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Neste trabalho foi estudada a influência da forma de preparo do inóculo na morfologia do microrganismo e na produção de glicoamilase por Aspergillus awamori, em cultivo submerso. Foram realizados ensaios variando-se a concentração de esporos utilizada no preparo do inóculo para o fermentador (pré-cultivo de células em incubador rotativo a 200 rpm e 35 oC, por 24 horas) no intervalo entre 9,5 exp 03 e 1,8 exp 07 esporos/mL. Os inóculos preparados com concentração de esporos entre 9,5 exp 03 e 9,5 exp 05 esporos/mL apresentaram-se na forma de uma suspensão de “pellets" e conduziram a um crescimento na forma filamentosa, em fermentador. A produção de glicoamilase, nos ensaios com concentração inicial de substrato de 40 g/L, realizados em fermentador, não foi influenciada pela concentração de esporos nessa faixa, mantendo-se entre 2100 e 2200 U/L. O aumento da concentração de esporos utilizada no preparo do inóculo para 1,8 exp 07 esporos/mL conduziu à formação de um inóculo na forma filamentosa contendo muitos aglomerados de esporos não germinados, que levaram a um crescimento, em fermentador, na forma de “pellets", reduzindo a produção de glicoamilase para cerca de 1600 U/L e mostrando que o crescimento na forma de “pellets" não é indicado para a produção de glicoamilase. Foram estudadas, também, outras formas de preparo do inóculo, variando-se as condições de germinação dos esporos (pH e tempo de pré-cultivo do inóculo). A forma de preparo do inóculo que conduziu a uma maior produção de glicoamilase, em fermentador, foi o cultivo de esporos em incubador rotativo, a pH 2,5 durante 7 horas, o que evitou a aglomeração de esporos durante a etapa de germinação e a formação de “pellets" em fermentador, conduzindo a um crescimento na forma filamentosa e a uma alta produção de glicoamilase. / The influence of the inoculum preparation on Aspergillus awamori morphology and glucoamylase synthesis in submerged cultures has been investigated. A series of runs were performed, varying the spore concentration of the inoculum (inoculum size) in the range from 9.5 exp 03 to 1.8 exp 07 spores/mL. The inoculum was cultivated in shaker, at 35 oC and 200 rpm, for 24 hours. The inoculum prepared with a spore concentration in the range from 9.5 exp 03 to 9.5 exp 05 spores/mL was composed by a pellet suspension. This pellet suspension led to a filamentous growth in fermenter, but did not influence the glucoamylase production, which reached values from 2,100 to 2,200 U/L. The use of a higher spore concentration (1.8 exp 07 spores/mL) produced an inoculum composed by dispersed hyphae and many spores agglomerates, which led to pellet formation in the fermenter and reduced glucoamylase production to 1,600 U/L. Thus, pellet formation is not recommended in the process of glucoamylase synthesis. Some forms of inoculum preparation have been studied, varying spore germination conditions (pH and time of inoculum culture). The form of inoculum preparation that led to the highest glucoamylase activity in fermenter was the spore germination in shaker at pH 2.5 for 7 hours, which avoided pellet formation in the reactor and conducted to a high glucoamylase production.

Neste trabalho foi estudada a influência da forma de preparo do inóculo na morfologia do microrganismo e na produção de glicoamilase por Aspergillus awamori, em cultivo submerso. Foram realizados ensaios variando-se a concentração de esporos utilizada no preparo do inóculo para o fermentador (pré-cultivo de células em incubador rotativo a 200 rpm e 35 oC, por 24 horas) no intervalo entre 9,5 exp 03 e 1,8 exp 07 esporos/mL. Os inóculos preparados com concentração de esporos entre 9,5 exp 03 e 9,5 exp 05 esporos/mL apresentaram-se na forma de uma suspensão de “pellets” e conduziram a um crescimento na forma filamentosa, em fermentador. A produção de glicoamilase, nos ensaios com concentração inicial de substrato de 40 g/L, realizados em fermentador, não foi influenciada pela concentração de esporos nessa faixa, mantendo-se entre 2100 e 2200 U/L. O aumento da concentração de esporos utilizada no preparo do inóculo para 1,8 exp 07 esporos/mL conduziu à formação de um inóculo na forma filamentosa contendo muitos aglomerados de esporos não germinados, que levaram a um crescimento, em fermentador, na forma de “pellets”, reduzindo a produção de glicoamilase para cerca de 1600 U/L e mostrando que o crescimento na forma de “pellets” não é indicado para a produção de glicoamilase. Foram estudadas, também, outras formas de preparo do inóculo, variando-se as condições de germinação dos esporos (pH e tempo de pré-cultivo do inóculo). A forma de preparo do inóculo que conduziu a uma maior produção de glicoamilase, em fermentador, foi o cultivo de esporos em incubador rotativo, a pH 2,5 durante 7 horas, o que evitou a aglomeração de esporos durante a etapa de germinação e a formação de “pellets” em fermentador, conduzindo a um crescimento na forma filamentosa e a uma alta produção de glicoamilase. / The influence of the inoculum preparation on Aspergillus awamori morphology and glucoamylase synthesis in submerged cultures has been investigated. A series of runs were performed, varying the spore concentration of the inoculum (inoculum size) in the range from 9.5 exp 03 to 1.8 exp 07 spores/mL. The inoculum was cultivated in shaker, at 35 oC and 200 rpm, for 24 hours. The inoculum prepared with a spore concentration in the range from 9.5 exp 03 to 9.5 exp 05 spores/mL was composed by a pellet suspension. This pellet suspension led to a filamentous growth in fermenter, but did not influence the glucoamylase production, which reached values from 2,100 to 2,200 U/L. The use of a higher spore concentration (1.8 exp 07 spores/mL) produced an inoculum composed by dispersed hyphae and many spores agglomerates, which led to pellet formation in the fermenter and reduced glucoamylase production to 1,600 U/L. Thus, pellet formation is not recommended in the process of glucoamylase synthesis. Some forms of inoculum preparation have been studied, varying spore germination conditions (pH and time of inoculum culture). The form of inoculum preparation that led to the highest glucoamylase activity in fermenter was the spore germination in shaker at pH 2.5 for 7 hours, which avoided pellet formation in the reactor and conducted to a high glucoamylase production.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Rhizoctonia solani inoculum can be present either as soil- or tuber-borne sclerotia or hyphae. Although both inoculum sources play a role in disease development, it is not clear which of the two is more important. Successive cultivation of potato crops increases R. solani soil inoculum load resulting in an escalation in disease incidence and severity. The use of tolerant cultivars, however, can effectively reduce inoculum levels thereby decreasing disease intensity. Four pot trials were conducted; the objective of the first two pot trials was to determine the effect of tuber and soil-borne inoculum and stolon inoculations on disease development in sandy and clay loam soils. The second two pot trials were aimed at determining susceptibility levels of five cultivars. Two field trials were planted over two growing seasons in the same soils, using three inoculum levels. Results from the pot trials showed that tubers harvested from inoculated sandy soils developed significantly more disease than those harvested from clay loam soils. Of the three inoculum sources, stolon inoculation and seed-borne inoculum resulted in significantly more disease on progeny tubers than those from R. solani spiked soils. Although none of the cultivars proved to be tolerant to R. solani, BP1 was less susceptible to R. solani at temperatures between 21-26oC. More severe disease symptoms were observed under cooler temperatures on all cultivars. Results from the field trial showed the cultivation of potatoes in the same soil over two growing seasons resulted in an increase in diseased (black scurf) tubers. Furthermore, black scurf was most severe on tubers from soils infested with the highest concentration of inoculum. There were significant disease severity differences, with initial soil inoculum levels being directly proportional to final disease severity. Future studies in South Africa should focus on investigating the genetic composition of various cultivars; the effect of soil type and pH on the pathogenicity of R. solani and the use of molecular diagnostic tools to detect and quantify R. solani in soils. / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

This thesis is an investigation of detecting and quantifying airborne inoculum of Sclerotinia sclerotiorum (Lib.) de Bary to improve the Sclerotinia rot of carrot (SRC) forecast model. A quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify DNA from airborne ascospores of S. sclerotiorum. The qPCR assay was evaluated on air samples collected using a Burkard Sampler, and showed that ascospores of S. sclerotiorum were specifically detected among a pool of foreign DNA. The concentration of detected ascospores was related to the observed incidence of SRC to suggest a preliminary threshold of 2 to 4 ascospores m-3 of air for SRC development. Evaluation of an Andersen Sampler, the blue plate test (BPT) and the qPCR assay showed that the latter two methods were equally effective in detecting and quantifying ascospores of S. sclerotiorum and consistently detected greater numbers of ascospores than an Andersen Sampler. Three days are required to confirm the presence of S. sclerotiorum using the BPT, while results from the qPCR assay can potentially provide results within five hours of air sampling. The choice of detection method depends on the available resources and need for a quick result. Analysis of data from nine years of air sampling using the BPT indicated that a single air sampling site is sufficient to detect ascospores when counts are low, increasing to two sites during periods when ascospores are detected near threshold levels and crop and environmental conditions are conducive to disease. Chitosan and canopy trimming were evaluated to manage SRC under field conditions. Chitosan reduced area under the disease progress curve (AUDPC) by 55 and 42% in 2009 and 2011, respectively, which was comparable to a standard fungicide. Trimming enhanced chitosan efficacy, reducing AUDPC by 88 and 82% in 2009 and 2011, respectively. Trimming as a stand-alone treatment reduced AUDPC by 66% in 2011. Under controlled environmental conditions, chitosan inconsistently enhanced defense responses against S. sclerotinia. The results show that chitosan has potential to be integrated into SRC management systems, particularly when combined with foliar trimming in years with moderate to high disease risk. / National Research Council of Canada; University of Guelph; Department of Plant Agriculture; Ontario Ministry of Agriculture, Food and Rural Affairs

Este trabalho pretende avaliar o efeito da salinomicina no consumo de feno coast cross e sal proteinado, na digestibilidade total, ruminal in situ e in vitro, taxas de produção de gases e taxas de fermentação. Para a avaliação da digestibilidade e degradabilidade in situ foram utilizados 4 novilhos Holandeses com peso médio de 350 kg ± 43 kg. O delineamento experimental utilizado foi o quadrado latino com 4x4. No ensaio in vitro foram utilizados 4 substratos *8 inóculos *2 corridas com delineamento em blocos e arranjo fatorial. Os tratamentos estudados foram: 0; 0,1; 0,2; 0,3 mg de salinomicina /kg de peso vivo. Não foram encontradas diferenças significativas (p>0,05) no consumo de MS(1,92; 1,73; 1,60; 1,63 %PV), digestibilidade aparente da MS (81,79; 80,32; 80,32 e 80,49), na degradação in situ da MS (52; 51,25; 49,20 e 51,38) e na degradação in vitro da MS (45,31; 45,64; 45,77; 45,03) entre os vários tratamentos. Os únicos parâmetros onde foram identificadas alterações significativas entre os tratamentos foram no volume final de gases in vitro e nas curvas de taxas de fermentação. Os valores encontrados de para o volume final de gases foi de 231,8; 204; 168 e 157,78 ml /g de MS para a dose de 0; 0,1; 0,2 e 0,3 mg de salinomicina/ kg de PV, respectivamente. O tratamento 3 foi o que teve menores perdas de energia. / This experiment was conducted to evaluate the effect of graded levels of salinomycin on feed intake, apparent di estibility, gas production and fermentation rate, degradability in situ and in vitro of cynodon hay supplemented with proteic salt with 0, 0.1, 0.2 and 0,3mg of salynomicin per kg LW. Intake , digestibility and degradabilty studies were analysed as a latine square design 4x4 and in vitro trials followed the block randomized design with a two factors (inocula and substrate). No differences (p>)0,05) were found between the treatments for DM intake (1.92, 1.73, 1.60, 1.63 per % LW), DM digestibility (81.79, 80.32, 80.32 and 80.49), DM in situ degradability (52, 51.25, 49.20 and 51.38) and in vitro DM degradability (45.31, 45.64, 45.77 and 51.38). Differences between treatments were found only for potencial gas production in vitro (231.8, 204, 168 and 157.78 ml/g DM) for 0, 0.1, 0.2 and 0,3 mg of salinomycin/kg LW. Therefore, wasting energy was minimized at treatment 3.

Este trabalho pretende avaliar o efeito da salinomicina no consumo de feno coast cross e sal proteinado, na digestibilidade total, ruminal in situ e in vitro, taxas de produção de gases e taxas de fermentação. Para a avaliação da digestibilidade e degradabilidade in situ foram utilizados 4 novilhos Holandeses com peso médio de 350 kg ± 43 kg. O delineamento experimental utilizado foi o quadrado latino com 4x4. No ensaio in vitro foram utilizados 4 substratos *8 inóculos *2 corridas com delineamento em blocos e arranjo fatorial. Os tratamentos estudados foram: 0; 0,1; 0,2; 0,3 mg de salinomicina /kg de peso vivo. Não foram encontradas diferenças significativas (p>0,05) no consumo de MS(1,92; 1,73; 1,60; 1,63 %PV), digestibilidade aparente da MS (81,79; 80,32; 80,32 e 80,49), na degradação in situ da MS (52; 51,25; 49,20 e 51,38) e na degradação in vitro da MS (45,31; 45,64; 45,77; 45,03) entre os vários tratamentos. Os únicos parâmetros onde foram identificadas alterações significativas entre os tratamentos foram no volume final de gases in vitro e nas curvas de taxas de fermentação. Os valores encontrados de para o volume final de gases foi de 231,8; 204; 168 e 157,78 ml /g de MS para a dose de 0; 0,1; 0,2 e 0,3 mg de salinomicina/ kg de PV, respectivamente. O tratamento 3 foi o que teve menores perdas de energia. / This experiment was conducted to evaluate the effect of graded levels of salinomycin on feed intake, apparent di estibility, gas production and fermentation rate, degradability in situ and in vitro of cynodon hay supplemented with proteic salt with 0, 0.1, 0.2 and 0,3mg of salynomicin per kg LW. Intake , digestibility and degradabilty studies were analysed as a latine square design 4x4 and in vitro trials followed the block randomized design with a two factors (inocula and substrate). No differences (p>)0,05) were found between the treatments for DM intake (1.92, 1.73, 1.60, 1.63 per % LW), DM digestibility (81.79, 80.32, 80.32 and 80.49), DM in situ degradability (52, 51.25, 49.20 and 51.38) and in vitro DM degradability (45.31, 45.64, 45.77 and 51.38). Differences between treatments were found only for potencial gas production in vitro (231.8, 204, 168 and 157.78 ml/g DM) for 0, 0.1, 0.2 and 0,3 mg of salinomycin/kg LW. Therefore, wasting energy was minimized at treatment 3.

<p>Bistable switches are commonly observed in the regulation of critical processes such as cell cycles and differentiation. The switches possess two fundamental properties: memory and bimodality. Once switched ON, the switches can remember their ON state despite a drastic drop in stimulus levels. Furthermore, at intermediate stimulus levels with cellular noise, the switches can cause a population to exhibit bimodal distribution of cell states. Till date, experimental studies have focused primarily on cellular mechanisms that generate bistable switches and their impact on cellular dynamics. </p><p>Here, I study emergent bistability due to bacterial interactions with either synthetic gene circuits or antibiotics. A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. I illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. The results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.</p><p>In the context of antibiotic treatment, I investigate bistability as the underlying mechanism of inoculum effect. The inoculum effect refers to the decreasing efficacy of an antibiotic with increasing bacterial density. Despite its implication for the design of antibiotic treatment strategies, its mechanism remains poorly understood. Here I show that, for antibiotics that target the core replication machinery, the inoculum effect can be explained by bistable bacterial growth. My results suggest that a critical requirement for this bistability is sufficiently fast turnover of the core machinery induced by the antibiotic via the heat shock response. I further show that antibiotics that exhibit the inoculum effect can cause a "band-pass" response of bacterial growth on the frequency of antibiotic treatment, whereby the treatment efficacy drastically diminishes at intermediate frequencies. The results have implications on optimal design of antibiotic treatment.</p> / Dissertation