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Tripartite rhizosphere (host, fungus, and rhizobacterial) interactions were studied to determine the mechanism(s) associated with lack of oospore germination and host colonization by Pythium aphanidermatum at soil temperatures below 27°C. Results indicate that rhizobacterial competition for nutrients was responsible for the general supression of pathogen activity at low soil temperature. In general less than 25% host colonization occurred at 20°C whereas greater than 90% colonization recorded at 27°C. However, when bacterial competition was reduced or eliminated by the addition of vancomycin, host colonization at 20°C increased to 83%. Competition between the fungus and the resident rhizobacterial population was also shown to occur prior to any significant increase in bacterial multiplication. The generation time of bacteria in undisturbed rhizosphere soil was estimated at about 8 hrs at both 20 and 27°C. Whereas both the rate and percentage germination of oospores were increased significantly at both 20 and 27°C in the rhizosphere soil if bacterial competition was inhibited by the addition of vancomycin.

Ready-to-eat vegetable salads containing lettuce as a main ingredient have become popular food items in recent years. Microorganisms associated with these products determine their shelf-life, sensory appeal and safety. This thesis investigates the bacterial ecology of lettuce, aspects of their pre-harvest contamination with microorganisms, and the presence of antimicrobial constituents in such produce. Commercial pesticides (insecticides, herbicides, fungicides), used during lettuce cultivation were examined as potential sources of microbial contaminants. None of the pesticide concentrates contained viable microorganisms. After reconstitution in water, two of the pesticides supported growth of inoculated species of Pseudomonas, Salmonella and Escherichia coli. Pesticides reconstituted in agricultural waters (bore, dam and river) supported the growth of microorganisms (e.g. Pseudomonas, Acinetobacter, Aeromonas spp. and coliforms) naturally present in these waters. Unless properly managed, pesticide application could contribute microbial contaminants to vegetable produce, thereby affecting their quality. Bacterial species associated with retail samples of lettuce were examined by plate culture on Tryptone Soy Agar and PCR-DGGE analysis. Macerates and rinses of lettuce sub-samples with and without addition of Tween 80 were examined to maximize bacterial recoveries. Predominant bacteria isolated by agar culture included species of Pseudomonas, Agrobacterium, Curtobacterium and Burkholderia, at populations of 103-106 cfu/g. PCR-DGGE was unable to recover the same incidence of species as agar culture and failed to detect bacteria in many samples. In some samples, PCR-DGGE detected species of Bacillus, Pseudomonas, Serratia and Acinetobacter, not found by culture. Failure of the PCR-DGGE analyses was attributed to interference by plant chloroplast DNA. Preparative agarose gel electrophoresis of lettuce macerates was necessary to remove chloroplast DNA before application of PCR-DGGE analysis. Thirty percent of lettuce samples contained Acinetobacter species at 101-104 cfu/g when examined after culture on minimal salts agar or enrichment in Baumann enrichment medium. Other Acinetobacter media failed to give reliable isolation of these species from lettuce and salad vegetables. Lettuce could be an environmental source of Acinetobacter nosocomial infections. Juices, solvent extracts and supercritical fluid carbon dioxide extracts of lettuce and capsicum samples did not exhibit antimicrobial action against a range of food spoilage and pathogenic bacteria.

Ready-to-eat vegetable salads containing lettuce as a main ingredient have become popular food items in recent years. Microorganisms associated with these products determine their shelf-life, sensory appeal and safety. This thesis investigates the bacterial ecology of lettuce, aspects of their pre-harvest contamination with microorganisms, and the presence of antimicrobial constituents in such produce. Commercial pesticides (insecticides, herbicides, fungicides), used during lettuce cultivation were examined as potential sources of microbial contaminants. None of the pesticide concentrates contained viable microorganisms. After reconstitution in water, two of the pesticides supported growth of inoculated species of Pseudomonas, Salmonella and Escherichia coli. Pesticides reconstituted in agricultural waters (bore, dam and river) supported the growth of microorganisms (e.g. Pseudomonas, Acinetobacter, Aeromonas spp. and coliforms) naturally present in these waters. Unless properly managed, pesticide application could contribute microbial contaminants to vegetable produce, thereby affecting their quality. Bacterial species associated with retail samples of lettuce were examined by plate culture on Tryptone Soy Agar and PCR-DGGE analysis. Macerates and rinses of lettuce sub-samples with and without addition of Tween 80 were examined to maximize bacterial recoveries. Predominant bacteria isolated by agar culture included species of Pseudomonas, Agrobacterium, Curtobacterium and Burkholderia, at populations of 103-106 cfu/g. PCR-DGGE was unable to recover the same incidence of species as agar culture and failed to detect bacteria in many samples. In some samples, PCR-DGGE detected species of Bacillus, Pseudomonas, Serratia and Acinetobacter, not found by culture. Failure of the PCR-DGGE analyses was attributed to interference by plant chloroplast DNA. Preparative agarose gel electrophoresis of lettuce macerates was necessary to remove chloroplast DNA before application of PCR-DGGE analysis. Thirty percent of lettuce samples contained Acinetobacter species at 101-104 cfu/g when examined after culture on minimal salts agar or enrichment in Baumann enrichment medium. Other Acinetobacter media failed to give reliable isolation of these species from lettuce and salad vegetables. Lettuce could be an environmental source of Acinetobacter nosocomial infections. Juices, solvent extracts and supercritical fluid carbon dioxide extracts of lettuce and capsicum samples did not exhibit antimicrobial action against a range of food spoilage and pathogenic bacteria.

Runoff from agricultural lands carrying microorganisms from livestock manure can contaminate the food and water supplies of both animals and humans. Planning and design of animal waste best management practices (BMPs) thus becomes more important as livestock populations become more concentrated. A computer model is proposed to predict the effects of animal waste BMPs on the bacteria concentration of runoff from agricultural lands. The model uses Monte Carlo simulation to combine the deterministic relationships resulting from previous modeling efforts with statistical knowledge concerning rainfall events and temperature variation. Model output is in the form of monthly maximum and minimum log bacteria concentrations of runoff resulting from a storm assumed to occur immediately after manure is applied to the land. The effects of implementing such BMPs as waste storage, filter strips, and incorporation of manure into the soil can be compared. Data and information collected from the Owl Run watershed in Fauquier County, Virginia is used to demonstrate the model applicability and potential. Long-term manure storage is determined to be the most appropriate practice for reducing bacteria concentrations for the study site. Incorporation of manure is as effective as long-term storage, but requires additional labor. Buffer strips significantly reduce bacteria concentrations, but not as effectively as long-term storage or incorporation. Additional efforts are needed to investigate the most influential variables and to make the temperature simulation submodel more computationally efficient. Once BMPs have been implemented on the study site, more data should be collected to test the accuracy of the model. / Master of Science

Orientador: Wanderley José de Melo / Coorientador: Lúcia Maria Carareto Alves / Banca: Thiago Assis Rodrigues Nogueira / Banca: Estevam Guilherme Lux Hoppe / Resumo: O lodo de esgoto é uma alternativa como fertilizante orgânico na agricultura, porém em sua composição pode apresentar patógenos que oferecem risco ao homem e ao ambiente. Objetivou-se, com o presente estudo, avaliar a fertilidade do solo, e a presença de ovos viáveis de helmintos, coliformes termotolerantes, Escherichia coli para os patótipos EHEC, EPEC e STEC e a atividade enzimática das enzimas protease, redutase do nitrato e urease no solo após dezoito anos de aplicações anuais de lodo de esgoto em um Latossolo Vermelho eutroférrico (LVef) e Latossolo Vermelho distrófico (LVd). O lodo utilizado foi obtido na SABESP de Franca, São Paulo e o experimento foi instalado em delineamento de blocos cazualiados, sendo 4 tratamentos e 5 repetições. Os tratamentos foram T1: controle, apenas com aplicação de adubação mineral, T2: 5, T3: 10 e T4: 20 Mg ha-1 de LE. Antes de ser incorporado ao solo, realizou-se análise do lodo para ovos viáveis de helmintos e coliformes termotolerantes. Aos 40 dias, coletou-se amostras de solo na profundidade de 0-10 cm para avalição de ovos viáveis de helmintos no solo. Aos 70 dias, coletou-se amostras de solo na profundidade de 0-20cm para a análise da fertilidade. Para a análise de coliformes termotolerantes, seguindo a técnica de tubos múltiplos, as amostras foram coletadas no dia 0, 26, 40 e aos 78 dias. Para a realização da reação em cadeia da polimerase (PCR) para identificar a presença de Escherichia coli, coletou-se amostras de solo antes do iní... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sewage sludge is an alternative as organic fertilizer to use in agriculture, but in its composition may have pathogens that offer to humans and the environment risks. The present study objective was to evaluate soil fertility, and the presence of viable helminth eggs, fecal coliforms, Escherichia coli for pathotypes EHEC, EPEC and STEC and the enzymatic activity of protease enzymes, nitrate reductase and urease in the soil after eighteen years of annual applications of sewage sludge in an Oxisol (LVef) and Oxisol (LVd). The sludge used was obtained in SABESP Franca, São Paulo and the experiment was installed in designing cazualiados blocks, 4 treatments and 5 repetitions. Treatments were T1: control, only with application of mineral fertilizer, T2: 5, T3: T4 10 and 20 Mg ha-1 LE. Before being incorporated into the soil, there was sludge analysis for viable helminth eggs and fecal coliforms. At 40 days, it is collected soil samples at a depth of 0-10 cm for viable helminth eggs evaluation in the soil. After 70 days it is collected soil samples at a depth of 0-20cm for fertility analysis. For fecal coliforms analysis, following the technique of multiple pipes, the samples were collected at day 0, 26, 40 and 78 days. To carry out the polymerase chain reaction (PCR) for the presence of Escherichia coli was collected from soil samples before the beginning of the experiment at day 0, after 26 days 40, 58, 78, 110 and 146 days . For the evaluation of enzyme activity, samples wer... (Complete abstract click electronic access below) / Mestre

Modern pig farming production may over burden the environment with organic substances, exposure of bacterial pathogens and introduction of resistance gene. This may be caused by the pig’s droppings, lack of seepage management or accidental spillage of seepage which may impact on the environment and its physicochemical parameters. The objective of this study is to determine and assess the level of bacteriological pollution emanating from the pig farm and their impact on the physicochemical parameters of soil and water as well as to identify the presence of antibiotic resistance gene of these prevailing bacteria. Soil and water samples were collected monthly for a period of six months (March- August 2013). Samples were collected at pig enclosures, soil 20 m and 100 m away from pig enclosures, constructed wetland used for treating pig farm wastewater, soil 20m and 100 m away from constructed wetland. Procedure followed for analysing soil and water samples includes physicochemical analyses, viable cell counts of 10-1 to 10-8 dilutions, identification of bacteria using API 20E test kit, antibiotic susceptibility analyses, and identification of resistance gene using molecular procedures. The media that were used for viable cell counts were, Nutrient agar, MacConkey Agar, Xylose Lysine Deoxycholate agar (XLD agar), and Eosin Methylene Blue (EMB). Physicochemical parameters of water showed unacceptable high levels of analysed parameters for BOD (163 mg/L to 3350 mg/L), TDS (0.77 g/L to 6.48 mg/L), COD (210 mg/L to 9400 mg/L), NO3 (55 mg/L to 1680 mg/L), NO2 (37.5 mg/L to 2730 mg/L), and PO43− (50 mg/L to 1427 mg/L) were higher than the maximum permissible limits set by Department of Water Affairs and Forestry (DWAF). For soil samples TDS (0.01g/L to 0.88 g/L), COD (40 mg/L to 304 mg/L), NO3 (32.5 mg/L to 475 mg/L), and NO2 (7.35 mg/L to 255 mg/L) and PO43- (32.5 mg/L to 475 mg/L ) were observed to be higher than recommended limits set by Federal Ministry for the Environmental (FME). The viable cells in soil samples 30cm depth ranged from 0 cfu/mL to 2.44 x 1010cfu/mL, in soil 5cm depth ranged from 1.00 x 101 cfu/mL to 1.91 x 1010 cfu/mL, and in water samples viable cells ranged from 5.00 x 101 to 5.05 x 109. Pseudomonas luteola (Ps. luteola), Escherichia vulneris (E. vulneris), Salmonella choleraesuis spp arizonae, Escherichia coli 1(E. coli 1), Enterobacter cloacae, Pseudomonas flourescens/putida (Ps. flourescens/putida), Enterobacter aerogenes, Serratia ordoriferal, Pasteurella pneumotropica, Ochrobactrum antropi, Proteus vulgaris group, Proteus vulgaris, Salmonella spp, Aeromonas Hydrophila/caviae/sobria1, Proteus Mirabillis, Vibrio fluvials, Rahnella aquatillis, Pseudomonas aeruginosa (Ps. aeruginosa), Burkholderia Cepacia, Stenotrophomonas maltophilia (St. maltophilia), Shwenella putrefaciens, Klebsiela pneumonia, Cedecea davisa, Serratia liquefaciens, Serratia plymuthica, Enterobacter sakaziki, Citrobacter braakii, Enterobacter amnigenus 2, Yersinia pestis, Serratia ficaria, Enterobacter gergoriae, Enterobacter amnigenus 1, Serratia marcescens, Raoutella terrigena, Hafnia alvei 1, Providencia rettgeri, and Pantoa were isolated from soil and water samples from the pig farm. Isolates were highly resistant to Penicillin G, Sulphamethaxazole, Vancomycin, Tilmocozin, Oxytetracycline, Spectinomycin, Lincomycin, and Trimethoprim. The most resistance genes detected in most isolates were aa (6’)-le-aph (2”)-la, aph (2”)-lb, aph (3”)-llla, Van A, Van B, Otr A and Otr B. Pig farm seepage is causing bacterial pollution which is impacting negatively on the natural environment in the vicinity of pig farm by introducing bacterial pathogens that have an antibiotic resistance gene and is increasing the physicochemical parameters for soil and water in the natural environment at the pig farm. It is therefore recommended that pig farms should consider the need to implement appropriate regulatory agencies that may include the regular monitoring of the qualities of final effluents from waste water treatment facilities. In addition there is a need to limit soil pollution in order to safe guard the natural environment in the vicinity of pig farm from bacteriological pollution and introduction of antibiotic resistance gene. It is also recommended that more advanced technologies should be introduced that will assist pig farms to manages the seepage properly. / Environmental Sciences / M. Sc. (Environmental Sciences)

Modern pig farming production may over burden the environment with organic substances, exposure of bacterial pathogens and introduction of resistance gene. This may be caused by the pig’s droppings, lack of seepage management or accidental spillage of seepage which may impact on the environment and its physicochemical parameters. The objective of this study is to determine and assess the level of bacteriological pollution emanating from the pig farm and their impact on the physicochemical parameters of soil and water as well as to identify the presence of antibiotic resistance gene of these prevailing bacteria. Soil and water samples were collected monthly for a period of six months (March- August 2013). Samples were collected at pig enclosures, soil 20 m and 100 m away from pig enclosures, constructed wetland used for treating pig farm wastewater, soil 20m and 100 m away from constructed wetland. Procedure followed for analysing soil and water samples includes physicochemical analyses, viable cell counts of 10-1 to 10-8 dilutions, identification of bacteria using API 20E test kit, antibiotic susceptibility analyses, and identification of resistance gene using molecular procedures. The media that were used for viable cell counts were, Nutrient agar, MacConkey Agar, Xylose Lysine Deoxycholate agar (XLD agar), and Eosin Methylene Blue (EMB). Physicochemical parameters of water showed unacceptable high levels of analysed parameters for BOD (163 mg/L to 3350 mg/L), TDS (0.77 g/L to 6.48 mg/L), COD (210 mg/L to 9400 mg/L), NO3 (55 mg/L to 1680 mg/L), NO2 (37.5 mg/L to 2730 mg/L), and PO43− (50 mg/L to 1427 mg/L) were higher than the maximum permissible limits set by Department of Water Affairs and Forestry (DWAF). For soil samples TDS (0.01g/L to 0.88 g/L), COD (40 mg/L to 304 mg/L), NO3 (32.5 mg/L to 475 mg/L), and NO2 (7.35 mg/L to 255 mg/L) and PO43- (32.5 mg/L to 475 mg/L ) were observed to be higher than recommended limits set by Federal Ministry for the Environmental (FME). The viable cells in soil samples 30cm depth ranged from 0 cfu/mL to 2.44 x 1010cfu/mL, in soil 5cm depth ranged from 1.00 x 101 cfu/mL to 1.91 x 1010 cfu/mL, and in water samples viable cells ranged from 5.00 x 101 to 5.05 x 109. Pseudomonas luteola (Ps. luteola), Escherichia vulneris (E. vulneris), Salmonella choleraesuis spp arizonae, Escherichia coli 1(E. coli 1), Enterobacter cloacae, Pseudomonas flourescens/putida (Ps. flourescens/putida), Enterobacter aerogenes, Serratia ordoriferal, Pasteurella pneumotropica, Ochrobactrum antropi, Proteus vulgaris group, Proteus vulgaris, Salmonella spp, Aeromonas Hydrophila/caviae/sobria1, Proteus Mirabillis, Vibrio fluvials, Rahnella aquatillis, Pseudomonas aeruginosa (Ps. aeruginosa), Burkholderia Cepacia, Stenotrophomonas maltophilia (St. maltophilia), Shwenella putrefaciens, Klebsiela pneumonia, Cedecea davisa, Serratia liquefaciens, Serratia plymuthica, Enterobacter sakaziki, Citrobacter braakii, Enterobacter amnigenus 2, Yersinia pestis, Serratia ficaria, Enterobacter gergoriae, Enterobacter amnigenus 1, Serratia marcescens, Raoutella terrigena, Hafnia alvei 1, Providencia rettgeri, and Pantoa were isolated from soil and water samples from the pig farm. Isolates were highly resistant to Penicillin G, Sulphamethaxazole, Vancomycin, Tilmocozin, Oxytetracycline, Spectinomycin, Lincomycin, and Trimethoprim. The most resistance genes detected in most isolates were aa (6’)-le-aph (2”)-la, aph (2”)-lb, aph (3”)-llla, Van A, Van B, Otr A and Otr B. Pig farm seepage is causing bacterial pollution which is impacting negatively on the natural environment in the vicinity of pig farm by introducing bacterial pathogens that have an antibiotic resistance gene and is increasing the physicochemical parameters for soil and water in the natural environment at the pig farm. It is therefore recommended that pig farms should consider the need to implement appropriate regulatory agencies that may include the regular monitoring of the qualities of final effluents from waste water treatment facilities. In addition there is a need to limit soil pollution in order to safe guard the natural environment in the vicinity of pig farm from bacteriological pollution and introduction of antibiotic resistance gene. It is also recommended that more advanced technologies should be introduced that will assist pig farms to manages the seepage properly. / Environmental Sciences / M. Sc. (Environmental Sciences)