<p>Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, <i>Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria</i>). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera <i>Pantoea</i> were found to inhibit growth of <i>E. coli</i> O157:H7 <i>in vitro</i>. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface.</p> / Master of Science

Le système fluvial Saigon-Dong Nai (SG- DN) est la plus importante source d'eau pour les douze villes et provinces du sud du Vietnam. Il est aujourd'hui gravement pollué par les activités humaines, agricoles, industrielles et domestiques, constituant une menace pour la vie de millions de personnes. Le ministère vietnamien des Ressources naturelles et de l'environnement a rapporté que les rivières ont reçu environ 1,54 milliard de litres d'eaux usées provenant de 70 parcs industriels par jour, dont 35% de déchets médicaux non traités, et que des tests effectués depuis 2006 ont montré des niveaux élevés de pollution, en particulier de substances toxiques organiques. Jusqu'à présent, il n'y a pas de données sur la diversité microbienne dans le système fluvial SG-DN, en particulier dans les sédiments, où la plus grande partie de la biomasse microbienne est généralement localisée. Les échantillons de sédiments ont été recueillis, réseau hydrographique national SG-DN, à 13 endroits dans les rivières représentant des emplacements pollués. Afin de caractériser les populations microbiennes présentes sur nos sites choisis, l'ADN total des échantillons environnementaux a été extrait et amplifié dans les régions V3 à V1 de l'ADNr 16S. L'étude a révélé que la population microbienne changeait de l'amont vers l'aval au niveau du phylum, du genre et de l'OTU après avoir traversé la zone de population industrielle et dense. De plus, les canaux du bassin versant SG-DN sont fortement pollués par de fortes concentrations de composés organiques (PAH) et possèdent différentes communautés bactériennes par rapport aux échantillons des rivières. / The SaiGon-DongNai (SG-DN)river system is the most important major water source for all twelve Southern Vietnam cities and provinces and is now dramatically polluted by industrial and living activities, giving “a threat” to the lives of millions people sharing this water source. The Ministry of Natural Resources and Environment of Vietnam reported that the rivers received around 1.54 billion liters of waste water from 70 industrial parks per day, including 35 percent of untreated medical waste, and tests since 2006 have found pollution in this river has increased to “serious levels”, an especially high concentration of organic toxic substances. Until now, there is no data on the microbial diversity in SG-DN river system especially in the sediments, where most of the microbial biomass is generally located. The sediment samples were collected in 13 locations across the rivers representing warning polluted locations done by Mr. Nguyen Thanh Hung of the National Water Qualifying in SG-DN river system. In order to characterize the microbial populations present at our chosen sites, the total DNA from the environmental samples were extracted and amplified at the V3 to V1 regions of the 16S rDNA. The study revealed that microbial population changed from upstream to downstream at the phylum, genus and OTUs levels after running through the industrial and dense population zone. Moreover, the canals of the SG-DN river catchment are heavily polluted with high concentrations of organic compounds (PAHs) and possessed different bacterial communities compared to the samples from the rivers.

No description available.

Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, <i>Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria</i>). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera <i>Pantoea</i> were found to inhibit growth of <i>E. coli</i> O157:H7 <i>in vitro</i>. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface. / Master of Science in Life Sciences / Master of Science

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Vermicomposts (VCs) are the solid excreta of earthworms, known to contain plant available nutrients, large amounts of microbial life and diversity, and plant growth regulating hormones. VCs may play an integral role in the nitrogen nutrition of Lupinus angustifolius and function to reduce the reliance of legume crops on chemical fertilizers. Furthermore, the effects of the combination of VC solids and VC teas on legume growth and N nutrition, is unknown. The aim of the present study was to determine the role of varying concentrations of chicken manure VC, with and without the additions of varying VC tea concentrations, on the substrate bacterial functional diversity, plant biomass and N nutrition of the legume, L. angustifolius. In the first experiment the plants were grown in pots under glasshouse conditions and VC was substituted into the quartz sand growth media at rates of 5%, 10% and 100%. Furthermore, rhizobia inoculated and non-inoculated groups were established within the VC treatments. The plants were harvested after 30 days and analysed for tissue nutrient concentrations and biomass production. The VC-containing substrates were assessed for wide-spectrum soil analyses, nematode diversity and microbial diversity via Biolog EcoPlates. In the second experiment the plants were grown under similar conditions for 50 days and amended with 5% VC in the growth media as well as 50ml of aerated VC teas. The teas were brewed for 24 hours in concentrations of 4%, 10% and 20% (v/v), with molasses and kelp extract added as sugar sources. The combined treatments of 5% VC and rhizobia inoculation yielded the greatest biomass response. Furthermore the addition of VC allowed for bacterial nitrogen fixation within non-rhizobia treatments. However, changes in VC concentration had no effect on bacterial guild structure but were found to effect nematode funtional diversity. The additions of VC teas to soil media containing 5% VC had no effect on biomass production but were found to influence bacterial nitrogen fixation. Lower concentration teas increased BNF while the 20% tea reduced this parameter significantly over 50 days. The 20% tea also contained significantly greater bacterial functional diversity than the 5% and 10% teas. The findings of study indicate that the combined treatment of VC solids and teas do not increase the plant biomass of L. angustifolius, but that the additions of 20% teas result in greater microbial diversity in the soil. This in turn may lead to increases in soil fertility. Furthermore, additions of high concentration vermicompost teas (20%) shift the dependence of the plant from atmospheric N sources to soil N sources. / AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar

<p>Although there are many examples of microbial biogeography, few microbes have been studied at high taxonomic resolution over large spatial scales. As a result, the environmental and ecological processes that drive niche partitioning, diversity, composition, and activity of microbial taxa are often poorly understood. To address this gap, I examine the most abundant phytoplankton in the global ocean, Prochlorococcus sp., a marine cyanobacterium. Using amplicon libraries of the Prochlorococcus internal transcribed spacer (ITS) region and 23S rRNA gene as markers, I demonstrate several key differences between the two major high light (HL) clades of Prochlorococcus. First, by examining ITS amplicon libraries at high taxonomic resolution it is revealed that “sub-ecotype” clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Whereas unique combinations of environmental traits drive the distribution of the HL-I sub-ecotype clades, the HL-II sub-ecotype clades appear ecologically coherent. Second, using 23S rRNA and rDNA libraries I show that activity (rRNA) and abundance (rDNA) are highly correlated for Prochlorococcus across all sites and operational taxonomic units (OTUs) in the surface ocean, demonstrating a tight coupling between activity and abundance. Finally, I investigate the associations between Prochlorococcus and the rest of the microbial community in the North Pacific and find region-specific trends in both strength and sign. Associations with other microbes are strongest for HL-I in the temperate region and strongest for HL-II in the sub-tropical gyre. This dissertation clarifies the relative importance of the environment, geography, community, and taxonomy in terms of their role in creating complex assemblages of Prochlorococcus and helps improve our understanding of how marine microbial communities are assembled in situ.</p> / Dissertation

O conhecimento das associações entre os microrganismos componentes da microbiota dos solos é de grande interesse no meio científico, principalmente relacionado aos microrganismos que se associam de forma benéfica com as plantas. Neste contexto, destaca-se a micorriza arbuscular, que é a associação entre os fungos micorrízicos arbusculares (FMAs) e uma amplitude de espécies vegetais. Além desta íntima interação entre estes organismos, torna-se necessário conhecer a importância dos demais componentes do sistema solo, como a microbiota formada por fungos e bactérias dos solos, para o estabelecimento desta interação. Os objetivos dos estudos componentes desta tese se concentraram na avaliação de FMAs (D. heterogama, R. clarus e Gi. rosea) inoculados em sistemas alterados quanto à composição das comunidades microbianas dos solos, promovidas pela metodologia de \'diluição para extinção\'. No primeiro estudo foram encontradas respostas diferenciais na capacidade de colonização micorrízica (%CM) de plantas de cana-de-açúcar pelos FMAs inoculados nos diferentes sistemas, além do efeito diferencial dos FMAs quanto às alterações nas comunidades de fungos e bactérias do solo. Num estudo mais detalhado, desenvolvido apenas com R. clarus em plantas de milho, foi verificado que a maiores diversidades microbianas do solo resultaram em maior colonização do hospedeiro, principalmente no período inicial de desenvolvimento das plantas. Neste experimento foi descrita a correlação direta da capacidade de colonização do FMA com a riqueza e a diversidade filogenética da microbiota dos solos. A descrição dos perfis metabólicos dos solos contendo diferentes comunidades microbianas revelou a capacidade diferencial destes solos em utilizar diferentes fontes de carbono, além de demonstrar um aumento no metabolismo (evidenciado pelo consumo total de fontes de carbono) devido à inoculação do FMA. Em conjunto, os dados obtidos nos dois estudos indicam que a micorrização das plantas depende da ação de outros microrganismos do sistema solo, que atuam como um terceiro fator nesta simbiose e que a microbiota do solo responde a inoculação de um organismo exógeno, primordialmente aumentando seu metabolismo. Deve-se, portanto, considerar que a degradação biológica dos solos, com perda de sua biodiversidade, pode ter papel determinante no funcionamento de interações específicas e benéficas às plantas. / Knowledge about the associations between microbial components of soil microbiota is of great interest in the scientific community, primarily related to microorganisms that are associated beneficially with plants. In this context, there is the arbuscular mycorrhiza, which is made of the association between mycorrhizal fungi (AMF) and a range of plant species. In this close interaction between these organisms, it is necessary to describe the importance of other components of the soil system, such as the microbiota formed by fungi and bacteria from the soil, to establish this interaction. The objectives of the studied that compose this thesis focused on the evaluation of AMF (D. heterogama, R. clarus and Gi. rosea) fitness, when inoculated in modified systems on the composition of the microbial communities in the soil, with alterations promoted by the \'dilution to extinction\' methodology. In the first study, differential responses were found in root colonization capacity (% CM) of sugarcane by AMF inoculated in different systems, and the differential effect of AMFs, changing the communities of fungi and bacteria of soil. In a more detailed study, designed only to R. clarus in corn plants, it was found that higher microbial diversity of soil resulted in higher colonization of the host, especially in the initial period of plant development. In this experiment it was described the direct correlation of AMF colonization capacity with richness and phylogenetic diversity of soil microbiota. The description of the metabolic profiles of soil containing various microbial communities revealed the differential ability of these soils utilize different carbon sources, in addition to demonstrating an increase in metabolism (as evidenced by the total consumption of carbon sources) due to inoculation of the AMF. Together, the data from the two studies indicate that colonization of plants by AMF depends on the action of other microorganisms soil system, which act as a third factor in this symbiosis, and that the soil microbes respond to inoculation of an exogenous organism, primarily increasing its metabolism. One should therefore consider that the biological degradation of the soil, with loss of biodiversity, may have crucial role in the functioning of specific and beneficial interactions to plants.

O uso de microrganismos como promotores de crescimento vegetal tem se apresentado como uma alternativa promissora, sobretudo para a ciclagem de nutrientes, especialmente o fósforo. Dessa forma, objetivou-se avaliar o papel da diversidade microbiana na disponibilização do fósforo, oriundo de fontes de diferente solubilidade, para as plantas de milho no ambiente rizosférico. A hipótese é que quanto mais diverso for o microbioma da rizosfera e do solo, maior será a disponibilidade de fósforo para as plantas e por conseguinte melhor o seu desenvolvimento. O experimento foi instalado em casa de vegetação utilizando a metodologia de diluição para extinção. Foram estabelecidos três fontes de fósforo (superfosfato triplo, fitato de cálcio e fosfato de Araxá) em um gradiente de diversidade microbiana, correspondentes às diluições 10-1, 10-3, 10-6 e 10-9. Adicionalmente foi mantido um tratamento controle sem adição de fósforo (P) e todas as análises compreendiam o ambiente solo e rizosfera. O papel da diversidade microbiana frente a disponibilidade de P foi acessado por meio de metodologias dependentes de cultivo (BIOLOG) e metodologias independente de cultivo (qPCR e PCR-DGGE). As plantas adubadas com fosfato de Araxá apresentaram menor teor de P foliar e os sintomas mais severos de deficiencia de P (raquitismo e arroxeamento), sobretudo nas maiores diluições (menor diversidade). O percentual de P-lábil em relação ao P-total foi bastante variável entre as diluições dentro de cada fonte e ambiente avaliado. Entretanto, o tratamento adubado com fosfato de Araxá apresentou menor proporção de P-lábil (18-55%). Em relação ao gene phoD, no ambiente rizosfera as comunidades menos diversas apresentaram maiores números de cópia nos tratamentos, indicando que a abundância deste gene pode ter sido enriquecida nos tratamentos menos diversos. A atividade enzimática (principalmente para a fosfatase em pH 6,5) diminuiu frente a redução da diversidade da comunidade microbiana. A abundância do gene phoD e a atividade da fosfatase em pH 6,5 se relacionaram apenas no ambiente rizosférico. A estrutura e organização da comunidade bacteriana (gene rrs) e solubilizadora de fósforo (phoD) diferiram entre as diluições (R>0,75; p<0,05). Em relação ao perfil de consumo de fontes de carbono, as fontes avaliadas apresentaram perfis distintos entre si. Foi possivel observar que as fontes de fósforo foram mais atuantes do que o gradiente de diversidade no processo de disponibilidade de P para as plantas. Isso pode estar relacionado à redundância funcional da microbiota. / The use of microorganisms as promoters of plant growth has been presented as a promising alternative, especially for the cycling of nutrients, especially phosphorus. The objective of this study was to evaluate the role of microbial diversity in the availability of phosphorus from sources of different solubility for maize plants in the rhizospheric environment. The hypothesis is that as more diverse the rhizosphere and soil microbiome, the greater will be the availability of phosphorus to the plants and therefore better their development. The experiment was installed in a greenhouse using the dilution methodology for extinction. Three sources of phosphorus (triple superphosphate, calcium phytate and Araxá phosphate) were established in a microbial diversity gradient, corresponding to the dilutions 10-1, 10-3, 10-6 and 10-9. In addition, a control treatment was maintained without addition of P and all analyzes comprised the soil and rhizosphere environment. The role of microbial diversity against P availability was accessed through culture - dependent methodologies (BIOLOG) and independent culture methodologies (qPCR and DGGE). Araxá phosphate fertilized plants had lower leaf P content and P deficiency symptoms (rickets and purplishness), especially in the higher dilutions (lower diversity). The percentage of P-labile in relation to the P-total was quite variable between the dilutions within each source and evaluated environment. However, the Araxá phosphate fertilizer treatment had a lower proportion of P-labile (18-55%). In relation to the phoD gene, in the environment rhizospherein the less diverse communities presented higher copy numbers in the treatments, indicating that the abundance of the gene may have been enriched in the less diverse treatments. Enzymatic activity (mainly for phosphatase at pH 6.5) decreased as the diversity of microbial community decreased. The abundance of the phoD gene and phosphatase activity at pH 6.5 were related only in the rhizosphere environment. The structure and organization of the bacterial community (16S rRNA) and phosphorus solubilizer (phoD) differed at the dilutions (R> 0.75; p <0.05). The consumption of carbon source was variable in relation to evaluated treatments. It was possible to observe that the phosphorus sources were more active than the diversity gradient in the P availability process for the plants. This may be related to the functional redundancy of the microbiota.

O sedimento marinho da Península Antártica representa uma área sensível a mudanças ambientais. No entanto, pouco se conhece sobre as comunidades microbianas que habitam esse ecossistema, incluindo a sua diversidade, distribuição e variações temporais. O objetivo foi determinar a estrutura das comunidades microbianas nos sedimentos marinhos da Baía do Almirantado, Ilha Rei George, Península Antártica. Sedimentos da Baía apresentam uma predominância dos Filos Proteobacteria, Firmicutes e Actinobacteria. Análise temporal revelou que comunidades microbianas em sedimentos próximos à estação Ferraz são mais estáveis quando comparadas aos sedimentos em áreas de menor atividade antrópica. No gradiente de profundidade foi observado que a estrutura de comunidade não mudou, indicando tolerância a variações de pressão hidrostática. Organismos heterotróficos dos gêneros Psychrobacter, Psychromonas e Loktanella foram os mais abundantes, sugerindo uma alta concentração de matéria orgânica disponível. O enriquecimento de culturas metanogênicas produziu até 1,70 mmol de CH4 após 120 dias de incubação. Este estudo sugere que as condições dos sedimentos favorecem organismos psicrofílicos de metabolismo heterotrófico. / Marine sediment of the Antarctic Peninsula is a susceptible area to environmental changes. However, little is known about the microbial communities inhabiting this ecosystem, including its diversity, distribution and variations over time. The aim of this study was to determine the structure of microbial communities present in marine sediments of Admiralty Bay, King George Island, on the Antarctic Peninsula. Sediments from Admiralty Bay shown a predominance of the Proteobacteria, Firmicutes, and Actinobacteria phyla. Temporal analysis revealed that microbial communities in sediment, near Ferraz station, are more stable compared to that in the sediments in areas of lower human activity. No variation on the community structure was observed in depth gradient, indicating tolerance to hydrostatic pressure variations. Heterotrophic organisms of the genera Psychrobacter, Psychromonas and Loktanella were the most abundant, suggesting a high concentration of organic matter in the sediment. Enrichment of methanogenic cultures enrichment yielded 1.70 mmol of CH4. This study suggests that conditions in sediments favoring metabolism of heterotrophic and psychrophilic organisms.