The bacterium Vibrio vulnificus is an autochthonous inhabitant of estuarine waters and also found in shellfish such as oysters. It is a human pathogen of importance in the seafood industry, and can also infect recreational water users. Currently, recognized methods of detection rely upon isolation of pure cultures which requires at least 24 hours. To reduce the time needed for identification of the pathogen and simultaneously ascertain the virulence potential of the strains present, real-time PCR assays and sample processing procedures were developed (Chapter 1). These assays discriminate between type A (environmental, generally lower virulence) and type B (clinical, higher virulence) isolates. The genetic relationships between environmental V. vulnificus strains isolated from permitted and prohibited shellfish harvesting areas was determined using BOX-PCR genomic fingerprinting coupled with sequence analysis of three proposed virulence markers: (1) the virulence correlated gene (vcg), (2) 16S rRNA type and (3) presence/absence of the vulnibactin gene (viuB) (Chapter 2). The real-time PCR assays were able to detect the presence of seeded V. vulnificus in environmental water at a concentration of 160 cells 100·ml-1. In seeded oyster homogenates, the assays were able to detect a minimum of 10³ cells and 10² cells per reaction of type A and type B respectively. The phylogenetic analysis separated the majority of type A/ vcgE strains isolated from permitted shellfish harvesting areas from those isolated from prohibited harvesting areas. The genomic (BOX-PCR) fingerprints of type A and type AB isolates were more similar to one another than to type B isolates. Only one type A/ vcgE isolate contained the viuB gene; however, eight type B/ vcgC isolates had that gene. No obvious grouping was discerned between type B/ vcgC isolates from permitted versus prohibited shellfish harvesting areas or between those possessing the viuB gene versus those lacking viuB. These data provide insight into the ecology and correlation between population biology and general water quality, as gauged by the classification of the shellfish growing area waters. The 16S typing assays can be used for routine rapid typing to aid in risk assessment and reduce infection frequency through consumption of contaminated seafood.

Degradation of the several million tons of solid waste produced in the U.S. annually is microbially mediated, yet little is known about the structure of prokaryotic communities actively involved in the waste degradation process. In the first study, leachates generated during degradation of municipal solid waste (MSW) in the presence (co-disposal) or absence of biosolids were analyzed using laboratory-scale bioreactors over an eight-month period. Archaeal and bacterial community structures were investigated by denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes. Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and temporal trends. Methanogen sequences from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales. Effect of moisture content on indicator organism (IO) survival during waste degradation was studied using culture-based methods. Fecal coliform and Enterococcus concentrations in leachate decreased below detection limits within fifty days of bioreactor operation during the hydrated phase. IOs could be recovered from the bioreactor leachate even after a prolonged dry period. This study advances the basic understanding of changes in the microbial community during solid waste decomposition. The purpose of the second study was to compare the ability of BOX-PCR to determine genetic relatedness with that of the "gold standard" method, 16S rRNA gene sequencing. BOX-PCR typing could clearly differentiate the strains within different Enterococcus species but closely related genera were not as distinguishable. In contrast, 16S rRNA gene sequencing clearly differentiates between closely related genera but cannot distinguish between different strains of Enterococcus species. This study adds to our knowledge of genetic relationships of enterococci portrayed by two separate molecular methods. The incidence of vancomycin resistant enterococci (VRE) in environmental matrices, residential and hospital wastewater was also investigated. Low-level VRE ( vanC genotype) were isolated from environmental matrices and residential wastewater. VRE isolates from hospital wastewater were identified as E. faecium and demonstrated resistance to ampicillin, ciprofloxacin and vancomycin (vanA genotype), but sensitivity to chloramphenicol and rifampin. Although no high-level VRE were isolated from surface waters, the high proportion of low-level VRE in environmental matrices is a cause for concern from the public health perspective.

Black rot caused by Xanthomonas campestris pv. campestris (X. c pv. campestris) is a major disease constraint to cabbage production. The control of black rot is difficult and resistant cultivars could play an important role in reducing the losses due to the disease. Information on the distribution and diversity of X. c pv. campestris is critical before any meaningful disease resistance screening can be done. However, little is known about the diversity and international significance of South African X. c pv. campestris strains. To assess the genetic diversity and international significance of X. c pv. campestris strains in South Africa, strains of the pathogen were obtained from cabbage growing districts in Gauteng, Mpumalanga and North West Provinces of South Africa in 2010. International strains were obtained from international culture collections. Isolates from South Africa were purified and race typed using differential sets of Brassica spp according to Nickerson–Zwaan protocols. Four races, race 1(14%), race 3 (7%), race 4 (68%) and race 6 (10%) of the pathogen were identified. Repetitive DNA polymerase chain reaction–based fingerprinting using Eric– and Box–primers were used to assess the genetic diversity. Polyacrylamide gel electrophoresis allowed clear and reproducible differentiation of the PCR products. Of the amplified loci for South African isolates 5 loci were present in at least 90 % of the isolates for Eric–profiles and 6 in at least 80% of the isolates for Box–profiles. Of these prominent loci, none had corresponding high presence in international isolates. While no loci had a presence greater than 51% and 61% for Eric– and Box– profiles in international isolates, respectively, several loci among South African isolates were unique to isolates from specific geographic origin. Generated fingerprints of X. c pv. campestris were similar for the South African isolates and distinguishable from those of X. c pv. armoraciae and X. c pv. raphani reference strains. However, when international X. c pv. campestris were considered, no profile pattern was observed to be unique to international X. c pv. campestris isolates as was the case with South African isolates. Eric– and Box–PCR profiles of international isolates varied widely with some isolates having profile patterns similar to those of reference strains. Cluster analysis divided X. c pv. campestris into two major groups, the South African group and the international isolates group. The South African group could be divided into subgroups, which clustered according to the geographical origin of the isolates. The same was observed for international isolates, which generally clustered isolates according to country of origin. However, isolates from different countries also clustered together. A few X. c pv. campestris strains of international origin clustered with the South African isolates group. Furthermore, a few South African isolates were clustered in the international isolate group. Although X. c pv. campestris distribution may be unique to its geographical origin, our findings, based on the present data set, suggest wide spread of the pathogen both at national and international level. The existence of different races, genetic variability and international distribution of the pathogen should be considered when resistant crucifer cultivars are bred to control black rot of crucifers / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2011.

Black rot caused by Xanthomonas campestris pv. campestris (X. c pv. campestris) is a major disease constraint to cabbage production. The control of black rot is difficult and resistant cultivars could play an important role in reducing the losses due to the disease. Information on the distribution and diversity of X. c pv. campestris is critical before any meaningful disease resistance screening can be done. However, little is known about the diversity and international significance of South African X. c pv. campestris strains. To assess the genetic diversity and international significance of X. c pv. campestris strains in South Africa, strains of the pathogen were obtained from cabbage growing districts in Gauteng, Mpumalanga and North West Provinces of South Africa in 2010. International strains were obtained from international culture collections. Isolates from South Africa were purified and race typed using differential sets of Brassica spp according to Nickerson–Zwaan protocols. Four races, race 1(14%), race 3 (7%), race 4 (68%) and race 6 (10%) of the pathogen were identified. Repetitive DNA polymerase chain reaction–based fingerprinting using Eric– and Box–primers were used to assess the genetic diversity. Polyacrylamide gel electrophoresis allowed clear and reproducible differentiation of the PCR products. Of the amplified loci for South African isolates 5 loci were present in at least 90 % of the isolates for Eric–profiles and 6 in at least 80% of the isolates for Box–profiles. Of these prominent loci, none had corresponding high presence in international isolates. While no loci had a presence greater than 51% and 61% for Eric– and Box– profiles in international isolates, respectively, several loci among South African isolates were unique to isolates from specific geographic origin. Generated fingerprints of X. c pv. campestris were similar for the South African isolates and distinguishable from those of X. c pv. armoraciae and X. c pv. raphani reference strains. However, when international X. c pv. campestris were considered, no profile pattern was observed to be unique to international X. c pv. campestris isolates as was the case with South African isolates. Eric– and Box–PCR profiles of international isolates varied widely with some isolates having profile patterns similar to those of reference strains. Cluster analysis divided X. c pv. campestris into two major groups, the South African group and the international isolates group. The South African group could be divided into subgroups, which clustered according to the geographical origin of the isolates. The same was observed for international isolates, which generally clustered isolates according to country of origin. However, isolates from different countries also clustered together. A few X. c pv. campestris strains of international origin clustered with the South African isolates group. Furthermore, a few South African isolates were clustered in the international isolate group. Although X. c pv. campestris distribution may be unique to its geographical origin, our findings, based on the present data set, suggest wide spread of the pathogen both at national and international level. The existence of different races, genetic variability and international distribution of the pathogen should be considered when resistant crucifer cultivars are bred to control black rot of crucifers / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2011.

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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.

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.

All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX-PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. / MSc Environmental Sciences, North-West University, Potchefstroom Campus, 2015

All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX-PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. / MSc Environmental Sciences, North-West University, Potchefstroom Campus, 2015

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-21T19:29:33Z No. of bitstreams: 1 2016 - Caroline Barra Sales Khayat da Costa.pdf: 5220401 bytes, checksum: 553248261c960b6b5fb62cadb1b26ebf (MD5) / Made available in DSpace on 2018-09-21T19:29:33Z (GMT). No. of bitstreams: 1 2016 - Caroline Barra Sales Khayat da Costa.pdf: 5220401 bytes, checksum: 553248261c960b6b5fb62cadb1b26ebf (MD5) Previous issue date: 2016-07-26 / CAPES / One of the most known and important process in nature is the biological nitrogen fixation (FBN). Few classes of microorganisms perform this process; named diazotrophics that associated with plants is an important alternative to enhance nitrogen nutrition in agricultural systems. Diazotrophic bacterial population in association with sugarcane improves the yield due to its ability to colonize the inner roots of the host plant and do not provoke any pathogenicity signal. Quantitative analyzes have indicated that selection of combinations of endophytic diazotrophic strains when associated with sugarcane varieties can improve the yield and therefore varieties of sugarcane may be better exploited in order to benefit this association for agricultural purposes. Thus, this work proposes to monitor the establishment of diazotrophic bacteria present in the inoculant of Embrapa, during the initial development of sugarcane plants. Micro-propagated sets of the sugarcane variety RB867515 were inoculated individually and with a mixture of five nitrogen fixing bacteria strains that compose the sugarcane inoculant: Nitrospirillum amazonense (CBAmC) Paraburkholderia tropica (Ppe8) Gluconacetobacter diazotrophicus (Pal5) Herbaspirillum seropedicae (HRC54) and Herbaspirillum rubrisubalbicans (HCC103). Plants were grown in a greenhouse and harvested 30 days after planting. Fresh root material was separated for evaluation of the establishment of the strains by bacterial counting using the technique of Most Probable Number (MPN), absolute quantification by Real Time PCR (qPCR), besides the identification of the inoculated strains through analysis of the rDNA profile of the pellicle formed in the respective semisolid media using the BOX-PCR technique. The results of bacterial count diazotrophic present in the roots by NMP showed that the treatments inoculated in the control (native population) showed higher population of bacterial cells around 105 cells by fresh root mass, but not statistically significant. The methodology qPCR permitted quantitation of the number of 16S rDNA copies in the order of 105 bacterial cells g - 1 fresh weight root, showing that there were differences in the population of endophytic species that colonize the roots of sugarcane. The profiles of BOX -PCR of the respective pellicles formed in semisolid media did not show high similarity (> 80 %) with the profile of the species inoculated for most of the treatments. The results obtained indicate that the qPCR technique showed the establishment of some of the inoculated strains in sugar cane buds while the NMP technique showed no significant difference between treatments inoculated and non-inoculated possibly due to the lower sensitivity of the method. / Um dos processos mais conhecidos e importantes na natureza ? a fixa??o biol?gica de nitrog?nio atmosf?rico (FBN). Este processo ? realizado por algumas classes de microrganismos, denominados diazotr?ficos, que associados com esp?cies vegetais s?o uma alternativa importante para aprimorar a nutri??o nitrogenada nos sistemas agr?colas. As popula??es de bact?rias fixadoras de nitrog?nio atmosf?rico quando associadas ? cultura da cana-de-a??car melhoram a produ??o e, ao colonizarem o interior das ra?zes promovem benef?cios ?s plantas hospedeiras. An?lises quantitativas t?m indicado que a sele??o das combina??es de estirpes de bact?rias diazotr?ficas endof?ticas e variedades de cana-de-a??car podem ser melhor exploradas com o objetivo de aperfei?oar a associa??o para finalidades agr?colas. Assim, o presente trabalho prop?e monitorar o estabelecimento das bact?rias diazotr?ficas presentes no inoculante para cana-de- a??car da Embrapa, durante o desenvolvimento inicial planta. Toletes propagados da variedade RB867515 de cana-de-a??car foram inoculados individualmente e com a mistura das cinco estirpes de bact?rias diazotr?ficas: Nitrospirillum amazonense (CBAmC), Paraburkholderia tropica (PPe8), Gluconacetobacter diazotrophicus (Pal5), Herbaspirillum seropedicae (HRC54) e Herbaspirillum rubrisubalbicans (HCC103). As plantas foram crescidas em casa de vegeta??o e coletadas 30 dias ap?s o plantio, sendo separado o material vegetal para avalia??o do estabelecimento das estirpes pela t?cnica do N?mero Mais Prov?vel (NMP) e quantifica??o absoluta por PCR em Tempo Real (qPCR). Adicionalmente foi feita a identifica??o das estirpes inoculadas atrav?s da an?lise do perfil de rDNA das pel?culas bacterianas formadas nos respectivos meios semiss?lidos pela t?cnica de BOX-PCR. Os resultados obtidos da contagem de bact?rias diazotr?ficas presente nas ra?zes por NMP mostrou que os tratamentos inoculados em rela??o ao controle (popula??o nativa), apresentaram uma maior popula??o de c?lulas bacterianas em torno de 105 c?lulas por massa fresca de raiz, por?m n?o significativa. A metodologia de qPCR permitiu a quantifica??o do n?mero de c?pias do 16S rDNA, da ordem de 105 c?lulas bacterianas g-1 massa fresca de raiz, mostrando que houve diferen?a na popula??o das esp?cies diazotr?ficas endof?ticas que colonizam as ra?zes de cana-de-a??car. Os perfis obtidos por BOX-PCR das pel?culas formadas nos respectivos meios semiss?lidos n?o mostraram alta similaridade (>80%) com o perfil das esp?cies inoculadas para a maioria dos tratamentos inoculados. Os resultados obtidos indicam que a t?cnica de qPCR permitiu mostrar o estabelecimento de algumas estirpe do inoculante nos toletes de cana-de a??car enquanto que a t?cnica de NMP n?o mostrou diferen?a significativas entre os tratamentos inoculados e n?o inoculados possivelmente pela menor sensibilidade da metodologia.