ENGLISH : The use of different characteristics (the polyphasic approach) to describe bacterial taxa is a prerequisite for a stable classification. The taxonomy of root- and stem-nodulating rhizobia is in a state of transition. As more legumes are studied, new species and genera of rhizobia are described. It is important to study the indigenous South African rhizobia, as without them a complete rhizobial taxonomy is not possible. Furthermore, strains with superior nitrogen fixation abilities may be discovered. Indigenous strains better adapted to the harsh South African environment are possible candidates for commercial inoculants for cropped legumes.Only two local studies have been done on the diversity of the indigenous rhizobia. These studies revealed the diversity of rhizobia existing in the South African context. As part of a polyphasic approach used to identify and determine the diversity of the indigenous rhizobia, 16S rDNA sequencing analysis was performed on some selected rhizobial and putative rhizobial isolates. The aim of the study was to characterise and identify the indigenous isolates by 16S rDNA sequencing analysis and compare our data with those available in the GenBank database. Results showed that most of the indigenous isolates were slow-growers belonging to the genus Bradyrhizobium. Two isolates from supposedly non-nodulating legume genera (Cassia and Senna) were found to belong to the genus Bradyrhizobium. Some of the isolates were shown to belong to the genera Mesorhizobium, Rhizobium and Sinorhizobium. The identity of five isolates was not clear and further studies need to be performed to unequivocally determine their taxonomic position. Partial sequence analysis of 16S rDNA proved a valuable tool to characterise and identify the indigenous isolates. However, the method was unable to clearly distinguish between closely related species and strains. AFRIKAANS : 'n Stabiele klassifikasiesisteem vir die beskrywing van bakteriese taksa is slegs moontlik deur verskillende eienskappe (die poli-fasiese benadering) te gebruik. Die taksonomie van die wortel- en stamnodulerende rhizobiums verander gedurig. 'n Volledige rhizobiumtaksonomie is slegs moontlik indien die inheemse Suid-Afrikaanse rhizobiums bestudeer word. Geharde inheemse rasse met voortreflike stikstofbindende vermoens kan ontdek word. Hierdie rasse is kandidate vir kommersiele inokulums vir verboude peulplante. Net twee plaaslike studies is gedoen om die diversiteit van die inheemse rhizobiums te bepaal. Die studies het bewys dat die inheemse rhizobiums baie divers is. As deel van die polifasiese benadering om die diversiteit van die inheemse rhizobiums te identifiseer en te bepaal, is 16S rDNS volgordebepaling gedoen op uitgesoekte rhizobia en sogenaamde rhizobia isolate. Die doel van die studie was die karakterisering en identifisering van die inheemse isolate deur 16S rDNS volgordebepaling en die vergelyking van die data met die beskikbaar in die GenBank databasis. Die resultate wys dat die meeste inheemse isolate stadige groeiers is en dus behoort aan die genus Bradyrhizobium. Twee isolate vanaf sogenaamde nie-nodulerende peulplantgenusse (Cassia en Senna) behoort ook tot die genus Bradyrhizobium. Sommige isolate behoort tot die genusse Mesorhizobium, Rhizobium en Sinorhizobium. Die identiteit van vyf isolate was nie duidelik nie en verdere studies is nodig om hul taksonomiese posisie ondubbelsinnig te bepaal. Die gedeeltelike volgordebepaling van die 16S rDNS was 'n waardevolle hulpmiddel om die inheemse isolate mee te karakteriseer en te identifiseer, alhoewel die metode nie tussen nabyverwante spesies en rasse kon onderskei nie. Copyright / Dissertation (MSc (Microbiology))--University of Pretoria, 1999. / Microbiology and Plant Pathology / unrestricted

Bactérias endofíticas são aquelas que habitam o interior de tecidos vegetais, sem causar dano aparente aos mesmos, além de desempenhar funções importantes no processo de adaptação das plantas. Especial interesse tem sido dado a tais bactérias devido ao seu potencial no controle biológico. Por isso, é muito importante estudar a diversidade genética de endófitos, além de avaliar o impacto da introdução de endófitos geneticamente modificados no ambiente. Estudos vêm sendo feitos nesse sentido, mas não com bactérias endofíticas da família Enterobacteriaceae. Desta forma, o presente trabalho teve como objetivos estudar a diversidade genética de bactérias endofíticas da família Enterobacteriaceae isoladas de plantas de cacau, cana-de-açúcar, citros, eucalipto e soja, utilizando diferentes técnicas moleculares. Análises por ARDRA e seqüenciamento do rDNA 16S identificaram 20 haplótipos e revelaram que os isolados pertenceram aos gêneros Enterobacter, Erwinia e Pantoea, sendo este último o mais freqüente. Tais estudos revelaram ainda que a diversidade dos isolados variou de acordo com a planta hospedeira. A técnica de BOX-PCR foi também utilizada para avaliar a diversidade dos isolados. Um total de 23 diferentes OTUs (operational taxonimic units) obtidas indicaram que o total de isolados avaliados compreenderam pelo menos 23 espécies diferentes. Dois isolados foram transformados com pPAgfp, um plasmídio contendo os genes de resistência ao antibiótico ampicilina e o gene gfp, que codifica a proteína verde fluorescente. Tais isolados foram inoculados em plântulas de Catharantus roseus (vinca) e foi feito reisolamento de bactérias endofíticas em dois períodos diferentes após a inoculação. O impacto desta inoculação na diversidade da comunidade microbiana natural de vinca foi avaliado utilizando-se a técnica de ARDRA, a qual mostrou que os endófitos expressando gfp colonizaram as raízes e caules das plantas inoculadas, sem causar qualquer sintoma de doença. Além disso, os colonizadores endofíticos não levaram à diminuição da diversidade da população microbiana natural de vinca. Os resultados obtidos poderão contribuir para a compreensão sobre a interação entre Enterobacteriaceae endofítica e planta hospedeira, além de ajudar a responder questões sobre o papel ecológico dos endofíticos e seu potencial biotecnológico. / Endophytic bacteria have been defined as those that reside within living plant tissues, or extracted from inner plant parts without causing apparent damage to them. They also are able to play an important role in the process of plant adaptation. There is an increasing interest to endophytic bacteria and its potential in the biological control and many studies has been done in order to evaluate the diversity and the impact of endophytes and genetically modified endophytes (GME) released into environment. In this way, information about the diversity of endophytic bacteria has been obtained, except for bacteria exclusively from Enterobacteriaceae family belonged to different host plants. Thus, the aim of the present work was study the diversity of Enterobacteriaceae bacteria isolated from citrus, cocoa, eucalypti, soybean and sugar cane by different molecular approaches. The 16S rDNA of each isolate was amplified by PCR and the isolates were grouped into 20 clusters by analysis of restriction patterns of the PCR-amplified 16S rDNA (ARDRA). These analysis showed a variety of organisms, with 5 different genera encountered: Pantoea was most frequently encountered followed by Enterobacter and Erwinia, which isolates presented the great bacterial diversity according to host plants. Through cluster analysis of the BOX-PCR technique profiles, 23 different OTUs (Operational Taxonomic Units) were distinguished, the presence of 23 OTUs could indicate that isolates comprised at least 23 different species. Two isolates were transformed with pPAgfp, a plasmid harboring the ampicillim resistance gene and the gfp gene, which encodes for the green fluorescent protein. These two isolates were inoculated in seedlings of Catharantus roseus (vinca) and re-isolation of endophytic was performed in two times after inoculation. The impact of endophytes inoculation was evaluated by using the ARDRA technique. It showed that endophytes expressing gfp colonized roots and shoots of inoculated plants without causing any symptom of disease. Besides, the endophytes colonizers do not decreased the diversity of the vinca’s natural microbiota. The results obtained here provided important insights into the endophytic Enterobacteriaceae-host relationship that will be useful for further answer basic questions about the ecological role of the endophytes and its biotechnological potential.

Bactérias endofíticas são aquelas que habitam o interior de tecidos vegetais, sem causar dano aparente aos mesmos, além de desempenhar funções importantes no processo de adaptação das plantas. Especial interesse tem sido dado a tais bactérias devido ao seu potencial no controle biológico. Por isso, é muito importante estudar a diversidade genética de endófitos, além de avaliar o impacto da introdução de endófitos geneticamente modificados no ambiente. Estudos vêm sendo feitos nesse sentido, mas não com bactérias endofíticas da família Enterobacteriaceae. Desta forma, o presente trabalho teve como objetivos estudar a diversidade genética de bactérias endofíticas da família Enterobacteriaceae isoladas de plantas de cacau, cana-de-açúcar, citros, eucalipto e soja, utilizando diferentes técnicas moleculares. Análises por ARDRA e seqüenciamento do rDNA 16S identificaram 20 haplótipos e revelaram que os isolados pertenceram aos gêneros Enterobacter, Erwinia e Pantoea, sendo este último o mais freqüente. Tais estudos revelaram ainda que a diversidade dos isolados variou de acordo com a planta hospedeira. A técnica de BOX-PCR foi também utilizada para avaliar a diversidade dos isolados. Um total de 23 diferentes OTUs (operational taxonimic units) obtidas indicaram que o total de isolados avaliados compreenderam pelo menos 23 espécies diferentes. Dois isolados foram transformados com pPAgfp, um plasmídio contendo os genes de resistência ao antibiótico ampicilina e o gene gfp, que codifica a proteína verde fluorescente. Tais isolados foram inoculados em plântulas de Catharantus roseus (vinca) e foi feito reisolamento de bactérias endofíticas em dois períodos diferentes após a inoculação. O impacto desta inoculação na diversidade da comunidade microbiana natural de vinca foi avaliado utilizando-se a técnica de ARDRA, a qual mostrou que os endófitos expressando gfp colonizaram as raízes e caules das plantas inoculadas, sem causar qualquer sintoma de doença. Além disso, os colonizadores endofíticos não levaram à diminuição da diversidade da população microbiana natural de vinca. Os resultados obtidos poderão contribuir para a compreensão sobre a interação entre Enterobacteriaceae endofítica e planta hospedeira, além de ajudar a responder questões sobre o papel ecológico dos endofíticos e seu potencial biotecnológico. / Endophytic bacteria have been defined as those that reside within living plant tissues, or extracted from inner plant parts without causing apparent damage to them. They also are able to play an important role in the process of plant adaptation. There is an increasing interest to endophytic bacteria and its potential in the biological control and many studies has been done in order to evaluate the diversity and the impact of endophytes and genetically modified endophytes (GME) released into environment. In this way, information about the diversity of endophytic bacteria has been obtained, except for bacteria exclusively from Enterobacteriaceae family belonged to different host plants. Thus, the aim of the present work was study the diversity of Enterobacteriaceae bacteria isolated from citrus, cocoa, eucalypti, soybean and sugar cane by different molecular approaches. The 16S rDNA of each isolate was amplified by PCR and the isolates were grouped into 20 clusters by analysis of restriction patterns of the PCR-amplified 16S rDNA (ARDRA). These analysis showed a variety of organisms, with 5 different genera encountered: Pantoea was most frequently encountered followed by Enterobacter and Erwinia, which isolates presented the great bacterial diversity according to host plants. Through cluster analysis of the BOX-PCR technique profiles, 23 different OTUs (Operational Taxonomic Units) were distinguished, the presence of 23 OTUs could indicate that isolates comprised at least 23 different species. Two isolates were transformed with pPAgfp, a plasmid harboring the ampicillim resistance gene and the gfp gene, which encodes for the green fluorescent protein. These two isolates were inoculated in seedlings of Catharantus roseus (vinca) and re-isolation of endophytic was performed in two times after inoculation. The impact of endophytes inoculation was evaluated by using the ARDRA technique. It showed that endophytes expressing gfp colonized roots and shoots of inoculated plants without causing any symptom of disease. Besides, the endophytes colonizers do not decreased the diversity of the vinca’s natural microbiota. The results obtained here provided important insights into the endophytic Enterobacteriaceae-host relationship that will be useful for further answer basic questions about the ecological role of the endophytes and its biotechnological potential.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-31T14:41:08Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_Mestrado_Roberto_Carvalho_2016.pdf: 4128480 bytes, checksum: d7d2a90680981c944fb1c8f51356fdf0 (MD5) / Made available in DSpace on 2017-07-31T14:41:08Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_Mestrado_Roberto_Carvalho_2016.pdf: 4128480 bytes, checksum: d7d2a90680981c944fb1c8f51356fdf0 (MD5) Previous issue date: 2016-06-28 / FACEPE / Neste trabalho foi realizado um estudo da ecologia microbiana com o objetivo de entender a composição e a dinâmica da comunidade microbiana formada em dois reatores do tipo UASB, correlacionando os parâmetros ambientais obtidos no monitoramento prévio com os parâmetros biológicos identificados ao longo da pesquisa. Um dos reatores foi planejado para ser operado em regime totalmente anaeróbio e o outro em condição microaerofílica. Ambos foram operados com efluente têxtil sintético durante 3 fases (Fase I – condições normais do efluente sintético; Fase II – adição de salinidade no afluente; Fase III – Adição de sulfato no afluente), ao final de cada fase foram coletadas amostras da biomassa da manta de lodo, como também uma amostra da biomassa aderida ao aerador apenas na fase III e por fim amostra do inóculo utilizado. Através das técnicas de PCR-DGGE foi possível obter uma pré-visualização da diversidade das amostras servindo como uma ferramenta para selecionar as amostras que seguiriam para sequenciamento do 16S rDNA utilizando a plataforma Illumina. As amostras coletadas foram exploradas geneticamente e analisadas por ferramentas de bioinformática (filtros de qualidade, análise de cluster, taxonomia entre outros), e por fim foram inferidas as informações fenotípicas das culturas identificadas. As amostras apresentaram índice de diversidade de microrganismos Simpson 1-D entre 0,8751 e 0,9806, onde os filos mais abundantes em todas as fases de operação foram Proteobacteria (13,17 – 44,21%), Firmicutes (7,12 - 41,94%), Bacteroidetes (13,05 – 26,17%) e Chloroflexi (2,51 – 4,77%). Os gêneros Trichococcus, Syntrophus, Methanosaeta foram influenciados positivamente pela adição de salinidade e sulfato com aumento significativo na abundância relativa nas fases II e III. Foram identificados gêneros aptos a catalisar a quebra do corante presente no efluente, como também foram encontrados exclusivamente na biomassa do reator micro aerado, espécies do gênero Brevundimonas, reportados na literatura por possuir algumas espécies aptas a realizar mineralização de alguns tipos de aminas aromáticas que são subprodutos tóxicos da degradação do corante. / In this paper is presented a study of microbial ecology with the objective to understand the composition and dynamics of microbial community formed in two UASB reactors, correlating the environmental parameters obtained in early monitoring with the biological parameters identified during the research. One of them is completely anaerobic (R1) and the other is microaerophilic (R2). The reactors were operated with synthetic textile effluent along 3 operational phases (Phase I - normal conditions of the synthetic effluent; Phase II - addition of salinity in the inffluent; Phase III – sulphate addition in the inffluent). At the end of each phase, biomass samples were collected from the sludge blanket, also, a sample of biomass attached to the aerator was collected ate the end of phase III, and finally, a sample of inoculum, used in both reactors, was collected. Through PCR -DGGE techniques was able a preview of the diversity of samples serving as a tool to select which samples went to the 16S rRNA sequencing using the Illumina platform. The samples were explored genetically and analyzed by bioinformatic tools (quality filters, cluster analysis, taxonomy among others), and finally was inferred the phenotypic information about the identified cultures. The samples showed good diversity of microorganisms (Simpson 1-d between 0.8751 and 0.9806), the most abundant phyla in all operating phases were Proteobacteria (13.17% to 44.21%), Firmicutes (7.12% to 41.94%), Bacteroidetes (13.05% to 26.17%) and Chloroflexi (2.51% to 4.77%). The genera Trichococcus, Syntrophus, Methanosaeta were positively influenced by the addition of salinity and sulfate with a significant increase in relative abundance in phases II and III. These genres have metabolic characteristics that complement each other indicating a possible syntrophic environment between these genera. Some genera able to catalyze the breakdown of dye were identified, as well, was identified in the sample of the biomass attached to aerator, species of genus Brevundimonas, reported in literature as bacterial species able to degrading some types of aromatic amines, which are toxic byproducts of azo dye degradation.