade de linhagens de Ralstonia solanacearum provenientes de solanáceas, plantas ornamentais e eucalipto, em plantas de batata, tomate e fumo, bem como caracterizar as linhagens por meio de técnicas moleculares. Vinte e duas linhagens foram utilizadas nos ensaios de avaliação da agressividade, em experimentos conduzidos em casa-de-vegetação evidenciaram alta severidade da doença pelas linhagens de R. solanacearum quando inoculadas em plantas de tomate e batata, sendo a batata mais afetada nas inoculações. Todas as linhagens mostraram-se agressivas, sendo que o fumo mostrou baixa suscetibilidade ao ataque das bactérias. As linhagens mais agressivas em plantas de tomate foram IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 e IBSBF 2000, pertencentes às biovares I, II e III. As linhagens mais agressivas às plantas de fumo foram IBSBF 309, IBSBF 2131 e IBSBF 292T, pertencentes à biovar I. Foi efetuado também ensaio de microbiolização in vitro em sementes de eucalipto, a fim de se identificar possíveis linhagens patogênicas a esta espécie vegetal e concluiu-se que todas as linhagens utilizadas infectaram plantas de eucalipto ou afetaram seu crescimento. A caracterização molecular de 41 linhagens de Ralstonia solanacearum, provenientes de diversas plantas hospedeiras, incluindo solanáceas, bananeira, helicônia, plantas ornamentais e eucalipto, foi efetuada empregando-se ERIC e BOX-PCR e os resultados mostraram grande diversidade genética entre as linhagens. A análise de PCR-RFLP da região espaçadora 16S-23S DNAr permitiu distinguir os isolados pertencentes à biovar III das demais biovares (I, II, IIA e IIT), quando digeridos com as enzimas Taq I e Hin6 I. A análise de sequenciamento de parte dos genes Endoglucanase (Egl) e MutS possibilitou a classificação em filotipos e os resultados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the aggressiveness of strains of Ralstonia solanacearum from solanaceus, ornamental and eucalyptus plants, on potato, tomato and tobacco, and to characterize the strains through molecular techniques. Twenty-two strains were used in this study to evaluate the aggressiveness and, the experiments conducted in a greenhouse revealed the high susceptibility of tomato and potato plants, with the potato being the most affected on through the inoculations. All isolates proved to be aggressive and higher tolerance to the attack of bacteria was verified on tobacco plants. Strains more aggressive on tomato were IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 and IBSBF 2000, belonging to biovars I, II and III. The more aggressive strains on the tobacco plants were IBSBF 309, IBSBF 292T and IBSBF 2131 belonging to biovar I. Tests in vitro of microbiolization of eucalyptus seeds were also performed in order to identify possible pathogenic strains to this species and the results showed that all strains used cause infection on emerging plants or affected their growth. To molecular characterization of 41 strains of Ralstonia solanacearum from several host plants including solanaceous, banana, heliconia, ornamentals and eucalyptus were employed to ERIC and BOX-PCR, and the results showed high genetic diversity among strains. The analysis of PCR-RFLP of 16S-23S spacer region rDNA allowed us to distinguish the isolates belonging to biovar III from the others (biovars I, II, IIA and IIT) when digested with enzymes Taq I and Hin6 I. The sequence analysis of the partial of Endoglucanase (Egl) and MutS genes allowed the classification in phylotypes and the results revealed a predominance of the phylotype II in Brazil, and four isolates were classified in the phylotype I, all belonging to biovar III / Orientador: Antonio Carlos Maringoni / Coorientador: Suzete Aparecida Lanza Destéfano / Banca: Valdemar Atilio Malavolta Junior / Banca: Ivan Paulo Bedendo / Mestre

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-21T17:26:17Z No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-21T18:17:40Z (GMT) No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-21T18:17:40Z (GMT). No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Research on biological nitrogen fixation (BNF) in common bean had a great progress in recent years, especially in the knowledge of microsimbiont and exploring new approaches seeking greater variability in macrosimbiont for efficiency of BNF. Studies of bacterial diversity and taxonomy, especially applied to common bean symbionts showed a great evolution due to new molecular methodologies for evaluation and characterization. This study aimed to evaluate the tolerance to salinity and temperature, to characterize based on molecular markers and to evaluate the symbiotic efficiency of bacterial isolates obtained from nodules of common bean cultivated on soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were evaluated for salinity and temperature on YMA medium with different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28ºC, 33ºC, 38ºC, 43ºC and 48ºC). For molecular characterization based on BOX-PCR and REP-PCR profiles the isolates were grown in liquid YMA for 24 hours and then DNA extraction was performed. Evaluation of symbiotic efficiency of the isolates was conducted under greenhouse conditions in Leonard jars. Seeds of common bean (var. Pérola) were inoculated with different isolates selected in the previous analysis. Nodule number (NN), dry mass of nodules (DMN), specific mass of nodules (SMN), root dry weight (RDW), dry matter of aerial part (DMAP), relation root/shoot (R/S), total nitrogen (N) and leaf area (LA) were evaluated. It was observed that 41.12% of the isolates grew in more restrictive conditions than standard strains SEMIA 4077, SEMIA 4080 and SEMIA 4088, and 29.90% of the isolates grew in less restrictive conditions than SEMIAs strains.BOX-PCR and REP-PCR profiles showed high genetic diversity among the evaluated isolates, demonstrating a high degree of polymorphism. JPrG8A7 and JPrG8A6 isolates exhibited superior performance compared to standard strains when compared the NN , SMN and DMN. The latter showed a positive correlation with the DMAP, Total-N and LA. It was observed that some isolates showed competitive features equal or superior than commercial standards strains, with results that can improve the process of symbiosis between plant and bacteria, thereby generating greater productivity for the common bean cultivation. / A pesquisa sobre a fixação biológica de nitrogênio (FBN) no feijoeiro teve bastante progresso nos últimos anos, especialmente no conhecimento do microsimbionte e no estudo de novas abordagens buscando maior variabilidade no macrosimbionte para maior eficiência da FBN. Os estudos da diversidade e taxonomia bacteriana, especialmente aplicados aos simbiontes do feijoeiro-comum apresentou uma grande evolução devido às novas metodologias moleculares de avaliação e caracterização. Este trabalho teve como objetivo avaliar a resistência à salinidade e temperatura, caracterizar molecularmente e avaliar a eficiência simbiótica de isolados de nódulos de feijoeiro-comum oriundos dos estados de GO, MG e PR. Os isolados foram avaliadas quanto à salinidade e temperatura em meio YMA com diferentes concentrações de NaCl (0%; 1%; 2%; 4% e 6%) em diferentes temperaturas (28ºC; 33ºC; 38ºC; 43ºC e 48ºC). Para a caracterização molecular os isolados foram crescidos em meio YMA líquido por 24 horas e logo em seguida foi realizada a extração do DNA. Foram avaliados perfis BOX-PCR e REP-PCR. A avaliação da eficiência simbiótica dos isolados foi conduzida em casa-de-vegetação com vasos tipo Leonard com a cultivar Pérola inoculada com diferentes isolados selecionados na análise anterior. Foi avaliado o número de nódulos (NN), massa seca de nódulos (MSN), massa específica de nódulos (MEN), massa seca de raiz (MSR), matéria seca da parte aérea (MSPA), relação raiz/parte aérea (R/PA), nitrogênio total (N) e área foliar (AF). Observou-se que 41,12% dos isolados cresceram em condições mais restritivas que as estirpes padrão SEMIA 4077, SEMIA 4080 e SEMIA 4088, e 29,90% dos isolados cresceram em condições menos restritivas que as SEMIAs. Os perfis BOX-PCR e REP-PCR apresentaram grande diversidade genética entre os isolados avaliados, demonstrando um alto grau de polimorfismo. Os isolados JPrG8A7 e JPrG8A6 apresentaram desempenho superior as estirpes padrão quando comparados o NN, MEN e MSN. Esta última apresentou correlação positiva com a MSPA, N-Total e AF. Foi observado que alguns isolados apresentaram características competitivas iguais ou superiores as estirpes-padrões comerciais, apresentando resultados que podem melhorar o processo de simbiose entre a planta e a bactéria, gerando assim uma maior produtividade para a cultura do feijoeiro-comum.

The study was focused on molecular characterization of 42 clostridial strains. DNA was isolated by fenol-chloroform extraction procedure and precipitated with ethanol. After DNA isolation, PCR amplifications with specific primer sets were used for genus and species identification. Finally 19 strains were clasified as Clostridium tyrobutyricum and 3 strains were clasified as Clostridium butyricum. Presence of hydrogenase gene hydA was tested by PCR amplification using specific primer set HGf and HGr. Presence of hydrogenase gene was detected within 21 strains. (GTG)5 primer (rep-PCR) and Pr1 and Pr6 primers (RAPD) were used for differentiation of clostridial strains. Next, the cultivation of Clostridium tyrobutyricum S5 was studied under different conditions. The cultivation was carried out in liquid Reinforced Clostridial Medium (RCM) with lactose and cheese whey instead of glucose under anaerobe conditions. Growth was observed at laboratory temperature (20 to 23 °C) and at 37 °C, pH values ranging from 4.0 to 8.0 with 0.5 unit.